犬骨髓基质干细胞向成骨细胞诱导分化及细胞片层的制备

Study of induced differentiation of bone marrow stromal stem cells into osteoblasts and preparation of cell sheets in dogs

目的分离培养犬骨髓基质干细胞(bone marrow stromal cells,BMSCs)并向成骨细胞诱导分化,制备骨髓基质干细胞的细胞片层。方法密度梯度离心法分离犬骨髓基质干细胞,接种于DMEM成骨诱导培养基,向成骨细胞诱导分化,利用温度反应性培养皿的温度感应性,制备细胞片层。观察犬BMSCs、成骨细胞诱导分化及细胞片层形态学特点与生物学特性。结果分离培养出犬BMSCs,成功地向成骨细胞实现诱导分化,并观察到钙结节。利用温度反应性培养皿的温度感应性,收获了完整的细胞片层。结论细胞片层技术与传统的骨组织工程方法相结合,将有望构建出含板层骨结构的大块组织工程骨。

Objective To isolate,culture and induce dog bone marrow stromal cells into osteoblasts in vitro,and to prepare cell sheets with induce-differentiated bone marrow stromal cells.Methods Bone marrow stromal cells were seeded in DMEM medium after isolated by density gradient centrifugation,and induced differentiation into osteoblasts,temperature-responsive culture dish was used to prepare cell sheets.Morphological features and biological properties of osteoblasts and cell sheets were observed by assay of methyl thiazolyl tetrazolium（MTT）,in-verted phase contrast microscope,alkaline phosphorase（ALP） stain and stain of calcified nodules.Results Dog bone marrow stromal cells were isolated and cultured,and were successfully induced into osteoblasts;calcium nodules was also observed.Complete cell sheets were harvested with temperature-responsive culture dishes.Conclusion It is expected to build larger tissue-engineered bone which has cortical bone structure by traditional methods of bone tissue engineering combing with cell-sheet engineering approach.