【作者】 花奇凯；

【导师】 赵劲民；

【作者基本信息】 广西医科大学 ， 骨科， 2004， 硕士

【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。更多还原

【Abstract】 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. <WP=6>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.更多还原