Strontium- and magnesium-doped hydroxyapatite scaffolds enhance bone regeneration by promoting macrophage polarization toward the M2 phenotype.

The development of synthetic bone grafts that integrate osteogenic and immunomodulatory functions represents a critical strategy in regenerative orthopedics. In this study, we fabricated strontium- and magnesium-doped hydroxyapatite (HA/Mg/Sr) scaffolds derived from natural bovine cancellous bone. Physicochemical characterization confirmed the preservation of a three-dimensional interconnected porous architecture (~80% porosity, ~350 μm pore size) with a homogeneous nanoparticulate surface. Doping with Mg and Sr enhanced scaffold degradation, sustained the release of bioactive ions (Ca2+, Mg2+, Sr2+), and significantly increased compressive strength to levels comparable with human cancellous bone. HA/Mg/Sr scaffolds exhibited excellent biocompatibility, promoting adhesion and proliferation of both Bone marrow-derived mesenchymal stem cells (BMSCs) and bone marrow-derived macrophages (BMMs). While Mg primarily facilitated direct osteogenic differentiation of BMSCs, Sr played a dominant role in directing macrophage polarization toward the anti-inflammatory M2 phenotype, as evidenced by increased CD206 and IL-10 expression alongside reduced CCR7 and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Notably, macrophage-conditioned medium from HA/Mg/Sr cultures substantially enhanced BMSC osteogenesis, highlighting immunomodulation as a central mechanism. Transcriptomic analysis indicated that the HA/Mg/Sr scaffolds promoted macrophage polarization toward the M2 phenotype and modulated immune responses through the NF-κB signaling pathway, which was validated by Western blot. In a rat femoral condyle defect model, HA/Mg/Sr scaffolds-particularly those with 10% Sr content (HA/Mg/Sr2)-demonstrated superior bone regeneration, characterized by bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and enhanced new bone ingrowth and integration, accompanied by elevated M2 macrophage infiltration in vivo. Overall, these findings establish that HA/Mg/Sr scaffolds promote bone repair not only via direct osteoconduction but, more importantly, through immunomodulation-fostering a pro-healing M2 macrophage microenvironment in an NF-κB-dependent manner, thereby offering a promising dual-functional biomaterial strategy for critical-sized bone defect reconstruction. This study underscores the therapeutic relevance of targeting macrophage polarization in bone repair and delineates a mechanistic framework for Sr-based immunomodulatory biomaterial design.