软骨细胞与骨髓间充质干细胞隔离共培养构建组织工程化软骨
Isolated co-culture of bone mesenchymal stem cells and auricular chondrocytes to construct cartilage
吴俊 1薛珂 2刘凯 2孙俊英3
作者信息
- 1. 苏州大学附属第一医院骨科,江苏苏州215006;苏州大学附属第三医院骨科,江苏常州213003
- 2. 上海交通大学医学院附属第九人民医院整形外科,上海200011
- 3. 苏州大学附属第一医院骨科,江苏苏州215006
- 折叠
摘要
目的 探讨隔离共培养条件下,软骨细胞诱导骨髓间充质干细胞(BMSCs)向软骨细胞分化并形成软骨组织的能力.方法 分别提取并培养兔BMSCs和耳软骨细胞,以5.0×107/ml的终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架上并置于Transwell室内.实验组Transwell下方为贴壁软骨细胞,对照组Transwell下方为DMEM培养液(含10％胎牛血清).两组BMSCs-支架复合物体外培养8周后植入裸鼠皮下,继续培养6周后取材.通过大体观察、逆转录-聚合酶链反应(RT-PCR)检测、组织学及免疫组织化学等相关检测对新生组织进行评价.结果 体外培养期间,实验组和对照组BMSCs在支架上黏附良好并能分泌细胞外基质,RT-PCR检测示实验组Ⅱ型胶原和蛋白聚糖有较强的表达,而对照组两者的表达较低或检测不到.裸鼠皮下培养6周,实验组BMSCs-支架复合物能基本保持原形,组织学染色及免疫组织染色显示新生组织有明显的软骨陷窝形成并表达软骨特异性细胞外基质.对照组BMSCs-支架复合物逐渐皱缩变形,未见形成软骨样组织.结论 采用Transwell隔离共培养的方法,软骨细胞能够诱导BMSCs向软骨细胞分化.
Abstract
Objective To explore the feasibility of chondrogenesis by isolated co-culture of BMSCs and chondrocytes so as to confirm the hypothesis that chondrocytes can provide chondrogenic differentiation of BMSCs. Methods Rabbit BMSCs (5.0 × l07/ml) were seeded onto a polyglycolic acid/polyactic acid (PGA/PLA) scaffold. The cell-scaffold constructs were put into a transwell, under transwell were adherent chondrocytes, as experimental group. The control group had no adherent chondrocytes under transwell (containing 10% fetal calf serum). All specimens were harvested after in vitro culture for 8 weeks and implanted subcutaneously in the dorsum of athymic nude mice for 6 weeks. Gross observation, RT-PCR, histology and immunohistolochemistry were used to evaluate the results. Results In experimental and control groups,the cells adhered to the scaffold well and produced abundant extracellular matrices after one week of in vitro culture. RT-PCR shows type Ⅱ collagen and aggrecan demonstrate stronger expression in experimental group, while that express little in control group after 8 weeks of in vitro culture, which suggest BMSCs in experimental group underwent chondrogenic differentiated stage. After 6 weeks of subcutaneous implantation into nude mice, specimen in experimental group could form cartilage-like tissue with typical cartilage lacuna, and maintain the original size and shape. The cell-scaffold constructs in control group shrunk gradually and could not form cartilage. The results were further supported by histological feature and immunohistochemistry. Conclusion Chondrocytes can promote the chondro-genesis of BMSCs by isolated coculture.
关键词
软骨细胞/骨髓间充质干细胞/共培养Key words
chondrocytes/BMSCs/co-culture引用本文复制引用
基金项目
常州市卫生局重大课题资助项目(2D201008)
出版年
2012
NETL
NSTL
维普
万方数据