兔骨髓基质干细胞与软骨细胞混和培养体内成软骨(英文)

In vivo chondrogenesis by co-culture of rabbit bone marrow-derived mesenchymal stem cells and chondrocytes

背景：诱导因子及软骨微环境是影响骨髓基质干细胞成软骨分化及软骨形成的主要因素。目的：利用少量软骨细胞以共培养方式诱导骨髓基质干细胞体内成软骨。设计、时间及地点：2004-09/2005-03在解放军第四军医大学口腔医院病理教研室完成的随机对照动物实验。材料：选择清洁级新西兰兔15只用于细胞支架复合物的种植,随机分为混合细胞组、软骨细胞组、骨髓基质干细胞组,5只/组。取新生1-3d龄新西兰兔5只用于骨髓基质干细胞、软骨细胞的分离培养。聚羟基乙酸无纺网支架为上海易括公司产品,材料直径15μm,平均间距150-200μm,孔隙率97%,厚2mm。方法：混合细胞组将骨髓基质干细胞与软骨细胞按3：1混匀,调整细胞密度为6.0×10^10·L^-1,接种于经培养液预湿的塑形聚羟基乙酸5mm×5mm的支架上,然后在复合物周围滴加含胎牛血清的DMEM液培养1周。软骨细胞组、骨髓基质干细胞组的细胞密度调整为6.0×10^10·L^-1后同法接种于支架上。各组兔麻醉后于背部一侧皮下组织植入对应的细胞-支架复合物。主要观察指标：植入后第8周行新生软骨大体观察和苏木精-伊红、Masson三色染色。结果：各组细胞与聚羟基乙酸支架黏附情况良好。植入8周后,混和培养组和软骨细胞组均形成了成熟的软骨样组织,并基本保持复合物初始的大小和形状,两组新生软骨外观及组织学特征较接近。骨髓基质干细胞组在体内培养过程中未形成软骨组织,而是形成了纤维结缔组织。结论：骨髓基质干细胞与软骨细胞按3：1混合形成的微环境,能有效诱导骨髓基质干细胞向软骨细胞分化,并促进骨髓基质干细胞的体内成软骨。

BACKGROUND： Inducing factor and chondrogenic microenvironment is a primary factor, which influences chondrogenic differentiation and chondrogenesis of bone marrow-derived mesenchymal stem cells （MSC s）. OBJECTIVE： To explore the feasibility of in vivo chondrogenesis by co-culture of bone marrow-derived MSCs and chondrocytes. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed at Department of Pathology, Stomatological Hospital, Fourth Military Medical University of Chinese PLA between September 2004 and March 2005. MATERIALS： Fifteen New Zealand rabbits of clean grade were used for cell-scaffold construct transplantation. The rabbits were randomly divided into co-culture, chondrocyte, and bone marrow-derived MSC groups, with 5 rabbits in each group. Five neonatal New Zealand rabbits, aged 1-3 days, were used for isolation and culture of bone marrow-derived MSCs and chondrocytes. Polyglycolic acid （PGA） scaffold material （Shanghai Yikuo Company, China） has a fiber diameter of 15 μ m, with an average interval of 150-200 μ m, an interval porosity of 97% and 2-ram thickness. METHODS： In the co-culture group, bone marrow-derived MSCs and chondrocytes were mixed at a ratio of 3： 1. The mixed cells were seeded onto a pre-wetted PGA scaffold （5 mm × 5 mm ）at the ultimate concentration of 6.0×10^10· L^-1. Dulbecco＇s modified Eagle＇s medium （DMEM） supplemented with fetal bovine serum was dropwise added to peripheral compound for I week of culture In the chondrocyte, and bone marrow-derived MSC groups, chondrocytes and bone marrow-derived MSCs of the same ultimate concentration were seeded respectively onto the PGA scaffold. Then, the cell-scaffold constructs were transplanted into subcutaneous tissue of adult rabbits. MAIN OUTCOME MEASURES： Gross observation and hematoxylin-eosin ＆ Masson staining of neo-cartilage were performed after in vivo culture for 8 weeks. RESULTS： Cell in all groups had a fine adhesion to the scaffold. In both co-culture and chondrocyte groups, the cell-scaffold constructs could maintain the original size and shape during in vivo culture and formed homogenous mature cartilage after 8 weeks of in vivo culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features. In the bone marrow-derived MSCs group, connective tissue rather than cartilage was found during in vivo culture. CONCLUSION： Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of bone marrow-derived MSCs and thus promote the chondrogenesis of bone marrow-derived MSCs in vivo.