犬骨髓基质细胞和蚕丝丝素的体外生物相容性

Biocompatibility of silk fibroin with dog bone marrow stromal cells in vitro

目的探讨体外培养的犬骨髓基质细胞(BMSCs)与蚕丝丝素材料的生物相容性,寻找BMSCs组织工程化神经的支架材料。方法通过差速贴壁法体外分离、培养犬骨髓基质细胞,与丝素共培养后,通过光镜(经免疫荧光染色)、扫描电镜观察细胞在丝素上黏附和生长情况。利用丝素浸出液培养BMSCs后,通过透射电镜观察细胞内部超微结构,用四甲基偶氮唑盐(MTT)法检测丝素、羟基磷灰石、有机锡浸出液和普通IMDM完全培养基培养细胞12、24、48、72h和7d的细胞活力,每组重复12次。流式细胞术检测丝素浸出液培养BMSCs的细胞周期及表型,实验重复3次。结果通过光镜、扫描电镜观察,发现BMSCs紧紧黏附于丝素材料,并沿着丝素纤维延伸,黏附于丝素纤维的细胞呈圆形、椭圆形及呈梭形。与普通IMDM完全培养基培养的细胞相比,透射电镜下可见丝素浸出液培养后的BMSCs内部结构未见异常;MTT检测丝素和羟基磷灰石浸出液对骨髓基质细胞的活力无显著性影响(P>0.05);流式细胞术检测丝素浸出液对骨髓基质细胞周期和表型无明显影响。结论蚕丝丝素材料与犬BMSCs生物相容性好,且未见丝素对BMSCs有毒性作用,可作为BMSCs组织工程化神经的支架材料。

Objective To investigate the biocompatibility of dog bone marrow stromal cells （BMSCs） in vitro with silk fibroin material and to explore the possibility of using silk fibroin as a novel material in the fabrication of tissue- engineered nerve scaffolds combined with the introduction of BMSCs. Methods Dog bone marrow stromal cells were isolated from other cells by adherence to plastic. The dog BMSCs were then cultured on the substrate silk fibroin fibers and the cell attachment and growth observed using immunofluorescence light microscopy and scanning electron microscopy. The dog BMSCs were also cultured in the silk fibroin extract fluid. The cell ultrastructure was observed by transmission electron microscopy. MTT test was used to detect cell viability in different medium after 12, 24, 48 and 72 hours or 7 days in culture （n = 12 for each group）. Flow cytometry was employed to detect BMSCs cell cycle and phenotypes （n = 3 ）. Results BMSCs were tightly attached to the silk fibroin fibers and grew along the silk fibroin as demonstrated by immunofluorescence light microscopy and scanning electron microscopy, some of which exhibited a spherical, oval or spindle shape. The results of transmission electron microscopy, MTT test and flow cytometry analysis showed that there were no significant morphological, cell viability, proliferation and phenotypes differences between BMSCs cultured in the silk fibroin extract fluid and those in plain IMDM medium. Conclusion These data indicate that silk fibroin has good biocompatibility with BMSCs and does not exert any significant cytotoxic effects on their phenotype, thus it is a potential scaffold material combined with the introduction of BMSCs in the fabrication of tissue-engineered nerve.