胶原/壳聚糖/β-磷酸三钙层状修复体负载骨髓间充质干细胞体外培养  

In vitro culture of bone mesenchymal stem cells on the layered collagen-chitosan-beta-tricalcium phosphate composite scaffold

作　　者：朱书涛[1] 朱立新[1] 余磊[2] 严乐平[3] 于巧莲[2] 黄锐[1] 张世浩[1] 王九限[1] 

机构地区：[1]南方医科大学珠江医院骨科中心,广东省广州市510282 [2]南方医科大学临床解剖研究所,广东省组织工程构建与检测重点实验室,广东省广州市510515 [3]华南理工大学材料科学和工程学院,广东省广州市510640

出　　处：《中国组织工程研究与临床康复》2008年第10期1815-1818,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省科技计划项目基金资助2003C34002~~

摘　　要：目的:观察胶原/壳聚糖/β-磷酸三钙层状梯度修复体对经诱导骨髓间充质干细胞的吸附作用及对细胞生物学性状的影响,评价其作为关节软骨工程支架的可行性。方法:实验于2007-09/2008-03在广东省组织工程构建与检测实验室进行。①实验方法:以胶原、壳聚糖、β-磷酸三钙制备复合支架,孔隙率≥90%,孔径100μm;体外培养新西兰大白兔骨髓间充质干细胞并成骨诱导3周,经检测诱导成功后,使之吸附于多孔胶原/壳聚糖/β-磷酸三钙层状梯度修复体上三维立体培养4周。②观察指标:通过倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测修复体三维立体培养对成骨细胞的表型、增殖及功能的影响。结果:①骨髓间充质干细胞经成骨诱导,检测碱性磷酸酶染色阳性,钙结节染色阳性,证实诱导成功。②胶原/壳聚糖/β-磷酸三钙复合支架亲水性好,经诱导的骨髓间充质干细胞吸附于支架表面24h后,顺孔隙迁徙至支架内部,倒置显微镜和扫描电镜观察显示细胞在孔壁贴附良好。③组织学及免疫组织化学检测表明成骨细胞在复合支架上增殖和表型维持稳定,能分泌细胞外基质,Ⅰ型胶原免疫组织化学染色阳性。结论:胶原/壳聚糖/β-磷酸三钙层状梯度修复体与经成骨诱导的骨髓间充质干细胞相容性良好,为修复关节软骨下层骨的进一步研究提供了实验依据。AIM： To evaluate the feasibility of the layered cylindric collagen-chitosan- β-tricalcium phosphate composite as a scaffold for the articular cartilage tissue engineering after an observation of how it absorbs the induced bone mesenchymal stem cells and affects the cell biological behaviors. METHODS： The experiment was carried out in the Key Laboratory of Tissue Construction and Detection of Guangdong Province from September 2007 to March 2008.①The collagen-chitosan- β -tricalcium phosphate composite was prepared in a pore diameter of 100 μ m and porosity ≥ 90%. Rabbit bone mesenchymal stem ceils were cultured and induced in vitro, then the cells were seeded onto the porous collagen-chitosan-β-tricalcium phosphate composite scaffold and were cultured in a three-dimensional environment for 4 weeks.②The effects of the composite scaffold on the phenotype, proliferation, and function of osteoblasts were observed by the inverted phase contrast microscopy, histology, scanning electron microscopy, and immunohistochemistry. RESULTS： ①The induced bone mesenchymal stem ceils were positive for both alkaline phosphatase stain and calcification tubercles stain, which all indicated successful induction.②Due to good hydrophilicity of collagen-chitosan- 13 -tricalcium phosphate composite scaffold, the induced bone mesenchymal stem cells adhered to the surface of the scaffold by the inverted phase contrast microscopy and scanning electron microscopy, then cells migrated into the scaffold 24 hours later.③The results of histology and immunohistochemistry revealed that osteoblasts proliferated on the composite scaffold, maintained a rounded morphology and could secrete the extracellular matrix. The immunohistochemistry stain of collagen Ⅰ was positive. CONCLUSION： The layered cylindric collagen-chitosan-β -tricalcium phosphate composite scaffold has a good compatibility with the induced bone mesenchymal stem cells, and it will provide experimental evidences for the further study of article cartilage repai

关 键 词：胶原 壳聚糖 磷酸三钙 关节软骨 细胞相容性 组织工程 生物材料 

分 类 号：R318[医药卫生—生物医学工程]