【摘要】 采用初生山羊羔股骨骨膜,通过植块培养法、完全消化法和半消化植块法分离、培养成骨细胞。结果表明,植块培养法、完全消化法和半消化植块培养法的贴壁时间分别为4、24、4 h;培养4 d时的细胞数量分别为少量细胞、少量细胞、大量细胞,细胞汇合时间分别为21、19、10 d。传代后第7 d即可汇合,第14 d出现矿化结节,茜素红染色呈橘红色,Von Kossa染色呈黑色;姬姆萨和HE染色后可见体外培养的细胞呈三角形、多角形,胞质丰富,胞核卵圆形,单核,有1~3 个核仁;碱性磷酸酶染色后胞质呈黑色颗粒或块状沉淀;扫描电镜观察胞突发达,伸出的突起与临近细胞的突起相连。体外培养传至15代时细胞的生物学性状未发生变化。试验结果显示,半消化植块培养周期短,细胞损害小,有利于细胞贴壁,是一种可行的骨组织工程研究方法。更多还原

【Abstract】 The caprine osteoblasts of fetus were obtained from long bone periosteum, and cultured in DMEM at 37℃ with different bone membrane explants, explants with complete enzyme digestion and incomplete digestion,respectively. The results indicated that adhesion time, cell numbers and confluence time were 4h, 24h, 4h, respectively; few, few, large number of cells; and 21d, 19d,10d, respectively. The merging osteoblasts were found from passage culture on day 7, and there were apparent mineralized nodules on day 14. Von kossa and Alizarin red staining showed positive with jacinth and black respectively. Triangular, polyangular cells and obvious cell process were observed. While the cells have large nuclei and 1-3 nucleoli by Giemsa and H.E. staining. There were black deposits through Alkaline phosphatase(ALP) staining. Rough cell process was observed, which join and overlap cells under scanning electron microscope. Compared with primary culture, no bio-characteristic changes were observed from the 15th (filial) cells. It was concluded that incomplete digestion has less harmful to osteoblasts and is a kind of feasible method for bone tissue engineer, and the osteoblasts have good ability of attachment, fairly short culture period.更多还原