海藻酸钠载体培养成年兔软骨细胞的生物学性状研究

Biological Characteristic Study of Culture of Chondrocytes Combined with Alginate Gel from Joint Articular in Rabbits in Vitro

目的 :探索以海藻酸钠为载体的成年兔软骨细胞构建工程化软骨的可行性。方法 :取 3 2周龄新西兰兔膝关节软骨 ,酶消化法得到高纯度软骨细胞 ,与海藻酸钠混合 ,种植密度为 4× 10 6/ml,通过硅胶盘模制成圆形柱状细胞盘 (2 0ul/个 ) ,CaCl2 溶液中凝胶化 10min ,DMEM /F12 无血清培养基加 2 0 %FBS (fatalbloodsolution)于 2 4孔培养板中培养。于 2、 4、 6、 8、 10、 12周取细胞盘 ,行HE、AB PAS染色及免疫组化分析 ,测定细胞盘中蛋白多糖含量 ,并作投射电镜观察。结果 :成年软骨细胞在海藻酸钠中呈丛状或球状增殖 ,4周时达增殖高峰 ,盘中Ⅱ型胶原及蛋白多糖的含量随培养时间延长逐渐增加 ,无I型胶原产生。电镜观察软骨细胞超微结构无异常改变。结论 :成年兔软骨细胞在海藻酸钠中可良好生长增殖 ,海藻酸钠可保留软骨细胞分泌的基质 ,成功构建工程化软骨。

Objective:To observe the biological characteristics of chondrocytes cultured in alginate.Methods:Chondrocytes from the knee joints of 34-week-old New Zealand white rabbits were obtained using collagenase enzyme.The chondrocytes were mixed with alginate in a cell density of 4×10 6/ml.The suspension was gelled in a shape of disc in CaCl 2 solution and each disc contains 20ul suspensions.The discs of chondrocytes and alginate compound were cultured in 24-Multiwell.The discs were taken at the end of 2,4,6,8 and 10 weeks after culture,and were used for evaluation of proteoglycan and collagen II with HE and AB-PAS staining,biochemical analysis,immunohistochemistry and transmission electron microscopy.Result:Chondrocytes from the knee joints of 34-week-old New Zealand white rabbits were obtained using collagenase enzyme.The chondrocytes were mixed with alginate in a cell density of 4×10 6/ml.The suspension was gelled in a shape of disc in CaCl 2 solution and each disc contains 20ul suspensions.The discs of chondrocytes and alginate compound were cultured in 24-Multiwell.The discs were taken at the end of 2,4,6,8 and 10 weeks after culture,and were used for evaluation of proteoglycan and collagen II with HE and AB-PAS staining,biochemical analysis,immunohistochemistry and transmission electron microscopy.Conclusion:The chondrocytes from the adult rabbits cultured in alginate can keep the phenoshape and construce engineered cartilage tissue in vitro.