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J.Health Sci., 56(6), 738-744, 2010
-Rapid Communication-
Cell Shape and Matrix Production of Fibroblasts Cultured on Fibroin-organized Silk Scaffold with Type-II β-turn Structured (Ala-Gly-Ala-Gly-Ser-Gly)n Sequences
Michiko Hirata, Megumi Kobayashi,
Chiho Matsumoto, Chisato Miyaura,*
Tetsuo Asakura, and Masaki Inada
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei 184-8588, Japan
Tissue engineering for skin regeneration is closely related to advances in carrier materials for fibroblast. To investigate the optimal fibroblast scaffold, we developed a silk protein containing multiple (Ala-Gly-Ala-Gly-Ser-Gly)n sequences, and used it for in vitro evaluations of cell shape, adhesion and matrix production. NIH-3T3 fibroblasts were cultured on silk scaffold and on control culture plates. At 2 hr, fibroblasts cultured on silk scaffold showed round-rose shape, but cells spread on control plates. The numbers of adhesive fibroblasts were the same in both scaffolds, indicating that silk scaffold affects cell shape but not adhesive efficiency. To examine the effects of silk scaffold on cytoskeletal changes of fibroblasts, cells were applied to actin staining. Fibroblasts cultured for 2 hr on silk scaffold showed dense actin fibers which formed ring-like structures, while actin stress fibers were formed in cells cultured on control plates. In a long-term culture of 14 days, piled matrices produced by fibroblasts have been shown distinctly in cells cultured on silk scaffold compared with cells cultured on control plates. The expression of fibronectin mRNA was elevated in fibroblasts cultured on silk scaffold compared with control, but the expression of collagens, type I and type III, mRNAs was similar in fibroblasts cultured on both scaffold. These results indicate that silk scaffold influences cell shape and actin fiber, and enhances matrix production with increased fibronectin. Therefore, silk protein may be an useful scaffold for regenerative therapy in various skin wounds.
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