Preparation And Characterization Of Three-dimensional Scaffold Containing Antheraea Pernyi Silk Fibroin Nano/Submicron Fibers

When silk fibroin porous materials are used as dermal regeneration scaffolds, the problem that how to promote the formation of capillaries and microvascular network in the silk fibroin scaffold, and improve the survival rate after silk fibroin porous materials transplanted into the dermal wound need to be further solved. By the contact guidance effect of nano/submicron fibers on cells, scaffold nano/submicron wall of hole similar to natural extracellular matrix and connected pore structure can be endowed, physical stimulation signal and microenvironment for the adhesion, migration and growth of vascular endothelial cells can be provided. It is a vaible way to increase the speed of Antheraea pernyi engineering scaffold vascularization. In this article, with hexafluoroisopropanol as a solvent, high-voltage electrostatic spinning technology was used to prepare Antheraea pernyi silk fibroin nano/submicron fibers. Then, freeze-drying was used to prepare silk fibroin scaffolds containing nano/submicron Antheraea pernyi silk fibroin fibers. The scaffold contained natural RGD tripeptide sequence of Antheraea pernyi silk fibroin and carried chemical signals which can stimulate the adhesion and growth of vascular endothelial cells. And there were nano/submicron fibers in the wall of hole, better connectivity between the holes, physical signals which can stimulate the adhesion and growth of vascular endothelial cells were carried. We studied the influences of the content and length of Antheraea pernyi silk fibroin nanometer/submicron fibers on the structure of scaffolds, and the influence of the content of fibers on growth of cells within three-dimensional scaffolds by culturing human umbilical vein endothelial cells EA.hy926in vitro.First, the influences of the spinning solution concentration, electrostatic voltage and receiving distance on the diameter of the Antheraea pernyi silk fibroin nano/submicron fibers prepared by high-voltage electrostatic spinning with hexafluoroisopropanol as a solvent were studied. The results showed that when the solution injection speed, needle size, electrostatic voltage and receive distance were fixed to constant values respectively, the diameter of the electrostatic spinning Antheraea pernyi silk fibroin fibers increased at7-9%of the spinning solution concentration. On the condition that the solution injection speed, needle size, spinning solution concentration and receive distance remained unchanged, the average diameter of the electrospun fibers with a electrostatic voltage of12kV is smaller than that of8kV or16kV. However, when the solution injection speed, needle size, electrostatic voltage and spinning solution concentration were all fixed, the receive distances were set to7,10and13cm respectively, the result indicated that with the increase of the receive distance, the average diameter of Antheraea pernyi silk fibroin fibers became larger.Then, casting method and freeze-drying were used to prepare silk fibroin films and porous silk fibroin materials containing nano/submicron Antheraea pernyi silk fibroin fibers. The influence of the content and length of nano/submicron Antheraea pernyi silk fibroin fibers on the structure and properties of silk fibroin films and porous silk fibroin materials were studied. The results showed that:when the content of Antheraea pernyi silk fibers within the silk fibroin films changed from3to20percent, the tensile breaking strength and Young’s modulus of the silk fibroin films increased. And when the content of Antheraea pernyi silk fibers was more than20percent, the strength, elongation, and Young’s modulus of the silk fibroin films decreased. The length of Antheraea pernyi silk fibroin fibers had no significant influences on the strength, elongation, and Young’s modulus of the silk fibroin films. When the content of Antheraea pernyi silk fibroin fibers within porous silk fibroin materials was more than50%, the number of microporous on the wall of hole was significantly increased, and the connectivity between holes was significantly improved. When the content of Antheraea pernyi silk fibroin fibers within porous silk fibroin materials was more than100%, part of walls of holes were full composed of Antheraea pernyi silk fibroin fibers, the number of microporous on the wall of hole became more, and the connectivity between holes had a more significant improvement. The length of Antheraea pernyi silk fibroin fibers within porous silk fibroin materials had no significant influence on the structure of pore. Crosslinked by polyethylene glycol diglycidyl ether, the aggregation structure of silk fibroin films and porous silk fibroin materials changed from amorphous Silk Ⅰ to Silk Ⅱ structure. When the content of fibroin fibers was5%, there had no aggregation structure change which can be detected on silk fibroin films and porous silk fibroin materials.Finally, by culturing cells in vitro, we studied the influences of three-dimensional scaffold whose Antheraea pernyi silk fibroin fibers content was0,50,150,250%on the adhesion and growth of EA.hy926cells. Confocal laser and alma blue law were used to test and analyse the cytocompatibility of the three-dimensional scaffold containing Antheraea pernyi silk fibroin fibers. The results showed that EA.hy926cells on the three-dimensional scaffold containing Antheraea pernyi silk fibroin fibers can adhere, proliferate and growth, and the cell number increased with the prolongation of the culturing time. EA.hy926cells had a stronger capacity to proliferate than others, when the content of Antheraea pernyi silk fibroin fibers was150%.In this article, silk fibroin films and porous silk fibroin materials containing nano/submicron Antheraea pernyi silk fibroin fibers were prepared by casting method and freeze-drying, respectively. The influences of the content and length of nano/submicron Antheraea pernyi silk fibroin fibers on the structure and properties of silk fibroin films and porous silk fibroin materials were studied. The effects of the three-dimensional scaffold containing nano/submicron Antheraea pernyi silk fibroin fibers on adhesion and proliferation of EA.hy926cells were investigated by culturing cells in vitro. The three-dimensional scaffold containing nano/submicron Antheraea pernyi silk fibroin fibers can promote the adhesion, proliferation and growth of EA.hy926cells, it can be used as a new regeneration scaffold to promote the vascularization during dermal regeneration re-epithelialization process.