Preparation of rhBMP2 /BCB and assay of its osteoinductivity
Keywords:bone morphogenetic proteins(BMP);xenograft;osteogenesis
Abstract:AIM To develop a new grafting material of bone xenograft with strong bone inductive and conductive capaci┐ty.METHODS Based on successful clinical application of the reconstituted bone xenograft(RBX)，a new xenograft containing rhBMP2 (rhBMP2 /BCB)was made by combining recombinant human BMP2 (rhBMP2 )with antigen-free bovine cancellous bone(BCB).The ectopic induction of rhBMP2 /BCB was analyzed by using thigh muscle pouch in mice.RE┐SULTS Histological examination showed that rhBMP2 /BCB induced chondrogenesis on d7，with woven bone formed on d14，and lamellar bone and marrow on d21，while BCB failed to induce chondrogenesis or osteogenesis on d7，14and21.ALP activitis and calcium content in the rhBMP2 /BCB group were higher than those in the BCB group with very singificant difference on d7，14and21.CONCLUSION rhBMP 2 /BCB is an ideal grafting material with strong bone inductive and conductive capacity without evoking immune reaction.
复合rhBMP2 的异种骨的研制及成骨活性
袁志，马平，胡蕴玉，罗卓荆，赵广跃，吕荣，孙梁，李丹
关键词: 骨形态发生蛋白质类;移植，异种;骨生成
摘 要:目的 研制新型具有诱导成骨活性的异种骨植骨材料. 方法 在重组合异种骨(RBX)基础上，以基因重组人BMP2 (rhBMP 2 )取代从牛皮质骨中提取的牛BMP，与去抗原牛松质骨载体(BCB)复合，制成复合rhBMP2的异种骨(rhBMP2 /BCB)，并采用小鼠股部肌袋模型，通过组织学、骨计量学方法检测rhBMP2 /BCB的诱导成骨活性. 结果 实验组(rhBMP2 /BCB组)术后7d在小鼠肌袋可诱导软骨生成，14d形成编织骨，21d改建成板状骨并形成大量骨髓;对照组(BCB组)于术后各时间点均未见有软骨及骨形成.实验组的碱性磷酸酶活性和钙含量均高于对照组，有非常显著性差异(P<0.01). 结论 rhBMP2 /BCB具有较好的骨诱导能力，是一种较理想的植骨材料.
INTRODUCTION
BMP(bone morphogenetic protein)has more possi-bilities in developing grafting materials that possess osteoinductive capacity[1，2] .We combined recombi-nant human BMP2 (rhBMP2 )with an antigen-free bovine cancellous bone(BCB)as a carrier to make a new grafting material-a xenograft containing rhBMP
2 (rhBMP2 /BCB).Then the ectopic bone in-duction of rhBMP2 /BCB was analyzed by using thigh muscle pouch in mice.
MATERIALS AND METHODS
Bioassay of rhBMP 2 inductivity Mature peptide of rhBMP2 with a molecular mass of12ku which is ex-pressed by E.coli was provided by the Academy of Medical Sciences of PLA.Two male BALB/c mice with20～23g body mass were implanted with0.2mg of rhBMP2 ，each in unilateral femoral muscle.The specimens retrieved the10th postoperative day were fixed with40g L-1 neutral formalin，routinely sectioned，HE stained and observed for the osteoin-ductivity of rhBMP2 under the light microscope.
Preparation of rhBMP 2 /BCB chips Fresh calf can-cellous bone was collected from proximal humerus and was morseled into4mm×4mm×4mm chips.The bone chips were then repeatedly washed with pressurized water，dried up in the air，and defatted using chloroform and methanol(1V 1V)for12h，soaked in8.8mol L-1 hydrogen peroxide to be de-proteinized for48h.Finally the chips were partially decalcified by immersing them in0.6mol L-1 hy-drochloric acid for5min，freeze-dried and packed.The BCB carriers thus prepared were recombined in vacuum with0.2mg of rhBMP
2 adsorbed to each chip.The resulting composite was dialyzed against distilled water，lyophilized，sterilized with ethylene oxide and stored ready for use.Some of rhBMP 2 /BCB chips and BCB chips were observed under scan-ning electron microscopy(SEM).
Bioassay of rhBMP2 /BCB inductivity Sixty male BALB/c mice with20to23g body mass and were randomized into study group of30，and control group of30.Each group was then evenly distribut-ed in3subgroups for observation at set time points of7，14and21d postoperatively.Mouse femoral muscle pouch model was used.Incision was made over the left proximal lateral aspect of the thigh to prepare a quadriceps muscle pouch where a rhBMP2 /BCB chip was implanted in the study group and a BCB chip in the control group.Postoperativ-ely，the mice were kept in cages under the same conditions.The mice were killed on d7，14and21after implantation.Every implanted sample harvest-ed was cut evenly into2parts.One partion of the sample was immersed in40g L-1 neutral formalin，then decalcified in a decalcifying admixure for72h，followed by gelatin embedding，frozen section，HE staining for light microscopic examination.Another portion of the cut-in-half specimen was cleaned of muscle tissue，weighed，minced，homogenized in1mL of saline and centrifuged at4000r min-1 ×15min.The ALP activity per unit tissue mass was measured of the supernatant.And the residue cen-trifuged was digested with hydrochloric acid prior to measurement of calcium content using an atomic ab-sorption spectrophotometer(Hitachi7500)，fol-lowed by calculation of the calcium content in unit mass of the specimen.
RESULTS
Histological findings of rhBMP 2 inductivity Histo- logical examination of the specimens retrieved the10th postoperative day from the mice implanted with0.2mg rhBMP2 showed that many mesenchy-mal cells proliferated，many chondrocytes occurred and became mature，with cartilage matrix mineral-ized，indicated that rhBMP2 itself had strong bone-inductive capacity(Fig1).
SEM observation of rhBMP2 /BCB and BCB A porous structure of BCB with smooth pore walls，and the pores were homogeneously filled up with rhBMP2 (Fig2).
Bioassay of rhBMP2 /BCB inductivity Macroscopic observation The study group:On d7，at the site of rhBMP2 /BCB implant there appeared a mass the size of0.6cm across with moderate tenaci-ty，and a sectional view showed that it was sur-rounded by greyish tissue with identifiable bound-ary.On d14，the mass slightly enlarged to0.8cm which was tough on palpation，presenting as yel-lowish tissue indiscernible from the surrounding tis-sue.On d21，the mass increased in consistency but was seen with no significant increase in size，with white cortex and red marrow easily discernible.The control group:The mass were much smaller with low consistency on d7，14and21，and the BCB granules were wrapped up by homogeneous greyish tissue.
Histological observation In study group，on d7，under the light microscope numerous undifferenti-ated mesenchymal cells were seen aggregating and proliferating along with a number of chondrocytes，with mineralized cartilage matrix and osteoid formed which had a diffuse focal distribution(Fig3).On d14，chondrocytes increased in number and merged with each other，and woven bone occurred(Fig4).On d21，mature lamellar bone and marrow tissue was seen(Fig5).In control group，connective tis-sue was seen enclosing BCB and filling up the pores of BCB，no bone and cartilage tissue were seen on d7，14and21.
Biochemical determination After implantation，the ALP activities and calcium content in the study group were higher than those in the control group(P<0.01，Tab1). atrix and osteoid were formed HE ×200 b P<0.01，vs control group.ALP:alkaline phosphatase.
DISCUSSION
In1965，Urist successfully used demineralized bone matrix to induce ectopic bone formation in muscle tissue，which led to the discovery of bone morpho-genetic protein(BMP).He also showed that the major biological effect of BMP is manifested by its stimulation of aggregation，proliferation and differ-entiation of undifferentiated mesenchymal cells，re-sulting in new bone formation.Many scholars had gained success in the clinical treatment of bone de-fects and nonunions with BMP from animal bone.Especially，HU et al first developed the reconstitut-ed bone xenograft(RBX)by recombining the anti-gen-free bovine cancellous bone as a carrier with crude extract of BMP from bovine cortical bone.RBX possesses strong osteoinductivty without evok-ing immune rejection as demonstrated by serial ex-perimental and clinical studies，which provides a successful solution to the difficult problem of xeno-geneic grafting[1] .However，BMP extract from ani-mal bone is not a protein constituent inherent to hu-man，and it will evoke an immune response to a cer-tain extent，although it is only weakly immuno-genetic.As far as a biomaterial is concerned，this is a major drawback which could not be ignored.Fur-thermore，with the minimal content of BMP in ani-mal bone tissue，e.g.1mg kg-1 (wet mass)in bovine bone and0.76mg kg
-1 (wet mass)in porcine bone，it is difficult to meet the demand in clinical practice using the natural occurring product.Also the extraction process of BMP is a labour-in-tensive and time consuming，which must go through multiple steps，with the purity of BMP differing from batch to batch [2] .In late1980's，with the ad-vent of gene engineering technology of BMP and subsequent improvements in the procedure，produc-tion of recombinant human BMP(rhBMP)has be-come a subject of intensive study to which much at-tention has been devoted.Wozney et al in1988first reported cloning and expression of cDNA of rhBMP1 ，rhBMP2A ，rhBMP 2B and rhBMP
3 .Subse-quently16rhBMPs were cDNA cloned，among which rhBMP2 ，rhBMP4 and rhBMP
7 are more os-teoinductive than the others，but for the present more experience has been gained with the use of rhBMP 2 ，which would promise fine prospects for its clinical use[3-6] .
In the present study we empolyed rhBMP 2 which is cheaper，high-yielding and exhibits more stable biological activities [7，8] in place of the BMP crude extract from bovine cortical bone in RBX，and combined it with BCB as a carrier to make rhBMP 2 /BCB and studied its osteoinductive ability on mouse muscle pouch model.The results showed:rhBMP2 / BCB induced chondrogenesis on d7，with woven bone formed on d14，and lamellar bone and marrow on d21.So，rhBMP2 /BCB was proved to be a ideal grafting material with strong bone-inductive and conductive capacity without evoking immune reac-tion，while BCB is a ideal carrier of rhBMP2 .
ACKNOWLEGEMENT:Prof.Pan BR in Oncology Center，Xijing Hospital，has great offort for this article.
REFERENCES:
[1]Luo ZJ，Hu YY，Lu R，Yang G，Li XB，Li D，Zhu YS.The experimental study on osteoinductive activity of massive recon-stituted bone xenograft [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1996;17(6):468-469.
[2]Chen SM.Significance and expression of human BMP gene [A].In:Yang LJ，Jin Y，Hu YY.Bioactive materials in stomatology and orthopaedics [M].Xi'an，Shaanxi Science and Technology Inc.1993;243-250.
[3]Pu Q，Chen SM，Chen NC.Expression of recombinant mature peptide of human bone morphogenetic protein in Escherichia coli [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1998;19(1):8-11.
[4]Du JJ，Hu YY，Luo ZJ，Lu R，Ma ZZ.Synergism of rhBMP2and TGF-βin osteoinduction and osteogenesis [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1999;20(7):555-558.
[5]Wang D，Zhao GY，Hu YY，Zhong CQ，Xie KN，Yang G，Lu R.Preparation ofβ-TCP/rhBMP2 at different degradation rates and assay of their osteoinductive activity [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1998;19(2):198.
[6]Zhou Y，Fan QY，Jing WZ，Cai HP，Wen YH.Bone induction of compound material of decalcified bone matrix with rhBMP2impregnated bone cement [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1999;20(12):1085-1087.
[7]Yuan Z，Ma P，Hu YY，Luo ZJ，Jin GL，Lu R，Wang J.Ex-periment study on combined free periosteum grafting and new re-constituted bone xenograft in repairing segmental defects [J].J Med Coll PLA，2000;15(4):283-287.
[8]Yuan Z，Ma P，Hu YY，Luo ZJ，Jin GL，Lu R，Li D，Wang J.Combined use of rhBMP2/BCB and vascularized periosteum in repairing segmental defects in radii of rabbits [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，2001;22(11):1013-1018.
Biography:YUAN Zhi(male，born in1966in Liaoyuan County，Jilin Province)，M D candidate in Institute of Orthopaedics，Xijing Hos-pital.Tel.(029)3375289 Email.Orth1@fmmu.edu.cn
(第四军医大学西京医院全军骨科研究所，陕西西安710033)
(Institute of Orthopaedics of Chinese PLA，Xijing Hospital，Fourth Military Medical University，Xi'an710033，China)
Editor XU Fu-Ming
Received date:2001┐06┐30, http://www.100md.com(YUAN Zhi，MA Ping，HU Yun-Yu，LUO Zhuo-Jing，ZHAO Guan)
Abstract:AIM To develop a new grafting material of bone xenograft with strong bone inductive and conductive capaci┐ty.METHODS Based on successful clinical application of the reconstituted bone xenograft(RBX)，a new xenograft containing rhBMP2 (rhBMP2 /BCB)was made by combining recombinant human BMP2 (rhBMP2 )with antigen-free bovine cancellous bone(BCB).The ectopic induction of rhBMP2 /BCB was analyzed by using thigh muscle pouch in mice.RE┐SULTS Histological examination showed that rhBMP2 /BCB induced chondrogenesis on d7，with woven bone formed on d14，and lamellar bone and marrow on d21，while BCB failed to induce chondrogenesis or osteogenesis on d7，14and21.ALP activitis and calcium content in the rhBMP2 /BCB group were higher than those in the BCB group with very singificant difference on d7，14and21.CONCLUSION rhBMP 2 /BCB is an ideal grafting material with strong bone inductive and conductive capacity without evoking immune reaction.
复合rhBMP2 的异种骨的研制及成骨活性
袁志，马平，胡蕴玉，罗卓荆，赵广跃，吕荣，孙梁，李丹
关键词: 骨形态发生蛋白质类;移植，异种;骨生成
摘 要:目的 研制新型具有诱导成骨活性的异种骨植骨材料. 方法 在重组合异种骨(RBX)基础上，以基因重组人BMP2 (rhBMP 2 )取代从牛皮质骨中提取的牛BMP，与去抗原牛松质骨载体(BCB)复合，制成复合rhBMP2的异种骨(rhBMP2 /BCB)，并采用小鼠股部肌袋模型，通过组织学、骨计量学方法检测rhBMP2 /BCB的诱导成骨活性. 结果 实验组(rhBMP2 /BCB组)术后7d在小鼠肌袋可诱导软骨生成，14d形成编织骨，21d改建成板状骨并形成大量骨髓;对照组(BCB组)于术后各时间点均未见有软骨及骨形成.实验组的碱性磷酸酶活性和钙含量均高于对照组，有非常显著性差异(P<0.01). 结论 rhBMP2 /BCB具有较好的骨诱导能力，是一种较理想的植骨材料.
INTRODUCTION
BMP(bone morphogenetic protein)has more possi-bilities in developing grafting materials that possess osteoinductive capacity[1，2] .We combined recombi-nant human BMP2 (rhBMP2 )with an antigen-free bovine cancellous bone(BCB)as a carrier to make a new grafting material-a xenograft containing rhBMP
2 (rhBMP2 /BCB).Then the ectopic bone in-duction of rhBMP2 /BCB was analyzed by using thigh muscle pouch in mice.
MATERIALS AND METHODS
Bioassay of rhBMP 2 inductivity Mature peptide of rhBMP2 with a molecular mass of12ku which is ex-pressed by E.coli was provided by the Academy of Medical Sciences of PLA.Two male BALB/c mice with20～23g body mass were implanted with0.2mg of rhBMP2 ，each in unilateral femoral muscle.The specimens retrieved the10th postoperative day were fixed with40g L-1 neutral formalin，routinely sectioned，HE stained and observed for the osteoin-ductivity of rhBMP2 under the light microscope.
Preparation of rhBMP 2 /BCB chips Fresh calf can-cellous bone was collected from proximal humerus and was morseled into4mm×4mm×4mm chips.The bone chips were then repeatedly washed with pressurized water，dried up in the air，and defatted using chloroform and methanol(1V 1V)for12h，soaked in8.8mol L-1 hydrogen peroxide to be de-proteinized for48h.Finally the chips were partially decalcified by immersing them in0.6mol L-1 hy-drochloric acid for5min，freeze-dried and packed.The BCB carriers thus prepared were recombined in vacuum with0.2mg of rhBMP
2 adsorbed to each chip.The resulting composite was dialyzed against distilled water，lyophilized，sterilized with ethylene oxide and stored ready for use.Some of rhBMP 2 /BCB chips and BCB chips were observed under scan-ning electron microscopy(SEM).
Bioassay of rhBMP2 /BCB inductivity Sixty male BALB/c mice with20to23g body mass and were randomized into study group of30，and control group of30.Each group was then evenly distribut-ed in3subgroups for observation at set time points of7，14and21d postoperatively.Mouse femoral muscle pouch model was used.Incision was made over the left proximal lateral aspect of the thigh to prepare a quadriceps muscle pouch where a rhBMP2 /BCB chip was implanted in the study group and a BCB chip in the control group.Postoperativ-ely，the mice were kept in cages under the same conditions.The mice were killed on d7，14and21after implantation.Every implanted sample harvest-ed was cut evenly into2parts.One partion of the sample was immersed in40g L-1 neutral formalin，then decalcified in a decalcifying admixure for72h，followed by gelatin embedding，frozen section，HE staining for light microscopic examination.Another portion of the cut-in-half specimen was cleaned of muscle tissue，weighed，minced，homogenized in1mL of saline and centrifuged at4000r min-1 ×15min.The ALP activity per unit tissue mass was measured of the supernatant.And the residue cen-trifuged was digested with hydrochloric acid prior to measurement of calcium content using an atomic ab-sorption spectrophotometer(Hitachi7500)，fol-lowed by calculation of the calcium content in unit mass of the specimen.
RESULTS
Histological findings of rhBMP 2 inductivity Histo- logical examination of the specimens retrieved the10th postoperative day from the mice implanted with0.2mg rhBMP2 showed that many mesenchy-mal cells proliferated，many chondrocytes occurred and became mature，with cartilage matrix mineral-ized，indicated that rhBMP2 itself had strong bone-inductive capacity(Fig1).
SEM observation of rhBMP2 /BCB and BCB A porous structure of BCB with smooth pore walls，and the pores were homogeneously filled up with rhBMP2 (Fig2).
Bioassay of rhBMP2 /BCB inductivity Macroscopic observation The study group:On d7，at the site of rhBMP2 /BCB implant there appeared a mass the size of0.6cm across with moderate tenaci-ty，and a sectional view showed that it was sur-rounded by greyish tissue with identifiable bound-ary.On d14，the mass slightly enlarged to0.8cm which was tough on palpation，presenting as yel-lowish tissue indiscernible from the surrounding tis-sue.On d21，the mass increased in consistency but was seen with no significant increase in size，with white cortex and red marrow easily discernible.The control group:The mass were much smaller with low consistency on d7，14and21，and the BCB granules were wrapped up by homogeneous greyish tissue.
Histological observation In study group，on d7，under the light microscope numerous undifferenti-ated mesenchymal cells were seen aggregating and proliferating along with a number of chondrocytes，with mineralized cartilage matrix and osteoid formed which had a diffuse focal distribution(Fig3).On d14，chondrocytes increased in number and merged with each other，and woven bone occurred(Fig4).On d21，mature lamellar bone and marrow tissue was seen(Fig5).In control group，connective tis-sue was seen enclosing BCB and filling up the pores of BCB，no bone and cartilage tissue were seen on d7，14and21.
Biochemical determination After implantation，the ALP activities and calcium content in the study group were higher than those in the control group(P<0.01，Tab1). atrix and osteoid were formed HE ×200 b P<0.01，vs control group.ALP:alkaline phosphatase.
DISCUSSION
In1965，Urist successfully used demineralized bone matrix to induce ectopic bone formation in muscle tissue，which led to the discovery of bone morpho-genetic protein(BMP).He also showed that the major biological effect of BMP is manifested by its stimulation of aggregation，proliferation and differ-entiation of undifferentiated mesenchymal cells，re-sulting in new bone formation.Many scholars had gained success in the clinical treatment of bone de-fects and nonunions with BMP from animal bone.Especially，HU et al first developed the reconstitut-ed bone xenograft(RBX)by recombining the anti-gen-free bovine cancellous bone as a carrier with crude extract of BMP from bovine cortical bone.RBX possesses strong osteoinductivty without evok-ing immune rejection as demonstrated by serial ex-perimental and clinical studies，which provides a successful solution to the difficult problem of xeno-geneic grafting[1] .However，BMP extract from ani-mal bone is not a protein constituent inherent to hu-man，and it will evoke an immune response to a cer-tain extent，although it is only weakly immuno-genetic.As far as a biomaterial is concerned，this is a major drawback which could not be ignored.Fur-thermore，with the minimal content of BMP in ani-mal bone tissue，e.g.1mg kg-1 (wet mass)in bovine bone and0.76mg kg
-1 (wet mass)in porcine bone，it is difficult to meet the demand in clinical practice using the natural occurring product.Also the extraction process of BMP is a labour-in-tensive and time consuming，which must go through multiple steps，with the purity of BMP differing from batch to batch [2] .In late1980's，with the ad-vent of gene engineering technology of BMP and subsequent improvements in the procedure，produc-tion of recombinant human BMP(rhBMP)has be-come a subject of intensive study to which much at-tention has been devoted.Wozney et al in1988first reported cloning and expression of cDNA of rhBMP1 ，rhBMP2A ，rhBMP 2B and rhBMP
3 .Subse-quently16rhBMPs were cDNA cloned，among which rhBMP2 ，rhBMP4 and rhBMP
7 are more os-teoinductive than the others，but for the present more experience has been gained with the use of rhBMP 2 ，which would promise fine prospects for its clinical use[3-6] .
In the present study we empolyed rhBMP 2 which is cheaper，high-yielding and exhibits more stable biological activities [7，8] in place of the BMP crude extract from bovine cortical bone in RBX，and combined it with BCB as a carrier to make rhBMP 2 /BCB and studied its osteoinductive ability on mouse muscle pouch model.The results showed:rhBMP2 / BCB induced chondrogenesis on d7，with woven bone formed on d14，and lamellar bone and marrow on d21.So，rhBMP2 /BCB was proved to be a ideal grafting material with strong bone-inductive and conductive capacity without evoking immune reac-tion，while BCB is a ideal carrier of rhBMP2 .
ACKNOWLEGEMENT:Prof.Pan BR in Oncology Center，Xijing Hospital，has great offort for this article.
REFERENCES:
[1]Luo ZJ，Hu YY，Lu R，Yang G，Li XB，Li D，Zhu YS.The experimental study on osteoinductive activity of massive recon-stituted bone xenograft [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1996;17(6):468-469.
[2]Chen SM.Significance and expression of human BMP gene [A].In:Yang LJ，Jin Y，Hu YY.Bioactive materials in stomatology and orthopaedics [M].Xi'an，Shaanxi Science and Technology Inc.1993;243-250.
[3]Pu Q，Chen SM，Chen NC.Expression of recombinant mature peptide of human bone morphogenetic protein in Escherichia coli [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1998;19(1):8-11.
[4]Du JJ，Hu YY，Luo ZJ，Lu R，Ma ZZ.Synergism of rhBMP2and TGF-βin osteoinduction and osteogenesis [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1999;20(7):555-558.
[5]Wang D，Zhao GY，Hu YY，Zhong CQ，Xie KN，Yang G，Lu R.Preparation ofβ-TCP/rhBMP2 at different degradation rates and assay of their osteoinductive activity [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1998;19(2):198.
[6]Zhou Y，Fan QY，Jing WZ，Cai HP，Wen YH.Bone induction of compound material of decalcified bone matrix with rhBMP2impregnated bone cement [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，1999;20(12):1085-1087.
[7]Yuan Z，Ma P，Hu YY，Luo ZJ，Jin GL，Lu R，Wang J.Ex-periment study on combined free periosteum grafting and new re-constituted bone xenograft in repairing segmental defects [J].J Med Coll PLA，2000;15(4):283-287.
[8]Yuan Z，Ma P，Hu YY，Luo ZJ，Jin GL，Lu R，Li D，Wang J.Combined use of rhBMP2/BCB and vascularized periosteum in repairing segmental defects in radii of rabbits [J].Di-si Junyi Daxue Xuebao(J Fourth Mil Med Univ)，2001;22(11):1013-1018.
Biography:YUAN Zhi(male，born in1966in Liaoyuan County，Jilin Province)，M D candidate in Institute of Orthopaedics，Xijing Hos-pital.Tel.(029)3375289 Email.Orth1@fmmu.edu.cn
(第四军医大学西京医院全军骨科研究所，陕西西安710033)
(Institute of Orthopaedics of Chinese PLA，Xijing Hospital，Fourth Military Medical University，Xi'an710033，China)
Editor XU Fu-Ming
Received date:2001┐06┐30, http://www.100md.com(YUAN Zhi，MA Ping，HU Yun-Yu，LUO Zhuo-Jing，ZHAO Guan)