Mai Watanabe^†‡, Ujjal K. Bhawala^¶, Shinji Takemoto^&, Norihiro Nishiyama^%, Yuichi Nakahara^∞, Ken-ichiro Tatematsu^∞, Hideki Sezuts^∞, Nobuo Kuwabara^ę, Tamiko Minamisawa^†, Kiyotaka Shiba^†* and Tetsuo Asakura^‡*

^†Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto, Tokyo 125-8550, Japan, ^‡Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan, ^¶Department of Biochemistry and Molecular Biology, and Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakae-cho Nishi, Matsudo, Chiba 271-8587, Japan, ^&Department of Biomedical Engineering, Iwate Medical University, Iwate 028-3694, Japan,^%Department of Dental Biomaterials, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakae-cho Nishi, Matsudo, Chiba 271-8587, Japan, ^∞Transgenic Silkworm Research Unit, National Agriculture and Food Research Organization,1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan,　^ęGunma Sericultural Technology Center, 2326osteo-2 Soja-machi, Maebashi, Gunma 371-0852, Japan

Silk fibroin (SF) from Bombyx mori has superior properties as both a textile and a biomaterial, and has been used to functionalize the surfaces of various medical inorganic materials including titanium (Ti). In this paper, we endowed SF with reversible binding ability to Ti by embedding a titanium binding motif (minTBP-1, RKLPDA). Artificial SF proteins were first created by conjugating gene cassettes for SF motif (AGSGAG) and minTBP-1 motif with different ratios, which have been shown to bind reversibly to Ti surfaces in quartz crystal microbalance analyses. Based on these results, the functionalized SF (TiBP-SF) containing the designed peptide [TS[(AGSGAG)₃AS]₂RKLPDAS]₈was prepared from the cocoon of transgenic B. mori , which accelerates the ossific differentiation of MC3T3-E1 cells when coated on titanium substrates. Thus, TiBP-SF presents an alternative for endowing the surfaces of titanium materials with osseointegration functionality, which would allow the exploration of potential applications in the medical field.