骨髓间质干细胞体外定向诱导分化为软骨细胞的实验研究

目的：探讨分离骨髓间充质干细胞（MSCs）并诱导其向软骨细胞转化的体外培养方法，为软骨组织工程的种子细胞来源提供实验依据。方法：抽取兔髂骨骨髓液，经梯度离心法和贴壁法进行体外培养，贴壁细胞传代，取第3代细胞在培养基中添加软骨分化诱导剂含转化生长因子（TGF-β2）10ng／ml、地塞米松10^7mol／L、维生素C50μmol／L，经7、14、21d诱导培养后，倒置显微镜观察细胞形态，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。将诱导细胞与软骨支架材料-聚磷酸钙纤维／左旋聚乳酸（CPP／PLLA）复合，1周后终止培养，扫描电镜观察细胞黏附情况。结果：诱导后细胞体外扩增能力显著降低，细胞形态由成纤维样梭形向多角形、多边形或类圆形转变，诱导21d后细胞形态变化最为显著，Ⅱ型胶原免疫组化染色深而均匀。诱导后的MSCs可在支架材料内良好黏附生长。结论：体外培养的MSCs可定向诱导分化为软骨细胞，分泌软骨细胞特异性基质，可用作软骨组织工程的种子细胞。

Inducing Bone Marrow-derived Mesenchymal Stem Cells Into Chondrocytes in Vitro

Objective： To explore the method of isolation, culture and chondrocytic phenotype differentiation of mesenchymal stem cells （MSCs） from the bone marrow of rats in vitro and to offer experimental reference for the resources of seeding cells in cartilage tissue engineering. Methods： MSCs were isolated from bone marrow and purified by density gradient centrifuge and cultured in vitro. The MSCs attachment formed and those in passage 3 were chosen to induce into chondrogenic differentiation. After 7, 14, 21 days, immunohistochemical techique was applied to detect the expression of collagen type Ⅱ. The differentiated cells were implanted on the CPP/PLLA composites. After the cell-scaffold complex was cultured in vitro for one week, the uhrastructure of the scaffold was observed with scanning electron microscopy. Results： The differentiated cells changed from a spindle-like fibroblastic appearance to a polygonal shape, the capability of proliferation was down markedly. Immunohistochemical staining of collagen Ⅱ were positive for the pass age, especially in the 21st days. Induced MSCs were well adherent to the scaffold composites and the cells were embedded by the cell-matrix. Conclusion： Under the induced medium, MSCs could differentiate into chondrogenic phenotype and secrete specificity matrix of cartilage in vitro. MSCs can likely be served as optimal cell source for cartilage tissue engineering.