Materials Science and Engineering C 30 (2010) 763–769

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Materials Science and Engineering C

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Maxillary sinus floor augmentation on humans: Packing simulations and 8 monthshistomorphometric comparative study of anorganic bone matrix and β-tricalciumphosphate particles as grafting materials

A. Martinez a,⁎, J. Franco b, E. Saiz b,1, F. Guitian c

a Facultad de Medicina y Odontología, University of Santiago de Compostela, Rua San Francisco s/n; 15782 Santiago de Compostela, Spainb Lawrence Berkeley National Laboratory, 1 Cyclotron Rd Mail stop 62R0203 Berkeley, CA 94720, USAc Instituto de Ceramica de Galicia, Av Mestre Mateo s/n, 15782 Santiago de Compostela, Spain

⁎ Corresponding author.E-mail address: arturo.martinez@usc.es (A. Martinez

1 Current address: UK Centre for Advanced StructuMaterials, Imperial College, London.

0928-4931/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.msec.2010.03.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 December 2009Received in revised form 24 February 2010Accepted 18 March 2010Available online 24 March 2010

Keywords:Grafting particulatesCalcium phosphatesHistomorphometryPacking simulationsSinus floor augmentation

The present study compares the behaviour of an anorganic bone matrix material and a synthetic β-Tricalcium phosphate employed as grafting materials in a sinus floor augmentation two step protocol inhumans. In order to estimate the initial occupation level for the two materials, an ‘in vitro’ simulation hasbeen performed to analyse macroporosity created due to particle packing in terms of porosity andinterparticle distances. Grafting in the sinus floor augmentation was performed by filling the defects onlywith pure grafting materials without autogenous bone addition. The new-bone generated is 100% based onthe osteoconductive properties of the grafted materials in contact with physiological fluids. The implantswere placed 8 months after the grafting procedure. All the implanted positions were biopsied and embeddedin methacrylate resin. Histomorphometric analyses were done over thin film undecalcified sections. Packingsimulations allow establishing a comparison of the resorbed volumes related to the initial occupancy of thegrafting materials inside the defect. The nature of this interconnected pore network is very alike for eithermaterial so new-bone generated was similar (∼35 vol.%).

).ral Ceramics, Department of

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The human body possesses intrinsic mechanisms which allow selfhealing, but ‘restitutio ad integrum’ is not frequent. Biomaterials canenhance the natural capability of healing and can be successfully used torestore some body functions [1]. Dental implants have become asatisfactory option to restore masticatory functions in patients withedentulous spaces [2]. Long term survival and success of dental implantsrequire primary stability and appropriate bone volume [3]. The scarringprocess in the uppermaxilla usually entails atrophic alveolar ridges, thatlack thevolume for apredictablefixtureplacement [4]. The employmentof guided bone regeneration procedures allows the improving of thebone crest volume. This increasing of bone volume in the posteriorupper maxilla has been achieved by combining various procedures andmaterials [5–7]. Sinus augmentation is one of the most predictableprocedures to increase the amount of bone in posterior edentulousmaxillae [8]. In cases where alveolar ridge height is less than 4 mm, atwo step protocol is recommended because it improves osteointegra-tion and long term success of the implants [9,10]. In a first surgical

procedure a sinus floor elevation is performed and the graft is placed.The material implanted under the Schneiderian membrane starts topromote bone formation and is replaced by the bone. In a secondsurgery, implants are placed in this medium, surrounded by the newformed bone and partially resorbed grafting material [11].

Autogenous bone grafts are still considered the gold standard for boneregeneration in implant dentistry [12]; they have the best behaviour interms of osteogenesis but they also have significant disadvantages. Thereis limited bone supply in the oral cavity and frequently bone should beharvested from an extraoral site. This procedure might require theemployment of general anaesthesia and is frequently associated withmorbidity at the donor site, and patient discomfort [13]. Differentmaterials have been proposed as bone substitutes; those materials areused as a scaffold that allows development of new-bone by maintainingan initial volumewhich is progressively replaced bynew-bone. Anorganicbone grafts (ABG) are different xenogenic materials (mainly from bovineorigin) fromwhich all organic components have been removed [5,14–19].This material and human cancellous bone are very alike in structure,having 75% to 80% porosity and a crystal size of approximately 10 nm [5].The interconnecting pore system of macropores, mesopores and micro-pores facilitates the vascular colonization and the osteoblasts appositionalgrowth [5,16]. ABG show high biocompatibility and osteoconductivitywhenused in sinus elevation procedures [16,17,20–22]. However, there issome controversy regarding the kinetics of resorption and new-bone

Table 1Commercial bone graft particulates.

Product Composition Origin Particle sizeinterval

Cerasorb® (Curasan, Germany) β-TCP Synthetic 100–500 µm500–1000 µm1000–2000 µm

Vitoss® (Orthovita, USA) β-TCP Synthetic 100–1000 µmCeros® granules (Mathys AG Bettlach,Switzerland)

β-TCP Synthetic 500–700 µm700–1400 µm

KeraOs® (Keramat, Spain) β-TCP Synthetic 250–1000 µmR.T.R® (septodont, UK) β-TCP Synthetic 500–1000 µmBio-Oss® (Geistlich, Switzerland) Anorganic bone

matrixBovine 250–1000 µm

Osteobiol®Apatos (TecnossDental, Italy)

Anorganic bonematrix

Porcine 600–1000 µm

Gen-ox® Inorganic(Baumer, Brazil)

Anorganic bonematrix

Bovine 250–1000 µm

Biogen® (Bioteck, Italy) Anorganic bonematrix

horse 500–1000 µm

Endobon® (Biomet, Switzerland) Anorganic bonematrix

Bovine 500–1000 µm

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formation. Many authors consider that due to its slow resorption ABG is along lastingmaterial [5,13,23,24]. Consequently the final result of an ABGimplanted site is a composite formed by new-bone and the remainingmaterial with controversial mechanical implications [21,25]. On the otherhand some authors think that this long term stability avoids undesirablebone volume resorption due to the bone remodelling process [22,26].Long term volumetric stability in sinus graft procedures has confirmedlong lasting presence of ABG particles [19]. Beta-tricalcium Phosphate(TCP) particles have been employed successfully as alloplastic graftingmaterials in sinus elevation procedures [27–30]. These particles exhibit amicro porous surface which facilitates the anchorage of proteins and cellsto the surface of the graft [31], have good osteoconductive properties andpresumably resorb faster than ABG [20]. Particulate grafts exhibit a goodbehaviour due to the growth and development of a vascular networkwithin the interparticle voids and have been successfully used in bonereplacement procedures with or without seeding stem cells [32]. Incontrast, graft blocks, might be rejected due to poor revascularization[8,33] andhave tobeadapted, prior to implantation, to thedefect,whereasparticulate grafts can easily fill the defect.

To facilitate bone growth into a porous scaffold,macropores should bebigger than 50 µmwith an optimum value over 300 µm to achieve goodosteogenesis [34]. Particle size affects the effective packing of the graftinside the defect;when employing particulatematerialsmacroporosity isdictated by particle packing [35]. Small particles might diminish the sizeof the interparticulate voids and should not be smaller than 10 µm inorder to avoid inflammatory reactions from the multinucleated giantforeign cells macrophagic activity [19]. On the other hand, large particlesmay retard the graft substitution and the formation of bone bridges [22].When these particulate grafts are used as scaffolds for bone regeneration,de novo bone formation process occurs as a consequence of a biologicalcascade of events during the initiation of bone formation by newlydifferentiating population of osteogenic cells, over particulate surfaces[36]. In order to provide the best environment possible for this process totake place, an effective particle packing should create the macroporosityrequired to allow the growth of vascular buds and eventually basicmulticellular units (BMU) inside the material [37].

Althoughmany different graftmaterials have beenproposedwe oftenlack systematic ‘in vivo’ studies that will define and assess quantitativelythe key parameters which determine the performance of the graft. It isnecessary to determine what these parameters are and relate theirevolution to the physicochemical characteristics of the material (compo-sition, resorbability, micro andmacro porosity and chemistry…). The aimof this study is to compare the resorption and bone generation of twodifferent particulate grafting materials at opposite extremes of theresorbability spectrum, 8 months after a sinus floor elevation procedure.ABG are considered long lasting materials whereas TCP is moreresorbable. Both materials were physically and chemically characterized.As long as biopsies are only harvested at a single time of 8 months afterimplantation it would not be possible to talk about resorption times. Inorder to establish a time zero situation of the materials when packedinside the defect, packing simulations have been performed to analyse ‘invitro’ particle arrangement and therefore their resulting porosity. Dataobtained from packing simulations allow estimating the resorptionvolumes for the grafted materials in combination with the resultsobtained from the histomorphometric measurements. This simulationalso gives information about the amount and distribution of macro-porosity available for new-bone growth.

2. Materials and methods

2.1. Graft characterization

There aremany available commercial bone graft particulates eitherfor ABG and TCP (Table 1). Amongst all thematerials the choice of twoparticular brands was made in terms of the same particle size intervaland their ease of availability. The calcium phosphate grafting

materials employed in the maxillary sinus floor augmentation arean anorganic bovine-bone derived (ABB) (Bio-Oss 200–1000 µm,Geistlich, Switzerland) and synthetic 100% β-Tricalcium Phosphate(TCP) (KeraOs, 200–1000 µm, Keramat, Spain). To guarantee enoughmaterial volume for physicochemical characterization and packingsimulation procedures, 4 commercial batches of each material weremixed and homogenized. No sieving or any kind of classification wasperformed so the evaluated and grafted materials were the same. Thecharacterization was made by Scanning Electron Microscopy (S-4300SE/N, Hitachi America Ltd., USA), X-ray Diffraction (XRD, Cu Kα1λ=1.5406 Å, 30 mA/40 kV. Siemens D-5000, Germany), Differentialthermal and gravimetric analysis (DTA-TG, PL-1640 STA, PolymerLabs, UK), Specific surface area with the Brunauer, Emmett and Tellermethod (BET, Gemini 2360, Micromeritics Instrument Corporation,GA, USA) and Mercury porosimetry (Autopore II 9220, MicromeriticsInstrument Corporation, GA, USA). The Ca/P molar ratio wasdetermined by quantitative X-ray diffraction analysis [38] of ‘asreceived’ material for the synthetic material, and over 1000 °C 1 hthermally treated ABB. In order to determine what is the effectivepacking porosity, particles of both materials were introduced inside5 mmdiameter glass test tubes. The tubes were filled with epoxy resin(Bepox 1159, Gairesa, Spain) under vacuum and remaining trappedair bubbles were eliminated by centrifugation at 3000 rpm for 15 min(H-900, Kokusan, Japan). Neither particle breakage nor packingimprovements have been detected because of the centrifugationprocess; the initial level of the materials inside the test tubesremained constant and SEM examination did not find evidence ofparticle breakage. Twenty axial slices were cut from the epoxy-infiltrated specimens and polished (Isocut, Ecomet 4, Buehler LTDLake Bluff, IL. USA). The effective packing porosity was measuredusing image analysis (Image J v. 1.40 g, NIH, USA) over backscatteredelectron (BSE) images. Interparticle void image analysis has also beendone over BSE images measuring distances between particles alongthe lines of an overlaid grid.

2.2. Patient selection

The selected patients were partially edentulous in the upper post-canine region. Previous exclusion criteria are shown in Table 2. A totalamount of 20 sinus augmentations were performed in 16 patientswith severely resorbed alveolar process (range 1–5 mm) and a meanof 3.8 mm of remaining bone. Themean age of patients was 49.5 years(range 38–67. Because of the dissimilarities in the alveolar ridgedefects, each sinus was considered independently (even in those

Table 2Patient exclusion criteria.

Exclusion criteria

Drug abuse or any significant systemic diseaseAffection of previous pathology in maxillary sinusSmokersUnder biphosphonates treatmentActive periodontitis

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patients who underwent a bilateral elevation) and the choice of thegraft nature was randomized. All the sinus elevation procedures wererequired in order to obtain enough amount of bone to allow theimplant placement. All patients were carefully informed about thesurgical procedure, the bone substitute materials and the implantprosthetic solutions. Full informed consent was obtained from all thepatients. Panoramic radiographswere taken before and after the sinusaugmentation, and after implant placement. The University ofSantiago/Institute Ethics Committee approved the study.

2.3. Sinus floor elevation procedure

The sinuses were randomly distributed into two groups; 10 sinuseswere filled with ABB and 10 with TCP. Both materials were mixed withphysiologic serum to facilitate manipulation. The surgical protocolemployedwithbothmaterialswas the same.All thepatientswereunderantibiotic prophylaxis before sinus elevation procedure: 2 g of Amox-icillin 1h before surgery. Surgery was performed on all patients underlocal anaesthesia (Ultracain 05 epinefrin, Normon, Spain). The tech-nique was very strict avoiding any damage to soft and hard tissues. Thesurgical procedure is well described in the literature [8]. The protocolemployed was a two-stage delayed approach. In all the cases a two-stage lateral approach was used. Longitudinal mucosal incision wasdisplaced palatally following the variation of overlapped flap, distalreleasing incision was made buccally, and the mesial releasing incisionwas avoided if possible. Sinus elevation procedure was carried outfollowing the antero-lateral approach. Sinus was filled up withparticulate grafts to a 13.1±1.8 mm height in order to bridge buccaland palatine walls. The average volume of graft particulates was 1.6±0.4 cm3. Integrity of Schneiderianmembranewas tested inall patientsand in none of the studied cases did perforation of themembrane occur.Anthrotomywindowwas closed, in all cases, with a resorbable collagenmembrane (Bio-Guide, Geistlich, Switzerland) covering the packedmaterial. Surgical wounds were closed with tension free sutures,releasing periostium, employing PGA (Monosin, Braum, Germany)and Gore-Tex sutures (W.L. Gore & Associates, Flagstaff, USA). Thefollowing post-operative protocol was common to all patients:Amoxicillin 1 g/8 h for 7 days to avoid infection, Ibuprofen-Arginine600 mg/8 h for 4 days, to reduce pain and swelling and Rinobanedif(Bayer, Barcelona, Spain) with endonasal application as a decongestiveto facilitate the sinus drainage and epithelisation. A 0.5 mm plasticvacuum-formed maxillary splint (Henry Schein, UK) was prepared atthe end of the surgical procedure, for each patient, and applied in theirupper maxilla to promote the mucous healing and protect the palatalsutures from the tongue, establishing an effective barrier between thesinus and the mouth. Sutures and splint were permanently removed2 weeks after surgery. Post-operatory complications were limited tolocal swelling. Postsurgical visitswere scheduled atmonthly intervals tocheck the healing process.

Table 3Physicochemical properties of the grafted materials.

Material BET (m2/g) Porosity (%) Mean Pore size (µm)

ABM 63.33 56.3 0.02TCP 0.37 17.8 1.09

2.4. Biopsy retrieval and implants placement

After a healing period of 8 months, and at the time that implantswere placed in each patient, alveolar bone samples were retrieved.The surgical procedure was made under local anaesthesia by doingsmall releasing incisions and with a supracrestal longitudinal incision.Biopsies were taken using a trephine (outer diameter 2.8 mm, andinner diameter 1.9 mm. Hu-Friedy Mfg. Co. Chicago, USA) undercopious 4–5 °C sterile saline irrigation. A biopsy has been obtained forevery implanted position. The depth of the biopsy was approximately10 mm. The biopsies were harvested in the site were the implants hadto be placed. The biopsy specimens were washed in sterile salinesolution and immediately immersed in 10% phosphate bufferedformalin solution, and kept refrigerated at 7 °C. The implants (MKIII, Nobel Biocare, SE) were placed following the specific protocol.Implants were covered again suturing the mucoperiostal flap.

2.5. Histology

The biopsy specimens were processed immediately to obtainundecalcified thin ground sections, following Donath's method [39].The preparations were dyed with Harris Hematoxiline (Papanicolau,Merck, Germany) and Wheatley's modification of thrichromic stain(Chromotrope2R,Newcommersupply, USA) andpreservedwith Canadabalsam solution (Fluka Biochemika, USA). Preparations were examinedby using a transmitted light microscope (Optiphot, Nikon, Japan)equippedwith a digital Camera (DP-12, Olympus, Japan).Morphometricstudy was carried out using Image J software. The retrieved biopsieswere divided into three zones, apical, central and occlusive. The amountof bone, remaining material and bone marrow was evaluated in themiddle third of thebiopsies. Themost apical thirdwas rejected because amore disaggregated appearance of the granulate materials and theocclusive one because it contained mature cortical bone.

2.6. Statistics

KS test was employed to verify the normality of the samples. Twotails T-test was employed using statistical software package (SPSS.V17, Chicago, USA). Values of Pb0.05 were considered significant.

3. Results

Table 3 summarizes the physicochemical properties of the grafts.Twomainweight losses can be observed in the DTA-TG analysis of ABB.The first one (5.1 wt.%) occurs from 100 °C to 777 °C. The second(1.2 wt.%) takesplacebetween800 °C and940 °C. Theweight lossof TCPis less than 0.1 wt.% in the whole range from 100 °C to 1000 °C. Themicroporosity of the ABB granules measured using mercury porosime-try is 56.3 vol.% with an average pore size diameter of ∼20 nm. The TCPparticles exhibitmuch lowermicroporosity (17.8 vol.%)with anaveragepore sizeof∼1 µm. Theobtainedmicroporosities are consistentwith theSEM observations (Fig. 1) and specific surface areas measured by BET.The only detectable phase by XRD in the TCP syntheticmaterial is β-TCP(Fig. 2). Two broad diffraction peaks in the 31.7°–32.7° range can beobserved in the XRD diffractograms of ABB. These peaks match the twomain peaks of Hydroxiapatite (HA, JCPDS file 9-432). After heating at1000 °C for 1 h the ABB graft consists mostly of well-crystallizedhydroxyapatite with a small content of β-TCP (Fig. 2). The Ca/P molar

Weight loss (1000C) (%) Ca/P Effective packing (%)

6.33 1.66 42.6±3.90.1 1.5 50.1±4.3

Fig. 1. SEMmicrographs of the starting particulates A. TCP particles show globular aggregated aspect with concavities and convexities. The spaces between aggregates give as a resultthe formation of grooves. B. ABB particles have sharp edges presumably derived from grinding cancellous bone structures. C. Microporosity of TCP; the TCP particles are formed bymicron-sized grains sintered together and exhibit a microscopic porosity consistent with Hg porosimetry readings. D. ABB particles exhibit a submicron porosity resulting from theremoval of the collagen fibers from the bone matrix during the deproteinization process. This sub micron porosity is responsible for the large specific surface of ABB.

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ratio measured using XRD is 1.5 for TCP (only β-TCP phase present) and1.66 for ABB (XRD quantitative analysis) [38]. The average interparticlespace is bigger for TCP (382 µm) than for ABB (340 µm) with a

Fig. 2. XRD spectra. A). TCP; there are no detectable secondary phases. B). As receivedAMB; The small sizes of the apatite needles present in the sample are responsible for thebroadening of the peaks on the 31.7°–32.7° range. C). ABB after 1 h at 1000 °C; there is asmall peakwhichmatches theβ-TCPmainpeak. The Ca/P is 1.5 forβ-TCP and 1.66 for ABB.

significant difference (Pb0.04). However, the interparticle distancedistribution is very alike for either material (Fig. 3). In both cases morethan 97% of measured interparticle distances are bigger than 50 µm(lower limit for macroporosity [34]). Due to the small population ofpores, smaller than 50 µm, all the interparticle surface measurementsare associated to macroporosity. These measurements gave as a resultfrom packing ‘in vitro’ simulations that macroporosity is larger for ABB(58.4±3.9 vol.%) than for TCP (49.9±4.3 vol.%). The differencebetween the groups is statistically significant (Pb0.001). Fig. 4 showsa BSE micrograph comparing ABB and TCP effective packing.

Monthly check-ups and radiographic examination before implantplacement have shown that there is no evidence of particle migration orvolume loss in any of the grafted positions. Well differentiated bonesurrounding and connecting remaining particles from both materialswere observed. Around 35 vol.% of new-bonewas found for both ABB andTCP. The proportion of lamellar bone was 40.7±15.1 vol.% for TCP groupand 34.9±7.1 vol.% for ABB (Fig. 5). The difference between the groupsis not significant (PN0.1). The remaining presence of TCP particles was32.6±6.2% and 34.8±10.5 vol.% for ABB without significant differences(PN0.5). The proportion of bone marrow is larger and more evenlydistributed in the ABB group (30.4±14.2 vol.%) than in the TCP group(18.1±8.2 vol.%), with a significant difference (Pb0.01). The new-boneformed was more homogeneously distributed in the ABB group. No gapsbetween thematerial andbonewere observed in any group. Therewas anintimate apposition of bone in contact with particles in both materials(Fig. 6); neither of the materials were encapsulated by connective orfibrous tissue, nor exhibited chronic or acute cell infiltration. Frequentlyosteoblasts or extra cellular matrix (ECM) were found in both groups inintimate contact with the material surface. Those osteoblast-like cells aresurrounded by amatrix that shows progressivemineralization. In the TCPsamples BMU were observed colonizing the material surface, showingscattered multinucleated cells related to the material degradation. Thesecells are next to groups of osteoblast-like cells related to ECM formation(Fig. 7), as described by the modelling and remodelling of the cancellousbone [40]. Small fragments of TCP are usually incorporated into the

Fig. 3. Inter particle distance distribution. Besides the region b100 9 mwhere ABB has ahigher percentage than TCP, the macropores created due to particle packing are veryalike for both materials.

Fig. 5. Histomorphometric results for bone, marrow and remaining grafting material.When remainingmaterial presence results are normalized using the zero time hypothesisto the initial occupation volume, it shows that TCP particles have been significantly moreresorbed than ABB ones.

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mineralized ECM of new formed bone that grows all around without anykind of gap (Fig. 8). Fragments of TCP have been separated from themainparticles and become surrounded by rich-cell mesenchymal matrix.Particles of remaining ABB show less affinity to the dye and seem to be

Fig. 4. BSE micrographs. Polished surface of ABB and TCP particles embedded in epoxyresin. The black zone corresponds to the graft free area. Interparticle distances weremeasured along the lines of the overlaid grid. The overall macroporosity is higher forABB but the inter particle voids are similar for both materials. The scale bar is 2 mm.

tightly integrated into the new formed bone. Some peripheral graft-bonelacunae of the ABB particles are colonized by cells from the host (Fig. 9).

4. Discussion

According to the analysis, ABB are bone derived particles withoutremaining organic matter mainly precedent from crushed cancellousbone. The analysed batches were composed bymature bonewith small

Fig. 6. Optical transmitted light microphotography of undecalcified thin sections dyedwith Harris Hematoxilyn/Chromotrope 2R stain. There is new-bone (NB) in directapposition to the surface of both materials. Particles are surrounded by new formed bone.TheABBparticles are easily recognizable because of less affinity to thedye, probably due tothe lack of collagen inside the particles.

Fig. 7. Transmitted light microphotography of undecalcified thin sections dyed with HarrisHematoxilyn/Chromotrope 2R stain. Bone metabolic unit colonizing the TCP graft interface.Inside this BMU there are multinucleated cells invading the TCP surface (inside black circle)and in the opposite pole an osteoblast-like cell (inside black square) is being included in theextracellular matrix. Note the new-bone (NB) lacunae occupied by osteocites.

Fig. 9. Transmitted light microphotography of undecalcified thin sections dyed with HarrisHematoxilyn/Chromotrope 2R stain. ABB-bone interface detail. Note the osteocite like cellscolonizing the lacunae of an ABB particle (highlighted with white ellipses). NB (new-bone).

768 A. Martinez et al. / Materials Science and Engineering C 30 (2010) 763–769

quantities of carbonated species that decompose during heatingbetween 700 °C and 1000 °C. The elevated BET values are consistentwith the removal of the organic matter from the bone structure and aremainingmicroporositywith averagepore sizes of 20 nm. In the case ofTCP particles, themicropores have amedian pore size of 1 µmand a BETvalue b1 m2/g. The irregular shape of both graft materials allows thecreation of an internal macroporosity between particles when packedinside a defect. From our ‘in vitro’ simulations, this macro porosity is10 vol.% bigger forABBparticles.Nevertheless, as shown in Fig. 3, in bothcases there are gaps larger than 100 µm and their average interparticledistance measured by image analysis is bigger than 300 µm. This valuehas been pointed out as adequate for good osteogenesis because it is thecritical sizewhere capillaries can be observed [34]. Although the overallmacroporosity is larger for ABB particles, the macropore systems arevery alike for both materials (Fig. 3). Because of this similarity in thepore system, the main difference in the host response to the graftingmaterials should be attributed to the nature of the materials itself, interms of chemical solubility and physical morphology. Thoroughcharacterization of grafting particulate materials is of vital importancefor the determination of accurate interplays between their propertiesand biological response [30].

Fig. 8. Transmitted lightmicrophotography of undecalcified thin sectionsdyedwithHarrisHematoxilyn/Chromotrope 2R stain. Detail of a small TCP fragment included in the boneextracellular matrix (ECM) with close apposition to osteocites inside the lacunaesurrounding it. The new-bone (NB) ECM different mineralization gradation could beobserved: Pink to purple low mineralized bone. Bluish high mineralized bone.

Besides longitudinal studies [19], the absence of data for the initialoccupation of the grating material makes it impossible to discussresorption percentages from the histomorphometric measurements.According to ‘in vitro’ simulation, the initial occupation volumes were50.1 vol.% and 42.6 vol.% for TCP and ABB respectively. Raw analysis ofthe remaininggraft presencemakesnodifferencebetweenTCPandABB.In thisworkwehypothesize that the occupancy of thegraftingmaterialsobtained from the packing simulations can be taken as a zero timesituation. If we divide the remaining percentage of TCP and ABB by theinitial occupancypercentages (TCP=50.1 vol.%, ABB=42.6 vol.%), thenthe remaining graft percentages would transform into 65.1 vol.% and81.6 vol.% for TCP and ABB respectively (Fig. 5). These remainingpercentagesmean that TCP resorption is 34.9 vol.%andABB resorption is18.4 vol.%. These resorption percentages are consistent with thechemical composition of the materials; ABB particles due to theirmajor content in HA are less soluble than TCP particles [41], therefore itis reasonable that TCP resorption percentage be higher.

The present study agrees with previous ones showing that bothmaterials are osteoconductive and allow the formation of bone withoutinflammatory infiltrate even without harvesting autogenous bone[20,29]. After an 8 month grafting period, more than 30 vol.% of new-bone has been created in the two groups. This new-bone volume willprovide the required stability for the implants placed in a second step.Although TCP has resorbed more than ABB, there are no significantdifferences in the new-bone amount between sites grafted with ABB orTCPparticles. Inboth cases, thevolumepercentageofnew-bone is alwayssmaller than the initial macroporosity between particles and there isenough bone volume for the right placement of the implants, evenwithout the addition of autologous bone with the grafted materials. Thisavailability of space and the similarity in the interparticle distancesmightexplainwhy thebonevolumecreated is statistically the same forABBandTCP. The interconnected macroporosity allows a rapid interaction withthemedia creating an early clot rich in growth factors (GF) and proteins.The releasing of those GF and proteins will colonize the surface of bothmaterials and will start the formation of new-bone [42].

Themain goal in a maxillary sinus floor augmentation procedure is toobtain enough bone volume to place osseointegrated fixations. It is alsovery important that thisnewgeneratedbonebeable toexhibit a long termfavourable biomechanical response. ABB and TCP have shown excellentbiocompatibility and osteoconductive capabilities [5,14,17,19,27–30]. Themain difference between ABB and TCP particles response might beattributable mainly to their different solubility ‘in vivo’. The resorption ofABBparticles is slower thanTCPsonew-bone ismainly generatedover thesurface of the ABB particles. Considering the zero time hypothesis, in thecaseof TCPparticles the amountof resorbedmaterial is almost double thatof ABB. Data reported in the literature shows that after 6 months the

769A. Martinez et al. / Materials Science and Engineering C 30 (2010) 763–769

resorption of ABB and its eventual substitution by bone slows down andthe material may last for years [17,18,43]. The resulting structure is acomposite formed by new-bone and residual ABB particles [44]. Thiscomposite seems to have an efficient biomechanical response adequate tothe implant mechanical demands [25]. Although further biomechanicalassessments arenecessary, it is reasonable toassume that the residualABBparticles are accepted as a structural element in the bone remodellingprocess, and bone grows surrounding them [44]. We have found thepresence of cells from thehost inside the lacunae of theABB,mainly in theperipheral portions showing the full tolerance and compatibility of thisgraft material. Osteoblast-like cells have been found directly attached tothe surface of both materials with the formation of osteoid matrix. It isimportant to highlight that after an 8month grafting period, the interfacebetweenABB andnew-bone is stable,whereas for TCP there seems to be adynamic resorption front where thematerial is progressively replaced bynew-bone. The interface stability of ABB is supported by a slow solubilityof ABB particles and the use of the empty structures by the host cells(Fig. 9) as already reported by Mangano et al. [5].The osteoconductivecapability of β-TCP is well known [45–47] and the results we haveobtained agreewith other author'sfindingswhenworkingwith similarβ-TCP grafts in maxillary sinus floor augmentation procedures [27–30]. β-TCP is a resorbable material but there are still doubts about how thisprocess takes place. We have hardly found reticuloendothelial system(RES) cells in the interface. This might suggest that osteoclasts ormacrophagic cells do not play an important role in the resorption processof β-TCP grafts as previously reported by Knabe [29] and Zerbo [27]. Themobile resorption front does not affect the growth of new-bone, and thebone remodelling process takes place because of a dissolution process ofthematerial and eventually the surface colonization by capillaries and theconsequent BMU development.

5. Conclusions

The performance of packing simulations in order to establish thetime zero stage for grafting materials helps to evaluate the resorptionpercentage of the grafted materials from the carrying out of a biopsywhen the implants are placed. The standardization of this packinganalysis procedurewould avoid the ethical controversy of the executionof multiple biopsies on humans at different times. The ABB and TCPevaluated seem to work as a good scaffold for bone regeneration,allowing the migration and formation of new-bone over their surfaces.The use of these materials in sinus augmentation without addition ofautogenous bone seems to bring similar results. No implant failureswere reported during the follow-up period 2–3.5 years after placement.Eightmonths after grafting, the amount of new-bone ismore than1/3 ofthe total volume for either material, and presumably will increase withtime. This agrees with our evaluation that shows that the differences inresorption rates do not have significant effects on the kinetics of new-bone formation. In this respect, the kind of macroporosity (similar inboth materials) seems to be the key parameter. The behaviouraldifferences are attributable to the chemical composition and micropo-rosity of each grafting material. However, in order to fully assess howthese differences affect the performance of the graft, the next step willbe to perform a systematic biomechanical analysis of the regeneratedbone and the bone–particle construct.

Acknowledgements

This work was partially supported by the National Institutes ofHealth (NIH) under Grant no. 5R01 DE015633 and the Ministry ofIndustry and Energy of Spain project number PROFIT300100-2006/73.

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