Study On The Biocompatibility Of The Recombinant Spider Silk Protein Scaffolds

The recombinant spider silk protein as a new biomaterial had caused wide attention in tissue engineering. In our previous work, a new plasmid pNSR-32 containing RGD cell adhesion sites was constructed to express the higher molecular weight recombinant spider silk protein by E. coli BL21(DE3)pLysS. We optimized the purification conditions of the recombinant spider silk protein in this study. The conditions are ultrasound broken cells for 10min, heating for 10min at 80℃, adjusted to pH 5.5, sulfates saturation at 15%. The final purity of protein was above 85% and yield 1.5～2.5mg/g wet cells.The recombinant spider silk protein were made the scaffolds and evaluated the cytocompatibility. MTT assay was used to investigate the scaffolds cytotoxicity. The effects of cell growing, adhesion and differentiation on the scaffolds were discussed by culturing NIH-3T3 cells on scaffolds. The MTT experimental results were shown that the toxic rank of scaffolds below 1 level, were the qualified biomaterials. The SEM and HE staining showed the scaffolds with RGD had the better cell adhesion Cells can grow in the scaffolds and form the multi-layered cells on the surface of scaffolds. Immunohistochemistry results showed the cells on the scaffolds can proliferate and secret bFGF.By the back depth scalded of rat model, we studied the depth scald wound healing using scaffolds with NIH-3T3 cells. When the scald wound was repaired with pNSR-16 or pNSR-32 protein scaffold-NIH-3T3 cell complexes,the hydroxyproline contents of these two groups were more than others. HE staining showed that the scald wound repair of these two groups were more active than others in the initial wound healing. Immunohistochemistry showed that bFGF had the high expression on cell reproduction active areas during the scald wound healing. The bFGF expression of these two groups were higher than others. The preliminary results indicated that the recombinant spider silk protein scaffold-cell complexes group were better than simple material group and the control group. It had effect on promoting the deep scald wound healing.