异种生物骨衍生材料与大鼠骨髓基质细胞粘附的体外实验研究
|
摘要
目的：制备一种生物骨衍生材料，将此材料与大鼠骨髓基质细胞（BMSCs）联合培养，通过光镜及电镜对联合培养过程进行连续观察，了解该材料与大鼠骨髓基质细胞（BMSCs）的生物相容性及粘附情况，寻找一种有临床应用前景，适用于构建组织工程骨的支架材料。
方法：将新西兰大白兔的四肢长骨制成骨条，以改良的过氧化氢乙醚法处理骨条后消毒并－80℃冻存。使用percoll分离液密度梯度离心分离大鼠的骨髓基质细胞（BMSCs），在含10%小牛血清（fbs）的低糖DMEM培养液中，置于37℃，5%co2培养箱内进行体外培养，保留贴壁细胞传代，至第5代时，以胰蛋白酶消化细胞后吹打制成细胞悬液，调整细胞数量约为1×106/ml，。将细胞悬液滴入预先制好的生物骨衍生材料粗糙面上，使之充分渗入。加入10%小牛血清DMEM培养液进行联合培养。通过光镜及电镜对联合培养过程进行连续观察，了解该材料与大鼠骨髓基质细胞（BMSCs）的相容性及粘附情况。
结果：1、扫描电镜观察(SEM)可见生物衍生骨材料具有原骨组织的网状孔隙结构系统，其原骨组织骨盐支架的三维多孔结构存在。2、所分离提纯的大鼠骨髓基质细胞（BMSCs）经培养后呈成纤维细胞样生长，细胞轮廓清晰，胞浆折光性好，逐渐形成细胞集落，表现出骨髓基质细胞的生长特点且生长力旺盛。3、大鼠骨髓基质细胞（BMSCs）在生物骨衍生材料上粘附情况：倒置显微镜观察：联合培养第3日可见细胞从生物骨衍生材料向外长出，尤其向骨粒方向延伸显著。随培养时间延长，板内细胞进一步增多，井与骨粒上长出的细胞连接成片。扫描电镜观察：生物骨衍生材料表面粗糙，可见大小不等的裂隙与微孔。细胞贴附于骨粒表面，或向裂隙或微孔内生长。细胞为梭形或多边形，其表面有丝状突起。
结论：我们制备的这种生物骨衍生材料来源广泛，易获取，易制备，有三维立体多孔隙结构，并有良好的生物相容性和材料—细胞界面：大鼠骨髓基质细胞（BMSCs）能附着在支架上分裂、增殖，表明该材料是一种有临床应用前景，适用于组织工程骨构建的支架材料。
Objective: we prepared a kind of derived material of natural bone , and we cultured the mouse marrow stromal cells with this kind of material, observed the combined culture process with the microscope, to understand the consistent and the adhibit character. We want to find a kind of plank material that has bright clinical future and can be fit for the tissue-engineered bone constuction.
Method: we made the bone pieces from the New Zealand rabbits’ limb bone. The bone pieces were treated with H2O2 and aether, then sterilized and stored by 80℃ under zero. The BMSCS were isolated and purified by gradient centrifugation in Percoll. The BMSCS were collected and cultured in DMEM(LG) with 15% fatal bovine serum(FBS).Cultures were maintained at 37℃ in humidified atmosphere chamber containing 5% CO2 . The adhibit cells kept for passage. The five passage cells were digested by trypsin and the number of the cells were adjusted to about 1×106 /ml. The cells were dripped and filtered into the derived material’s coarse faces. Then the cells and the derived material were cultured together with 15% fatal bovine serum(FBS). We observe the combined culture process with the microscope, to understand the consistent and the adhibit character.
results: 1、we observed by SEM that the biological bone material has the original bone tissue’s reticulation hole structure system and the original bone tissue plank’s three dimension holes system. 2、 The BMSCs cultured were grown like fibroblast, the figure of the BMSCs were in focus, the cell plasm refract well, gradually gather together ,this can explain that the BMSCs grew bloom. 3、 The third day we saw the cells crept from the biological bone material, after several days the cells on the derived material increased, and grew together. We observed by SEM that the biological bone material has the coarse surface and crannies and microholes. The BMSCs adhibited the bone material or grew to the crannies and the microholes. The BMSCs had the shuttle figure or polygon with the threadlike prominency.
Conclusion: The derived material of natural bone can be got easily and has many sources. It has the three dimension hole stucture and dynam intension, and has good biological consistent character and the material-cell border; The BMSCs can split and proliferate on the plank, this proves that the material has a bright clinical future can be fit for the tissue-engineering bone.
方法：将新西兰大白兔的四肢长骨制成骨条，以改良的过氧化氢乙醚法处理骨条后消毒并－80℃冻存。使用percoll分离液密度梯度离心分离大鼠的骨髓基质细胞（BMSCs），在含10%小牛血清（fbs）的低糖DMEM培养液中，置于37℃，5%co2培养箱内进行体外培养，保留贴壁细胞传代，至第5代时，以胰蛋白酶消化细胞后吹打制成细胞悬液，调整细胞数量约为1×106/ml，。将细胞悬液滴入预先制好的生物骨衍生材料粗糙面上，使之充分渗入。加入10%小牛血清DMEM培养液进行联合培养。通过光镜及电镜对联合培养过程进行连续观察，了解该材料与大鼠骨髓基质细胞（BMSCs）的相容性及粘附情况。
结果：1、扫描电镜观察(SEM)可见生物衍生骨材料具有原骨组织的网状孔隙结构系统，其原骨组织骨盐支架的三维多孔结构存在。2、所分离提纯的大鼠骨髓基质细胞（BMSCs）经培养后呈成纤维细胞样生长，细胞轮廓清晰，胞浆折光性好，逐渐形成细胞集落，表现出骨髓基质细胞的生长特点且生长力旺盛。3、大鼠骨髓基质细胞（BMSCs）在生物骨衍生材料上粘附情况：倒置显微镜观察：联合培养第3日可见细胞从生物骨衍生材料向外长出，尤其向骨粒方向延伸显著。随培养时间延长，板内细胞进一步增多，井与骨粒上长出的细胞连接成片。扫描电镜观察：生物骨衍生材料表面粗糙，可见大小不等的裂隙与微孔。细胞贴附于骨粒表面，或向裂隙或微孔内生长。细胞为梭形或多边形，其表面有丝状突起。
结论：我们制备的这种生物骨衍生材料来源广泛，易获取，易制备，有三维立体多孔隙结构，并有良好的生物相容性和材料—细胞界面：大鼠骨髓基质细胞（BMSCs）能附着在支架上分裂、增殖，表明该材料是一种有临床应用前景，适用于组织工程骨构建的支架材料。
Objective: we prepared a kind of derived material of natural bone , and we cultured the mouse marrow stromal cells with this kind of material, observed the combined culture process with the microscope, to understand the consistent and the adhibit character. We want to find a kind of plank material that has bright clinical future and can be fit for the tissue-engineered bone constuction.
Method: we made the bone pieces from the New Zealand rabbits’ limb bone. The bone pieces were treated with H2O2 and aether, then sterilized and stored by 80℃ under zero. The BMSCS were isolated and purified by gradient centrifugation in Percoll. The BMSCS were collected and cultured in DMEM(LG) with 15% fatal bovine serum(FBS).Cultures were maintained at 37℃ in humidified atmosphere chamber containing 5% CO2 . The adhibit cells kept for passage. The five passage cells were digested by trypsin and the number of the cells were adjusted to about 1×106 /ml. The cells were dripped and filtered into the derived material’s coarse faces. Then the cells and the derived material were cultured together with 15% fatal bovine serum(FBS). We observe the combined culture process with the microscope, to understand the consistent and the adhibit character.
results: 1、we observed by SEM that the biological bone material has the original bone tissue’s reticulation hole structure system and the original bone tissue plank’s three dimension holes system. 2、 The BMSCs cultured were grown like fibroblast, the figure of the BMSCs were in focus, the cell plasm refract well, gradually gather together ,this can explain that the BMSCs grew bloom. 3、 The third day we saw the cells crept from the biological bone material, after several days the cells on the derived material increased, and grew together. We observed by SEM that the biological bone material has the coarse surface and crannies and microholes. The BMSCs adhibited the bone material or grew to the crannies and the microholes. The BMSCs had the shuttle figure or polygon with the threadlike prominency.
Conclusion: The derived material of natural bone can be got easily and has many sources. It has the three dimension hole stucture and dynam intension, and has good biological consistent character and the material-cell border; The BMSCs can split and proliferate on the plank, this proves that the material has a bright clinical future can be fit for the tissue-engineering bone.
引文
[1] Brown K L，Cruess R L．Bone and cartilage transplantation surgery[J].J Bone Jt Surg Am．1982，64(2)：270—279．
[2] Boer H． The history of bone grafts[J]． Clin Orthop，1998，（226）：292－305．
[3] Keith T，Cima LG，Yaremchuk，et al．Injectable cartilage[J]．Plast Reconstr Surg，1995，96（6）：1390-1399．
[4] Vacanti CA, Vacanti JP . Tissue-engineered morphojenesis of cartilage and bone by means of cell transplantation using synthetic biodegradable polymer matrices[J]. Clin plast surg, 1994,21(3):445-451
[5] Bruder S P, Fox B S.Tissue engineering of bone[J]. Cell based strategies.1999,367S:S68-83
[6] Langer R，VacantiJP．Tissue engineering[J] science，1993，260(14)：920—925
[7] 崔福斋，杜昶 ．骨组织工程，世界医疗器械，1999；5：50
[8] Ozaki T,Inoue H,Sugihara S,et al.Radiological long-term follow up of grafted xenoheneic bone in patients with bone tumors.Acta Med Okayama,1992; 46(2): 87
[9] Valentini P, Abensur D, Maxillary sinus floor elevation for implant placement with demineralized freeze－dried bone and bovine bone（Bio－Oss）： A clinlcal study of 20 patients. Int J Periodontics Restorative Dent． 1997： 17（3）： 232
[10] Valentini P，Abensur D，Densari D，et al．Histological evaluation of Bio－Oss in a 2－stage sinus floor elevation and implantation procedure：a human case report．Clin Oral Implants Res．1998：9（l）：59
[11] Kim BS，Mooney DJ．Development of biocompatible synthetic extracellular matries for tissue engineering[J] Trends Biotechnol，1998，16(5)：224—230．
[12]郑磊，王前，裴国献．骨组织工程中细胞外基质材料的选择．
中华外科杂志，2000，10：745-748
[13] Yoshikawa T，Ohgushi H，Tamai S．Immediate bone forming capability of prefabricated osteogenic hydrox-yapatite[J]． J Biomed Mater Res，1996，32（3）：481-492
[14] Bruder SP，Kraus KH，Goldberg VM，et al．The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects[J]．J Bone Joint Surg Am，1998，80（7）：985-996
[15] Breitbart S, Graride DA, keler R. tissue engineered bone repair of calvarial defects using cultured periosteal cells. Plast Reconstr Surg, 1998;101(3):567
[16] Ishaug SL, Crane GM, Gurlek A, et al. Ectopic bone formation by marrow stromal osteoblast transplantation using poly (DL-lactic-co-glycolic acid) foams implanted into the rat mesentery, J Biomed Mater Res,1997;36(1):1
[17] Isogai N, Landis W, Kim TH, et al. Formation of phalanges and small joint by tissue-engineering. J Bone Join Surg, 1999;81A:306
[18] Mikos AG，Lyman MD，Freed LE．Wetting of poly（L-lactic acid） and poly (LD-lactic-co-glycolic acid) foams for tissue culture．Biomaterials，1994，15（1）：55
[19] Sims CD, Butler PE, Cao YL,et al. Tissue engineered neocartilage using plasma derived polymer substrates and chondrocytes. Plast Reconstr Surg, 1998;101(6):1580
[20] Mizuno M，Shindo M，Kobayashi D，et al．Osteogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo[J]．Bone，1997，20（2）：101-107
[21] 罗卓荆，胡蕴玉，王茵．块状重组合异种骨修复犬桡骨骨缺损．中华骨科杂志，1998，18（6），363
[22] 杨志明，李彦林，解慧琪，等．生物衍生骨复合成骨细胞体内异位成骨研究．中华创伤杂志，2001，17（18），475-478
[23] 杨志明，李彦林，解慧琪，等．生物衍生骨支架材料的组织相容性研究．中华整形外科杂志，2002，18（1），6-8
[24] 杨志明，余希杰，屈 艺，等．I型胶原—整合素α2 β1系统对成骨细胞生物学特性的调控．华西医科大学学报，200013l(3)：28l
[25] 孔明学， 脱钙骨基质诱导成骨活性的因素分析 [J] 国外医学，外科学分册，2001，28（5）：286－8
[26] 李裕标，杨志明，等，成骨细胞与生物衍生材料联合培养的实验研究 [J] 中国修复重建外科杂志，2002，36 （3）：57－9
[27] Katoh T, Sato K, Kawamura M. Osteogenesis in sintered bone combined with bovine bone morphogenetic protein. Clin Orthop, 1993,287:266-275
[28] Meena C, Mengi S.A, Dashpande. S G. Biomedical and industrial applications of collagen proceedings of the Indian academy of sciences.Chemical Science, 1999, 111(2): 319-329
[29] 罗卓荆，胡蕴玉等. 去抗原异种松质骨移植的免疫反应研究中华外科杂志，1997，11（3）：690－693
[30] 高中礼，王金成等，不同时间脱蛋白异种骨抗原性和生物力学
相关性研究. 骨与关节损伤杂志，2002，17（2）：122－123
[31] 胡永康，安洪等. 四种脱蛋白骨组织学和生物力学比较研究
中华创伤杂志，1998，14：277
[32] 刘植珊，李光业，陈永裕，等．异体半关节移植术．中华骨
科杂志，1990；10 13．
[2] Boer H． The history of bone grafts[J]． Clin Orthop，1998，（226）：292－305．
[3] Keith T，Cima LG，Yaremchuk，et al．Injectable cartilage[J]．Plast Reconstr Surg，1995，96（6）：1390-1399．
[4] Vacanti CA, Vacanti JP . Tissue-engineered morphojenesis of cartilage and bone by means of cell transplantation using synthetic biodegradable polymer matrices[J]. Clin plast surg, 1994,21(3):445-451
[5] Bruder S P, Fox B S.Tissue engineering of bone[J]. Cell based strategies.1999,367S:S68-83
[6] Langer R，VacantiJP．Tissue engineering[J] science，1993，260(14)：920—925
[7] 崔福斋，杜昶 ．骨组织工程，世界医疗器械，1999；5：50
[8] Ozaki T,Inoue H,Sugihara S,et al.Radiological long-term follow up of grafted xenoheneic bone in patients with bone tumors.Acta Med Okayama,1992; 46(2): 87
[9] Valentini P, Abensur D, Maxillary sinus floor elevation for implant placement with demineralized freeze－dried bone and bovine bone（Bio－Oss）： A clinlcal study of 20 patients. Int J Periodontics Restorative Dent． 1997： 17（3）： 232
[10] Valentini P，Abensur D，Densari D，et al．Histological evaluation of Bio－Oss in a 2－stage sinus floor elevation and implantation procedure：a human case report．Clin Oral Implants Res．1998：9（l）：59
[11] Kim BS，Mooney DJ．Development of biocompatible synthetic extracellular matries for tissue engineering[J] Trends Biotechnol，1998，16(5)：224—230．
[12]郑磊，王前，裴国献．骨组织工程中细胞外基质材料的选择．
中华外科杂志，2000，10：745-748
[13] Yoshikawa T，Ohgushi H，Tamai S．Immediate bone forming capability of prefabricated osteogenic hydrox-yapatite[J]． J Biomed Mater Res，1996，32（3）：481-492
[14] Bruder SP，Kraus KH，Goldberg VM，et al．The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects[J]．J Bone Joint Surg Am，1998，80（7）：985-996
[15] Breitbart S, Graride DA, keler R. tissue engineered bone repair of calvarial defects using cultured periosteal cells. Plast Reconstr Surg, 1998;101(3):567
[16] Ishaug SL, Crane GM, Gurlek A, et al. Ectopic bone formation by marrow stromal osteoblast transplantation using poly (DL-lactic-co-glycolic acid) foams implanted into the rat mesentery, J Biomed Mater Res,1997;36(1):1
[17] Isogai N, Landis W, Kim TH, et al. Formation of phalanges and small joint by tissue-engineering. J Bone Join Surg, 1999;81A:306
[18] Mikos AG，Lyman MD，Freed LE．Wetting of poly（L-lactic acid） and poly (LD-lactic-co-glycolic acid) foams for tissue culture．Biomaterials，1994，15（1）：55
[19] Sims CD, Butler PE, Cao YL,et al. Tissue engineered neocartilage using plasma derived polymer substrates and chondrocytes. Plast Reconstr Surg, 1998;101(6):1580
[20] Mizuno M，Shindo M，Kobayashi D，et al．Osteogenesis by bone marrow stromal cells maintained on type I collagen matrix gels in vivo[J]．Bone，1997，20（2）：101-107
[21] 罗卓荆，胡蕴玉，王茵．块状重组合异种骨修复犬桡骨骨缺损．中华骨科杂志，1998，18（6），363
[22] 杨志明，李彦林，解慧琪，等．生物衍生骨复合成骨细胞体内异位成骨研究．中华创伤杂志，2001，17（18），475-478
[23] 杨志明，李彦林，解慧琪，等．生物衍生骨支架材料的组织相容性研究．中华整形外科杂志，2002，18（1），6-8
[24] 杨志明，余希杰，屈 艺，等．I型胶原—整合素α2 β1系统对成骨细胞生物学特性的调控．华西医科大学学报，200013l(3)：28l
[25] 孔明学， 脱钙骨基质诱导成骨活性的因素分析 [J] 国外医学，外科学分册，2001，28（5）：286－8
[26] 李裕标，杨志明，等，成骨细胞与生物衍生材料联合培养的实验研究 [J] 中国修复重建外科杂志，2002，36 （3）：57－9
[27] Katoh T, Sato K, Kawamura M. Osteogenesis in sintered bone combined with bovine bone morphogenetic protein. Clin Orthop, 1993,287:266-275
[28] Meena C, Mengi S.A, Dashpande. S G. Biomedical and industrial applications of collagen proceedings of the Indian academy of sciences.Chemical Science, 1999, 111(2): 319-329
[29] 罗卓荆，胡蕴玉等. 去抗原异种松质骨移植的免疫反应研究中华外科杂志，1997，11（3）：690－693
[30] 高中礼，王金成等，不同时间脱蛋白异种骨抗原性和生物力学
相关性研究. 骨与关节损伤杂志，2002，17（2）：122－123
[31] 胡永康，安洪等. 四种脱蛋白骨组织学和生物力学比较研究
中华创伤杂志，1998，14：277
[32] 刘植珊，李光业，陈永裕，等．异体半关节移植术．中华骨
科杂志，1990；10 13．