骨髓间充质干细胞复合纤维蛋白封闭剂体内构建可注射性组织工程软骨

目的 探讨利用人骨髓间充质干细胞（marrow mesenchymal stem cells，MSCs）与可注射性纤维蛋白封闭剂（fibrinsealant，FS）复合，在裸鼠体内构建组织工程软骨的可行性。方法 体外分离扩增健康人MSCs，以含有转化生长因子β1（transforming growth factor β1，TGF—β1）、地塞米松、维生素C的培养基进行成软骨诱导，诱导第7、14天分别检测软骨细胞特异的生物学特性。将诱导7d的MSCs与FS复合，接种于裸鼠背部皮下作为实验组，同时设单纯只注射FS或MSCs的支架对照组和细胞对照组。分别于接种后6、12周取材进行大体观察，行HE、阿尔新蓝染色和Ⅱ型胶原免疫组织化学染色评价其成软骨能力。结果 MSCs以特定的培养基诱导后由纺锤形变为多角形，并表达软骨细胞分泌的基质。复合物接种6和12周后，实验组均可形成软骨样组织块，6周时形成的组织块较小而质地柔韧，陷窝清楚，可检测到阳性阿尔新蓝及Ⅱ型胶原表达；12周形成的组织块较大，质地较硬，表面光滑，软骨细胞位于成熟的陷窝中，阿尔新蓝及Ⅱ型胶原免疫组化阳性染色较6周增强。两个对照组均无软骨样组织块形成。结论 MSCs复合FS可以作为一种较优良的可注射性组织工程软骨的构建方法。

CONDUCTION OF INJECTABLE CARTILAGE USING FIBRIN SEALANT AND HUMAN BONE MARROW MESENCHYMAL STEM CELLS IN VIVO

OBJECTIVE: To investigate the feasibility of the complex of the fibrin sealant (FS) and the bone marrow mesenchymal stem cells(MSCs) to create a new cartilage in the nude mice by the issue engineering technique. METHODS: The MSCs were isolated from healthy humans and were expanded in vitro. And then the MSCs were induced by the defined medium containing the transforming growth factor beta1 (TGF-beta1), dexamethasone, and ascorbic acid. The biomechanical properties of the chondrocytes were investigated at 7 and 14 days. The MSCs induced for 7 days were collected and mixed with FS. Then, the FS-MSCs mixture was injected by a needle into the dorsum of the nude mice in the experimental group. In the two control groups, only FS or MSCs were injected respectively. The specimens were harvested at 6 and 12 weeks,and the ability of chondrogenesis in vivo was investigated by the gross observation, HE, Alcian Blue staining, and type II collagen immunohistochemistry. RESULTS: The MSCs changed from a spindle-like fibroblastic appearance to a polygonal shape when transferred to the defined medium, and could be induced to express the chondrocyte matrix. After an injection of the mixture, the cartilage-like tissue mass was formed, and the specimens were harvested from the mass at 6 and 12 weeks in the experimental group. The tissue mass at 6 weeks was smaller and relatively firm in texture, which had a distinct lacuna structure. And glycosaminoglycan (GAG) and Type II Collagen expressions were detected. The tissue mass at 12 weeks was bigger, firmer and glossier with the mature chondrocytes lying in the lacuna structure. The positive Alcian blue and Collagen II immunohistochemistry stainings were stronger at 12 weeks than at 6 weeks. But there was no cartilage-like tissue mass formed in the two control groups. CONCLUSION: This study demonstrates that the fibrin sealant and the bone marrow mesenchymal stem cells can be successfully used in a constructing technique for the tissue engineered injectable cartilage.