模拟微重力环境下层状修复体与软骨细胞复合培养的实验研究

Study of chondrocyte on the layered scaffold under minic microgravity culture

[目的]探讨模拟微重力作为软骨组织工程培养方法的作用和胶原/壳聚糖/β-磷酸三钙(trical cium phosphate,TCP)层状梯度修复体作为关节软骨组织工程支架的可行性。[方法]体外培养新西兰大白兔关节软骨细胞并扩增,吸附于多孔胶原/壳聚糖/β-磷酸三钙层状梯度修复体上,模拟微重力和普通环境下三维立体分别培养3周,通过生长曲线、倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测微重力对软骨细胞培养的影响和支架在三维立体培养对软骨细胞的表型、增殖及功能的影响。[结果]软骨细胞/修复体体外培养3周,软骨细胞模拟微重力培养组明显比普通培养组在层状修复体上分布均匀,修复体中心软骨细胞数量明显较多,并分泌细胞基质,包裹在软骨细胞周围,Ⅱ型胶原免疫组织化学染色阳性。[结论]模拟微重力环境有利于软骨细胞在三维支架上的均匀增殖,有望成为软骨组织工程中的一种重要培养方法;胶原/壳聚糖/β-磷酸三钙层状梯度修复体,细胞相容性良好,有望成为一种比较理想的关节软骨组织工程支架材料。

[ Objective] To evaluate the feasibility of the minic microgravity as a method and the layered cylindric collagenchitosan-β-triealcium phosphate composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondroeytes and affects the cell behavior. [ Method ] The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous eollagen-chitosan-β-triealcium phosphate composite scaffold and were cultured in both minie mierogravity and ordinary environment for 3 weeks. The effects of the composite scaffold on the cell adhesivity,proliferation, morphological changes and synthesis of the extracellularmatrix were observed by the growth curve,phase-contrast microscopy, histology,scanning electron microscopy and immunohistochemistry. [ Result] The chondrocytes that adhered to the scaffold increased significantly and secreted extracellular matrix in the center of the porous scaffold around the chondrocytes under minic microgravity compared with ordinary environment. Immunohistochemistry of type Ⅱ collagen was positive. [ Conclusion ] The minic microgravity environment will be a good method for the cartilage tissue engineering. And the layered cylindric collagen-chitosan-β-tricalcium phosphate composite scaffold has a good cellular compatibility. It will be an ideal scaffold for the cartilage tissue engineering.