Study On RGD-recombinant Spider Silk Fibroin And Antheraea Pernyi Silk Fibroin As Tissue Engineering Scaffolds

Owning to its excellent strength, elasticity, flexibility, elongation and biodegradability, spider silk has caused great attention in tissue engineering scaffold material field. RGD (Arg-Gly-Asp) tripeptide sequence, which can promote cell adhesion, is the specific ligand to the integrin on the cell surface. Our lab staff has set up a method to prepare recombinant spider silk by gene engineering and high density fermentation technique. Antheraea pernyi silk is produced by wild A. pernyi silkworm cocoon. It is a precious resource in China. However, it is noly used as a raw material of textile industry presently. Reports on its application in tissue engineering scaffold were scarce. With its RGD tripeptide sequence, to be richly endowed by nature, A. pernyi silk fibroin has superiority in tissue engineering scaffold material field. A. pernyi silk fibroin can be knitted to a tendon-shaped braid, and it can also endure the elongation stress of the body with its outstanding mechanical properties. Therefore, the application of A. pernyi silk fibroin as tissue engineering tendon scaffold has a widely perspective.In this research, porous complex scaffolds of the two RGD-containing proteins (recombinant spider silk fibroin pNSR-16 and A. pernyi silk fibroin) were prepared. As a first, we obtained recombinant spider silk fibroin complex nanofiber scafflod by electrospinning. Application of Fourie transform infrared spectroscopy (FTIR) and mechanical property determination, the influencing factors to the protein molecular conformation and mechanical properties of the complex scaffold were discussed. Results showed that Formyl Acid promote the transformation of pNSR-16 to random coil, while LiBr, heating, denaturation and macromolecule material (PVA and CS) promte the transformation toβ-fold. It was also found that micromolecule elasticizer and PVA with high molecular weight promote the transformation toβ-fold of A. pernyi silk fibroin, while raising heating temperature, crosslinking and PEG destroy theβ-fold conformation of A. pernyi silk fibroin. According to the differences of mechanical properties between different materials, it can be concluded that PVA with high molecular weight, elasticizer glycerol, chemical crosslinking and denaturation by 75% ethyl alocohol enhance the mechanical properties of A. pernyi silk fibroin complex material. Cyto-compatibility of the porous complex scaffold of the two RGD-containing proteins was investigated by cytotoxicity determination of the extract of the scaffolds and preparation of Cell-scaffold constructs. According to the evaluation criterion in ISO10993, cytotoxicity of the extract of the two scaffolds is of ranking I and qualified. Results showed that both the scaffolds promote the adhesion and propagation of NIH3T3, namely, both the scaffolds have good cyto-compatibility.The feasibility of A. pernyi silk fibroin to be used as tissue engineering tendon scaffold was investigated in vitro and in vivo, respectively, utilizing tenocytes and tendon injury rabbit model. In vitro results showed that A. pernyi silk fibroin promote the adhesion and propagation of the tenocytes. Results on macrographic examination, histological examination, immunohistochemical examination, scanning electron micrograph examination and biomechanical determination reveled that A. pernyi silk fibroin promote the recovery of the injury tendon of the rabbit in vivo. Prelimanary, we conclude that the application of A. pernyi silk fibroin as tissue engineering tendon scaffold is feasible.