cell shape and and matrix production of

738Journal of Health Science,56(6)738–744(2010)

—Rapid Communication—Cell Shape and Matrix Production of Fibroblasts Cultured on

Fibroin-organized Silk Scaffold with Type-IIβ-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,2–24–16Nakamachi,Koganei184–8588,Japan

(Received April28,2010;Accepted August5,2010;Published online August13,2010)

Tissue engineering for skin regeneration is closely related to advances in carrier materials forfibrob-last.To investigate the optimalfibroblast 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-3T3fibroblasts were cultured on silk scaffold and on control culture plates.At2hr,fibroblasts cultured on silk scaffold showed round-rose shape,but cells spread on control plates.The numbers of adhesivefibroblasts were the same in both scaffolds,indicating that silk scaffold affects cell shape but not adhesive efficiency.To examine the ef-fects of silk scaffold on cytoskeletal changes offibrob-lasts,cells were applied to actin staining.Fibroblasts cultured for2hr on silk scaffold showed dense actin fibers which formed ring-like structures,while actin stressfibers were formed in cells cultured on control plates.In a long-term culture of14days,piled ma-trices produced byfibroblasts have been shown dis-tinctly in cells cultured on silk scaffold compared with cells cultured on control plates.The expression offi-bronectin mRNA was elevated infibroblasts cultured on silk scaffold compared with control,but the ex-pression of collagens,type I and type III,mRNAs was similar infibroblasts cultured on both scaffold.These results indicate that silk scaffold influences cell shape and actinfiber,and enhances matrix production with increasedfibronectin.Therefore,silk protein may be an useful scaffold for regenerative therapy in various ∗To whom correspondence should be addressed:Depart-ment of Biotechnology and Life Science,Tokyo University of Agriculture and Technology,2–24–16Nakamachi,Ko-ganei184–8588,Japan.Tel.&Fax:+81-42-388-7390;E-mail: miyaura@cc.tuat.ac.jp skin wounds.

Key words——cell scaffold,cell shape,matrix produc-tion,silkfibroin,skinfibroblast,tissue engineering

INTRODUCTION

Dermatological treatments for skin injury and defects need trials to develop graft materials us-ing several biosynthetic approaches.1)The autograft is the widely used method to heal certain wounds, however,the limitation of the autologous donor skin area is a crucial subject in terms of constant treatment.To establish a growth technique for autologous cells,supportive carrier materials have been widely investigated in thefield of tissue engi-neering.Natural polymers,including collagen and hyaluronan,generally form the solid-state scaffold applied to tissue engineering.2)

Silkfibroin,a natural polymer,forms a solid scaffold,and can be applied to tissue engineering because of itsflexibility for molding.Silkfibroin has been defined as an amino acid sequence contain-ing repeated motifs(Gly-Ala-Gly-Ala-Gly-Ser)n in the protein.3–5)Silkfibroin produced by Bombyx mori(B.mori),a silkworm,consists of two forms offibroin,named silk I(silk structure before spin-ning)and silk II(silk structure after spinning).6)We have found that the crystal structure of silk I con-sists of a repeatedβ-turn type II structure,whereas the biological activity of silk I protein as a scaffold forfibroblasts is still unknown.

Graft materials for skin wounds should have some features promoting cell viability and sta-bility for epithelial andfibroblastic cells.Re-

C 2010The Pharmaceutical Society of Japan