Bone matrix-derived TGF-β1–osteopontin complex is associated with FGF2-dependent mineralized matrix formation on titanium surfaces

Purpose Bioactive factors retained within the bone matrix contribute to bone regeneration; however, the mechanisms underlying their retention remain unclear. This study investigated BMP- and TGF-β–like activities in demineralized bone extracts, identified the non-collagenous proteins (NCPs) responsible for these activities, and evaluated their osteogenic potential. Methods Bone proteins were extracted from rat femora and tibiae and subsequently fractionated. BMP- and TGF-β–like activities were evaluated using alkaline phosphatase and luciferase reporter assays, respectively. Major acidic NCPs were identified using mass spectrometry, and TGF-β1 was quantified using enzyme-linked immunosorbent assay (ELISA). The molecular species of the major NCPs were further separated, and their association with TGF-β1 was analyzed using heterologous sandwich ELISA. Finally, the mineralization-inducing activity of the major NCP fraction was evaluated using human umbilical cord perivascular cells (HUCPVCs). Results BMP- and TGF-β–like activities were detected in all bone extract fractions. Mass spectrometry identified osteopontin (OPN) as the predominant NCP in the fraction exhibiting the highest TGF-β–like activity. Luciferase reporter assays confirmed TGF-β receptor–dependent transcriptional activity. ELISA detected that TGF-β1 was associated with both the 60 kDa and 64 kDa OPN molecular species. Notably, the OPN-rich fraction containing TGF-β1 promoted fibroblast growth factor 2 (FGF2)-dependent mineralization of HUCPVCs on titanium discs. Conclusions Demineralized bone matrix contains biologically active TGF-β1 associated with specific OPN molecular species. The OPN-rich fraction containing TGF-β1 exhibited FGF2-dependent osteogenic activity, suggesting that this association contributes to the osteogenic properties of bone graft materials used for bone regeneration and implant therapy.