人骨膜来源细胞在聚乳酸三维支架体外成骨的超微结构研究  

Biological activity and ultrastructure of human periosteum-derived osteoblasts in vitro cultured in poly lactic acid scaffolds

作　　者：贝抗胜[1] 刘延晓[2] 熊英辉[1] 吴礼杨[1] 卢林俊[1] 

机构地区：[1]粤北人民医院骨科,广东省韶关市512026 [2]浙江省衢州市人民医院骨科

出　　处：《中华创伤骨科杂志》2015年第12期1086-1092,共7页Chinese Journal of Orthopaedic Trauma

基　　金：广东省科技攻关项目（2005836001054）;韶关市科技局项目[韶科（卫）2013-13]

摘　　要：目的探讨人骨膜来源细胞在聚乳酸（PLA）三维支架体外培养成骨的可行性及其超微结构特征。方法取材于成人胫骨骨膜，组织块法分离出骨膜细胞并体外培养传至第3代后进行定向诱导分化为骨膜来源成骨细胞，相差显微镜观察细胞形态特征，分为诱导组和未诱导组，分别在5、10、15、20d取3个样本，采用碱性磷酸酶（ALP）试剂盒法及钙结节VonKossa染色法检测ALP和钙结节的表达。经诱导分化的成骨细胞随机分为实验组和对照组，分别接种至经含骨形态发生蛋白．7成骨诱导剂预处理的PLA三维支架和普通二维细胞培养板，1、3、5、7、9、11d采用CCKl8法测量两组细胞的增殖能力，并绘制生长曲线；3、6、9、12d电镜下观察细胞在PLA的生长情况和超微形态特征。结果骨膜来源细胞经诱导分化其形态与成骨细胞相似，诱导组的ALP和钙结节染色阳性率明显高于未诱导组，差异均有统计学意义（P〈0．05）。实验组和对照组细胞生长曲线均呈“S”形，实验组对数生长期增殖较快，平稳期细胞数目明显下降，对照组平稳期数目则有上升，差异有统计学意义（P〈0．05）。电子显微镜观察体外骨膜来源成骨细胞具有良好的超微形态学结构。结论体外骨膜来源成骨细胞与PLA三维支架复合有良好的生物相容性、细胞增殖力和超微形态学结构；体外复合成活后应在第8，9天及时转入体内生长以发挥骨修复作用。Objective To explore the feasibility of osteogenesis by human periosteum-derived osteoblasts in vitro cultured in poly lactic acid （PLA） three-dimensional scaffolds and the ultrastructural features of the human periosteal osteoblasts. Methods The cells derived from adult tibial periosteum were separated through tissue explants, cultured in vitro to the third generation, and directionally differentiated into the human periosteum-derived osteoblasts. Their morphological features were observed under a phase contrast microscope. After they were divided into an induction group and a non-induction group, 3 samples were taken from each group at 5, 10, 15 and 20 days for observation of the expression of alkaline phosphatase （ALP） and calcium nodules using ALP reagents and Von Kossa staining. After the osteogenically differentiated cells were randomly divided into an experimental group and a control group, and respectively seeded onto PLA scaffolds and ordinary two-dimensional cell culture plates. At 1, 3, 5, 7, 9, 11 days CCK-8 method was used to measure cellular proliferation in both groups and growth curves were drawn. At 3, 6, 9, 12 days, the growth and ultrastructural morphology of the cells were observed in both groups under an electron microscope. Results The morphology of the periosteum-derived cells was like that of osteoblasts after being induced and differentiated. The positive rates of ALP staining and calcium nodules in the induction group were significantly higher than in the non-induction group （ P 〈 0. 05）. The cell growth curve presented an S-shape in both the experimental and the control groups. The proliferation in the logarithmic growth phase in the experimental group was significantly faster than in the control group while the number of ceils decreased significantly in the stationary phase in the experimental group than in the control group （ P 〈 0. 05）. Electron microscopy showed fine ultrastructural morphology of the in vitro periosteum-derived osteoblasts. Conclusions The biocom

关 键 词：成骨细胞 细胞培养技术 组织支架 超微结构 

分 类 号：R318.08[医药卫生—生物医学工程]