人骨髓间充质干细胞接种纳米晶胶原骨构建组织工程骨

Tissue-enginered bone constructrd by seeding human messenchymal stem cells on nano-hydroxyapatite/collagen

目的观察体外培养的人骨髓间充质干细胞(mesenchymal stem cells,MSCs)与纳米晶羟基磷灰石/胶原骨(nano-hydroxyapatite/collagen,nHAC)的生物相容性及细胞在nHAC上的生长情况。方法全骨髓法体外培养骨髓间充质干细胞,应用成骨诱导剂诱导向成骨细胞表型转化,通过细胞活性、免疫组化鉴定诱导培养的成骨细胞的细胞学特性。通过倒置显微镜、扫描电镜观察细胞生长及其在nHAC上的生长情况。结果原代培养的骨髓细胞增殖迅速,10～12d左右即可稳定传代,传代细胞7～9d即可传代。经诱导培养的细胞的ALP染色阳性,Von Kossa染色阳性,可见钙化的基质沉积,呈现典型的成骨细胞形态和生物学特征。构建的MSCs与nHAC共培养的模型中,细胞可在nHAC表面良好贴壁。复合培养8天,分布于支架材料上的细胞大量增殖、分泌细胞外基质。第14天,大量细胞在材料表面和孔隙中生长。细胞之间广泛存在突起连接。结论nHAC适合种子细胞的贴附、生长和增殖,是组织工程良好的载体材料。

Objective To observe the biocompatibility ofmesenchymal stem cells cultured in vitro with nano-hydroxyapatite/collagen（nHAC） and its growth condition, To established a model of human mesenchymal stem cells differentiated into osteoblast together with nHAC in vitro with tissue-engineering techniques, To explore the best method of integrating MSCs into NHAC, Methods The adherent cells were selected as marrow stromal cells when marrow suspensions were cultured, and the growth curve was drawn accordingly, They were induced and differentiated into the phenotype of osteoblast by the revulsant, and the cytological characteristics of the osteoblast were identified through cells activity and immunohistochemistry method, The growth condition with or without nHAC was evaluated through inverted microscope and electronic scanning microscope respectively. Results The cultured marrow stromal cells proliferated into uniform fibroblast-like cells rapidly, MSCs reached confluence and started to form multilayers averaging from 10 to 12 days, passaged stably as well, Then the MSCs passaged from 7 to 9 days. Cytochemistry evaluation showed that MSCs in induced culture were positive for ALP and Von Kossa stain, and deposited calcified matrix. It showed a typical osteoblast feature in morphology and biology, In coculture model of bone marrow mesenchymal stem cells together with nHAC, MSCs would attach to the inner surface of nHAC after combination, 8 days after coculture, the osteoblasts proliferated in the nanometer crystal hydroxyapatite collagen material network, We saw the secretion of the matrix, Lots of cells adhered on the surface of nano-collagen basal bone and into its inside pores. There were extensive prominent connections among cells, and collagens were secreted in 14th day, Conclusion nHAC is easy to combine with bone marrow derived mesenchymal stem cells, and it was suitable for cells to adhere, grow and proliferate, and verified that the nano-collagen basal bone was a good carrier material for tissue engineering.