软骨细胞和骨髓间质干细胞混合培养构建组织工程软骨的实验研究  

The construction of engineered cartilage by co-culture of chondrocytes and bone mesenchymal stem cells in vitro

作　　者：陈刚[1] 崔维顶[1] 范卫民[1] 

机构地区：[1]南京医科大学第一附属医院骨科,210029

出　　处：《中华骨科杂志》2010年第7期684-690,共7页Chinese Journal of Orthopaedics

摘　　要：目的探讨软骨细胞和骨髓间质干细胞（bone mesenchymal stem cells，BMSCs）混合培养对构建组织工程软骨的影响，并确定两者的最佳比例。方法体外分离培养兔（1月龄）关节软骨细胞和BMSCs，按不同比例（软骨细胞和BMSCs的浓度比为：4：1、2：1、1：1、1：2、1：4及单纯软骨细胞）混合培养一代。以4×10^7／ml的细胞终浓度接种40μl于聚乳酸-羟基乙酸共聚物[poly（lactic—CO—glycolicacid），PLGA：4mm×4mm×2mm），静态培养2d，然后移至周期性压力场（压力0-200kPa，频率：0．1Hz，时间：8h／d）培养3周。收获组织工程软骨行大体观察，组织切片，定量检测糖胺聚糖幢lyeosaminoglycans，GAGs）含量、DNA含量及Ⅱ型胶原染色面积。结果软骨细胞和BMSCs混合培养组与单纯软骨细胞组相比，体积较大，表面光滑，有弹性，有光泽。组织学检查显示混合培养组结构致密，细胞外基质分布更均匀，其中软骨细胞和BMSCs的浓度比为2：1组可见软骨陷窝。混合培养组的Ⅱ型胶原染色面积、GAGs含量、DNA含量高于单纯软骨细胞组，其中软骨细胞和BMSCs的浓度比为2：1组含量最高，与其他比例混合组比较，差异有统计学意义。结论软骨细胞和BMSCs混合培养能提高组织工程软骨的质量，其中以软骨细胞和BMSCs的浓度比为2：1最佳。Objective To explore the effects of co-culture of chondroeytes and bone mesenehymal stem cells （BMSCs） on constructing engineered cartilages, and confirm the most suitable ratio of chondrocytes to BMSCs. Methods Chondroeytes and BMSCs were isolated from articulars cartilages of rabbit （1 month old） and expanded respectively in vitro. They were mixed at six different ratios （ehondrocytes to BMSCs： 4：1, 2：1, 1： 1, 1：2, 1：4, and chondrocytes alone） after the first passage, and co-cultured for another passage. The mixed cells （40 μl, 4×10^7/ml） were seeded into a poly（lactie-eo-glycolie acid） （PLGA） scaffold and cultured statically for 2 days. They were transferred into the cyclic pressures system, and cultured under cyclic pressures for 3 weeks. The engineered cartilages were harvested and examined by gross observation, histological staining, immunohistoehemisty of collagen Ⅱ, the content of glyeosaminoglycans, GAGs, DNA and the percentage of collagen Ⅱ dyeing area. Results The engineered cartilages of the eo-euhured groups grew bigger than those of the ehondroeytes alone group, and their surfaces were smooth and glossy. The distributions of cartilaginous extracellular matrices in the co-cultured groups were more homogenous than those of the ehondroeytes alone group. Cartilage lacunas were found in the 2：1 group only. The contents of GAGs, DNA and the percentages of collagen Ⅱ dyeing area of the co-cultured groups were higher than those of the ehondroeytes alone group. The contents and the percentages of the 2：1 group were the highest, and the differences were statistically significant. Conclusion Co-culture of ehondroeytes and BMSCs could improve the quality of engineered cartilages. The 2：1 ratio for rabbit ehondroeytes and rabbit BMSCs was of the best co-cultured effect.

关 键 词：软骨细胞 间质干细胞 骨髓细胞 组织工程 

分 类 号：R318[医药卫生—生物医学工程]