Background: Biological-derived hydroxyapatite is widely used as a bone substitute for addressing bone defects, but its limited osteoconductive properties necessitate further improvement. The osteo-immunomodulatory properties hold crucial promise in maintaining bone homeostasis, and precise modulation of macrophage polarization is essential in this process. Metabolism serves as a guiding force for immunity, and fluoride modification represents a promising strategy for modulating the osteoimmunological environment by regulating immunometabolism. In this context, we synthesized fluorinated porcine hydroxyapatite (FPHA), and has demonstrated its enhanced biological properties and osteogenic capacity. However, it remains unknown whether and how FPHA affects the immune microenvironment of the bone defects. Methods: FPHA was synthesized and its composition and structural properties were confirmed. Macrophages were cultured with FPHA extract to investigate the effects of FPHA on their polarization and the related osteo-immune microenvironment. Furthermore, total RNA of these macrophages was extracted, and RNA-seq analysis was performed to explore the underlying mechanisms associated with the observed changes in macrophages. The metabolic states were evaluated with a Seahorse analyzer. Additionally, immunohistochemical staining was performed to evaluate the macrophages response after implantation of the novel bone substitutes in critical size calvarial defects in SD rats. Results: The incorporation of fluoride ions in FPHA was validated. FPHA promoted macrophage proliferation and enhanced the expression of M2 markers while suppressing the expression of M1 markers. Additionally, FPHA inhibited the expression of inflammatory factors and upregulated the expression of osteogenic factors, thereby enhancing the osteogenic differentiation capacity of the rBMSCs. RNA-seq analysis suggested that the polarization-regulating function of FPHA may be related to changes in cellular metabolism. Further experiments confirmed that FPHA enhanced mitochondrial function and promoted the metabolic shift of macrophages from glycolysis to oxidative phosphorylation. Moreover, in vivo experiments validated the above results in the calvarial defect model in SD rats. Conclusion: In summary, our study reveals that FPHA induces a metabolic shift in macrophages from glycolysis to oxidative phosphorylation. This shift leads to an increased tendency toward M2 polarization in macrophages, consequently creating a favorable osteo-immune microenvironment. These findings provide valuable insights into the impact of incorporating an appropriate concentration of fluoride on immunometabolism and macrophage mitochondrial function, which have important implications for the development of fluoride-modified immunometabolism-based bone regenerative biomaterials and the clinical application of FPHA or other fluoride-containing materials. Graphical FPHA was successfully prepared through the chemical and thermal process. The immunomodulatory effects of FPHA were investigated through in vitro and in vivo studies, revealing its ability to induce a metabolic shift in macrophages from glycolysis to mitochondrial oxidative phosphorylation (OxPhos). This metabolic remodeling resulted in a notable suppression of M1 macrophage polarization and promotion of M2 macrophage polarization. Furthermore, FPHA was found to enhance osteogenic differentiation and facilitate bone repair. These findings underscore the promising potential of FPHA as a biomaterial for bone regenerative applications, providing valuable insights for the development of bioactive materials with metabolic-immunoregulatory properties: Graphical

Biological hydroxyapatite (BHA) has been extensively employed in alveolar socket preservation, yet its clinical application is often compromised by delayed bone healing triggered by macrophage-mediated pro-inflammatory responses. Building upon our previous work, in which we successfully incorporated fluorine into BHA to develop fluorinated biogenic hydroxyapatite (FBHA) with superior physicochemical and biological properties, this study systematically investigated the effects of fluorine doping on macrophage-mediated osteoimmunomodulation and socket bone healing. The synthesized FBHA was characterized using SEM, EDS, and fluoride ion release assays to confirm fluorine incorporation. In macrophage co-culture models, FBHA demonstrated significant advantages over BHA, effectively suppressing iNOS and TNFα gene expression, reducing NO release, and inhibiting phagocytic activity in M1 macrophages. RNAseq analysis revealed that the M1 phenotype suppression might be mediated through enhanced cellular antioxidant activity. Moreover, in macrophage-conditioned microenvironments, FBHA significantly upregulated osteogenic gene expression and ALP activity of pre-osteoblasts. In vivo experiments demonstrated FBHA’s superior performance in alveolar ridge preservation, especially in new bone formation and mineralization inside sockets. Fluorine doping significantly boosted socket bone healing via suppressing the inflammatory response of macrophages and enhancing osteogenic differentiation of pre-osteoblasts. These findings provide valuable insights into the development of next-generation biomaterials for alveolar socket preservation.