Aus der Poliklinik für Kieferorthopädie, Präventive Zahnmedizin und Kinderheilkunde
(Direktor: Prof. Dr. med. dent. T. Gedrange) Im Zentrum für Zahn-, Mund-, und Kieferheilkunde
(Geschäftsführender Direktor: Prof. Dr. med. dent. Dr. h.c.G. Meyer) der Medizinischen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald
CHANGES IN INSULIN LIKE GROWTH FACTORS, MYOSTATIN
AND VASCULAR ENDOTHELIAL GROWTH FACTOR
IN RAT MUSCULUS LATISSIMUS DORSI
BY POLY-3-HYDROXYBUTYRATE IMPLANTS
Inaugural-Dissertation
zur
Erlangung des akademischen Grades
Doktor der Zahnmedizin
(Dr. med. dent.)
der
Medizinischen Fakultät
der
Ernst-Moritz-Arndt-Universität
Greifswald
2009
vorgelegt von Dr.Tomasz Gredes
geboren am 20.08.1975
in Dzierżoniów (Polen)
Dekan: Univ.- Prof. Dr. rer. nat. H. K. Kroemer
Erster Gutachter: Prof. Dr. T. Gedrange
Zweiter Gutachter: PD Dr. A. Lupp
Ort, Raum: Greifswald, Hörsaal der Neuen Zahnklinik
Tag der Disputation: 17.02.2010
2
Moim Rodzicom, Babci i Siostrze
3
Contents List of abbreviations p. 5
1. Introduction p. 7
1.1. Regulation of bone remodelling p. 7
1.2. Bone formation p. 10
1.3. Bone surrogates p. 11
1.4. Poly-3-hydroxybutyrate – PHB p. 14
1.5. Aim of this study p. 16
2. Materials and Methods p. 18
2.1. Poly-3-hydroxybutyrate (PHB) p. 18
2.2. Experimental Design and Surgical Procedure p. 18
2.3. RNA-Isolation p. 20
2.4. Agarose gel electrophoresis p. 22
2.5. Reverse Transcriptase — Real-Time RT-PCR p. 23
2.6. Quantification of the gene expression - 2(-ΔΔCT) method p. 26
2.6.1. Derivation of the 2-ΔΔCT method p. 26
2.7. Statistical analysis p. 28
3. Results p. 29
3.1. Isolation of the total RNA from muscle samples p. 29
3.2. Examination of the primer specificity using standard RT-PCR p. 31
3.3. Quantification of the VEGF, IGF1, IGF2, and GDF8 mRNA expression p. 31
4. Discussion p. 35
5. References p. 39
6. Summary p. 48
7. Supplement p. 49
Legends of tables p. 56
Legends of figures p. 57
Affirmation p. 58
Acknowledgment p. 59
Curriculum vitae p. 60
Publication
4
List of abbreviations
bp base pairs
BMP bone morphogenetic protein
BMSC bone marrow stromal cell
BMU basic multicellular unit
cDNA complementary desoxyribonucleic acid
CSF colony stimulating factor
e.g. exempli gratia
etc. et cetera
CT threshold cycle
DNA desoxyribonucleic acid
FGF fibroblast growth factor
GDF8 growth differentiation factor 8
HB D,L-β-hydroxybutyrate
IGF insulin-like growth factor
IL interleukin
M. musculus
MMSC multipotent mesenchymal stem cell
mRNA messenger ribonucleic acid
MSC mesenchymal stem cell
MyHC myosin heavy chain
PCR polymerase chain reaction
PDGF platelet derived growth factor
PHB poly (3-hydroxybutyrate)
5
PMN polymorphonuclear neutrophil
RNA ribonucleic acid
rRNA ribosomal ribonucleic acid
RT reverse transcription
RT-PCR real-time polymerase chain reaction
Runx2 runt-related transcription factor 2
S.E.M standard error of the mean
SSC skeletal stem cell
TGF transforming growth factor
TNF tumour necrosis factor
VEGF vascular endothelial growth factor
vis. videlicet
vs. versus
6
1. Introduction
1.1. Regulation of bone remodelling
Bone is a dynamic tissue that constantly undergoes remodelling. The signal that initiates bone
remodelling has not been identified yet, but there is evidence that mechanical stress can alter
local bone architecture. This can be followed at osteocytes secreting paracrine factors such as
insulin-like growth factor (IGF-1) in response to mechanical forces (Lean et al. 1996).
This complex process requires interaction between different cell phenotypes regulated by
various biochemical and mechanical factors. This is a balance between the amount of bone
resorbed by osteoclasts and the amount of bone formed by osteoblast (Frost 1964). Bone
remodelling occurs in small packets of cells called basic multicellular units (BMU), which
turn over bone in multiple bone surfaces (Frost 1991). BMU consist of osteoblasts, other
bone-forming cells such as osteocytes and bone-lining cells, bone-resorbing cells osteoclasts,
the precursor cells of both, and their associated cells like endothelial and nerve cells
(Papachroni et al. 2009).
Osteoblasts are key components of the bone multi-cellular unit and play a seminal role in
bone remodelling, which is an essential function for maintenance of the structural integrity
and metabolic capacity of the skeleton. Osteoblasts originate from the non-hematopoietic part
of bone marrow which contains a group of fibroblast-like stem cells with osteogenic
differentiation potential, known as mesenchymal stem cells (MSCs) and also referred as
skeletal stem cells (SSCs), bone marrow stromal cells (BMSCs) or multipotent mesenchymal
stromal cells (MMSCs) (Abdallah and Kassem 2008; Heino and Hentunen 2008). MSCs are
capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts,
adipocytes, and chondrocytes (Dezawa et al. 2004; Luk et al. 2005).
Osteoblast growth and differentiation is determined by a complex array of growth factors and
signalling pathways. The following three families of growth factors influence the main
aspects of osteoblast activity and induce, mediate or modulate the effects of other bone
growth regulators:
— the transforming growth factor-β (TGF-β) family that promotes osteoblast
differentiation
— the insulin-like growth factors (IGFs), which induce osteoblastogenesis via activation
of Osterix gene expression
7
— bone morphogenetic proteins (BMPs), the autocrine and paracrine anabolic action
mediated by their specific receptors (Zhou et al. 1993; Mundy 1994; Bikle 2008).
Furthermore, other growth factors, such as the vascular endothelial growth factor (VEGF) as
well as platelet derived growth factor (PDGF) are involved in osteoblast differentiation and
summarized in table 1.
Table 1. Main growth factors and its function in osteoblast differentiation.
Growth factor Effects on osteoblasts References
IGFs
IGF-1 triggers osteoblast proliferation, increases bone collagen synthesis and decreases collagen degradation
IGF-I and -II promote Osterix (Osx) expression in osteoblastic cells, trigger osteoblast induction in vitro and a transient increase in bone mass in vivo
(Tang et al. 2006; Bikle 2008)
TGF-β family
Stimulate the production and deposition of ECM proteins
Potent inducers of committed bone cell replication and osteoblast matrix production
(Ahdjoudj et al. 2002; Ito and Miyazono 2003)
BMPs
Autocrine and paracrine action mediated by their kinase receptors
Induce early precursor bone cell replication and osteoblast commitment
(Duncan 1995; Zhao et al. 2000)
VEGF
Regulates vascularization of developing bone and osteoclast activity, involved in bone repair
Key component of a chondrocyte survival pathway, controls osteoblastic activity
(Zelzer and Olsen 2005; Papadopoulou et al. 2007)
PDGF
Potent mitogen and chemoattractant for target cells such as diploid fibroblasts and osteoblasts
(Kim et al. 2007)
8
Mechanisms that promote skeletal tissue specificity are necessary, because none of these
growth factors are specific for cells in the osteoblastic lineage. They involve interactions with
other circulating hormones (including glucocorticoids, sex steroids, parathyroid hormone or
prostaglandin E2) in addition to the action of specific intracellular mediators on bone-specific
transcription factors. It is certain that bone remodelling is regulated by systemic hormones
and by local factors (table 2 and 3) (Canalis 1983), which affect cells of both the osteoclast
and osteoblast lineages and exert their effects on the replication of undifferentiated cells, the
recruitment of cells, and the differentiated function of cells (Hill 1998).
Table 2. Local factors that regulate bone remodelling.
Growth factors that regulate bone remodelling
Fibroblast growth factors (FGF) Selected cytokines of the interleukin (IL)
Tumour necrosis factor (TNF) Colony-stimulating factor (CSF)
Table 3. Summary of bone remodelling regulating hormones.
Hormones that regulate bone remodelling
Polypeptide hormones
Parathyroid hormone Calcitonin
Insulin Growth hormone
Steroid hormones
1,25-Dihydroxyvitanin D3 Glucocorticoids
Sex steroids
Thyroid hormones
9
Local factors have effects on cells of the same class (autocrine factors) or on cells of another
class within the tissue (paracrine factors). The presence of local factors is not unique to the
skeletal system, because non-skeletal tissues also synthesize, and respond to autocrine and
paracrine factors. Growth factors are also present in the circulation and may act as systemic
regulators of skeletal metabolism, but the locally produced factors have more direct and
important functions in cell growth (Hill et al. 1997).
Besides growth factors and regulating hormones, expression of transcription factors is
necessary and sufficient for mesenchymal cell differentiation. Runx2 is an essential bone-
specific transcription factor. It was recently shown, that the complete Runx2 gene inactivation
in transgenic mice leads to complete lack of intramembraneous and endochondral ossification
owing to lack of mature osteoblasts (Komori et al. 1997). In addition, heterozygous Runx2
mice demonstrate specific skeletal abnormalities that are also characteristic for the human
heritable skeletal disorder cleidocranial dysplasia (Otto et al. 1997).
1.2. Bone formation Bone formations results from a complex cascade of events involving proliferation of primitive
mesenchymal cells (osteoinduction), differentiation into osteoblast precursor cells, maturation
of osteoblasts, formation of matrix, and finally mineralization figure 1). Osteoblasts converge
at the bottom of the resorption cavity and form osteoid which begins to mineralize after 13
days. The osteoblasts continue to form and mineralize osteoid until the cavity is filled. At the
bottom of the cavity osteoblasts are plump and vigorous, they have tall nuclei, and they make
a thick layer of osteoid. The cells gradually flatten and become quiescent lining cells. Some of
the osteoblasts differentiate into osteocytes and become embedded in the matrix. The initial
event must be chemotactic attraction of osteoblasts or their precursors to sites of the
resorption defect. This is likely to be mediated by local factors produced during the resorption
process. The second event involved in the formation phase of the coupling phenomenon is
proliferation of osteoblast precursors. This is likely to be mediated by osteoblast-derived
growth factors and those growth factors released from bone during the resorption process (see
tables 1-2). The third event in the formation phase is the differentiation of the osteoblast
precursor into the mature cell. Several of the bone-derived growth factors can cause the
appearance of markers of the differentiated osteoblast phenotype, including expression of
alkaline phosphatase activity, type I collagen, and osteocalcin (Hill et al. 1997).
10
Bone Formation
Figure 1. Regulation of bone formation (Roodman 2004).
1.3. Bone surrogates
One of the difficult clinic problems is a bone defect. These defects can be caused by
inflammation, congenital malformation, trauma or oncological surgery. The bone defects can
be a limiting factor in achievement of optimal orthodontic treatment. The conventional
biological methods of bone-defect management include autografting and allografting
cancellous bone, applying vascularised grafts of the fibula and iliac crest. The standard
treatments were continually improved and additionally new methods were searched (Burg et
al. 2000). The autografts often require longer operating time with the probability of infection,
pain, and hematoma. The allografting introduces the risk of disease and/or infection. It may
cause a lessening or complete loss of the bone inductive factors. Furthermore, the vascularised
grafts require a major elaborate microsurgical operative procedure (Bostrom and Mikos
1997).
Nowadays diverse bone substitutes were used for the creation of new bone in the patient. For
bone regeneration four components are essential: a morphogenetic signal, responsive host
cells response to the signal, a suitable carrier of this signal that can deliver it to specific sites
serving as scaffolding for the growth of the responsive host cells, and a viable, well
vascularised host bed (Harakas 1984; Croteau et al. 1999). The changes in material
11
technology provide to development of bone tissue engineering. Materials used as bone tissue-
engineered scaffolds may be injectable or rigid, the latter requiring an operative implantation
procedure.
Bone tissue-engineered scaffolds are divided into acellular and cellular with drug delivery
overlapping in both areas (Burg et al. 2000). Materials on or in which no additional cellular
component is cultured will be classified as acellular. The cellular materials are classified as
scaffolds to which a cellular component is added prior to implantation. The materials
commonly used in all three approaches are ceramics, polymers or composites (Burg et al.
2000). The ceramics and polymers are either absorbable or non-absorbable, and the polymers
can be naturally derived or synthesized materials (figure 2).
Biomaterials cellular / acellular
synthesized materials
naturally derived materials
absorbable nonabsorbable
nonabsorbable
absorbable
Figure 2. The classification of biomaterials used for bone tissue engineering modified
according to Burg (Burg et al. 2000).
Bone tissue-engineering systems include demineralized bone matrix, collagen composites,
fibrin, calcium phosphate, polylactide, poly(lactide-co-glycolide), polylactide-polyethylene
glycol, hydroxyapatite, dental plaster, and titanium (Tsuruga et al. 1997; DeGroot et al. 2004).
The mechanisms by which bone can be repaired or regenerated using bone surrogates are
osteoinduction, osteoconduction, and osteointegration. Osteoinduction is defined as the ability
to stimulate the proliferation and differentiation of pluripotent cells. The stem cells directly
differentiate into osteoblasts, which form bone through direct mechanisms. In endochondral
bone formation, stem cells differentiate into chondroblasts and chondrocytes, subsequently
lay down a cartilaginous extracellular matrix, which then calcifies and remodels into lamellar
bone. Osteoinduction is routinely stimulated by osteogenic growth factors. Osteoinduction is a
basic biological mechanism that occurs regularly, e.g. in fracture healing but also in implant
incorporation (Albrektsson and Johansson 2001). An osteoinductive material allows the bone
12
repair in a location that would normally not heal if left untreated (Ishaug et al. 1997; Helm
and Gazit 2005). Osteoconduction means that bone grows on a surface and is defined as the
ability to stimulate the attachment, migration, and distribution of vascular and osteogenic cells
within the graft material (Albrektsson and Johansson 2001). Several physical characteristics
can affect the graft osteoconductivity, including porosity, pore size, and three-dimensional
architecture. In addition, direct interactions between matrix proteins and their appropriate cell
surface receptors play a major role in the host response to the graft material. An
osteoconductive material guides repair in a location where normal healing will occur if left
untreated (Kulkarni et al. 1971; Helm and Gazit 2005). Osteointegration is defined as a direct
contact between living bone and implant, which means the formation of bony tissue around
the implant without the growth of fibrous tissue at the bone-implant interface. The
osteointegration is not an isolated phenomenon, but depends on previous osteoinduction and
osteoconduction (Albrektsson and Johansson 2001).
The ability of a graft material to produce bone independently is termed its direct osteogenic
potential. The main critical considerations in bone tissue-engineering scaffold design are
summarized in table 4.
Table 4. Selected critical consideration in bone tissue-engineering scaffold design (Peter et al. 1998; Burg et al. 2000)
Desirable qualities of a bone tissue-engineering scaffold
Available to surgeon on short notice Absorbs in predictable manner in concert with bone growth Adaptable to irregular wound site, malleable Maximal bone growth through osteoinduction and/or osteoconduction Correct mechanical and physical properties for application Good bony apposition
Promotes bone in-growth Does not induce soft tissue growth at bone/implant interface Average pore sizes approximately 200-400 μm No detrimental effects to surrounding tissue due to processing Sterilizable without loss of properties Absorbable with biocompatible components
13
To have direct osteogenic activity the graft must contain cellular components that induce bone
formation directly. Polymers have been shown to be an excellent substrate for cellular or
bioactive molecule delivery. They can differ in their molecular weight, polydispersity,
crystallinity, and thermal transitions, allowing different absorption rates. Their relative
hydrophobicity and percent crystallinity can affect cellular phenotype (Hollinger and Schmitz
1997). Various types of biomaterials (minerals and non-mineral based materials as well as
natural and artificial polymers) with different characteristics have been used for studying
ossification and bone formation. For example, calcium phosphate ceramics include a variety
of ceramics such as hydroxyapatite, tricalcium phosphate, calcium phosphate cement, etc.
Local tissue responses to polymers in vivo depend on the biocompatibility of the polymer as
well as its degradative by-products (Hollinger and Battistone 1986). The mentioned ceramics
have excellent biocompatibility and bone bonding or bone regeneration properties. They have
been widely used in no or low load-bearing applications (Milosevski et al. 1999).
Furthermore, natural polymers like collagen have been used for bone tissue engineering
purposes (Hutmacher 2001; Lauer et al. 2001). Recently non-biodegradable and degradable
membranes have been tested for their appliance in bone defects (Zhao et al. 2000). Scores of
artificial polymers of diverse character are already in use for bone supply. One of them,
poly(3)hydroxybutyrate (PHB) with little inflammatory response after implantation due to its
form stability, may serve as a scaffold for tissue engineering (Gogolewski et al. 1993;
Schmack et al. 2000).
1.4. Poly-3-hydroxybutyrate — PHB
The lipidic polymer, poly-3-hydroxybutyrate (PHB) is found in the plasma membrane of
Escherichia coli in complex with calcium polyphosphorate (Reusch and Sadoff 1983).
Different types of microorganisms produce PHB from renewable sources from sugar and
molasses as intracellular storage materials. PHB is polyester (figure 3) with optical activity
and very good barrier properties. It has a high melting temperature (175°C), glass transition
temperature (15°C), and a high degree of crystallinity and is low permeable for O2, CO2, and
H2O (Holmes 1987; Miguel et al. 1997). This polymer is perfectly isotactic viz. the monomers
have all branch groups on the same side of the polymeric chain and are oriented in the same
way. This linear flexible molecule bearing electron-donating carbonyl ester oxygens at
intervals allows multiple bonding between the polymer chain and cation (Armand 1987; Gray
14
1997). PHB does not contain any residues of catalysts like other synthetic polymers (Holmes
1987; Miguel et al. 1997).
a.
b.
–{ O – CH ( CH3 ) – ( C = O ) }n –
Figure 3. The polyester linkage creates a molecule which has 3-carbon segments separated by
oxygen atoms. The remainder of the monomer (a) becomes a side chain of the main
backbone of the polymer (b). In PHB the monomer unit is hydroxybutyric acid and
the side chain is a methyl group. PHB, with its short methyl side chain, is a very
crystalline and very brittle polymer. Industrially, it is difficult to use because the
temperature at which it melts is very close to the temperature at which it begins to
decompose. Its high degree of crystallinity causes it to crack easily.
PHB stiffness and brittleness depends on the degree of crystallinity, microstructure, and glass
temperature. The longer it is stored at room temperature, the more it becomes brittle. Because
of these findings its application is limited. Further application limitations are the poor process
ability and the low degradation (Mack et al. 2008).
15
However, PHB will be used for the following applications:
1. In pharmacology as a material for cell and tablet packaging or microcapsules during
therapy.
2. In medicine and dentistry as surgical implant because of its compatibility with the
tissues of mammals and an undisturbed metabolism in the human blood.
3. In packaging industries as a biodegradable plastic for solving environmental pollution,
hygiene and textile.
Because of its unique combination of biodegradability and biocompatibility it is of great
interest for medical applications (Vogel et al. 2006). PHB completely degrades releasing a
normal component of blood and tissue, D, L-β-hydroxybutyrate (HB) and it is an ideal
biomaterial characterized by stability, lack of toxicity, compatibility in contact with tissue and
low inflammatory response after implantation (Gogolewski et al. 1993; Sevastianov et al.
2003; Mai et al. 2006; Suwantong et al. 2007; Shishatskaya et al. 2008).
The biocompatibility of PHB has been confirmed in vitro in cultures of cells of various
origins (Saad et al. 1999; Shishatskaya et al. 2004; Volova et al. 2004) and surrounding
muscle tissues (Mack et al. 2008).
1.5. Aim of this study
The amount of free microvascular bone grafts is limited due to lack of donor sites with
sufficient bone volume of adequate quality. A bone substitute creation ectopically in an
anatomical area having good vascular supply and an adequate vascular pedicle could reduce
this limitation (Warnke et al. 2004; Mai et al. 2006). Ectopic bone formation has been long-
time tested in several animal studies (Kusumoto et al. 1996; Buma et al. 2004; Meyer et al.
2004; Wiesmann et al. 2004; Kroese-Deutman et al. 2005). A bone surrogate human
application created from an osteoconductive biomaterial of bovine origin in combination with
growth factors and additional autogenous spongiose bone has also been described (Warnke et
al. 2004).
Muscles are known to have a considerable potential of adaptation. The extracellular matrix of
the muscle tissue surrounding the implant can integrate changes of the mechanical load of the
muscle and hereupon induce signalling cascades with a following adaptation of protein
synthesis and arrangement of the cytoskeleton (Kjaer 2004). Recently was shown, that
latissimus dorsi muscles are used for the growth and preparation of bone grafts for subsequent
16
transplantation (Warnke et al. 2004). It was demonstrated that heterotopic bone induction and
custom vascularization is possible to form a bone replacement inside the latissimus dorsi
muscle in a human.
The aim of this study was to examine the effects of PHB implants on factors that regulate
vascularization or interaction with the extracellular matrix of the surrounding muscle tissue.
For this the mRNA expression of VEGF, IGF1, IGF2 as well as GDF8 should be analyzed
using quantitative RT-PCR in muscle tissue specimens from the Musculus latissimus dorsi of
rats. The muscle specimens were collected after subcutaneous implantation of PHB scaffolds
for six and twelve weeks.
.
17
2. Materials and Methods
All used materials, chemicals, buffer, and equipment are summarized in table a-d in the
section “supplement”.
2.1. Poly-3-hydroxybutyrate
Fully biodegradable biotechnologically produced polyester PHB was used in powder form as
raw material. The powder with a molecular weight of 540 000 g/mol was granulated using a
twin screw extruder equipment. The PHB multifilaments were produced using a high-speed
melt spinning and spin drawing process (Mai et al., 2006; Schmack et al., 2000). Round
embroidery patches with a thickness of 1.2 mm and a diameter of 12 mm were generated
using an embroidery automat followed by coating with calf skin collagen type I. The average
macro mesh pore size of the embroidery was 200 µm and the total weight of the implant was
approximately 12 mg. A total of 24 implants were prepared. All implants were ultrasonically
cleaned in 70 % ethanol for 15 minutes and sterilised by ethylene oxide before the surgical
procedure.
2.2. Experimental Design and Surgical Procedure
The experiments were performed on twelve six-weeks-old adult male Wistar-King rats, with
approximately 200 g body mass. All surgical and experimental procedures were approved by
the Animal Welfare Committee of the State Government (no. 24-9168.11-1-2004-2).
For surgery, each rat was anesthetized with an intraperitoneal injection of pentobarbital at an
approximate dosage of 75 mg/kg. For the subcutaneous implants a 3 cm sagittal incision was
made in the skin in the midline of the back. A blunt dissection away from the midline
cranially and caudally to the left and the right of the spine was used to form subcutaneous
pockets on the surface of the Musculus latissimus dorsi (figure 4a.). In one of these pockets a
PHB embroidery was inserted (Figure 4b). The subcutaneous pocket was then closed around
the implant with a resorbable suture and the skin closed with a continuous suture. Eight rats
without any intervention served as control group.
18
a. b.
Figure 4. Pictures of the surgical procedure.
a) The subcutaneous muscle pocket on the surface of the Musculus latissimus dorsi.
b) The PHB scaffold in a subcutaneous muscle pocket.
At the end of each time period (6 and 12 weeks after implantation) six rats were euthanized
with carbon dioxide. After euthanasia the implants with the surrounding tissues were retrieved
and prepared for molecular genetic evaluation (figure 5). Following a blunt dissection the
PHB surrounding tissue was carefully removed and frozen in liquid nitrogen.
skin
fascia
PHB scaffold
removed muscle tissue
M. latissimus dorsi
Figure 5. Schematic illustration of the PHB location and the removed muscle tissue.
19
2.3. RNA-Isolation The total RNA was isolated using guanidinium-isothiocyanate (RNeasy Fibrous Tissue Mini
Kit, Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction.
To reduce the viscosity of the lysates produced by disruption in Buffer RLT the fresh frozen
muscle tissue was homogenized. Homogenization shears high-molecular-weight genomic
DNA and other high-molecular-weight cellular components to create a homogeneous lysate.
Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin column
membrane and therefore significantly reduced RNA yields.
For disruption the muscle tissue was immediately frozen in liquid nitrogen and ground to a
fine powder under liquid nitrogen using a mortar and pestle. The lysis buffer was added and
the homogenization was continued as quickly as possible in QIAshredder columns
(Qiashredder, Qiagen, Valencia, CA, USA). Total RNA purification from fibrous tissues,
such as skeletal muscle, can be difficult due to the abundance of contractile proteins,
connective tissue, and collagen. RNeasy Fibrous TissueMini Kits contain proteinase K, which
removes these proteins. 10 μl proteinase K were added to the homogenized lysate and the
solution was incubated at 55°C for 10 min. After centrifugation a small pellet of tissue debris
was formed. Thereafter ethanol was added to the supernatant to create conditions that promote
selective binding of RNA to the RNeasy membrane. The samples were then applied to the
RNeasy Mini spin column. Total RNA bound to the membrane, contaminants were efficiently
washed away, and high-quality RNA was eluted in RNase-free water (figure 6).
20
Figure 6. Schematic illustration of the RNeasy Fibrous Tissue Mini Kit Procedure (RNeasy Fibrous Tissue Handbook 11706, Qiagen).
The quality and yield of the RNA was determined by spectrophotometry at 260 nm and the
integrity examined by agarose gel electrophoresis with ethidium-bromide staining. The
quantification of the total RNA was performed using a NanoDrop®ND-1000 UV-Vis
Spectrophotometer (NanoDrop Technologies) which minimized the loss of RNA material
during the measurement procedure. Because of this only 2 μl RNA solution were used per
sample. After the RNA isolation 1µg of the total RNA was reverse transcribed in cDNA. The
cDNA was stored at -20°C.
21
2.4. Agarose gel electrophoresis
The integrity and size distribution of the purified total RNA was checked by electrophoreses
on a denaturing at agarose gel. Both can be analysed by the survey of the respective ribosomal
RNAs (18S and 28S ribosomal RNA).
To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration, and then heated in a microwave oven until completely melted. Ethidium
bromide was added to the gel (final concentration 0.5 µg/ml) at this point to facilitate
visualization of DNA after electrophoresis. After cooling of the solution to about 60°C it was
poured into a casting tray containing a sample comb and allowed to solidify at room
temperature. After the gel has solidified the comb was removed and the gel was covered with
buffer, and samples containing RNA mixed with loading buffer were then filled into the
sample wells, the lid and power leads were placed on the apparatus, and a current was applied
(figure 7). The RNA was migrated towards the positive electrode. To visualize the RNA, the
gel was placed on an ultraviolet transilluminator.
Figure 7. Illustration of the pouring and loading of a horizontal agarose gel.
22
2.5. Reverse Transcriptase-polymerase chain reaction — Real-Time RT-PCR
Changes in the mRNA amount could be measured by Real-Time RT-PCR using gene specific
primers according to the schema on figure 8. The concentrations of the primers were adjusted
as described in a manual for the Power Sybr-Green Master Mix (Applied Biosystems, Foster
City, CA, USA).
RNA isolation and analysis
First-strand cDNA synthesis
Real-Time PCR amplification
Continuous fluorescent measurement of PCR product during each cycle of PCR
Analysis of data
Figure 8. The procedure of the mRNA quantification using Real-time RT-PCR.
Because of the instability of RNAs and its digestion by ubiquitous RNases, the total-RNA
was transcribed into a more resistant form viz. complementary DNA (cDNA) (figure 9). The
High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA)
was used for this procedure. Reverse Transcription (RT reaction) is a process in which single-
stranded RNA is reverse transcribed into cDNA by using total cellular RNA or poly(A) RNA,
a reverse transcriptase enzyme, specific primers, dNTPs and an RNase inhibitor. The resulting
cDNA can be used in RT-PCR reaction.
23
The High Capacity cDNA Reverse Transcription Kit delivers extremely high quantitative
single-stranded cDNA from total RNA. Although designed for short or long term archival of
cDNA, it yields very quantitative reverse transcription from 0.02 to 2 μg of total RNA for 20
μl.
Figure 9. Reverse transcription (The science of biology, 7th Edition, fig.16.8- schematic).
To quantify the expression of rat IGF1, IGF2, GDF8, VEGF and β-actin genes we applied the
Sybr-Green PCR Core Reagents (Applied Biosystems). Gene-specific PCR primers for the
genes were purchased from Qiagen (table 5).
Table 5. Information about RT-PCR primers. The sets of primers used for RT-PCR were obtained from Qiagen.
Gene Accession number Amplicon length
β-actin NM_031144 145 bp
IGF1 NM_001082479 135 bp
IGF2 XM_001064965 96 bp
VEGF NM_001110335 68 bp
GDF8 NM_019151 102 bp
24
Parallel PCR assays for each gene target with cDNA samples and a “no-template control”
with water were performed parallel in all experiments on each 96-well plate. Reaction
mixtures contained in each well 12.5 µl of the Master Mix (Sybr-Green PCR Core Reagents)
and 300 nM of each primer. 2 µl of 1:50 diluted cDNA of each sample served as a template.
The specifity of the reaction was examined by creating a dissociation curve for each sample
and finally by checking the PCR products by agarose gel-electrophoresis. Quantitative real-
time polymerase chain reaction (RT-PCR) using the Applied Biosystems 7500 Real-Time
PCR System provided an accurate method for determination of levels of specific DNA
sequences in tissue samples. It is based on the detection of a fluorescent signal produced
proportionally during amplification of a PCR product (figure 10).
1. Double-stranded DNA denaturation at
2. Primer annealing at 60°C
New target sequences;
Replication (step 1 to 3).
40x
3. DNA elongation at 72°C
RT-PCR RT-PCR with SYBR® -Green Dye
Denaturation — when the DNA is denatured,
the SYBR®Green I Dye is released and fluorescence is
drastically reduced.
Polymerization — during extension, primers anneal and a PCR product is generated.
The SYBR®Green I Dye
fluoresces when bound to double-stranded DNA, fluorescence detected by the instrument. Figure 10. Standard PCR reaction and Sybr-Green Real-Time PCR; schematic illustration (van der Velden et al., 2003).
25
2.6. Quantification of the gene expression — 2(-ΔΔCT) method
To determine the quantity of the target-gene specific transcripts present in treated cells in
relation to untreated ones, their respective CT values were first normalized by subtracting the
CT value obtained from the β-actin control. The concentration of the gene-specific mRNA in
treated cells relative to untreated cells was calculated by subtracting the normalized CT values
obtained for untreated cells from those obtained from treated samples and the relative
concentration was determined (Livak and Schmittgen, 2001).
2.6.1. Derivation of the 2-ΔΔCT method
The equation that describes the exponential amplification of PCR is
Xn = X0 × (1 + Ex)n
where Xn - the number of target molecules at cycle n of the reaction,
X0 - the initial number of target molecules. EX is the efficiency of target
amplification,
n - the number of cycles. The threshold cycle (CT) indicates the fractional cycle
number at which the amount of amplified target reaches a fixed threshold.
Thus,
XT = X0 × (1 + Ex)CT,X = Kx
where XT - the threshold number of target molecules,
CT,X - the threshold cycle for target amplification,
Kx - a constant.
A similar equation for the endogenous reference (internal control gene) reaction is
RT = R0 × (1 + ER)CT,R = KR
26
where RT - the threshold number of reference molecules,
R0 - the initial number of reference molecules,
ER - the efficiency of reference amplification,
CT,R - the threshold cycle for reference amplification,
KR - a constant.
Dividing XT by RT gives the expression:
XT / RT = X0 × (1 + Ex)CT,X / R0 × (1 + ER)CT,R = Kx / KR = K
For real-time amplification using TaqMan probes, the exact values of XT and RT depend on a
number of factors including the reporter dye used in the probe, the sequence context effects
on the fluorescence properties of the probe, the efficiency of probe cleavage, purity of the
probe, and setting of the fluorescence threshold. Therefore, the constant K does not have to be
equal to one. Assuming efficiencies of the target and the reference are the same,
EX = ER = E
(X0 / R0) × (1 + E)CT,X - CT,R = K
or XN × (1 + E)ΔCT = K
where XN - equal to the normalized amount of target (X0 / R0) and
ΔCT - equal to the difference in threshold cycles for target and reference (CT,X- CT,R)
Rearranging gives the expression
XN = K × (1 + E)-ΔCT
The final step is to divide the XN for any sample q by the XN for the calibrator (cb):
XN,q / XN,cb = K × (1 + E)-ΔCT,q / K × (1 + E)-ΔCT,cb = (1 + E)-ΔΔCT
Here : ΔΔCT = - (ΔCT,q - ΔCT,cb )
27
For amplicons designed to be less than 150 bp and for which the primer and Mg2+
concentrations have been properly optimized, the efficiency is close to one. Therefore, the
amount of target normalized to an endogenous reference and relative to a calibrator, is given
by
amount of target = 2-ΔΔCT.
2.7. Statistical analysis
Statistical analysis was performed using the SigmaPlot Software (Systat Software, Inc.1735,
Technology Drive, Sn Jose, CA 95110, USA). The obtained values for the groups were
compared using Student’s unpaired t-test. Data are given as means ± SEM. p < 0.05 was
considered statistically significant.
28
3. Results
In this study 20 age-matched adult male Wistar rats were used. Eight animals served as
controls without PHB scaffolds on the surface of the M. latissimus dorsi. The remaining 12
animals received subcutaneous PHB scaffolds. All animals recovered from the operation and
healed uneventfully until the end of the experiments.
A good biocompatibility of the PHB implants could be observed macroscopically, because
one of the early responses to the PHB implants was capillary generation in the muscle tissue
surrounding the implants (figure 11).
Figure 11. PHB scaffold after 6 weeks of subcutaneous implantation with newly built capillaries.
3.1. Isolation of the total RNA from muscle samples
For the quantification of the different gene specific mRNAs four different muscle tissue
samples surrounding the PHB scaffold were prepared from six animals 6 and 12 weeks after
implantation, respectively. The total RNA was isolated from about 30 mg muscle tissue
according to the manufacturer’s instructions. The quality and yield of the total RNA was
determined by spectrophotometry at 260 nm using the NanoDrop® ND-1000 UV-Vis
spectrophotometer. Figure 12 shows exemplary the electropherogram of one muscle tissue
RNA probe.
29
Figure 12. The electropherogram at 260 nm using the NanoDrop® ND-1000 UV-Vis spectrophotometer of one muscle tissue RNA probe isolated from Musculus latissimus dorsi.
The integrity of the isolated total-RNA was examined by agarose gel electrophoresis with
ethidiumbromide staining. The electrophoresis of two different RNA probes shows two clear
bands of the respective size of the 18S and 28S rRNA. Furthermore, no degradation products
were found (figure 13).
28 S 18 S
Figure 13. Examples of RNA isolated from muscle run on an agarose gel and strained with ethidium bromide. 28S and 18S ribosomal RNA bands are indicated.
30
3.2. Examination of the primer specificity using standard RT-PCR
Using standard RT-PCR we detected signals for all gene transcripts mentioned above in the
M. latissimus dorsi. Standard RT-PCR yielded PCR products of the expected size. All
products were exemplary sequenced and revealed the expected DNA sequence. Figure 14
shows the RT-PCR results for VEGF, IGF1, IGF2,GDF8, and β-actin, respectively.
β-
β-actin IGF1 IGF2 VEGF GDF8
marker 125 bp 75 bp
Figure 14. Agarose gel electrophoresis of all used genes samples after RT-PCR for the verification of the purity and length of the amplicons.
3.3. Quantification of the VEGF, IGF1, IGF2, and GDF8 mRNA expression
Gene-specific RT-PCR was performed to quantify the expression of the VEGF, IGF1, IGF2
and GDF8 genes in M. latissimus dorsi specimens with and without PHB scaffold
implantation.
The relative expression pattern of the four tested genes in muscle tissue specimens
surrounding PHB scaffold after 6 and 12 weeks is shown in figures 15-18. Copy numbers of
the gene transcripts are given in relation to those of β-actin. A “no-template control” with
water was performed parallel in all experiments. Each series of experiments was performed
twice.
In untreated muscle tissue the relative copy number of VEGF was 3.9 ± 1.0. After six weeks
of implantation a significant increase in the expression level of VEGF was observed (treated
31
vs. non-treated: 7.9 ± 2.0 vs. 3.9 ± 1.0, p<0.05). In muscle tissue samples surrounding PHB
scaffolds a 2 fold increase of the VEGF mRNA amount was found compared to non-treated
muscle samples (Figure 15). Furthermore, a 1.6 fold up-regulation of the VEGF mRNA
expression was found after 12 weeks of the implantation of the PHB scaffolds (treated vs.
non-treated: 6.2 ± 1.6 vs. 3.9 ± 1.0, p<0.05). It is to note, that the level of the VEGF mRNA
expression was higher at 6 than at 12 weeks after treatment, but the mRNA amount was not
significantly decreased after 12 weeks compared to that after six weeks of implantation.
VEGF
0
2
4
6
8
10
12
1 control 6 weeks 12 weeks
VEG
F/be
ta-a
ctin
*
*
Figure 15. Relative expression of the VEGF mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M., Student’s t-test, * p< 0.05
The expression of the IGF1 gene was also significantly increased as compared to the controls
over the observed time period. In muscle tissue samples surrounding PHB scaffolds a 1.5 fold
increase of the IGF1 mRNA amount was found compared to non-treated muscle samples,
respectively (6 weeks, treated vs. non-treated: 29.0 ± 7.0 vs. 14.0 ± 3.0, p<0.05; 12 weeks,
treated vs. non-treated: 19.2 ± 3.0 vs. 14.2 ± 3.0, p<0.05, Figure 16). Like VEGF, the IGF1
mRNA expression decreased 12 weeks after implantation compared to 6 weeks after
treatment.
32
IGF1
0
5
10
15
20
25
30
1 control 6 weeks 12 weeks
IGF1
/bet
a-ac
tin
*
*
Figure 16. Relative expression of the IGF1 mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M., Student’s t-test, * p< 0.05
In case of the IGF2 mRNA, the expression was significantly up-regulated after 6 weeks
(p<0.05), but not significantly increased after 12 weeks (p>0.05). The IGF2 mRNA amount
was 1.5 times increased in M. latissimus dorsi surrounding PHB scaffolds compared to un-
treated muscle tissue specimens (treated vs. non-treated: 8.2 ± 1.8 vs. 5.5 ± 1.8; p<0.05; figure
17).
IGF2
0
2
4
6
8
10
12
1 control 6 weeks 12 weeks
IGF2
/bet
a-ac
tin
*
Figure 17. Relative expression of the IGF2 mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M.,Student’s t-test, * p< 0.05
33
In contrast to all other tested genes, we observed a significantly decreased GDF8 gene
expression (p<0.05) after 6 and 12 weeks, respectively. Moreover, the GDF8 mRNA levels
were identically after 6 and 12 weeks (6 weeks, treated vs. untreated: 2.9 ± 1.0 vs. 4.8 ± 1.6,
p<0.05; 12 weeks, treated vs. non-treated: 3.3 ± 0.8 vs. 4.8 ± 1.6; p<0.05).
GDF8
0
1
2
3
4
5
6
7
1 control 6 weeks 12 weeks
GD
F8/b
eta-
actin
* *
Figure 18. Relative expression of the GDF8 mRNA in the M. latissimus dorsi of control rats and in the M. latissimus dorsi 6 and 12 weeks after PHB scaffold implantation. Mean ± S.E.M., Student’s t-test, * p< 0.05
34
4. Discussion
In this study we have demonstrated for the first time that the natural polyester
polyhydroxybutyrate (PHB) has an influence on the expression of growth factors such as
vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF) as well as
myostatin also known as growth differentiation factor 8 (GDF8). We have found, that
subcutaneous implantation of PHB scaffolds increased the VEGF, IGF1, and IGF2 mRNA
expression in the Musculus latissimus dorsi of rats, whereas the myostatin mRNA was
significantly decreased. These reactions could be caused by the immunological adaptation of
the implant or by muscle regeneration / adaptation.
Wound healing, or wound repair, is an intricate process in which an organ or tissue repairs
itself after injury (Nguyen et al. 2009). The classic model of wound healing is divided into
three or four sequential, yet overlapping, phases: (1) hemostasis, (2) inflammation, (3)
proliferative and (4) remodeling phase. Within one hour after wounding, polymorphonuclear
neutrophils (PMNs) arrive at the wound site and become the predominant cells for the first
two days after injury. About two days after injury macrophages replace PMNs as the
predominant cells in the wound. It is well known that PHB shows an excellent
biocompatibility as evidenced by lack of toxicity, compatibility in contact with tissue and
blood (Saito et al. 1991; Clarotti et al. 1992; Gogolewski et al. 1993). The polymer was
implanted subcutaneously in different species and no abscess formation, or tissue necrosis
were observed (Gogolewski et al. 1993). Furthermore, PHB scaffolds expressed post-
traumatic inflammation with mononuclear macrophages, proliferating fibroblasts, and mature
vascularized fibrous capsules as typical tissue response (Gogolewski et al. 1993; Shishatskaya
et al. 2004; Qu et al. 2006; Shishatskaya et al. 2008).
Macrophages and other leukocytes such as helper T cells secrete cytokines resulting in
enhanced T cell division and increase of inflammation. It is known that pro-inflammatory
cytokines stimulate the expression of the nerve growth factor (Abe et al. 2007). Furthermore it
was shown that the cytokines transforming growth factor (TGF)-beta1, and interleukin (IL)-
1beta stimulate the production of VEGF from cultured conjunctival fibroblasts (Asano-Kato
et al. 2005). In our study an increased VEGF mRNA expression was detected in muscles
surrounding the PHB scaffold. It is to speculate that the increased VEGF mRNA expression
after implantation of the PHB scaffolds could be stimulated by cytokines released from
macrophages.
35
The up-regulation of VEGF in the implant surrounding muscle tissue is necessary for wound
healing. One characteristic event of the proliferative phase of wound healing is angiogenesis,
the process of development and growth of new capillary blood vessels from pre-existing
vessels and the interaction with the extracellular matrix. Proliferation of capillary endothelial
cells is stimulated by VEGF (Klagsbrun and D'Amore 1996). The detection of the VEGF
mRNA in rat skeletal muscle is in agreement with previous studies. VEGF was identified to
be involved in the regulation of angiogenesis in skeletal muscles (Skorjanc et al. 1998;
Milkiewicz et al. 2001). Other angiogenesis factors, which are responsible for the maturation
of vascular networks in many tissues, have not been found in skeletal muscles so far.
However, the VEGF mRNA expression decreased over the entire investigation period and
resulted in delayed neo-vascularization of the implant. This time-dependent decrease of the
VEGF mRNA expression was also found after implantation of allogenous, acellular dermal
matrices on the gracilis muscle from rats (Mueller et al. 2009). Furthermore, recent studies
suggest that newly formed vessels are deleted by natural elimination (Peirce et al. 2004). The
VEGF induced growth of vessels is not accompanied by increased metabolic demand,
therefore durable presence is not essential for the function of the tissue.
Beside wound healing and immunological adaptation on the implant it is also possible that the
surrounding muscle tissue adapts and regenerates, because skeletal muscle has a remarkable
capacity to regenerate following exercise or injury. Muscle regeneration is a complex process
requiring the coordinated interaction between the myogenic satellite cells, growth factors,
cytokines, inflammatory components, vascular components and the extracellular matrix.
Differentiation, maturation, maintenance, and repair of skeletal muscle require ongoing
cooperation and coordination between an intrinsic regulatory programme controlled by
myogenic transcription factors and signalling pathways activated by hormones and growth
factors (Lassar and Munsterberg 1994; Naya and Olson 1999).
Important growth factors for muscle regeneration and differentiation are IGF1 for positive
adjustment of cell proliferation and myostatin as a negative growth factor. Besides, IGF2 is
also involved in muscle development and though IGF2 plays a crucial role in muscle
development (DeChiara et al. 1990; Marsh et al. 1997), its role in the repair / regeneration
process is less central and more secondary to other growth factors, for example IGF1. Age-
associated decrements in muscle repair process have also been shown to be associated with
the level of IGF gene expression. IGF1 might play a predominant role in protecting cells from
death mediated by myostatin (Yang et al. 2007). Thus, in the presence of IGF1, cells would be
arrested at G1/S (mitosis) and do not undergo apoptosis in response to myostatin. Myostatin
36
and IGF1 may regulate each other with a negative feedback mechanism to maintain
physiological homeostasis between cell growth and cell death during normal development.
This means that augmented IGF1 growth signal may require more myostatin-inhibitory
function to reach the balance between cell growth and cell death and vice versa. According to
other studies we could confirm that IGFs are expressed in muscles (Beck et al. 1987; Han et
al. 1987) and show that muscle fibres express increased IGF mRNA amounts after
implantation of PHB scaffolds. It is well established that endogenously produced IGF1 and
IGF2 can exert a strong positive effect on skeletal muscle differentiation (Florini et al. 1991;
Montarras et al. 1996). In contrast to these findings and the observation that elevated levels of
IGFs are sufficient to promote interstitial cell proliferation in otherwise untreated adult
skeletal muscle (Caroni and Grandes 1990), other findings support the hypothesis that the
early production of IGF1 by the inactive muscle fibre is involved in the initiation of the
proliferation reaction of muscle (Caroni et al. 1994). IGF1 was identified as a possible
initiator of restorative reactions in injured muscles (Caroni et al. 1994).
Recent results suggest that myostatin (GDF8) is a potent regulator of cell-cycle progression
and functions by regulating both the proliferation and differentiation steps of myogenesis
(Thomas et al. 2000; Langley et al. 2002; McCroskery et al. 2005). The role of myostatin has
been demonstrated in several studies not only during embryonic myogenesis, but also in
postnatal muscle growth. It is not known whether myostatin influences only muscle formation
or has also a function in the regulation of muscle metabolism (Ji et al. 1998). Lack of
myostatin results in accelerated regeneration and reduced fibrosis (McCroskery et al. 2005).
We observed 6 as well as 12 weeks after implantation a significantly decreased myostatin
expression. The decrease was about the same at both times. Several studies indicate that
myostatin might function as an inhibitor of satellite cell proliferation, suggesting a role of
myostatin in postnatal muscle growth and repair (Carlson et al. 1999; Wehling et al. 2000).
Consistent with this hypothesis, recent results from McCroskery et al. (McCroskery et al.
2003) indicate that myostatin is indeed expressed in satellite cells. Myostatin is known to
block hematogenesis and enhance chondrogenesis as well as epithelial cell differentiation
(Cieslak et al. 2003).
Moreover, muscles adapt to stress by change of fibre types and respective mRNA content.
Recently, Mack et al. (Mack et al. 2008) have shown that PHB implants can have an influence
on the myosin heavy chain (MyHC) isoform composition of the surrounding muscles. It was
found that MyHC isoform I increased significantly after the implantation of a PHB scaffold,
whereas the expression of the fast MyHC isoforms remains to be unchanged or decreased
37
(Mack et al. 2008). Accordingly, the basically fast rat Musculus latissimus dorsi adapted to a
slower phenotype with a more efficient energy utilisation and greater potential for
regeneration (Gedrange et al. 2003). Furthermore, in the course of adaptation and muscle
regeneration processes skeletal muscle are capable to change their anatomical characteristics.
These changes are often associated with changes in the intensity, duration, and frequency of
muscle activation by the central nervous system. It is known that the growth factors IGF1 and
IGF2 are candidates for muscle-derived nerve sprouting activity. These factors have been
shown to promote neurite outgrowth from sympathetic and sensory neurons in vitro (Recio-
Pinto et al. 1986) as well as innervated adult skeletal muscle (Caroni and Grandes 1990).
Muscle denervation or botulinum toxin-induced paralysis of adult rat skeletal muscle rapidly
led to elevated levels of IGF1 and IGF2 mRNA in the treated muscles (Ishii 1989). The
increase in the mRNA expression of both genes in our study could also cause nerve sprouting
and changes of the muscle activation.
Our results show that PHB implants in rat Musculus latissimus dorsi interact with the
surrounding muscle tissue and have influence on factors that regulate vascularization and
muscle adaptation. These changes in the mRNA expression of VEGF, GDF8, IGF1, and IGF2
are time-dependent. All changes occurred within six weeks and persisted up to twelve weeks
after implantation. These findings indicate on one hand a normal wound healing and on the
other hand improved muscle regeneration, stating that there is a synergistic effect between
PHB scaffolds and the surrounding muscle tissue. This synergistic effect should be further
elucidated.
38
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47
6. Summary
Bone defects can be a limiting factor in achievement of optimal orthodontic treatment.
Diverse bone substitutes were already used for the creation of new bone in the patient, such as
collagen composites, calcium phosphate, and titanium. Muscles are known to have a
considerable potential of adaptation. The latissimus dorsi muscle was used for the growth and
preparation of bone grafts for subsequent transplantation.
The aim of the present study was to identify the synergistic effect between the poly-3-
hydroxybutyrate (PHB) bone substitute and surrounding muscle tissue. To describe this effect,
changes of insulin like growth factors (IGF1, IGF2), myostatin (GDF8) and vascular
endothelial growth factor (VEGF) mRNA content were examined in the Musculus latissimus
dorsi of 12 Wistar-King rats after 6 and 12 weeks of PHB scaffold implantation. At each time
point six rats were killed and implants and surrounding tissues prepared for genetic
evaluation. Eight rats without any implants served as controls. RNA was extracted from
homogenized muscle tissue and reverse transcribed. Changes in the mRNA content were
measured by Real-Time RT-PCR using specific primers for IGF1, IGF2, GDF8, and VEGF.
The VEGF mRNA level was significantly increased (p<0.05) in muscle tissue specimens after
6 and 12 weeks of implantation compared to the controls. The expression of the IGF1 gene
was also significantly increased as compared to the controls over the observed time period
(p<0.05). Furthermore, the VEGF and IGF1 mRNA levels were higher after 6 than after 12
weeks of treatment. In the case of the IGF2 gene, the expression was significantly elevated
after 6 weeks (p<0.05), but not significantly increased after 12 weeks. In contrast to all other
tested genes we observed a significantly decreased GDF8 gene expression (p<0.05) both after
retrieval of implants after 6 as well as after 12 weeks. Moreover, the mRNA levels of GDF8
after 6 and 12 weeks were comparable the same.
Our results show that PHB implants in rat Musculus latissimus dorsi interact with the
surrounding muscle tissue and have influence on factors that regulate vascularization and
muscle adaptation. These findings indicate on one hand a normal wound healing and on the
other hand improved muscle regeneration, stating that there is a synergistic effect between
PHB scaffolds and the surrounding muscle tissue.
48
7. Supplement
Beta-actin
Rn_Actb_1_SG QuantiTect Primer Assay (200) (QT00193473)
Official gene name actin, beta
Official gene symbol Actb
Species Rat (Rattus norvegicus)
Entrez Gene ID 81822
Detected transcript NM_031144
Ensembl Transcript ID ENSRNOT00000001480
Length of detected transcript 1296 bp
Amplified exons*: 2/3
Amplicon length: 145 bp
Dye label / detection SYBR Green
Assay type QuantiTect Primer Assay
49
VEGF
Rn_RGD:619991_1_SG QuantiTect Primer Assay (200) (QT00198954)
Official gene name vascular endothelial growth factor A
Official gene symbol Vegfa
Species Rat (Rattus norvegicus)
Entrez Gene ID 83785
Detected transcript NM_001110335
Ensembl Transcript ID ENSRNOT00000026637
Length of detected transcript 2616 bp
Amplified exons*: 6/7
Amplicon length: 68 bp
Dye label / detection SYBR Green
Assay type QuantiTect Primer Assay
50
IGF2
Rn_Igf2_1_SG QuantiTect Primer Assay (200) (QT00195594)
Official gene name insulin-like growth factor 2
Official gene symbol Igf2
Species Rat (Rattus norvegicus)
Entrez Gene ID 24483
Detected transcript XM_001064965
Ensembl Transcript ID ENSRNOT00000027602
Length of detected transcript 3803 bp
Amplified exons*: 2/3
Amplicon length: 96 bp
Dye label / detection SYBR Green
Assay type QuantiTect Primer Assay
51
GDF8
Rn_Mstn_1_SG QuantiTect Primer Assay (200) (QT00189406)
Official gene name myostatin
Official gene symbol Mstn
Species Rat (Rattus norvegicus)
Entrez Gene ID 29152
Detected transcript NM_019151
Ensembl Transcript ID ENSRNOT00000038093
Length of detected transcript 1131 bp
Amplified exons*: 2/3
Amplicon length: 102 bp
Dye label / detection SYBR Green
Assay type QuantiTect Primer Assay
52
IGF1
Rn_Igf1_2_SG QuantiTect Primer Assay (200) (QT01745373)
Official gene name insulin-like growth factor 1
Official gene symbol Igf1
Species Rat (Rattus norvegicus)
Entrez Gene ID 24482
Detected transcript NM_001082479
Ensembl Transcript ID ENSRNOT00000004136
Length of detected transcript 1827 bp
Amplified exons*: 4/5
Amplicon length: 134 bp
Dye label / detection SYBR Green
Assay type QuantiTect Primer Assay
53
Material
Kits and biomaterials
Table a. Summary of all used kits and biomaterials.
material Manufacturer
High Capacity cDNA Archive Kit Applied Biosystems
AmpliTaq Gold DNA Polymerase Applied Biosystems
GeneAmp 10×PCR Gold Puffer Applied Biosystems
GeneAmp dNTP Mix Applied Biosystems
Power SYBR Green PCR Master Mix Applied Biosystems
25bp ladder Invitrogen
100bp ladder Invitrogen
RNeasy Fibrous Tissue Mini Kit Qiagen
Qiashredder Qiagen
Equipment Table b. Summary of all used equipments.
equipment manufacturer
7500 Real Time PCR System Applied Biosystems
Thermocycler Gene Amp PCR system 2400 Applied Biosystems
Geldetektionssystem Gel Doc 2000 Biorad
Centrifuge 5417 R Eppendorf
Photometer Eppendorf
Netzgerät EV 231 Consort
Biofuge Heraeus
Waage Portable Sartorius
Vortexer Vortex Genie 2.0 Scientific Industries
Microwelle Siemens
Pipettboy acu Tecnomaro
Gelkammern, Modell AGT-2-1 VWR International
54
Chemicals Table c. Summary of all used chemicals.
chemical manufacturer
Aqua ad injectabila B.Braun
Borsäure Roth
Bromphenolblau Roth
EDTA Merck
Ethanol absolut Roth
Ethidium bromide Roth
Liquid nitrogen
Glycerin Roth
2-Mercaptoethanol Roth
Natrium chloride Merck
peqGold Universal Agarose PeqLab
TRIS ICN Biomedicals
Table d. Buffer composition.
TBE buffer Loading buffer for agarose gels
107,8g Tris
55,0g boric acid
5,8g EDTA
pH 8,0; add 1000 ml aqua dest.
50mM EDTA
30% glycerin
Small amount of bromophenol blue
55
Legends of tables
Table 1. Main growth factors and its function in osteoblast differentiation.
Table 2. Local factors that regulate bone remodelling.
Table 3. Summary of bone remodelling regulating hormones.
Table 4. Selected critical consideration in bone tissue-engineering scaffold design.
Table 5. Information about RT-PCR primers.
Table a. Summary of all used kits and biomaterials.
Table b. Summary of all used equipments.
Table c. Summary of all used chemicals.
Table d. Buffer composition.
56
Legends of figures
Figure 1. Regulation of bone formation.
Figure 2. The classification of biomaterials used for bone tissue engineering.
Figure 3. The structure of the PHB monomer and polymer.
Figure 4. Pictures of the surgical procedure.
a) The subcutaneous muscle pocket on the surface of the Musculus latissimus dorsi.
b) The PHB scaffold in a subcutaneous muscle pocket.
Figure 5. Schematic illustration of the PHB location and the removed muscle tissue.
Figure 6. Schematic illustration of the RNeasy Fibrous Tissue Mini Kit Procedure.
Figure 7. Illustration of the pouring and loading of a horizontal agarose gel.
Figure 8. The procedure of the mRNA quantification using Real-time RT-PCR.
Figure 10. Standard PCR reaction and Sybr-Green Real-Time PCR.
Figure 11. PHB scaffold after 6 weeks of subcutaneous implantation with newly built
capillaries.
Figure 12. The electropherogram at 260 nm using the NanoDrop® ND-1000 UV-Vis
spectrophotometer of one muscle tissue RNA probe isolated from Musculus
latissimus dorsi.
Figure 13. Example of RNA isolated from muscle run on an agarose gel and strained with
ethidium bromide. 28S and 18S ribosomal RNA bands are indicated.
Figure 14. Agarose gel electrophoresis of all used genes samples after RT-PCR for the
verification of the purity and length of the amplicons.
Figure 15. Relative expression of the VEGF mRNA.
Figure 16. Relative expression of the IGF1 mRNA.
Figure 17. Relative expression of the IGF2 mRNA.
Figure 18. Relative expression of the GDF8 mRNA.
57
Eidesstattliche Erklärung / Affirmation Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig verfasst und keine
anderen als die angegebenen Hilfsmittel benutzt habe.
Die Dissertation ist bisher keiner anderen Fakultät vorgelegt worden.
Ich erkläre, dass ich bisher kein Promotionsverfahren erfolglos beendet habe und dass eine
Aberkennung eines bereits erworbenen Doktorgrades nicht vorliegt.
Datum Unterschrift
58
Danksagung / Acknowledgment
An vorrangiger Stelle möchte ich mich bei meinem Doktorvater Herrn Prof. Dr. Tomasz
Gedrange für die interessante Aufgabenstellung, die Anleitung und das ständige Interesse am
Fortgang der Arbeit sehr herzlich danken.
Ein ganz besonderer Dank geht an Frau Dr. Christiane Kunert-Keil für die großartige
Betreuung während meiner Doktorarbeit und ihre konstruktive Kritik, die in einem sehr
großen Maße zum Gelingen dieser Arbeit beitrugen. Sie hatte stets ein offenes Ohr für Fragen
und Probleme und stand mir jederzeit mit fachlichem Rat zur Seite.
Bei Frau Ingrid Pieper möchte ich mich ganz herzlich bedanken. Sie hat mich im Labor nicht
nur mit Hinweisen und Ideen unterstützt, sondern auch dafür gesorgt, dass der Spaß an der
Arbeit nicht verloren ging. Ich danke ihr ebenfalls für die Gespräche nicht nur über berufliche
Belange.
Meinem Kollegen, Herrn Dr. Phillip Eigenwillig danke ich für die grafische Unterstützung
dieser Arbeit und ein nettes und kollegiales Arbeitsklima in unserem Behandlungszimmer.
Den Mitgliedern der Abteilung für Kieferorthopädie der ZMKK danke ich für ihre
Hilfsbereitschaft, Freundlichkeit und die überaus angenehme Arbeitsatmosphäre.
Bei allen Mitarbeitern der Klinik mit Poliklinik für Innere Medizin C, Hämatologie,
Onkologie und Transplantationszentrum des Greifswalder Klinikums bedanke ich mich für
das gute Arbeitsklima und die Bereitstellung des Real-Time PCR Gerätes in der
Forschungsgruppe von Prof. Dr. Gottfried Dölken und Prof. Dr. Christian Schmidt.
Bei Herrn Prof. Dr. Alexander Wöll und Frau Dr. Silke Lucke möchte ich mich bedanken für
die freundliche Bereitschaft zum Korrekturlesen und ermunternde Worte.
Ein ausdrücklicher Dank gilt meinen Freunden: Herrn Dr. Ingo Merkl, Frau Dr. Margarita
Ruiz-Bamberg, Kantor Martin Rost, Herrn Dr. Thomas Klinke, Herrn André Wendel und
Familie Junge, für die moralische, verständnisvolle und auch anderweitig währende
Unterstützung.
Meiner Schwester Dr. Marzena Gredes danke ich für ihr Verständnis, ihre Unterstützung und
Ermutigungen.
Insbesondere möchte ich auf diesem offiziellen Weg meinen Eltern und meiner Oma, denen
diese Doktorarbeit gewidmet ist, danken. Sie haben mir jederzeit in jeder Hinsicht zur Seite
gestanden und mich nicht zuletzt fortlaufend ermuntert, diese Dissertation fertig zu stellen.
Prace ta dedykuj mojej Rodzinie, która zawsze we mnie wierzy i wspiera. Wam Kochani,
dedykuje ta prace.
59
D R . T O M A S Z J A K U B G R E D E S PERSÖNLICHE ANGABEN
Familienstand: ledig
Staatsangehörigkeit: Polen
Nationalität: polnisch
Geburtsdatum: 20. August 1975
Geburtsort: Dzierżoniów (Polen) AUSBILDUNG
09/1982 – 06/1990 Grundschule in Dzierżoniów
09/1990 – 06/1994 Lyzeum Nr.2 in Dzierżoniów, Abitur
10/1995 – 07/2000 Studium an der Wrocławer Universität, Fachrichtung Chemie (Diplom-Chemiker) 10/2000 – 07/2005 Studium an der Medizinischen Piastów-Śląskich-Universität in Wrocław, Fakultät für Zahnmedizin (Staatsexamen - Approbation als Zahnarzt)
12/2007 Promotion zum Dr.rer.med. an der EMAU Greifswald, Poliklinik für Kieferorthopädie Thema: Untersuchung der Genaktivität im Kiefer-gelenkknorpel des Schweins (Sus scrofa domesticus) nach Vorverlagerung des Unterkiefers
Seit 2007 Ausbildung zum Fachzahnarzt in der Kieferorthopädie- EMAU Greifswald
W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D •
T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h ) E - M A I L : t h o m a s g r e d e s @ y a h o o . d e
60
D R . T O M A S Z J A K U B G R E D E S
11/2009 Abgabe und Annahme der Promotion zur Erlangung des akademischen Grades “Dr.med.dent.“ mit dem Titel “Changes in insulin like growth factors, myostatin and vascular endothelial growth factor in rat musculus latissimus dorsi by poly-3-hydroxybutyrate implants” durch das Dekanat der Medizinischen Fakultät der EMAU Greifswald Rigorosum voraussichtlich im Februar 2010
BERUFSERFAHRUNG 10/2005 – 09/2006 Niederschlesiesches Zentrum für Pädiatrie (Dolnośląskie Centrum Pediatryczne im. J. Korczaka, Wrocław) - Zahnmedizinisches Assistenzjahr Seit 2006 wissenschaftlicher Mitarbeiter an der Poliklinik für Kieferorthopädie, Kinderzahnheilkunde und Präventive Zahnheilkunde, EMAU Greifswald Seit 2007 Weiterbildungsassistent an der Poliklinik für Kieferorthopädie,
Kinderzahnheilkunde und Präventive Zahnheilkunde, EMAU Greifswald
FORSCHUNGSPROJEKTE ITI Foundation 2007-2008
Early loading of palatal implants (ortho-implant type II): a prospective multicenter
randomized controlled clinical trial. (Mainz/Dresden/Greifswald)
DOT 2008-2009
Untersuchung der Anwendung von Knochenersatzpaste bei ausgeprägten Knochendefekten.
W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D • T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h )
E - M A I L : t h o m a s g r e d e s @ y a h o o . d e
61
D R . T O M A S Z J A K U B G R E D E S KONGRESSTEILNAHMEN
- International Annual Meeting of the Anatomische Gesellschaft; 30.03.-2.04.2007,
Giessen
- I. Internationales Fachsymposium zur Problematik der „Schlafatemstörungen“;
Greifswald, 07.07.2007
- Bone substitutes and endoprosthetic materials for structure conservation; Brunico 11-
14.03.2008, Italien
- II. Internationales Fachsymposium zur Problematik der „Schlafatemstörungen“;
Greifswald, 12.07.2008
- 84th Congress of the European Orthodontic Society- EOS; Lissabon, 10.-14.06.2008,
Portugal
- Internationale Zahnmedizinische Konferenz in der Periodontologie, 18.-20.09.2008,
Białystok, Polen
- XIV Zahnmedizinische Konferenz Expo-Andre in Toruń, 17-18.10.2008, Polen
- Knochenersatzmaterialen in der Kieferorthopädie; Brunico 16-19.03.2009, Italien
- 109th American Association of Orthodontists Annual Session in Boston,
Massachusetts, 1.-5.05.09, USA
- 85th Congress of the European Orthodontic Society- EOS Helsinki, 10.-14.06.2009,
Finnland
- Polish-German Life Science Forum, Szczecin 24.-25.09.2009, Polen
- XV Zahnmedizinische Konferenz Expo-Andre in Toruń, 15.-17.10.2009, Polen
PUBLIKATIONEN UND KONGRESSBEITRÄGE Veröffentlichungen:
1. A novel post-and-core restoration system--results of three years of clinical application of the "Wuerzburg Post". Rottner K, Boldt J, Proff P, Spassov A, Gredes T, Mack F, Richter EJ, J Physiol Pharmacol. 2008 9; 5:105-15.
W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D • T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h )
E - M A I L : t h o m a s g r e d e s @ y a h o o . d e
62
D R . T O M A S Z J A K U B G R E D E S
2. Evaluation of shape and size changes of bone and remodelled bone substitute after different fixation methods. Gedrange T, Mai R, Mack F, Zietek M, Borsos G, Vegh A, Spassov A, Gredes T., J Physiol Pharmacol. 2008 59; 5:87-94.
3. Adaptation of myosin heavy chain mRNA expression after implantation of
poly(3)hydroxybutyrate scaffolds in rat m. latissimus dorsi. Mack HB, Mai R, Lauer G, Mack F, Gedrange T, Franke R, Gredes T., J Physiol Pharmacol. 2008; 9; 5:95-103.
4. Physiological functions of the human finger.
Dumont C, Burfeind H, Kubein-Meesenburg D, Hosten N, Fanghanel J, Gredes T, Nagerl H., J Physiol Pharmacol. 2008 Nov;59 Suppl 5:69-74.
5. Different bone sesitivity to malformations induced by procarbazine in fetal rats.
Weingartner J, Proff P, Fanghanel J, Kundt G, Gedrange T, Kubein-Meesenburg D, Gredes T., J Physiol Pharmacol. 2008 Nov;59 Suppl 5:17-25.
6. A new design for post and core restorations implementing positive locking.
Richter EJ, Boldt J, Groth S, Proff P, Gredes T, Rottner K., Biomed Tech (Berl). 2008;53(5):234-41.
7. The influence of the root cross-section on the stress distribution in teeth restored with a positive-locking post and core design: a finite element study. Schilling KU, Rottner K, Boldt J, Proff P, Gredes T, Richter EJ, Reicheneder C., Biomed Tech (Berl). 2008;53(5):255-8.
8. Increased oxidative stress in mdx mice masticatory muscles
Spassov A., Gredes T., Gedrange T., Pavlovic D., Lupp A., Kunert-Keil C.; Am J Orthod and Dent Orth, submitted
9. Histological changes in masticatory muscles of mdx mice
Spassov A., Gredes T., Gedrange T., Lucke S., Pavlovic D., Kunert-Keil C., Arch Oral Biol 2009, accepted
10. Differential expression of MyHC isoforms in masticatory muscles of mdx mice
Spassov A., Gredes T., Gedrange T., Lucke S.,Morgenstern S., Pavlovic D., Kunert-Keil C., Europ J Orthod, submitted
11. Caveolin-1, caveolin-3 and VEGF expression in masticatory muscles of mdx mice
Kunert-Keil C., Gredes T., Lucke S., Morgenstern S., Pavlovic D., Gedrange T., Spassov A., Histochem Cell Biol, submitted
W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D • T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h )
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12. Expression of muscle specific growth factors in mdx mice masticatory muscles Spassov A., Gredes T., Gedrange T., Pavlovic D., Lucke S., Kunert-Keil C., Europ J Orthod submitted
13. Histological changes and changes in the myosin mRNA content of the porcine
masticatory muscles after masseter treatment with botulinum toxin A. Gedrange T., Gredes T., Mai R., Kuhn U.D., Kunert-Keil C., Fanghänel J., Spassov A., Clinical Oral Invest, submitted
14. Comparison of reference points in different methods of temporomandibular joint
imaging Gedrange T., Spassov A., Hietschold V., Gredes T., Fanghänel J., Laniado M., J Visualization, submitted
15. Orthodontic tooth movement into jaw regions treated with synthetic bone substitute
Gedrange T., Mai R., Weingaertner J., Fanghänel J., Allegrini S., Spassov A., Gredes T., Rottner K.,. Proff P., Adv Med Sci, submitted
16. Morphological evaluation of bone defect regeneration after treatment with two
different bone substitution materials on the basis of BONITmatrix® Kunert-Keil C., Gredrange T., Mai R., Spassov A., Franke R., Lucke S., Klinke T., Habersack K., Gredes T., J Physiol Pharmacol., submitted
Vorträge:
1. „Kształtowanie dziąsła przy implantach NobelBiocare“; Internationale Zahnmedizinische Konferenz in der Periodontologie, 18.-20.09.2008, Białystok, Polen
2. „Zastosowanie medycyny regeneracyjnej w stomatologii”; XIV Zahnmedizinische Konferenz Expo-Andre in Toruń, 17-18.10.2008, Polen
3. „Regenerative Zahnmedizin“; Knochenersatzmaterialen in der Kieferorthopädie; Brunico 16-19.03.2009, Italien
4. “Gene expression investigation of the pigs condylar cartilage after anterior mandibular displacement”; 109th American Association of Orthodontists Annual Session, Boston, Massachusetts, 1.-5.05.09, USA
Zitierbare Abstrakts und Kurzbeiträge:
1. Eur J Orthod 2008 30: 1-199 “CEPHALOMETRIC CHARACTERIZATION OF AN
ORTHODONTIC PATIENT SAMPLE” EUROPEAN ORTHODONTIC SOCIETY, 84th Congress 2008, 10–14 June, Lisbon (Portugal)
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2. Eur J Orthod 2008 30: 1-199 “SOFT TISSUE INTEGRATION IN THE NECK AREA
OF IMPLANTS” EUROPEAN ORTHODONTIC SOCIETY, 84th Congress 2008, 10–14 June, Lisbon (Portugal)
3. Eur J Orthod 2009 31; e21; “FIBRE TYPE DISTRIBUTION AND OXIDATIVE
STATE IN MASTICATORY MUSCLES OF MDX-MICE.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)
4. Eur J Orthod 2009 31; e61; “ORTHODONTIC TOOTH MOVEMENT INTO THE
MANDIBULAR ALVEOLUS AFTER TOOTH EXTRACTION.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)
5. Eur J Orthod 2009 31; e63; “CHANGES IN GENE MATERIAL OF THE PIG
MANDIBULAR CONDYLAR CARTILAGE IN RESPONSE TO MANDIBULAR PROTRUSION.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)
6. Eur J Orthod 2009 31; e63; “INFLUENCE OF BONE SUBSTITUTE ON RAT
MUSCLE.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland) 7. Eur J Orthod 2009 31; e91; “CAVEOLIN-1, CAVEOLIN-3 AND VASCULAR
ENDOTHELIAL GROWTH FACTOR EXPRESSION IN MASTICATORY MUSCLES OF MDX MICE.” 85th EOS Tagung; 10-14.06.2009 Helsinki (Finland)
W A L T H E R - R A T H E N A U - S T R . 4 7 , 1 7 4 8 9 G R E I F S W A L D • T E L E F O N 0 3 8 3 4 / 8 6 7 5 4 3 ( d i e n s t l i c h )
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