人骨髓间充质干细胞复合两种填充材料体外生物相容性的比较

摘要

目的:对比膨体聚四氟乙烯(expanded polytetrafluoroethylene,ePTFE)与硅胶的体外生物相容性,为临床上ePTFE的广泛应用提供实验依据.方法:采用密度梯度离心法分离培养人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs),取第3代细胞通过流式检测、茜素红和油红O染色进行干细胞鉴定,将hMSCs分别与ePTFE和硅胶材料直接作用或与材料浸提液复合培养,观察细胞黏附、生长及增殖情况,计算细胞黏附率,CCK-8比色法检测细胞增殖.结果:成功分离培养hMSCs,形态上呈长梭形、纺锤状、类成纤维细胞样;流式检测结果显示第3代hMSCs高表达间充质干细胞表面标志物CD44(99.5%)、CD90(95.8%),低表达造血系细胞表面标志物CD45;且茜素红和油红O染色阳性;ePTFE组细胞黏附率(92.3%)明显高于硅胶组细胞黏附率(76.8%)(P<0.05);若两种材料直接作用培养后的细胞,随时间延长ePTFE组和空白对照组细胞活性逐渐增加,仍可保持正常的分裂增殖速度(P>0.05),而硅胶组活细胞吸光值明显低于ePTFE组(P<0.05).结论:ePTFE体外生物相容性优于假体硅胶,较适合作为支架材料应用于组织工程的构建.%Objective To compare the biocompatibility in vitro of expanded poly- tetrafluoroethylene(ePTFE) and silicone, which provides a reliable experimental basis for clinical application. Methods hMSCs were isolated by the methods of density gradient centrifugation. Flow cytometry, Alizarin red staining and Oil red O staining were used for identification of the 3rd passage of hMSCs.The cells were co- cultured with two filler materials directly or extracting solution respectively. The morphology and function of cells adhered to the scaffold were observed by inverted microscope. Cell adhesive rate and proliferation were also calculated by CCK-8 assay. Results We successfully isolated hMSCs. Morphologically, hMSCs had both long spindle-shaped and fibroblast-like morphology. Flow cytometry showed that the 3rd passage of hMSCs had high expressions of mesenchymal markers CD44 (99.5%) and CD90 (95.8%) and a low expression of hematopoietic cell surface markers CD45. And Alizarin red staining and Oil red O staining showed strong positive. The cell adhesive rate of the ePTFE group (92.3%)is significantly higher than that of the silicone group (76.8%)(P<0.05). When two filler materials directly acted on hMSCs, the ePTFE group revealed normal splitting proliferation rate as same as the control group(P>0.05), however OD value of the silicone group was less than that of the ePTFE group (P<0.05). Conclusion ePTFE has better biocompatibility than silicone and can be used as a vehicle for tissue engineering.