蛛丝蛋白/聚己内酯/壳聚糖复合纳米纤维支架与内皮细胞的相容性

Endothelial Cell Cytocompatibility of Spidroin/Polycaprolactone/Chitosan Composite Nanofiber Scaffolds

将RGD-重组蛛丝蛋白(pNSR32)、聚己内酯(PCL)和壳聚糖(CS)共混,应用静电纺丝技术制备复合纳米纤维支架(pNSR32/PCL/CS),进行细胞相容性的初步研究。选择体外细胞培养法,以内皮细胞为种子细胞,应用MTT比色法评价材料浸提液细胞毒性及材料对内皮细胞粘附、生长及增殖的影响,细胞免疫荧光法检测与支架复合培养的细胞表型表达。结果表明,支架对内皮细胞形态无明显影响,MTT细胞毒性试验显示细胞增殖率大于100%,毒级为0级,材料对细胞无明显毒性。粘附增殖试验结果显示pNSR32/PCL/CS的细胞粘附率大于100%,粘附以及增殖状况与阳性对照组(载玻片)相比差异显著(P<0.05),该材料能够很好地支持内皮细胞粘附、增殖。添加pNSR32的材料细胞增殖活力明显优于未添加pNSR32的材料(P<0.05),添加CS的材料3、5、7 d细胞增殖活力优于未添加CS的材料(P<0.05)。细胞免疫荧光结果表明,与pNSR32/PCL/CS支架复合培养的内皮细胞vWF、CD31因子呈阳性表达,表型特征保持良好。添加pNSR32及CS能够促进内皮细胞在材料表面的增殖作用。pNSR32/PCL/CS复合纳米纤维支架细胞相容性良好,适合内皮细胞粘附生长,有望成为一种新型的组织工程小直径血管支架。

This paper is aimed to evaluate the cytocompatibility of pNSR32/PCL/CS nanocomposites.Cytotoxity of extracts and effects of nanocomposites on endothelial cell adhesion,growth and proliferation were evaluated by MTT assay.Immunofluorescent staining was applied to observe the phenotype of cells seeded on scaffolds.The pNSR32/PCL/CS nanocomposites had not obvious effects on cell morphology.The average cell proliferation rate determined by MTT cytotoxity assay was over 100%,indicating the cytotoxity grade was non-toxic.Test of adhesion and proliferation displayed that the adhesion rate was over 100%,and there was statistical significance between the positive group（coverslip） and pNSR32/PCL/CS group（P0.05）,which indicated that the composite nanofibers supported adhesion and proliferation of the endothelium cells.The cell proliferation on the materials containing pNSR32 was better than that without pNSR32;and at 3,5,7 days the cell proliferation on the materials containing CS was better than that without CS（P0.05）.Immunofluorescent staining manifested that the endothelium cells seeded on pNSR32/PCL/CS scaffolds expressed vWF and CD31,with good phenotype characteristics.The cell proliferation on materials can be promoted by addition of pNSR32 and CS.The pNSR32/PCL/CS nanocomposites have good cytocompatibility,and it is a safe and prospective scaffold in vascular tissue engineering.