We have recently identified plasma-irradiated silk fibroin (P-AF) as a key regulator of bone matrix properties and composition. Bone matrix properties were tested in 48 femur critical size defects (3.25 mm in diameter) with the expression of osteoblast specific genes at 1 and 2 weeks after surgery. The scaffolds were characterized by various states of techniques; the scanning electronic microcopy revealed the large sized pores in the aqueous-based silk fibroin (A-F) scaffold and showed no alteration into the architecture by the addition of plasma irradiation. The contact angle measurements confirmed the introduction of plasma helped to change the hydrophobic nature into hydrophilic. The histological analyses confirmed the presence of silk fibroin in scaffolds and newly formed bone around the scaffolds. Immunohistochemical examination revealed the increased expression pattern in a set of osteoblast specific genes (TGF-beta, TGF-beta type III receptor, Runx2, type I collagen and osteocalcin). These data were the first to show that the properties of bone matrix are regulated, specifically through Runx2 pathway in P-AF group. Thus, an employment of P-AF increases several compositional properties of bone, including increased bone matrix, mineral concentration, cortical thickness, and trabecular bone volume. (c) 2014, The Society for Biotechnology, Japan. All rights reserved.