Study Of Vascular Cells Cocultured On Regenerated Silk Fibroin

Most cardiovascular diseases, such as thrombosis, atherosclerosis and vascular aging, need to replace vascular. Vascular substitutes, which has been used in clinical, are mainly of large diameter. There was no ideal substitute of small-caliber vessels (inner diameter <6mm) for clinical application. Regenerated silk fibroin has good biocompatibility and silk fibroin fiber has good mechanical properties. Taken regenerated silk fibroin and silk fibroin fiber as materials, Small-caliber tubular scaffolds were fabricated. This tubular scaffold got three layers, similar to the structure of native vascular. The structure and mechanical properties of tubular scaffold has been optimized by changing preparation conditions (freezing temperature, the concentration of silk fibroin solution, the proportion of cross-linking). In order to deeply analyze the mechanism of tissue reconstruction after the replacement of defect vessel with silk fibroin tubular scaffold，we conducted a study on in vitro co-culture of vascular cells on the regenerated silk fibroin materials.In this paper, we took regenerated silk fibroin film and regenerated silk fibroin tubular scaffold as study object. With vascular cells co-cultured on silk fibroin film or tubular scaffold, we study the influence of materials and co-culture cells on cell morphology, cell proliferation and function factor expression of cell.Firstly, we studied the change of co-cultured vascular cells on regenerated silk fibroin film. Cell morphology and cell proliferation were investigated through micropictures and DNA content analysis. Results showed that:vascular cells cultured on silk fibroin membrane maintain normal morphology during cultivation. The number of vascular cells cultured alone continued to increase, while the toal number of co-cultured vascular cells also continued to increase. And there was no significant difference between the two groups.Immunofluorescence analysis showed that:ECs and SMCs can grow together under co-culture condition and the number of ECs was more than SMCs",Similarly, HAFs and SMCs can grow together under co-culture condition and the number of HAFs was more than SMCs", Silk fibroin membrane do inhibit the expression of a-SMA in SMC; Co-culture with ECs can promote the expression of a-SMA in SMC; Silk fibroin membrane do inhibit the expression of CD31 in SMC.The impact of silk fibroin materials and interaction betweem vascular cells on the expression level of vascular cells, function factor was analysised through real-time fluorescence quantitative(RT-PCR). Results showed that:silk fibroin film had inhibitory effect on the expression of the functional factor of vascular smooth muscle cells (α-SMA and SM-MHC); vascular endothelial cells can promote the expression of the functional factor of vascular smooth muscle cells (α-SMA and SM-MHC); Silk fibroin film and vascular smooth muscle cells can promote the expression of the function factor of vascular endothelial cell (VEGF and CD31); vascular fibroblasts can promote the expression of the function factor of vascular smooth muscle cells (α-SMA and SM-MHC).Silk fibroin tubular scaffolds got different spatial structure with silk fibroin film. So we further study the effect of silk fibroin tubular scaffold and co-cultured vascular cells on vascular cells. The results showed that:Silk fibroin tubular scaffolds can support the adhesion, spreading and proliferation of vascular smooth muscle cells and vascular endothelial cell. The real-time fluorescence quantitative analysis showed that:with vascular endothelial cells co-cultured on scaffold can promote the expression of the functional factor of vascular smooth muscle cells (α-SMA and SM-MHC); with vascular smooth muscle cells co-cultured on scaffold can promote the expression of the function factor of vascular endothelial cell (VEGF and CD31); with vascular fibroblasts co-cultured on scaffold can promote the expression of the function factor of vascular smooth muscle cells (α-SMA and SM-MHC).