Surface Calcium Enrichment of Biogenic Hydroxyapatite Regulates Macrophage Inflammation via Toll-Like Receptor 4 Signaling.

Biogenic hydroxyapatite (BHA) is widely used for oral bone defect regeneration, yet its osteogenic efficacy is limited by slow osteogenesis and insufficient bone volume, which may be attributed to early inflammatory responses. The investigation of granular biomaterials is further challenged by conventional in vitro models that fail to ensure adequate cell-material interactions and efficient RNA extraction, limiting experimental reliability. Here, we developed an improved direct coculture system together with an optimized RNA extraction strategy to address these limitations. Using this platform, we characterized BHA-induced inflammatory responses at the gene, protein, and transcriptomic levels. Our results showed that the inflammatory activity of BHA is associated with its calcium-enriched surface, which activates the Toll-like receptor 4 signaling pathway. Furthermore, a calcium pre-adsorption strategy was introduced to modulate surface ion activity, which reduced calcium enrichment and attenuated inflammatory signaling. These findings suggest that modulating calcium enrichment capacity may offer a promising strategy to regulate the immune response of hydroxyapatite-based bone substitutes and improve their biological performance.