软骨细胞复合胶原/壳聚糖/β-磷酸三钙层状梯度修复体的体外培养  

In vitro culture of chondrocytes on the layered cylindric collagen/chitosan/beta-tricalcium phosphate composite scaffold

作　　者：张世浩[1] 朱立新[1] 靳安民[1] 严乐平[2] 黄锐[1] 王九现[1] 朱书涛[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]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41期8033-8036,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项2003C34002项~~

摘　　要：背景:关节软骨缺损的修复涉及到软骨和软骨下骨的双重修复,需要将骨与软骨组织工程结合起来,构建骨软骨双层支架,或者在同一支架上构建组织工程化骨软骨。目的:观察胶原/壳聚糖/β-磷酸三钙层状梯度修复体对软骨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。时间及地点:体外细胞学实验,于2007-12/2008-06在广东省构建与检测实验室进行。材料:以胶原、壳聚糖、β-磷酸三钙制备复合支架,上层以胶原/壳聚糖为主,下层以胶原/壳聚糖/β-磷酸三钙为主,孔隙率≥90%,孔径100μm。方法:体外培养新西兰大白兔软骨细胞并成骨诱导3周,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3周。主要观察指标: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:修复体/细胞体外培养3周,修复体上细胞数量明显增多,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:胶原/壳聚糖/β-磷酸三钙层状梯度修复体的细胞相容性良好,有望成为一种比较理想的关节软骨组织工程支架材料。BACKGROUND： Repair of articular cartilage defect involves double repair of cartilage and subchondral bone. Bone and cartilage tissue engineering should be combined to construct bone and cartilage double layer scaffold, or to construct tissue engineered bone and cartilage on the same scaffold. OBJECTIVE： To observe layered cylindric collagen/chitosan/β-tricalcium phosphate composite effects on adsorption and biological character of chondrocytes, and to evaluate the feasibility as a cartilage engineering scaffold. TIME AND SETTING： The experiment was performed at the Laboratory of Tissue Construction and Detection of Guangdong Province from December 2007 to June 2008. MATERIALS： Composite scaffold was made with collagen/chitosan/β-tricalcium phosphate. Upper layer was mainly collagen/chitosan, and lower layer was mainly collagen/chitosan/β-tricalcium phosphate. The factor of porosity was 90%, with antilinear of 100 μm. METHODS： The chondrocytes from New Zealand rabbits were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen/chitosan/β-tricalcium phosphate composite scaffold and were cultured in a three-dimensional environment for 3 weeks. MAIN OUTCOME MEASURES： The effects of the composite scaffold on the cell phenotype, proliferation and function of the extracellular matrix were observed by the growth curve, inverted phase-contrast microscopy, histology, scanning electron microscopy and immunohistochemistry. RESULTS： The chondrocytes that adhered to the scaffold were increased significantly and secreted the extracellular matrix in the porous scaffold around the chondrocytes at 3 weeks after in vitro culture. Immunohistochemistry of type Ⅱ collagen was positive. CONCLUSION： The layered cylindric collagen/chitosan/β-tricalcium phosphate composite scaffold has a good cellular compatibility, which can be an ideal scaffold for the cartilage tissue engineering.

关 键 词：胶原 壳聚糖 Β-磷酸三钙 软骨细胞 生物材料 

分 类 号：R318[医药卫生—生物医学工程]