Cobalt-Doped Biphasic Calcium Phosphate Orchestrates Osteogenesis-Angiogenesis Signals via Hypoxia-Mimetic Signaling

We engineered a cobalt-doped biphasic calcium phosphate (CoBCP) by combining monetite-derived pyrophosphate and β-tricalcium phosphate (β-TCP) with lattice-incorporated Co²⁺, and evaluated its physicochemical profile and in vitro bioactivity relevant to bone regeneration. Thermoanalytical (TGA/DTA), X-ray diffraction with Rietveld refinement, and FTIR/Raman confirmed monetite-pyrophosphate conversion after calcination, phase-pure β-TCP, and an approximately equimolar Co-containing biphasic composition in CoBCP; SEM/EDX verified appropriate microstructure and cobalt incorporation. Conditioned-medium assays in MC3T3-E1 pre-osteoblasts demonstrated cytocompatibility (MTT, crystal violet) and preserved collective migration (wound healing), with CoBCP, BCP, and CoCaP 1100 matching osteogenic medium in 24-h gap closure. Gene expression revealed that CoBCP selectively coordinated early proliferation and osteogenic-angiogenic programs: CDK2 was strongly induced at 3 and 7 days (≈300-fold at day 7 vs. control), RUNX2 rose across groups with BCP/CoBCP approximating osteogenic medium at day 7, and ALP was disproportionately elevated by CoBCP (~8-fold at day 3; ~400-fold at day 7). VEGF was sensitive among ceramics, with modest induction in CoCaP and CoBCP at day 3 and pronounced upregulation in TCP and BCP at day 7, while HIF-1α was highest in CoCaP at day 7, consistent with hypoxia-mimetic signaling. Cytoskeletal/adhesion transcripts diverged over time: β-TCP/BCP upregulated Src and Cofilin at day 7, whereas CoBCP selectively increased FAK, suggesting maturing focal-adhesion signaling. Gelatin zymography detected MMP-2/-9 activity across all groups with comparable magnitudes during the early window. Collectively, CoBCP provides a cytocompatible, bioactive milieu that synchronizes proliferation, adhesion, osteogenesis, and angiogenesis, supporting its potential use in orthopedic and dental bone regeneration; future work will map cobalt dose-response and ion release, and validate efficacy and safety in direct-contact and in vivo models.