Effects of RGDS sequence genetically interfused in the silk fibroin light chain protein on chondrocyte adhesion and cartilage synthesis.
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.
Kambe
Yusuke
Y
Department of Mechanical Engineering, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
Yamamoto
Koji
K
Kojima
Katsura
K
Tamada
Yasushi
Y
Tomita
Naohide
N
D000199:Actins / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D012831:Bombyx; D002356:Cartilage / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects*; D005349:Fibroins / Q000009:adverse effects / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D008855:Microscopy, Electron, Scanning; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011817:Rabbits; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D009842:Oligopeptides; D016596:Vinculin; C047981:arginyl-glycyl-aspartic acid; D005349:Fibroins; C046617:arginyl-glycyl-aspartyl-serine