Cell attachment, expansion, and migration in three-dimensional biomaterials are crucial steps for effective delivery of osteogenic cells into bone defects. Complexes composed of vitronectin (VN), insulin-like growth factors (IGFs), and insulin growth factor-binding proteins (IGFBPs) have been reported to enhance cell attachment, proliferation, and migration in a variety of cell lines in vitro. The aim of this study was to examine whether prebound complexes of VN and IGFs +/- IGFBPs could facilitate human osteoblast serum-free expansion in vitro and enhance cell attachment, proliferation, and migration in three-dimensional biomaterial constructs. Human osteoblasts derived from alveolar bone chips and the established human osteoblast cell line Saos-2 were used. These cells were seeded on tissue culture plates and porous scaffolds of type I collagen sponges and polyglycolic acid (PGA), which had been coated with VN +/- IGFBP-5 +/- IGF-I. Cell attachment, proliferation, and migration were evaluated by cell counting, confocal microscopy, and scanning electron microscopy. The number of attached human osteoblasts was significantly higher in VN-coated polystyrene culture dishes. Furthermore, significant increases in cell proliferation were observed when growth factors were bound to these surfaces in the presence of VN. In the two scaffold materials examined, greater cell attachment was found in type I collagen sponges compared with PGA scaffolds. However, coating the scaffolds with complexes composed of VN + IGF-I or VN + IGFBP-5 + IGF-I enhanced cell attachment on PGA. Moreover, the presence of VN + IGFBP-5 + IGF-I resulted in significantly greater osteoblast migration into deep pore areas as compared with untreated scaffolds or scaffolds treated with fetal calf serum. These results demonstrated that complexes of VN + IGFBP-5 + IGF-I can be used to expand osteoblasts in vitro under serum-free conditions and enhance the attachment and migration of human osteoblasts in three-dimensional culture. This in turn suggests a potential application in surface modification of biomaterials for tissue reconstruction.