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Bone Marrow Stromal Stem cells Bone Marrow Stromal Stem cells (bMSCs) (bMSCs) therapy for musculoskeletal therapy for musculoskeletal
problemsproblems ( disc cartilage and bone) ( disc cartilage and bone)
IsmailIsmail HD HD
bullDivision of Orthopaedic and Division of Orthopaedic and TTraumatology raumatology bull Department of SDepartment of Suurgery Faculty of Medicine University Of rgery Faculty of Medicine University Of IIndonesiandonesia
Musculoskeletal problemMusculoskeletal problem
bull Disc
bull Cartilage
bull Bone
Disc and boneDisc and bone
Cells matrix
Bone marrow stromal stem cellsBone marrow stromal stem cells
Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell or mesenchymal stem cellor mesenchymal stem cell
Bone problemBone problemTRAUMA NON-TRAUMA
FRACTURE
ACUTEFRESH NEGLECTED
METABOLIC
CONGENITAL ACQUIRED
DELAYEDNON-UNIONBONE GAPS
SHORTENING
Limb salvage RECONSTRUCTION in malignancy
bullachondroplasiabullCongenital
pseudoarthrosis of tibia
bullCoxa varabullAvascular necrosis
bullosteoporosis
Fracture repair
Reconstruction
BoneBone
Cells matrix
bullOsteoblastbullOsteoclastbullOsteocytes
bullOsteoprogenitor cells
Organic (40) Inorganic (60)
Collagen
Proteoglycans
Noncollagenous osteocalcin osteonectin osteopontin
Growth factor and Cytokines TGF-szlig IGF IL-1 IL-6 BMP
Calsium hydroxyapatite [Ca10(PO4)6(OH)2]
Osteocalsium Phosphate (Brushite)
Regardless of the technique tissue Regardless of the technique tissue engineering requires three engineering requires three components components
o a growth-inducing stimulus (growth a growth-inducing stimulus (growth factor)factor)
responsive cells and responsive cells and a scaffold to support tissue formationa scaffold to support tissue formation
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Musculoskeletal problemMusculoskeletal problem
bull Disc
bull Cartilage
bull Bone
Disc and boneDisc and bone
Cells matrix
Bone marrow stromal stem cellsBone marrow stromal stem cells
Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell or mesenchymal stem cellor mesenchymal stem cell
Bone problemBone problemTRAUMA NON-TRAUMA
FRACTURE
ACUTEFRESH NEGLECTED
METABOLIC
CONGENITAL ACQUIRED
DELAYEDNON-UNIONBONE GAPS
SHORTENING
Limb salvage RECONSTRUCTION in malignancy
bullachondroplasiabullCongenital
pseudoarthrosis of tibia
bullCoxa varabullAvascular necrosis
bullosteoporosis
Fracture repair
Reconstruction
BoneBone
Cells matrix
bullOsteoblastbullOsteoclastbullOsteocytes
bullOsteoprogenitor cells
Organic (40) Inorganic (60)
Collagen
Proteoglycans
Noncollagenous osteocalcin osteonectin osteopontin
Growth factor and Cytokines TGF-szlig IGF IL-1 IL-6 BMP
Calsium hydroxyapatite [Ca10(PO4)6(OH)2]
Osteocalsium Phosphate (Brushite)
Regardless of the technique tissue Regardless of the technique tissue engineering requires three engineering requires three components components
o a growth-inducing stimulus (growth a growth-inducing stimulus (growth factor)factor)
responsive cells and responsive cells and a scaffold to support tissue formationa scaffold to support tissue formation
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Disc and boneDisc and bone
Cells matrix
Bone marrow stromal stem cellsBone marrow stromal stem cells
Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell or mesenchymal stem cellor mesenchymal stem cell
Bone problemBone problemTRAUMA NON-TRAUMA
FRACTURE
ACUTEFRESH NEGLECTED
METABOLIC
CONGENITAL ACQUIRED
DELAYEDNON-UNIONBONE GAPS
SHORTENING
Limb salvage RECONSTRUCTION in malignancy
bullachondroplasiabullCongenital
pseudoarthrosis of tibia
bullCoxa varabullAvascular necrosis
bullosteoporosis
Fracture repair
Reconstruction
BoneBone
Cells matrix
bullOsteoblastbullOsteoclastbullOsteocytes
bullOsteoprogenitor cells
Organic (40) Inorganic (60)
Collagen
Proteoglycans
Noncollagenous osteocalcin osteonectin osteopontin
Growth factor and Cytokines TGF-szlig IGF IL-1 IL-6 BMP
Calsium hydroxyapatite [Ca10(PO4)6(OH)2]
Osteocalsium Phosphate (Brushite)
Regardless of the technique tissue Regardless of the technique tissue engineering requires three engineering requires three components components
o a growth-inducing stimulus (growth a growth-inducing stimulus (growth factor)factor)
responsive cells and responsive cells and a scaffold to support tissue formationa scaffold to support tissue formation
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Bone marrow stromal stem cellsBone marrow stromal stem cells
Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell Skeletal stem cells (SSCs) also known as bone-marrow stromal stem cell or mesenchymal stem cellor mesenchymal stem cell
Bone problemBone problemTRAUMA NON-TRAUMA
FRACTURE
ACUTEFRESH NEGLECTED
METABOLIC
CONGENITAL ACQUIRED
DELAYEDNON-UNIONBONE GAPS
SHORTENING
Limb salvage RECONSTRUCTION in malignancy
bullachondroplasiabullCongenital
pseudoarthrosis of tibia
bullCoxa varabullAvascular necrosis
bullosteoporosis
Fracture repair
Reconstruction
BoneBone
Cells matrix
bullOsteoblastbullOsteoclastbullOsteocytes
bullOsteoprogenitor cells
Organic (40) Inorganic (60)
Collagen
Proteoglycans
Noncollagenous osteocalcin osteonectin osteopontin
Growth factor and Cytokines TGF-szlig IGF IL-1 IL-6 BMP
Calsium hydroxyapatite [Ca10(PO4)6(OH)2]
Osteocalsium Phosphate (Brushite)
Regardless of the technique tissue Regardless of the technique tissue engineering requires three engineering requires three components components
o a growth-inducing stimulus (growth a growth-inducing stimulus (growth factor)factor)
responsive cells and responsive cells and a scaffold to support tissue formationa scaffold to support tissue formation
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Bone problemBone problemTRAUMA NON-TRAUMA
FRACTURE
ACUTEFRESH NEGLECTED
METABOLIC
CONGENITAL ACQUIRED
DELAYEDNON-UNIONBONE GAPS
SHORTENING
Limb salvage RECONSTRUCTION in malignancy
bullachondroplasiabullCongenital
pseudoarthrosis of tibia
bullCoxa varabullAvascular necrosis
bullosteoporosis
Fracture repair
Reconstruction
BoneBone
Cells matrix
bullOsteoblastbullOsteoclastbullOsteocytes
bullOsteoprogenitor cells
Organic (40) Inorganic (60)
Collagen
Proteoglycans
Noncollagenous osteocalcin osteonectin osteopontin
Growth factor and Cytokines TGF-szlig IGF IL-1 IL-6 BMP
Calsium hydroxyapatite [Ca10(PO4)6(OH)2]
Osteocalsium Phosphate (Brushite)
Regardless of the technique tissue Regardless of the technique tissue engineering requires three engineering requires three components components
o a growth-inducing stimulus (growth a growth-inducing stimulus (growth factor)factor)
responsive cells and responsive cells and a scaffold to support tissue formationa scaffold to support tissue formation
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
BoneBone
Cells matrix
bullOsteoblastbullOsteoclastbullOsteocytes
bullOsteoprogenitor cells
Organic (40) Inorganic (60)
Collagen
Proteoglycans
Noncollagenous osteocalcin osteonectin osteopontin
Growth factor and Cytokines TGF-szlig IGF IL-1 IL-6 BMP
Calsium hydroxyapatite [Ca10(PO4)6(OH)2]
Osteocalsium Phosphate (Brushite)
Regardless of the technique tissue Regardless of the technique tissue engineering requires three engineering requires three components components
o a growth-inducing stimulus (growth a growth-inducing stimulus (growth factor)factor)
responsive cells and responsive cells and a scaffold to support tissue formationa scaffold to support tissue formation
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Regardless of the technique tissue Regardless of the technique tissue engineering requires three engineering requires three components components
o a growth-inducing stimulus (growth a growth-inducing stimulus (growth factor)factor)
responsive cells and responsive cells and a scaffold to support tissue formationa scaffold to support tissue formation
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Tissue Engineering TriadTissue Engineering Triad
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Core conceptCore concept
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
factors regulating stem cell self-factors regulating stem cell self-renewal (Wntsrenewal (Wntshedgehog Dickopf) hedgehog Dickopf) versus differentiation (BMPsversus differentiation (BMPsTGF TGF beta OP-1)may have to be provided beta OP-1)may have to be provided in anin an appropriate temporal appropriate temporal sequencesequence
Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
bMSCs MSCbMSCs MSC differentiated differentiated undifferentiatedundifferentiated
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
there is no advantage of implantation of differentiated there is no advantage of implantation of differentiated MSCsMSCs
Transplantation of mesenchymal stromal cells on Transplantation of mesenchymal stromal cells on
mineralized collagen leads to ectopic matrix synthesis in mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation vivo independently from prior in vitro differentiation
Authors Authors Niemeyer PNiemeyer P11 Kasten P Kasten P22 Simank H- Simank H-GG22 Fellenberg J Fellenberg J22 Seckinger A Seckinger A33 Kreuz Pc Kreuz Pc11 Mehlhorn Mehlhorn AA11 Suumldkamp Np Suumldkamp Np11 Krause U Krause U33
SourceSource Cytotherapy Volume 8 Number 4 August 2006 Cytotherapy Volume 8 Number 4 August 2006 pp 354-366(13)pp 354-366(13)
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
In a study of geneIn a study of gene therapy targeting stem cells from therapy targeting stem cells from individuals with osteogenesisindividuals with osteogenesis imperfecta the transduced imperfecta the transduced cells were cultured forcells were cultured for 2 weeks in bonendashdifferentiation-2 weeks in bonendashdifferentiation-inducing media beforeinducing media before transplantation in vivo to show bone transplantation in vivo to show bone formationformation
TheseThese results imply that in vitro differentiation of stem cell results imply that in vitro differentiation of stem cell populationspopulations can improve bone formation Unfortunately can improve bone formation Unfortunately thisthis manipulation considerably manipulation considerably c complicates the process of omplicates the process of obtainingobtaining regulatory approval for clinical applicationregulatory approval for clinical application
bullChamberlain JR Schwarze U Wang P-R et al Gene targeting in stem cells from individuals with osteogenesis imperfecta Science 3031198ndash1201 2004 Sharp JG CLINICAL ORTHOPAEDICS AND RELATED RESEARCH2005Number 435 pp 52ndash61
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Induction of MSCs to Induction of MSCs to OsteoblastOsteoblast
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Characterization of Characterization of OsteoblastsOsteoblasts
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
mimics the extracellular matrix inmimics the extracellular matrix in regenerating bone environmregenerating bone environmentent NotNot a simple mechanical support but has to be lsquoinformativersquoa simple mechanical support but has to be lsquoinformativersquo to the cells to the cells A proper biomaterial should easily integrateA proper biomaterial should easily integrate with the adjacent bone and with the adjacent bone and
favor new bone tissuefavor new bone tissue ingrowth (osteo-conduction) Its architecture shouldingrowth (osteo-conduction) Its architecture should allow host blood vessels to colonize even the largestallow host blood vessels to colonize even the largest structures structures
biocompatiblebiocompatible and resorbable and resorbable
platelet-rich plasma (PRP)platelet-rich plasma (PRP) Collagen type 1Collagen type 1 HydroxyapatiteHydroxyapatite Calsium phosphateCalsium phosphate Polymers Poly(lactide-co-glycolide) (PLGA)Polymers Poly(lactide-co-glycolide) (PLGA)
scaffoldsscaffolds
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Bone Morphogenic Proteins (BMPs)Bone Morphogenic Proteins (BMPs) Osteogenic ProteinsOsteogenic Proteins TGF-szligTGF-szlig
Growth factorGrowth factor
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
TISSUE ENGINEERING APPROACH TO BONE REPAIR BY AUTOLOGOUS BMSCs LOADED ON SUPPORT SCAFFOLDS
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Topic study cells scaffold
The effects of transplantation of osteoblastic cells with bone morphogenetic protein (BMP)carrier complex on bone repair
In vitro and in vivo (Sprague-Dawley Rats)
Bone vol 29 no2 august 2001169-75
OsteoblasticBMP Poly-DL-lactic-co-glycolic acidgelatin sponge (PGS)
Engineered allogeinic mesenchymal stem cells repair femoral segmental defect in rats
Animal study (rat)
J Orthop Res 21 (2003) 44-53
MSC engineered with the gene for BMP-2
Collagen gel (vitrogen 100 95-98 type 1)
Calcium phosphate scaffold and bone marrow for bone reconstruction inirradiated area a dog study
animal model (dog)
Bone 36 (2005) 323ndash 330
Bone marrow macroporous biphasiccalcium phosphate bone substitute (MBCPk BiomatlanteVigneux de Bretagne France)
Treatment of Long Tubular Bone Defect of Rabbit Using AutologousCultured Osteoblasts Mixed with Fibrin
Animal model (NewZealand white rabbits )J of Korean Orthopaedic SocietyVolume 9 Number 1 May 2006
autologous cultured osteoblasts
fibrin
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Topic study cells scaffoldsBone Formation of Cultured Osteoblasts in Bone Defect of RadialShaft of Rabbit
Animal model (NewZealand white rabbits )
J of Korean Orthop Assoc 2005 40 453-459
autologous cultured osteoblasts
Theeffect of implants loaded with autologous mesenchymal stem cellson the healing of canine
segmental bone defects
non-union defect in adult dog femora
J Bone Joint Surg Am1998 80 985ndash996
Autologous MSC
hydroxyapatite beta-tricalciumphosphate (6535)
Autologous bonemarrow stromal cells loaded onto porous hydroxyapatite ceramicaccelerate bone repair in critical-size defects of sheep long bones
full-thickness gaps of tibia diaphysis in adult sheep
J Biomed Mater Res 200049 328ndash337
Autologous bonemarrow stromal cells
bioceramic composites
Transplantation of marrow-derived mesenchymal stem cells andplatelet-rich plasma during distraction osteogenesismdasha preliminary result of three cases
Clinical study achondroplasia patients bilaterally and three femoral and one tibiallengthenings were done in four patients because of a limb length discrepancy which was secondary to trauma (twolimbs) congenital pseudarthrosis of the tibia (one) and developmental coxa vara (one)Bone 35 (2004) 892ndash 898
osteoblast-like cells
platelet-richplasma (PRP)
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Topic study cells scaffolds
Enhanced Tibial Osteotomy Healing with Use ofBone Grafts Supplemented with PlateletGel or Platelet Gel and Bone Marrow Stromal Cells
A prospective randomized controlled study Thirty-three patients undergoing high tibial osteotomy to treat genu varum J Bone J SurgBr2007 11
Osteoblast
Platelet gel
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Topic study cells scaffolds
Treatment of Osteonecrosisof the Femoral Head withImplantation of AutologousBone-Marrow Cells
prospective cohort study Thirteen patients (eighteen hips) with stage-I or II osteonecrosis of the femoral head J Bone J SurgAm 2004
autologous bone-marrow mononuclear cells
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Study by Aziz NatherDepartment of orthopaedic
surgeryNational University of
Singapore
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
ConclusionConclusion1048708Addition of MSCs improved the biological
healing of the inert allografts1048708Addition of PRP did not have the same effect
1048708
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
A major unsolved problem possibly A major unsolved problem possibly lies in thelies in the development of the most development of the most appropriate matrix whichappropriate matrix which should be should be biodegradable yet resistant permit biodegradable yet resistant permit cell infiltrationcell infiltration and adequate and adequate survival proliferation and survival proliferation and differentiationdifferentiation of cells and also of cells and also provide integration with theprovide integration with the pre-pre-existing bony flankinexisting bony flanking the lesion g the lesion sitessites
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
a mixture or a temporal a mixture or a temporal administration of stem andadministration of stem and differentiated cell differentiated cell vs vs undifferentiated undifferentiated populations with populations with matrix andor growthmatrix andor growth factors might factors might prove to be the optimal approachprove to be the optimal approach
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Disc ProblemDisc Problem
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Disc Disc
Cells matrix
Nucleus pulposus
chondrocyte-like cell
Inner annulus
Chondrocyte-like cells
Outer annulus
fibroblast or fibrocyte-like-cells
End-plate
chondrocyte
Water and proteoglycans increase from outer the annulus to the
inner nucleus and in contrast collagens are inversely distributed
Proteoglycans aggrecans versican decorin biglycan fibromodulin lumican and perlecanCollagen type II
Collagen type Iproteoglycans
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Bone Marrow Stromal Stem cells (bMSCs) Bone Marrow Stromal Stem cells (bMSCs) transplantation and intervertebral disc transplantation and intervertebral disc
distraction to reverse Intervertebral Disc distraction to reverse Intervertebral Disc Degeneration Degeneration in rabbit modelin rabbit model
IsmailIsmail Hee Hwan Tak Nuryati C Siregar James Hee Hwan Tak Nuryati C Siregar James CH GohWong Hee Kit Subroto Sapardan CH GohWong Hee Kit Subroto Sapardan
bullDivision of Orthopaedic and traumatology Department of Sirgery Faculty of Division of Orthopaedic and traumatology Department of Sirgery Faculty of Medicine University Of ndonesiaMedicine University Of ndonesia
Department of Pathology Anatomy Faculty Of Medicine University Of Department of Pathology Anatomy Faculty Of Medicine University Of IndonesiaIndonesia
Department of Orthopaedic Surgery National University Hospital Department of Orthopaedic Surgery National University Hospital SingaporeSingapore
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Intervertebral Disc Intervertebral Disc Degeneration (IDD)Degeneration (IDD)
IDD is a multifaceted chronic process IDD is a multifaceted chronic process involving certain unfavorable involving certain unfavorable progressive changes in disc progressive changes in disc composition configuration and composition configuration and function occurring more quickly and or function occurring more quickly and or with greater gravity than those with greater gravity than those associated with normal aging and associated with normal aging and often associated with clinical often associated with clinical symptomssymptoms
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
alterationalteration
CELL EXTRACELLULER MATRIX
Failure of nutrient supply
bMSCs
Intervertebral disc distraction
bull restore disc height
bull uarr diffusion
bull uarr disc nutrition
Regenerationreparation bull Differentiated bMSCs can
be
survively transplanted in
degenerated intervertebral disc
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
ObjectivesObjectives
reverse intervertebral disc reverse intervertebral disc degeneration in rabbit model degeneration in rabbit model
Transplantation of bMSCs and or distraction of the intervertebral disc (IVD)
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
M E T H O D S
three sequential stages of three sequential stages of experiments were designed experiments were designed
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
STAGE 1 Pilot Study STAGE 1 Pilot Study STUDY TO STUDY TO CREATE INTERVERTEBRAL DISC CREATE INTERVERTEBRAL DISC DEGENERATION MODEL IN RABBIT DEGENERATION MODEL IN RABBIT MODELMODEL
Intervertebral disc degeneration was Intervertebral disc degeneration was created in rabbit model through the created in rabbit model through the application of controlled and application of controlled and quantified axial mechanical loadingquantified axial mechanical loading
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Study on Study on 1 Developing of external compression and distraction device1 Developing of external compression and distraction device
External compression device External distraction device
Stage 2 Interphase stage Stage 2 Interphase stage
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
2 Measuring of cross-sectional 2 Measuring of cross-sectional area of area of
intervertebral disc of the rabbit intervertebral disc of the rabbit
L0L1
Ln-1Ln
Ln + 1
L2
Mean of Mean of cross-sectional area = cross-sectional area = 7121 7121 ++ 64 mm 64 mm22 701 701 ++ 461 mm 461 mm22
computerized Manual
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
3 Establishing of method of isolation 3 Establishing of method of isolation culture and preparation of transplantation culture and preparation of transplantation
of of bone marrow stromal stem cellsbone marrow stromal stem cells
Illiac crest bone marrow
aspiration from rabbit donor Day 30 x 40
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Cultured bMSCs not more than at Cultured bMSCs not more than at
passage 1 are labeling passage 1 are labeling Carboxyfluorescein diacetate (CFDA) Carboxyfluorescein diacetate (CFDA) and embedded in atelocollagen and embedded in atelocollagen solution until a final cell density of 1 solution until a final cell density of 1 x 10x 1066 cellsml cellsml
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
In vitro Study In vitro Study Day 30 bMSCs embedded in atelocollagenDay 30 bMSCs embedded in atelocollagen
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Stage 3 Stage 3 intervention stage (bMSCs intervention stage (bMSCs transplantation and or distraction of transplantation and or distraction of
the intervertebral disc)the intervertebral disc)
DesignDesign Experimental Experimental post test only control post test only control
group designgroup design
LocationLocation Animal Holding Unit and NUSTEP (National University Animal Holding Unit and NUSTEP (National University
Tissue Engineering Project) Department of Orthopaedic Tissue Engineering Project) Department of Orthopaedic Surgery National University Hospital (NUH) and Eijkman Surgery National University Hospital (NUH) and Eijkman Institute Jakarta and Faculty of MedicineUniversity of Institute Jakarta and Faculty of MedicineUniversity of IndonesiaIndonesia
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
kept alive for 8 weeks
24 new zealand white rabbits average weight 334 kg
Group 1 Group 2 Group 4 Group 3
External compression device 23 MPa loaded into L4-L5 for 14 days
External compression device was removed
8 weeks unload
sacrificed
bMSCs transplantation Distraction of the intervertebral disc
bMSCs and IVD
distraction
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Tissue preparation 22 days
Lateral digital X-ray Micro-CT scan rarr disc height
Macroscopic morphological grade (Thomson et al)Microscopic histological score (Masuda and Booset al)
and proteoglycans grade (Norcross et al)
Cell viability (Tunel reaction) and cell labeling (CFDA)
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Surgical procedureSurgical procedure
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
axial stress to the disc 5 times the animalrsquos axial stress to the disc 5 times the animalrsquos body weight equivalent to 23 MPa for two body weight equivalent to 23 MPa for two weeksweeks
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
transplantation of 008 ml bMSCs solution are injected into transplantation of 008 ml bMSCs solution are injected into disc by disc by posterolateral approach and image intensifier guided posterolateral approach and image intensifier guided through a 25-gauge syringethrough a 25-gauge syringe
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Image intensifier-guidedImage intensifier-guided
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Cell was additionally analyzed Cell was additionally analyzed of viablepositive cells from of viablepositive cells from the fresh disc specimen by the fresh disc specimen by cell labeling of bMSCs with cell labeling of bMSCs with carboxyfluorescein diacetate carboxyfluorescein diacetate (CFDA) to know live cells are (CFDA) to know live cells are come from bMSCs come from bMSCs transplantation transplantation
Cell labeling
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
In vivo Cell labeling 8 weeks In vivo Cell labeling 8 weeks after transplantationafter transplantation
group 1 (bMSCs)
group 4 (distraction and bMSCs)
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Better disc height and higher Better disc height and higher proteoglycan content will be proteoglycan content will be obtained if bMSCs transplantation is obtained if bMSCs transplantation is combined with intervertebral disc combined with intervertebral disc distraction like what are shown in distraction like what are shown in group 4 group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) the disc height the disc height uarruarr 1665 on lateral view 1665 on lateral view
and 1084 on frontal viewand 1084 on frontal view
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Group 3 Group 1 Group 2 Group 4
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
In the group 4 (bMSCs and In the group 4 (bMSCs and distraction) distraction) pproteoglycan grade attained 75 on roteoglycan grade attained 75 on
the fourth grade and 25 on the fifth the fourth grade and 25 on the fifth gradegrade
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Axial distraction of the IVD will not Axial distraction of the IVD will not only only uarr uarr hidration but also increase disc hidration but also increase disc nutrition nutrition
Disc rehidration will Disc rehidration will uarruarr hidrostatic hidrostatic pressure that subsequently will pressure that subsequently will stimulate matrix gene expression stimulate matrix gene expression and and produce increased extra cellular produce increased extra cellular matrix protein expressionmatrix protein expression
Kroeber M Unglaub F Guehring T Nerlich A Hadi T Lotz J et al Spine 2005 2181-7 Guehring T Omlor GW Lorenz H Engelleiter K Richter W Kroeber M Spine 2006151658-65
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
The group 4 also showed the best The group 4 also showed the best histological score among all groups histological score among all groups
bMSCs transplantation followed by an bMSCs transplantation followed by an increase in disc rehidration gave a good increase in disc rehidration gave a good result on cells and matrix extra cellular result on cells and matrix extra cellular repairrepair
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
The group 2 (distraction of the IVD) The group 2 (distraction of the IVD) least dead cells count (7335 least dead cells count (7335 ++ 2155) 2155)
and significantly difference with and significantly difference with control group in all parameters control group in all parameters
Distraction of the IVD Distraction of the IVD darrdarr matrix-degradation enzyme such matrix-degradation enzyme such
as MMP-13 which can cause as MMP-13 which can cause apoptosis pathways to decreaseapoptosis pathways to decrease
Guehring T et al Spine 2006 15 1658ndash1665
Group 3
Group 2
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Clinical Implication and Further Clinical Implication and Further StudyStudy
Aspect of bMSCs
Aspect of IVD Distraction
bull Regeneration
bull Fusion
bull In vitro
bull In vivo
bullConservative
bullOperative
bullIn vitro
bullIn vivo
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
bMSCs bMSCs transplantatiotransplantatio
nn
distraction of distraction of the IVDthe IVD
to reverse intervertebral disc to reverse intervertebral disc degeneration in the rabbit degeneration in the rabbit
oror
bMSCs bMSCs transplantation transplantation and distraction and distraction
of the IVDof the IVD
bull uarr uarr disc height on lateral and frontal disc height on lateral and frontal viewviewbull Good and statistically significance Good and statistically significance in in histological score morphological histological score morphological and and proteoglycan grade (except bMSCs proteoglycan grade (except bMSCs group in morphological grade)group in morphological grade)bull darr darr the dead cell countthe dead cell count
uarr uarr restoration IDD toward restoration IDD toward regeneration regeneration in term of in term of lateral projection disc lateral projection disc height increase and height increase and histological score histological score
although not statistically although not statistically significant difference significant difference compared to bMSCs compared to bMSCs
transplantation only or transplantation only or IVD distractionIVD distraction
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Cell Therapy and Cell Therapy and Stem cells for Cartilage DefectStem cells for Cartilage Defect
Courtesy of Courtesy of Dr Andri Lubis Sp OTDr Andri Lubis Sp OT
Orthopaedic and Traumatology DivisionOrthopaedic and Traumatology Division
Faculty of Medicine University of Faculty of Medicine University of IndonesiaIndonesia
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
cartilage MFC denuded 3x5 cm
Osteochondral lesion
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
JBJS Am 2006882502-20
Autologous Chondrocyte Implantation (ACI)
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
JBJS Am 2006882502-20
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
ACI TechnologyACI Technology
Brittberg et al NEJM 1994 331889-95
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Matrix-carried Autologous Matrix-carried Autologous Chondrocyte Implantation Chondrocyte Implantation
(MACI)(MACI)
Cultivated autologous Cultivated autologous chondrocytes seeded in a 3-D chondrocytes seeded in a 3-D scaffold made of type IIII scaffold made of type IIII collagen membranecollagen membrane
Matrix is remodelled within a few Matrix is remodelled within a few months and replaced by the months and replaced by the extracellular matrix of the extracellular matrix of the regenerateregenerate
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Key Elements of MACIKey Elements of MACI
Autologous Chondrocytes controlled cell density Autologous Chondrocytes controlled cell density 10M 10M Collagen membrane bio-degradable gt6MCollagen membrane bio-degradable gt6M Fibrin glue promote cell migration and growth (Zheng Fibrin glue promote cell migration and growth (Zheng
et al 2005)et al 2005) Histology of MACI objective evidence (Zheng et al Histology of MACI objective evidence (Zheng et al
2006)2006) Functional Assessment achievement gt80 of good to Functional Assessment achievement gt80 of good to
excellent (Robinson et al 2007)excellent (Robinson et al 2007)
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
33rdrd Generation ACI Generation ACI
Do not need periosteal patchDo not need periosteal patch Example Fibrin Matrix ACIExample Fibrin Matrix ACI
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
J Kor Arthros Soc 2007111-5
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Bone Marrow-Derived Bone Marrow-Derived Mesenchymal Stem CellsMesenchymal Stem Cells
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
BMSCs StepsBMSCs Steps
Taking the stem cell from illiac crestTaking the stem cell from illiac crest Culture the stem cells Culture the stem cells 4 weeks lab 4 weeks lab Debridement the lessionDebridement the lession Taking tibial periosteumTaking tibial periosteum Suturing the periost to the defect Suturing the periost to the defect
implantation of stem cells implantation of stem cells completing the suturecompleting the suture
Fibrin glueFibrin glue
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Courtesy of Prof James Hui
National University Hospital Singapore
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Courtesy of Prof James Hui NUH
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Radiographics 2007 27207-22
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Eur Radiol 2007 17103ndash18
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
The success of Stem Cells The success of Stem Cells TherapyTherapy
Need good collaboration among Need good collaboration among surgeons laboratory technicians surgeons laboratory technicians and stem cells laboratory and stem cells laboratory
Dr Teo Cheng Peng
Parkway Cancer Center Srsquopore
Kompas March 16 2007 p 42
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
Tissue Engineering and Tissue Engineering and Cell Based TherapiesCell Based Therapies
a 21a 21stst Century Treatment Century Treatment
Dr Caplan
