Initial chondrocyte-silk fibroin interactions are implicated in chondrogenesis when using fibroin as a scaffold for chondrocytes. Here, we focused on integrin-mediated cell-scaffold adhesion and prepared cell adhesive fibroin in which a tandem repeat of the Arg-Gly-Asp-Ser (RGDS) sequence was genetically interfused in the fibroin light chain (L-chain) (L-RGDSx2 fibroin). We investigated the effects of the sequence on chondrocyte adhesion and cartilage synthesis, in comparison to the effects of fibronectin. As the physicochemical surface properties (e.g., wettability and zeta potential) of the fibroin substrate were not affected by the modification, specific cell adhesion to the RGDS predominately changed the chondrocyte adhesive state. This suggestion was also supported by the competitive inhibition of chondrocyte attachment to the L-RGDSx2 fibroin substrate with soluble RGD peptides in the medium. Unlike fibronectin, the expression of RGDS in the fibroin L-chain had no effect on chondrocyte spreading area but enhanced mRNA expression levels of integrins alpha5 and beta1, and aggrecan at 12 h after seeding. Although both the sequence and fibronectin increased cell adhesive force, chondrocytes grown on the fibroin substrate exhibited a peak in the force with time in culture. These results suggested that moderate chondrocyte adhesion to fibroin induced by the RGDS sequence was able to maintain the chondrogenic phenotype and, from the histology findings, the sequence could facilitate chondrogenesis.

An effective surface grafting method for chemically inert and elaborately porous medical expanded-polytetrafluoroethylene (ePTFE) was developed. Although surface graft polymerization onto basic polymeric biomaterials has been widely studied, successful modification of the ePTFE surface has been lacking due to its high chemical resistance. Herein, we succeeded in surface graft polymerization onto ePTFE through glycidyl methacrylate (GMA) as a bridge linkage following argon (Ar) plasma treatment. The epoxy group of GMA was expected to react with the peroxide groups produced on ePTFE by Ar plasma exposure, and its methacrylic groups can copolymerize with various monomers. In the present study, we selected 2-methacryloyloxyethyl phosphorylcholine (MPC) as a model monomer and the blood compatibility of modified ePTFE was evaluated. Two sequences of surface grafting were compared. In a two-step graft polymerization, GMA was first immobilized onto Ar plasma treated ePTFE, and then MPC was polymerized. In a one-step graft copolymerization, MPC and GMA were mixed and copolymerized simultaneously onto Ar plasma treated ePTFE, resulting in a poly(MPC-co-GMA) (PMG) graft surface. The roughness of the node-and-fibril structure of ePTFE was reduced by the uniform polymer layer, and the modified ePTFE had a good hydrophilic nature even after being stored in an aqueous environment for 30 days. The indispensable GMA in graft polymerization improved the surface grafting on ePTFE. The one-step and two-step graft polymerization methods could decrease the number of adhered platelets, and almost inhibit platelet activation. We concluded that graft polymerization with the GMA linker provides a novel strategy to modify the chemically inert ePTFE surfaces for functionalizing as new medical devices.

Transgenic silkworm technology has enabled the biological properties of silk fibroin protein to be altered by fusion to recombinant bioactive proteins. However, few studies have reported the fabrication of genetically modified fibroin proteins into three-dimensional spongy structures to serve as scaffolds for tissue engineering. We generated a transgenic silkworm strain that produces fibroin fused to basic fibroblast growth factor (bFGF) and processed the fibroin into a spongy structure using a simple freeze/thaw method. NIH3T3 mouse embryonic fibroblasts grown on bFGF-fused fibroin sponges proliferated and spread out well, showing half the population doubling time of cells cultured on wild-type fibroin sponges. Furthermore, the number of primary rabbit articular chondrocytes growing on bFGF-fused fibroin sponges was around five-times higher than that of the wild-type control at 3-days post cell-seeding. As the physical properties of wild-type and bFGF-fused fibroin sponges were almost identical, it is suggested that bFGF fused to fibroin retained its biological activity, even after the bFGF-fused fibroin was fabricated into the spongy structure. The bFGF-fused fibroin sponge has the potential for widespread application in the field of tissue engineering, and the method of fabricating this structure could be applicable to other recombinant bioactive fibroin proteins.

Random collagen fiber networks formed by a slowly degrading silk fibroin hydrogel injection prevented left ventricular enlargement after myocardial infarction.