| Objective:Combination of biomaterials with different properties has been widely used to create new ideal tissue engineering scaffolds. In this study, we combined collagen(COL) which has superior biocompatibility with silk fibroin(SF) which has unique mechanical property and used electrospinning to develop composite micro-nanofibrous scaffolds. The scaffolds were stabilized by glutaraldehyde(GTA) vapor. Properties of the composite scaffolds were studied to evaluate their potential application in tissue engineering.Methods:1. Choose appropriate solvent and electrospinning conditions for COL, SF and their blends.The electrospun fibers were crosslinked by GTA vapor. And choose appropriate crosslinking time according to the water-resistant ability and deformation degree of the scaffolds.2. Taking1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) as solvent, the composite fibrous scaffolds were prepared according to the mass ratio of COL/SF as100:0,70:30,50:50,30:70,0:100, and then crosslinked by GTA vapor for12h. The physical and chemical properties of the scaffolds were characterized by combined techniques of scanning electron microscopy (SEM), Fourier-transform infrared spectra (FTIR), X-ray diffraction (XRD), thermal analysis,tensile measure-ments and contact angle test.3. The initial blood compatibility and cytocompatibility of composite fibrous scaffolds were investigated by hemolysis test and cytotoxicity. The cell adhesion and proliferation were evaluated by SEM and MTT test after inoculating the scaffolds with fibroblasts (3T3and L929). The vivo biocompatibility was observed by subdermal implantation of the scaffolds in SD rats. Results:1. HFIP was found as an appropriate solvent for electrospinning of COL, SF and their blends. It was found that the average diameter of the electrospun fibers became thick with increases of the concentration of solution and the feed rate, and became fine with increases of the voltage and the distance. The optimal electrospinning fibers were appeared in the condition of10%concentration of solution,voltage of16kV, feed rate of2ml/h and distance of12cm. The composite scaffold can be insoluble in water for at least5days after crosslinked by GTA vapor for12h.2. Under the optimal electrospinning condition, the average fiber diameter of the electrospun fibers was in the range of550-1100nm, and the average pore size of the scaffolds is about in the range of8-13μm. The fibers were stabilized by GTA vapor. After crosslinking, the p-sheet structure, crystallinity and thermal stability of the nanofibers were improved. This improvement was more obvious with the SF content increases. The mechanical properties of crosslinked nanofibers were better than those of uncrosslinked fibers. The best average ultimate tensile strength was highest when SF content was70%and the best ultimate tensile elongation was highest when SF content was50%.3. The fibroblasts(3T3and L929) grew and proliferated well on the surface of the composite fibrous scaffolds, which indicate good cytocompatibility of the scaffolds. The results of the subdermal implantation showed that the COL/SF composite scaffolds had good biocompatibility in vivo. Conclusions:To mimic natural ECM, COL/SF complex microfibers were obtained by electrospinning. GTA vapor was found to be useful for stabilizing the morphologies of electrospun COL/SF fibers, blending of SF and crosslinking of GTA vapor could improve the physical and chemical properties of COL microfibers. The results of cell behavior on scaffolds showed that fibroblast grew and proliferated well on the microfibers. These results strongly support that the COL/SF composite scaffolds, have good biocompatibility both in vitro and in vivo, may be a better candidate for biomedical applications and tissue engineering scaffolds. |
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