Abstract

Effective reconstruction of critical-sized bone defect remains clinically challenging due to suboptimal osteogenic capacity, inadequate vascularization, and inflammatory complications associated with current autologous bone grafts or synthetic substitutes. To address these limitations, we developed a bioactive scaffold composed of hydroxyapatite doped with magnesium, strontium, and lithium, hereafter referred to as the Trio-Active Bone Scaffold (TABS), highlighting its tripartite osteo–angio–immune functions, engineered from bovine-derived cancellous bone via hydrothermal ion doping and controlled calcination. The resulting scaffold preserves the original three-dimensional interconnected porous structure and a nano-crystal, whisker microstructure formed on the surface. Comprehensive in vitro assessments reveal that TABS promote osteoblast and endothelial cell adhesion, proliferation, and lineage-specific differentiation while macrophage polarization toward an M2 phenotype. Transcriptomic analysis demonstrates that the scaffold activates key pathways involved in osteogenesis, angiogenesis, and immunoregulation. Mechanistically, the scaffold enhances osteogenesis via activation of Wnt/β-catenin signaling, stimulates angiogenesis through VEGF-dependent pathway, and modulates immune responses by inducing M2-type macrophage polarization through IL-4/JAK1/STAT6 signaling pathway. In a rat skull defect model, TABS accelerate bone regeneration with robust vascularization and improved local immune microenvironment. These findings highlight the osteo–angio–immune coordination enabled by TABS and support its potential as a bioactive scaffold for bone tissue engineering and clinical translation.