Int. J. Oral Maxillofac. Surg. 2008; 37: 275–281doi:10.1016/j.ijom.2007.11.010, available online at http://www.sciencedirect.com

Leading Research PaperTissue Enhancement

Alveolar bone regenerationusing absorbable poly(L-lactide-co-e-caprolactone)/b-tricalciumphosphate membrane andgelatin sponge incorporatingbasic fibroblast growth factor

Y. KinoshitaM. Matsuo, K. Todoki, S. Ozono S. Fukuoka, H. Tsuzuki, M. Nakamura, K.Tomihata, T. Shimamoto, Y. Ikada: Alveolar bone regeneration using absorbablepoly(L-lactide-co-e-caprolactone)/b-tricalcium phosphate membrane and gelatinsponge incorporating basic fibroblast growth factor. Int. J. Oral Maxillofac. Surg.2008; 37: 275–281. # 2007 International Association of Oral and MaxillofacialSurgeons. Published by Elsevier Ltd. All rights reserved.

0901-5027/030275 + 07 $30.00/0 # 2007 Interna

tional Association of Oral and Maxillofacial Surgeo

Y. Kinoshita1, M. Matsuo1,K. Todoki1, S. Ozono1,S. Fukuoka1, H. Tsuzuki1,M. Nakamura1, K. Tomihata2,T. Shimamoto2, Y. Ikada1,3

1Institute for Frontier Oral Science, KanagawaDental College, Yokosuka, Kanagawa, Japan;2Medical Material Center, Research &Development Center, Gunze Ltd, Japan;3Nara Medical University, Nara, Japan

Abstract. The aim of this study was to evaluate the effects of combining a porouspoly(L-lactide-co-e-caprolactone)/b-tricalcium phosphate membrane and gelatinsponge incorporating basic fibroblastic growth factor (bFGF) on bone regenerationin mandibular ridges. Four full-thickness saddle-type defects (10 mm long � 5 mmdeep) were symmetrically created in both edentulous mandibular alveolar ridges of6 beagles. The dome-shaped membrane was secured to each defect site, and agelatin sponge containing 200 mg bFGF was implanted on the left side of eachdefect (experimental group). Only the membranes (control group) were secured tothe defect sites on the right. Three and 6 months later, 3 animals were killed. Boneregeneration was analyzed by soft X-ray photographs, micro-computed tomography(CT) images, and peripheral quantitative CT (pQCT), and then examinedhistologically. Soft X-ray examination revealed an increase in new bone volume inthe experimental group 6 months postoperatively. pQCT showed that immaturebone density was higher in the experimental group. Micro-CT images revealed wellformed new bone along the original contour of the dome-shaped membrane in theexperimental group. Histologically, inflammatory infiltration of tissue surroundingthe membranes was slight. These results suggest that combining the poly(L-lactide-co-e-caprolactone)/b-tricalcium phosphate membrane and bFGF-gelatin sponge ispromising for alveolar ridge reconstruction.

Keywords: guided bone regeneration; poly(L-lactide-co-e-caprolactone); b- tricalcium phos-phate; basic fibroblast growth factor; gelatinsponge.

Accepted for publication 22 November 2007Available online 11 February 2008

ns. Published by Elsevier Ltd. All rights reserved.

276 Kinoshita et al.

Fig. 1. P(LLA-CL)/b-TCP mesh and PLLA pin.

Reconstruction of a thin or low-volumealveolar bone ridge is an important area ofimplant and prosthetic dentistry. Guidedbone regeneration (GBR) has attractedattention as a promising method. TheGBR membrane plays an essential rolein preventing the influx of soft tissue, andguides new bone formation into thedesired shape. Requirements include bio-compatibility, maneuverability, flexibil-ity, bioabsorbability, and sufficientmechanical strength to withstand soft-tis-sue compressive forces or occlusionforces during bone regeneration. So far,titanium mesh, extended polytetrafluor-oethylene, collagen and several biode-gradable polymers have been used asmembranes for GBR4,7,8,17. Titaniumand extended polytetrafluoroethylenemembranes are commonly used for con-venience, but are often associated withcomplications, including membraneexposure and infection, and they also haveto be removed by a second surgical pro-cedure after bone formation. Althoughmembranes made of collagen are cell-adhesive and absorbable, their mechan-ical strength is too poor to provide spacefor bone formation. While membranesmade of biodegradable synthetic poly-mers, such as poly(L-lactide) (PLLA),polyglycolide, polycaprolactone and theirco-polymers, do not require a secondsurgery, they present limitations regard-ing their ability to provide space for boneformation, early/late absorption, mechan-ical strength and inflammatory reactionduring biodegradation1,4,7,8,21.

Recently, to overcome the disadvan-tages of biodegradable syntheticpolymers while maintaining their advan-tages, polymer/calcium phosphate com-posites have been investigated5,9,10.Inorganic materials such as calcium phos-phates are expected to provide rigidity tothe soft polymer, a PH buffering effect inthe surrounding tissue, and X-ray imper-meability making it possible to monitorthe GBR membrane after implantation. Aporous biodegradable GBR membranemade of poly(L-lactide-co-e-caprolac-tone) (P(LLA-CL)) and b-tricalciumphosphate (TCP) has been manufactured.

In general, a GBR membrane does notitself enhance osteoblast proliferation andmigration, or extracellular matrix synth-esis. Use of a growth factor along with themembrane to promote bone formationmay be useful for reconstruction of thealveolar bone2,3,5,6,12,15,23. Basic fibroblastgrowth factor (bFGF) is known to enhancethe proliferation of undifferentiatedmesenchymal cells, resulting in promotionof bone formation14–16,22. When injected

in the free form, the growth factor does notstay at the injection site long enough toproduce the expected results. In an attemptto overcome this problem, TABATA et al.20

developed a biodegradable hydrogel com-posed of acidic gelatin to enable bFGF tobe released at the site of action for anextended time period. This hydrogel canionically interact with bFGF, and the in-vivo degradability of the growth factor canbe controlled by altering the extent ofgelatin cross linking (water content ofthe hydrogel). These authors reported that,when implanted in rabbit skull defects6 mm in diameter, the gelatin hydrogelsincorporating 100 mg of bFGF promotedbone regeneration at the defect in markedcontrast to free bFGF of the same dose.The objective of the present study was toevaluate the effects of combined treatmentwith a porous P(LLA-CL)/b-TCP mem-brane and bFGF-incorporated gelatinsponge on bone regeneration in a full-thickness saddle-type defect of the man-dibular alveolar bone ridge.

Materials and methods

Six beagles (1 year old, weighing 8–10 kg)were used. The animals had access to astandard laboratory diet and water untilthe beginning of the study. The AnimalCare Committee of Kanagawa Dental Col-lege approved the animal care protocol.

P(LLA-CL) (75:25) containing 30% b-TCP was pressed at 180 8C, to make a 0.3-mm-thick film. In addition, 0.2-mm-dia-meter holes were made at a distance of0.6 mm apart in the porous membrane(Fig. 1). PLLA (MW = 2–2.5 � 105) rodswere manufactured with an extruder. Therods were initially drawn using a ratio of2.5, and were shaped into pins. The pinshad a thread diameter of 3 mm and heightof 2.7 mm (Fig. 1).

The gelatin used had an isoelectric pointof 5.0 and a water content of 95% (Nitta

Gelatin Co., Japan). Human recombinantbFGF (Kaken Pharmaceutical Co., Japan)was used.

Surgical extraction

All animals operated on were premedi-cated with an intramuscular injection ofKetalal 10 mg/kg (Sankyo Co., Japan) andanesthetized using an intravenous injec-tion of Nembutal 25 mg/kg (DainipponSumitomo Pharma Co., Japan). The sec-ond, third, and fourth mandibular premo-lars on the left and right sides, and the firstmandibular molar were extracted in eachanimal. The sharp alveolar ridges afterteeth extraction were removed, followedby elevation of buccal and lingual muco-periosteal flaps. The flaps were then read-apted and sutured with 4-0 nylon sutures.The fourth premolars in the maxillary archwere also extracted to alleviate potentialtrauma to the experimental sites during thehealing phase.

Defect induction and treatment

Three months after teeth extraction, a totalof 4 saddle-type defects were symmetri-cally created in the left and right edentu-lous alveolar ridges of the mandible ineach of the 6 animals. Briefly, a mucoper-iosteal flap was elevated following gingi-vobuccal fold incisions. Next, 2 blocks ofbone (mesiodistal length of 10 mm atdepth of 5 mm) spaced 5 mm apart wereremoved from each side of the alveolarridge of the mandible. The bone blockswere outlined with a dental bur using ahand engine under irrigation with sterilewater, and were removed with a thin chi-sel. The defects were immediately sub-jected to the following treatments.

A P(LLA-CL)/b-TCP membraneshaped into a dome by heating to about70 8C was trimmed with scissors andadapted to the defect, and a 20-mg acidic

Alveolar bone regeneration using bFGF 277

hydrogel sponge containing 200 mg bFGF(bFGF-gelatin sponge) was implanted ineach of the left-side bone defects (theexperimental group). Then, each mem-brane was secured to the existent boneon the buccal site with 2 PLLA pins, whichwere press fitted into holes that wereguided into the bone and mesh by a dentalsteel bur (No. 700). The mucoperiostealflap was replaced and sutured with absorb-able threads (Vicryl, Ethicon Inc., USA)(Fig. 2). As the control group, P(LLA-CL)/b-TCP membranes only wereadapted and secured to the right-side bonedefects. All animals received an intra-muscular injection of broad-spectrumantibiotics (Viccillin, Meiji Co., Japan,50mg/kg/day) for 3 days postoperativelyand were fed a diet of soft canned food forthe first month postoperatively.

Post-surgery procedures

Every animal was clinically observed atleast once a week postoperatively. Threeanimals each were scheduled for euthana-sia at 3 and 6 months. The animals wereanesthetized and killed by an intravenousinjection of concentrated Nembutal. Theright and left mandibles were removedseparately and placed in a solution of10% buffered formalin for 3–5 days, afterwhich the muscle around the mandiblewas removed. Each mandible was treatedand examined as follows.

Soft X-ray photographs were taken bySofron (SRO-M50, Soken, Tokyo, Japan)at 50 Kv and 3 mA for 60 s, using electro-

Fig. 2. Schema of experimental procedure. P(LLdefect site and 20-mg acidic hydrogel spongesponge) was implanted in each of the left-sidmembrane was secured to the existent bone onP(LLA-CL)/b-TCP membranes were adapted andgroup).

scopic films (Fuji Photo Film Co., Ltd).All photographs were taken with an alu-minum step-wedge in order to correctunevenness in development. The photo-graphs were analyzed using a digitalimage-analyzing program (HC-2500/OL,WinROOF, Mitani Corp., Japan).

Bone regeneration of the defect siteswas analyzed by pQCT (XCT-ResearchSA+) and micro-CT (SMX-100CT, Shi-mazu Co., Japan).

For pQCT, each mandible was trimmedto a block of mesiodistal length 4 cmcontaining the bone-defect site, whichwas centrally located between the sourceof the scanner unit and the detector. Thesagittal plane of the center of the bone-defect site was scanned at 50 � 2 kv and0.55 mA with a voxel size of0.2 mm � 0.2 mm � 460 mm. UsingpQCT software (Version 5.21), immaturebone density was measured at a thresholdvalue of 217 mg/cm3, and the mature bonedensity (mg/cm3) was measured at athreshold value of 690 mg/cm3.

For micro-CT, the bone sample waspositioned on the stage so that the headshaft was vertical, and scanned at 65 kvand 40 mA. Each slice consisted of512 � 512 pixels and a slice thicknessof 0.52 mm, and the field of view xywas 30 mm. The volume of new bonewas measured using Mimics software(Materialise, Belgium). To determine therelative density of the threshold whichseparated the internal and externalregions, the relative densities of the airand bone portions were measured, and

A-CL)/b-TCP membrane was adapted to bone-containing 200 mg of bFGF (bFGF-gelatin

e bone defects (experimental group). Eachthe buccal site with 2 PLLA pins. Only thesecured to the right-side bone defects (control

their mean was taken as the threshold.The region-growing tool eliminated noiseand separated structures that were notconnected. Then, polygon mesh trianglesin an extraneous area were deleted, and thevolume of new bone in the defect site wasmeasured.

For histological examination, the bonesamples were decalcified with Plank andRychlo solution, embedded in paraffin,semi-serially sectioned at 6–8 mm in afrontal plane, and stained with hematox-ylin and eosin for microscopic observa-tion.

Statistical analysis

Data of soft X-ray analysis and micro-CTanalysis were expressed as a ratio of theexperimental group versus the controlgroup and analyzed by the Student’s t-test.Bone density data obtained by pQCT ana-lysis were expressed as means � error(SE) and analyzed by Student’s t-test.p < 0.05 was considered to be statisticallysignificant.

Results

Clinical observations

A part of the mesh was exposed in eachanimal of the control and experimentalgroups several days postoperatively,which had become gradually covered withnormal mucous membrane.

Soft X-ray observations

The bone-defect sites of both the controland experimental group showed new boneformation 3 months postoperatively(Fig. 3). The ratio of new bone volume(area � density) of the experimentalgroup versus that of the control group 3and 6 months postoperatively was0.97 � 0.10 and 1.34 � 0.13, respec-tively. New bone volume of the experi-mental group after 6 months wassignificantly higher than that of the controlgroup (p < 0.05) (Fig. 4).

pQCT observation

Bone density in the sagittal plane of thecenter of the bone-defect site was analyzed(Fig. 5). Three months postoperatively, theimmature bone density of the experimentalgroup and the control group was729.4� 30.3 mg/cm3 and 687.0�13.5 mg/cm3, respectively, and there wasno significant difference between the twogroups. Both values were significantlylower than that of the neighbouring normal

278 Kinoshita et al.

Fig. 3. Radiographs (soft X-ray) show bone regeneration of the defects 6 months postopera-tively. (a) P(LLA-CL)/b-TCP membrane alone (control group #3); (b) P(LLA-CL)/b-TCPmembrane + bFGF-gelatin sponge (experimental group #3). Arrows show bone regeneration inbone-defect sites.

alveolar bone (p < 0.05). Six months post-operatively, the immature bone density ofthe experimental group and control groupwas 760.6� 45.2 mg/cm3 and 662.5�35.9 mg/cm3, respectively. There was asignificant difference between the twogroups, and the former was most similarto that of the neighbouring alveolar bone.The mature bone density of the experimen-tal group and control group 3 months post-operatively was 872.0� 7.5 mg/cm3 and877.6.0 � 9.8 mg/cm3, respectively. Therewas no significant difference between thetwo groups and both values were signifi-

Fig. 4. Soft X-ray analysis. Y axis shows the r

cantly lower than that of normal alveolarbone (p < 0.05). Six months postopera-tively, the mature bone density of theexperimental group and control groupwas 887.0� 14.9 mg/cm3 and 879�9.6 mg/cm3, respectively. Both values weresignificantly lower than that of the neigh-bouring alveolar bone (p < 0.05).

Micro-CT observations

Most of the membranes maintained adome shape as a barrier for GBR 3 monthspostoperatively, but some of the meshes

elative volume (density � area). *P < 0.05.

were deformed. The membrane deforma-tion of the control group was more thanthat of the experimental group. Most of themeshes remained stable 6 months post-operatively, but some fragmentation wasobserved (Fig. 6). New bone was formedat the bone-defect sites in both the groups3 months postoperatively, and the trabe-cular bone became thick 6 months post-operatively. Comparing the experimentalgroup with the control group, the formershowed new bone formation along thecontour of the dome-shaped membranethat was implanted (Fig. 7). The ratio ofnew bone volume in the experimentalgroup versus that in the control group after3 and 6 months was 1.22 � 0.16 and1.34 � 0.05, respectively, and there wasstatistically no significant differencebetween the groups (Fig. 8).

Histological observation

Three months postoperatively, the mem-brane was surrounded with fibrous connec-tive tissue containing vessels that hadpenetrated the membrane pores. Gelatinsponge could not be observed, and newbone had formed inside the membrane inboth the groups. Six months postopera-tively, the membrane had partly degradedinto fragments and fibrous connective tis-sue was observed between the fragmentedmembranes and newly formed bone(Fig. 9). Inflammatory infiltration into thesurrounding tissue was slight. Inside themesh of both groups, matured trabecularbone was observed. The trabecular bone ofthe experimental group was thicker andmore mature than that of the control group.

Discussion

PLLA is the main component of variousbioabsorbable membranes due to its man-euverability, thermal plasticity, superiormechanical strength and biocompatibil-ity4,7,8,18,19. The polymer has the disad-vantage that it takes at least 4 years to beabsorbed completely7,8,11,13. It is desirablethat the membrane be absorbed within 1year at most. Co-polymers of lactide ande-caprolactone, lactide and glycolide, gly-colide and trimethylene carbonate, etc.were developed to shorten the time ofresorption4,7,8,23. These co-polymers alsohave disadvantages, including poor rigid-ity, insufficient mechanical strength tocombat gingival compression, andchanges in pH in relation to acid, due toresolution products of polymer, whichevoke inflammation of the surroundingtissue. Recently, addition of calcium phos-phate to polymer has been attempted to

Alveolar bone regeneration using bFGF 279

Fig. 5. pQCT analysis. Density of the sagittal slices in the center of the defects. *P < 0.05.

overcome these disadvantages9,10. KIKU-

CHI et al. developed b-TCP/co-polymer-ized poly-L-lactide composites (TCP/CPLA) as a GBR membrane10. Theyreported that TCP/CPLA decomposition

Fig. 6. Micro-CT images of frontal and sagipostoperatively. Squares show regenerated bone

Fig. 7. Micro-CT images of frontal and sagittapostoperatively. Squares show regenerated bone

regulated pH levels of the area surround-ing the composite at around 7.0. Thecomposite also retained mechanicalstrength longer than pure CPLA in phy-siological saline. This membrane was able

ttal section in control group #3, 6 monthsand arrows show degraded meshes.

l section in experimental group #3, 6 monthsand arrows show degraded meshes.

to guide new bone formation in largedefects in the mandible and tibia of dogs,whereas a pure CPLA membrane allowedinvasion of soft tissue into the defects.

The present authors developed a novelmacroporous GBR membrane made of co-polymer LLA and CL (75:25) containing30% b-TCP material [P(LLA-CL)/b-TCP]. This composite membrane has ther-moplasticity at 70 8C, moderate rigidity,X-ray opacity, which makes it possible totrace the membrane clinically, and can becut easily with scissors. In addition, con-nective tissue penetrating macropores pro-motes stability of the membrane insurrounding tissue and provides blood cir-culation both within the interior and exter-ior of the membrane. In the present study,the membrane was able to maintain thedome shape until bone was regenerated inthe alveolar ridge defects of the dogs. Thisimplies that P(LLA-CL)/b-TCP mem-brane has sufficient rigidity to withstandsoft-tissue compressive forces due to theaddition of b-TCP to polymer.

Exposure of the membrane was rarelyobserved. It is assumed that new bloodvessel formation penetrated membranepores, which supported gingiva blood cir-culation and ensured anchoring of bothtissue and membrane. The membrane par-tially degraded to fragments 6 monthspostoperatively. The absorption periodof P(LLA-CL)/b-TCP membrane mightbe longer than that of the individual mate-rial (copolymer or b-TCP), but inflamma-tory infiltration of the surrounding tissueand absorption of the new bone werebarely observable.

A pH-buffering effect of b-TCP in thetissue surrounding the membrane wouldcontribute to low inflammation and boneabsorption. An osteoconduction effect ofb-TCP was not in fact observed, probablybecause b-TCP was embedded in the poly-mer during new bone formation. Even if

280 Kinoshita et al.

Fig. 8. Volume of new bone 3 and 6 months postoperatively determined by micro-CT analysis.Y axis shows the relative volume of new bone.

the porous P(LLA-CL)/b-TCP membranedoes not have an osteoconductive effect, itcan be used as a GBR membrane becauseof its many other advantages.

Fig. 9. Histological microphotographs at 6months postoperatively. (a) control group#3; (b) experimental group #3. *: Membrane;NB: new bone. Bur: 200 mm. Hematoxylin &eosin staining.

Autogenous bone chips are often usedtogether with a GBR membrane to pro-mote bone formation or for their ability toprovide space for bone formation in themembrane. The application of growth fac-tors which enhance bone formation isattractive due to there being no patientmorbidity2,3,5,6,12,15,23. bFGF, selected inthis study, plays an important role in boneorganization and regeneration. It has beenproven that bFGF promotes proliferationof undifferentiated mesenchymal cells invitro, which has resulted in promotion ofbone formation in experiments invivo14,16,22. Regarding bone regeneration,a carrier that sustains release of bFGF atthe site of bone formation is indispensable.As reported elsewhere, bFGF is ionicallycomplexed to the acidic gelatin hydrogeland released in vivo as a result of hydrogeldegradation15,20. Changing hydrogeldegradability (water content of the hydro-gel) can be used to regulate the releaseperiod. The optimum water content forpromoting bone formation without dis-turbing it due to unabsorbed gelatin is90–95%. The water content of the hydro-gel in the present study was 95% and thebFGF release period associated withhydrogel degradation was about 4 weeks.In the present study, soft X-ray examina-tion showed that the new bone volume ofthe experimental group was significantlyhigher than that of the control group(p < 0.05) 6 months postoperatively.pQCT examination also showed that theimmature bone density (equivalent to thecancellous bone density) of the experi-mental group was significantly higher thanthat of the control group (p < 0.05) 6months postoperatively, being most simi-lar to that of the normal alveolar bone.Micro-CT examination revealed that boneformation in the experimental group wasmore abundant than that of the controlgroup 6 months postoperatively, and thatbone was formed along the original con-

tour of the dome-shaped membrane in theexperimental group (Fig. 7). These resultsshow that bFGF-hydrogels are effectivefor alveolar bone regeneration.

In conclusion, a macroporous biode-gradable P(LLA-CL)/b-TCP membranehas enough mechanical strength to enduresoft-tissue compressive forces, has suffi-cient maneuverability to form a desiredshape, provides space for bone formation,and causes a limited inflammatory reac-tion in the surrounding tissue, whichmakes it favorable for GBR membraneapplications. A combination of macropor-ous biodegradable P(LLA-CL)/b-TCPand bFGF-gelatin sponge is promisingfor reconstruction of the alveolar ridge.Future research is needed into decreasingthe biodegradation period of the P(LLA-CL)/b-TCP membrane, so that it may beabsorbed within 6 months when new bonemay be matured.

Acknowledgements. The authors thank Mr.Masayuki Kamegawa (Shimazu Co.) formicro-CT examination. This study wassupported by a grant (15390624) fromJapan Society for the Promotion ofScience.

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Address:Yukihiko Kinoshita82 Inaoka-ChoYokosuka 238-8580JapanTel: +81 46 822 9364Fax: +81 46 822 9364E-mail: kinoshit@kdcnet.ac.jp

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