明胶海绵/β-磷酸三钙复合物联合不同剂量碱性成纤维细胞生长因子诱导骨再生的评价
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摘要
背景:目前,常用的提高组织再生率的方法是将生长因子与支架材料相结合,因此基因增强的骨组织工程方法越来越受到重视。目的:评价明胶海绵/β-磷酸三钙复合物联合不同剂量碱性成纤维细胞生长因子的骨再生效果。方法:取40只雄性SD大鼠,制备颅骨缺损模型,随机分4组干预,于骨缺损处分别植入单纯明胶海绵/β-磷酸三钙复合物、载10,100,200ng碱性成纤维细胞生长因子的明胶海绵/β-磷酸三钙复合物。植入后8周取植骨材料及其下的颅骨,通过大体和组织学观察分析成骨情况。结果与结论:各组明胶海绵/β-磷酸三钙复合物被大量的纤维结缔组织包裹,纤维结缔组织中可见大量的新生血管及淋巴细胞,在β-磷酸三钙颗粒周围可见巨噬细胞,β-磷酸三钙颗粒未见明显吸收,β-磷酸三钙与颅骨间有新骨形成,新骨骨小梁之间有较多活跃的骨细胞,编织骨结构紊乱,编织骨下方为成熟的板层骨,是未破坏的原有颅骨;载100ng碱性成纤维细胞生长因子的明胶海绵/β-磷酸三钙复合物组新骨形成量最多,单纯明胶海绵/β-磷酸三钙复合物组新骨形成量最少。结果表明,碱性成纤维细胞生长因子联合明胶海绵/β-磷酸三钙复合物可促进骨缺损中的骨再生,其中以100 ng碱性成纤维细胞生长因子促进骨再生效果最好。
BACKGROUND: The current method of improving tissue regeneration is to combine growth factors with scaffold materials. Therefore, more and more attention has been paid to the gene-enhanced tissue engineering method.OBJECTIVE: To evaluate the effect of gelatin sponge/β-tricalcium phosphate composite mixed with different doses of basic fibroblast growth factor on bone regeneration. METHODS: Forty male Sprague-Dawley rats were selected to prepare skull defect models. Rat models were randomly divided into four groups, and implanted with gelatin sponge/β-tricalcium phosphate composite, gelatin sponge carrying 10, 100 and 200 ng of basic fibroblast growth factor/β-tricalcium phosphate composite. After 8 weeks of implantation, bone formation was observed by gross and histological observations. RESULTS AND CONCLUSION: In each group, the gelatin sponge/β-tricalcium phosphate composite was encapsulated by a large number of fibrous connective tissues, a large number of neovascularization and lymphocytes distributed in the fibrous connective tissue, and macrophages were visible around β-tricalcium phosphate granules. β-tricalcium phosphate granules were not obviously absorbed, and there was new bone formation between β-tricalcium phosphate and the skull. Many active bone cells existed among the new bone trabeculae, with disordered woven bone structure, the mature lamellar bone underneath the woven bone, and the undamaged original skull. The most active new bone formation was found in the basic fibroblast growth factor 100 ng+gelatin sponge/β-tricalcium phosphate composite group, and the new bone volume was the least in the solely used gelatin sponge/β-tricalcium phosphate composite. In conclusion, the combination of basic fibroblast growth factor with gelatin sponge/β-tricalcium phosphate composite can promote bone regeneration in the bone defect, and the best effect on bone regeneration is observed when 100 ng basic fibroblast growth factor is used.
BACKGROUND: The current method of improving tissue regeneration is to combine growth factors with scaffold materials. Therefore, more and more attention has been paid to the gene-enhanced tissue engineering method.OBJECTIVE: To evaluate the effect of gelatin sponge/β-tricalcium phosphate composite mixed with different doses of basic fibroblast growth factor on bone regeneration. METHODS: Forty male Sprague-Dawley rats were selected to prepare skull defect models. Rat models were randomly divided into four groups, and implanted with gelatin sponge/β-tricalcium phosphate composite, gelatin sponge carrying 10, 100 and 200 ng of basic fibroblast growth factor/β-tricalcium phosphate composite. After 8 weeks of implantation, bone formation was observed by gross and histological observations. RESULTS AND CONCLUSION: In each group, the gelatin sponge/β-tricalcium phosphate composite was encapsulated by a large number of fibrous connective tissues, a large number of neovascularization and lymphocytes distributed in the fibrous connective tissue, and macrophages were visible around β-tricalcium phosphate granules. β-tricalcium phosphate granules were not obviously absorbed, and there was new bone formation between β-tricalcium phosphate and the skull. Many active bone cells existed among the new bone trabeculae, with disordered woven bone structure, the mature lamellar bone underneath the woven bone, and the undamaged original skull. The most active new bone formation was found in the basic fibroblast growth factor 100 ng+gelatin sponge/β-tricalcium phosphate composite group, and the new bone volume was the least in the solely used gelatin sponge/β-tricalcium phosphate composite. In conclusion, the combination of basic fibroblast growth factor with gelatin sponge/β-tricalcium phosphate composite can promote bone regeneration in the bone defect, and the best effect on bone regeneration is observed when 100 ng basic fibroblast growth factor is used.
引文
[1]Masaoka T,Yamada T,Yuasa M,et al.Biomechanical evaluation of the rabbit tibia after implantation of porous hydroxyapatite/collagen in a rabbit model.J Orthop Sci.2016;21(2):230-236.
[2]Omata K,Matsuno T,Kazunari A,et al.Enhanced bone regeneration by gelatin-β-tricalcium phosphate composites enabling controlled release of bFGF.J Tissue Eng Regen Med.2014;8(3):604-611.
[3]Andreas S,Frank W,Marcus H,et al.Autonomic bone grafts in oralimplantology-is it still a"gold standard?"A consecutive review of279 patients with 456 clinical procedures.Int J Implant Dent.2017;46(1):3-23.
[4]Azi ML,Aprato A,Santi I,et al.Autologous bone graft in the treatment of post-traumatic bone defects:a systematic review and meta-analysis.BMC Musculoskelet Disord.2016;17(1):465.
[5]Rezende ML,Consolaro A,Sant'Ana AC,et al.Demineralization of the contacting surfaces in autologous onlay bone grafts improves bone formation and bone consolidation.J Periodontol.2014;85(5):121-129.
[6]Horowitz RA,Leventis MD.Bone grafting:history,rationale,and selection of materials and techniques.Compend Contin Educ Dent.2014;35(4):1-6.
[7]Liu J,Schmidlin P,Philipp A,et al.Novel bone substitute material in alveolar bone healing following tooth extraction:an experimental study in sheep.Clin Oral Implants Res.2016;27(7):762-770.
[8]Arima Y,Uemura N,Hashimoto Y,et al.Evaluation of bone regeneration by porous alpha-tricalcium phosphate/atelo-collagen sponge composite in rat calvarial defects.Orthod Waves.2013;72(3):23-29.
[9]Li P,Hashimoto Y.The effect of interferon-γand zoledronate treatment on alpha-tricalcium phosphate/collagen sponge-mediated bone-tissue engineering.Int J Mol Sci.2015;16(3):25678-25690.
[10]Ito T,Hashimoto Y,Baba S,et al.Bone regeneration with a collagen model polypeptides/-tricalcium phosphate sponge in a canine tibia defect model.Implant.Dent.2015;12(24):197-203.
[11]Li P,Hashimoto Y,Honda Y,et al.The effect of interferon-and zoledronate treatment on alpha-tricalcium phosphate/collagen sponge-mediated bone-tissue engineering.Int J Mol Sci.2015;23(16):25678-25690.
[12]Sun D,Wang W,Wang X,et al.bFGF plays a neuroprotective role by suppressing excessive autophagy and apoptosis after transient global cerebral ischemia in rats.Cell Death Dis.2018;9(2):172.
[13]Kakinoki S,Sakai Y,Fujisato T,et al.Accelerated tissue integration into porous materials by immobilizing basic fibroblast growth factor using a biologically safe three-step reaction.J Biomed Mater Res.2015;103(6):3790-3797.
[14]O'Keefe RJ,Mao J.Bone tissue engineering and regeneration:from discovery to the clinic--an overview.Tissue Eng Part B Rev.2011;17(6):389-392.
[15]Shrivats AR,McDermott MC,Hollinger J.Bone tissue engineering:state of the union.Drug Discov Today.2014;19(6):781-786.
[16]Venkatesan J,Nithya R,Sudha PN,et al.Role of alginate in bone tissue engineering.Adv Food Nutr Res.2014;45(73):45-57.
[17]Taktak R,Elghazel A,Bouaziz J,et al.Tricalcium phosphateFluorapatite as bone tissue engineering:Evaluation of bioactivity and biocompatibility.Mater Sci Eng C Mater Biol Appl.2018;1(86):121-128.
[18]Sionek A,Czwojdziński A,Kowalczewski J,et al.Hip osteonecroses treated with calcium sulfate-calcium phosphate bone graft substitute have different results according to the cause of osteonecrosis:alcohol abuse or corticosteroid-induced.Int Orthop.2018;42(7):1491-1498.
[19]Laverty DP,Kelly R,Addison O.Survival of dental implants placed in autogenousbonegrafts and bone flaps in head and neck oncology patients:a systematic review.Int J Implant Dent 2018;4(1):19.
[20]Dos Santos Pereira R,Boos FB,Gorla LF,et al.Maxillary Sinus Elevation Surgery with ChronOS and Autogenous Bone Graft:Immunohistochemical Assessment of RUNX2,VEGF,TRAP,and Osteocalcin.Int J Periodontics Restorative Dent.2017;37(6):e321-e327.
[21]Noguchi T,Sarukawa S,Tsuchiya Y,et al.Evaluation of postoperative changes in vascularized iliac bone grafts used for mandibular reconstruction.Int J Oral Maxillofac Surg.2018;47(8):990-997.
[22]Sarikaya B,Aydin HM.Collagen/Beta-Tricalcium Phosphate Based Synthetic Bone Grafts via Dehydrothermal Processing.Biomed Mater.2015;10(6):532-552.
[23]Fonseca H,Moreira-Gon?alves D,Coriolano HJ,et al.Bone quality:the determinants of bone strength and fragility.Sports Med.2014;44(1):37-53.
[24]Hamdy RC.Bone Mineral Density and Fractures.J Clin Densitom.2016;19(2):125-126.
[25]Zain NM,Seriramulu VP,Chelliah KK.Bone Mineral Density and Breast Cancer Risk Factors among Premenopausal and Postmenopausal Women A Systematic Review.Asian Pac J Cancer Prev.2016;17(7):3229-3234.
[26]Arahira T,Todo M.Variation of mechanical behavior ofβ-TCP/collagen two phase composite scaffold with mesenchymal stem cell in vitro.JMech Behav Biomed Mater.2016;16(61):464-474.
[27]Xu L,Zhang W.Peri-Implant Bone Regeneration Using rhPDGF-BB,BMSCs,andβ-TCP in a Canine Model.Clin Implant Dent Relat Res.2016;8(2):241-252.
[28]?ivadinovi?M,Andri?M,Milo?evi?V,et al.Histomorphometric evaluation of bone regeneration usingautogenous bone and beta-tricalcium phosphate in diabetic rabbits.Vojnosanit Pregl.2016;73(12):1132-1138.
[29]Ramesh N,Moratti SC,Dias GJ.Hydroxyapatite-polymer biocomposites for bone regeneration:A review of current trends.Journal of biomedical materials research.J Biomed Mater Res B Appl Biomater.2018;106(5):2046-2057.
[30]Huang YC,Chen CY,et al.Comparing morbidities of bone graft harvesting from theanterior iliac crest and proximal tibia:a retrospective study.J Orthop Surg Res.2018;13(1):115.
[31]Gross JB,Diligent J,Bensoussan D,et al.Percutaneous autologous bone marrow injection for treatment of delayed and non-union of long bone:a retrospective study of 45 cases.Biomed Mater Eng.2015;25(1):187-197.
[32]Lee DH,Ryu KJ,Kim JW,et al.Bone marrow aspirate concentrate and platelet-rich plasma enhanced bone healing in distraction osteogenesis of the tibia.Clin Orthop Relat Res.2014;472(12):3789-3797.
[33]Wu J,Cai XH,Qin XX,et al.The effects of sclerostin antibody plus parathyroid hormone(1-34)on bone formation in ovariectomized rats.Z Gerontol Geriatr.2018;51(5):550-556.
[34]Han Z,Bhavsar M,Leppik L,et al.Histological Scoring Method to Assess Bone Healing in Critical Size Bone Defect Models.Tissue Eng Part C Methods.2018;24(5):272-279.
[35]Akkaya S,Nazal?M,K?l??A,et al.Cefazolin-sodium has no adverse effect on fracture healing in an experimental rabbit model.Eklem Hastalik Cerrahisi.2012;23(1):44-48.
[36]Angle SR,Sena K,Sumner DR,et al.Healing of rat femoral segmental defect with bonemorphogenetic protein-2:a dose response study.JMusculoskelet Neuronal Interact.2012;12(1):28-37.
[37]Il'in DA,Arkhipov SA,Shkurupy VA.Analysis of IL-1α,bFGF,TGF-β1,IFNγ,MMP-1,and CatD Expression in Multinuclea Macrophages In Vitro.Bull Exp Biol Med.2018;164(4):456-458.
[38]Qu Y,Cao C,Wu Q,et al.The dual delivery of KGF and bFGF by collagen membrane to promote skin wound healing.J Tissue Eng Regen Med.2018;12(6):1508-1518.
[39]Xiao WL,Zhang DZ,Chen XJ,et al.Osteogenesis effect of guided bone regeneration combined with alveolar cleft grafting:assessment by cone beam computed tomography.Int J Oral Maxillofac Surg.2016;45(6):683-687.
[40]He B,Zhao J,Ou Y,et al.Biofunctionalized peptide nanofiber-based composite scaffolds for bone regeneration.Materials science&engineering.Mater Biol Appl.2018;9(2):728-738.
[41]Hiwatashi N,Hirano S,Mizuta M,et al.The efficacy of a novel collagen-gelatin scaffold with basic fibroblast growth factor for the treatment of vocal fold scar.J Tissue Eng Regen Med.2017;11(5):1598-1609.
[42]Lu J,Descamps M,Dejou J,et al.The biodegradation mechanism of calcium phosphate biomaterials in bone.J BiomedMater Res.2002;34(63):408-412.
[43]Bettach R,Guillaume B,Taschieri S,et al.Clinical performance of a highly porous beta-TCP as the grafting material for maxillary sinus augmentation.Implant Dent.2014;23(3):357-364.
[44]Castilho M,Rodrigues J,Pires I,et al.Fabrication of individual alginate-TCP scaffolds for bone tissue engineering by means of powder printing.Biofabrication.2015;7(1):4-15.
[45]Zhang C,Li Q,Deng S,et al.bFGF-and CaPP-Loaded Fibrin Clots Enhance the Bioactivity of the Tendon-Bone Interface to Augment Healing.Am J Sports Med.2016;44(8):1972-1982.
[46]Wang JS,Aspenberg P.Basic fibroblast growth factor enhances bone-graft incorporation:dose and time dependence in rats.J Orthop Res.1996;14(2):316-23.
[2]Omata K,Matsuno T,Kazunari A,et al.Enhanced bone regeneration by gelatin-β-tricalcium phosphate composites enabling controlled release of bFGF.J Tissue Eng Regen Med.2014;8(3):604-611.
[3]Andreas S,Frank W,Marcus H,et al.Autonomic bone grafts in oralimplantology-is it still a"gold standard?"A consecutive review of279 patients with 456 clinical procedures.Int J Implant Dent.2017;46(1):3-23.
[4]Azi ML,Aprato A,Santi I,et al.Autologous bone graft in the treatment of post-traumatic bone defects:a systematic review and meta-analysis.BMC Musculoskelet Disord.2016;17(1):465.
[5]Rezende ML,Consolaro A,Sant'Ana AC,et al.Demineralization of the contacting surfaces in autologous onlay bone grafts improves bone formation and bone consolidation.J Periodontol.2014;85(5):121-129.
[6]Horowitz RA,Leventis MD.Bone grafting:history,rationale,and selection of materials and techniques.Compend Contin Educ Dent.2014;35(4):1-6.
[7]Liu J,Schmidlin P,Philipp A,et al.Novel bone substitute material in alveolar bone healing following tooth extraction:an experimental study in sheep.Clin Oral Implants Res.2016;27(7):762-770.
[8]Arima Y,Uemura N,Hashimoto Y,et al.Evaluation of bone regeneration by porous alpha-tricalcium phosphate/atelo-collagen sponge composite in rat calvarial defects.Orthod Waves.2013;72(3):23-29.
[9]Li P,Hashimoto Y.The effect of interferon-γand zoledronate treatment on alpha-tricalcium phosphate/collagen sponge-mediated bone-tissue engineering.Int J Mol Sci.2015;16(3):25678-25690.
[10]Ito T,Hashimoto Y,Baba S,et al.Bone regeneration with a collagen model polypeptides/-tricalcium phosphate sponge in a canine tibia defect model.Implant.Dent.2015;12(24):197-203.
[11]Li P,Hashimoto Y,Honda Y,et al.The effect of interferon-and zoledronate treatment on alpha-tricalcium phosphate/collagen sponge-mediated bone-tissue engineering.Int J Mol Sci.2015;23(16):25678-25690.
[12]Sun D,Wang W,Wang X,et al.bFGF plays a neuroprotective role by suppressing excessive autophagy and apoptosis after transient global cerebral ischemia in rats.Cell Death Dis.2018;9(2):172.
[13]Kakinoki S,Sakai Y,Fujisato T,et al.Accelerated tissue integration into porous materials by immobilizing basic fibroblast growth factor using a biologically safe three-step reaction.J Biomed Mater Res.2015;103(6):3790-3797.
[14]O'Keefe RJ,Mao J.Bone tissue engineering and regeneration:from discovery to the clinic--an overview.Tissue Eng Part B Rev.2011;17(6):389-392.
[15]Shrivats AR,McDermott MC,Hollinger J.Bone tissue engineering:state of the union.Drug Discov Today.2014;19(6):781-786.
[16]Venkatesan J,Nithya R,Sudha PN,et al.Role of alginate in bone tissue engineering.Adv Food Nutr Res.2014;45(73):45-57.
[17]Taktak R,Elghazel A,Bouaziz J,et al.Tricalcium phosphateFluorapatite as bone tissue engineering:Evaluation of bioactivity and biocompatibility.Mater Sci Eng C Mater Biol Appl.2018;1(86):121-128.
[18]Sionek A,Czwojdziński A,Kowalczewski J,et al.Hip osteonecroses treated with calcium sulfate-calcium phosphate bone graft substitute have different results according to the cause of osteonecrosis:alcohol abuse or corticosteroid-induced.Int Orthop.2018;42(7):1491-1498.
[19]Laverty DP,Kelly R,Addison O.Survival of dental implants placed in autogenousbonegrafts and bone flaps in head and neck oncology patients:a systematic review.Int J Implant Dent 2018;4(1):19.
[20]Dos Santos Pereira R,Boos FB,Gorla LF,et al.Maxillary Sinus Elevation Surgery with ChronOS and Autogenous Bone Graft:Immunohistochemical Assessment of RUNX2,VEGF,TRAP,and Osteocalcin.Int J Periodontics Restorative Dent.2017;37(6):e321-e327.
[21]Noguchi T,Sarukawa S,Tsuchiya Y,et al.Evaluation of postoperative changes in vascularized iliac bone grafts used for mandibular reconstruction.Int J Oral Maxillofac Surg.2018;47(8):990-997.
[22]Sarikaya B,Aydin HM.Collagen/Beta-Tricalcium Phosphate Based Synthetic Bone Grafts via Dehydrothermal Processing.Biomed Mater.2015;10(6):532-552.
[23]Fonseca H,Moreira-Gon?alves D,Coriolano HJ,et al.Bone quality:the determinants of bone strength and fragility.Sports Med.2014;44(1):37-53.
[24]Hamdy RC.Bone Mineral Density and Fractures.J Clin Densitom.2016;19(2):125-126.
[25]Zain NM,Seriramulu VP,Chelliah KK.Bone Mineral Density and Breast Cancer Risk Factors among Premenopausal and Postmenopausal Women A Systematic Review.Asian Pac J Cancer Prev.2016;17(7):3229-3234.
[26]Arahira T,Todo M.Variation of mechanical behavior ofβ-TCP/collagen two phase composite scaffold with mesenchymal stem cell in vitro.JMech Behav Biomed Mater.2016;16(61):464-474.
[27]Xu L,Zhang W.Peri-Implant Bone Regeneration Using rhPDGF-BB,BMSCs,andβ-TCP in a Canine Model.Clin Implant Dent Relat Res.2016;8(2):241-252.
[28]?ivadinovi?M,Andri?M,Milo?evi?V,et al.Histomorphometric evaluation of bone regeneration usingautogenous bone and beta-tricalcium phosphate in diabetic rabbits.Vojnosanit Pregl.2016;73(12):1132-1138.
[29]Ramesh N,Moratti SC,Dias GJ.Hydroxyapatite-polymer biocomposites for bone regeneration:A review of current trends.Journal of biomedical materials research.J Biomed Mater Res B Appl Biomater.2018;106(5):2046-2057.
[30]Huang YC,Chen CY,et al.Comparing morbidities of bone graft harvesting from theanterior iliac crest and proximal tibia:a retrospective study.J Orthop Surg Res.2018;13(1):115.
[31]Gross JB,Diligent J,Bensoussan D,et al.Percutaneous autologous bone marrow injection for treatment of delayed and non-union of long bone:a retrospective study of 45 cases.Biomed Mater Eng.2015;25(1):187-197.
[32]Lee DH,Ryu KJ,Kim JW,et al.Bone marrow aspirate concentrate and platelet-rich plasma enhanced bone healing in distraction osteogenesis of the tibia.Clin Orthop Relat Res.2014;472(12):3789-3797.
[33]Wu J,Cai XH,Qin XX,et al.The effects of sclerostin antibody plus parathyroid hormone(1-34)on bone formation in ovariectomized rats.Z Gerontol Geriatr.2018;51(5):550-556.
[34]Han Z,Bhavsar M,Leppik L,et al.Histological Scoring Method to Assess Bone Healing in Critical Size Bone Defect Models.Tissue Eng Part C Methods.2018;24(5):272-279.
[35]Akkaya S,Nazal?M,K?l??A,et al.Cefazolin-sodium has no adverse effect on fracture healing in an experimental rabbit model.Eklem Hastalik Cerrahisi.2012;23(1):44-48.
[36]Angle SR,Sena K,Sumner DR,et al.Healing of rat femoral segmental defect with bonemorphogenetic protein-2:a dose response study.JMusculoskelet Neuronal Interact.2012;12(1):28-37.
[37]Il'in DA,Arkhipov SA,Shkurupy VA.Analysis of IL-1α,bFGF,TGF-β1,IFNγ,MMP-1,and CatD Expression in Multinuclea Macrophages In Vitro.Bull Exp Biol Med.2018;164(4):456-458.
[38]Qu Y,Cao C,Wu Q,et al.The dual delivery of KGF and bFGF by collagen membrane to promote skin wound healing.J Tissue Eng Regen Med.2018;12(6):1508-1518.
[39]Xiao WL,Zhang DZ,Chen XJ,et al.Osteogenesis effect of guided bone regeneration combined with alveolar cleft grafting:assessment by cone beam computed tomography.Int J Oral Maxillofac Surg.2016;45(6):683-687.
[40]He B,Zhao J,Ou Y,et al.Biofunctionalized peptide nanofiber-based composite scaffolds for bone regeneration.Materials science&engineering.Mater Biol Appl.2018;9(2):728-738.
[41]Hiwatashi N,Hirano S,Mizuta M,et al.The efficacy of a novel collagen-gelatin scaffold with basic fibroblast growth factor for the treatment of vocal fold scar.J Tissue Eng Regen Med.2017;11(5):1598-1609.
[42]Lu J,Descamps M,Dejou J,et al.The biodegradation mechanism of calcium phosphate biomaterials in bone.J BiomedMater Res.2002;34(63):408-412.
[43]Bettach R,Guillaume B,Taschieri S,et al.Clinical performance of a highly porous beta-TCP as the grafting material for maxillary sinus augmentation.Implant Dent.2014;23(3):357-364.
[44]Castilho M,Rodrigues J,Pires I,et al.Fabrication of individual alginate-TCP scaffolds for bone tissue engineering by means of powder printing.Biofabrication.2015;7(1):4-15.
[45]Zhang C,Li Q,Deng S,et al.bFGF-and CaPP-Loaded Fibrin Clots Enhance the Bioactivity of the Tendon-Bone Interface to Augment Healing.Am J Sports Med.2016;44(8):1972-1982.
[46]Wang JS,Aspenberg P.Basic fibroblast growth factor enhances bone-graft incorporation:dose and time dependence in rats.J Orthop Res.1996;14(2):316-23.