Biogenic hydroxyapatite is known for its osteoinductive potential due to its similarity to human bone and biocompatibility, but insufficient vascularization compared to autogenous bone during early implantation limits bone integration and osteogenesis. Fluorine has been shown to improve hydroxyapatite’s mechanical properties and the coupling of osteogenic and angiogenic cells. In this study, fluorine-modified biogenic hydroxyapatite (FPHA) with varying fluorine concentrations was prepared and tested for its ability to promote vascularized osteogenesis. FPHA prepared in this study retained the natural porous structure of biological cancellous bone and released F⁻ ions when immersed in cell culture medium. The extraction solutions of FPHA0.25 and FPHA0.50 promoted the formation of capillary-like tubes by human umbilical vein endothelial cells (HUVECs), with FPHA0.25 significantly upregulating vegf mRNA and VEGF protein levels in co-cultured human bone marrow mesenchymal stem cells (HBMSCs). Additionally, FPHA0.25 and FPHA0.50 upregulated pdgf-bb mRNA and PDGF-BB protein levels in HUVECs. In vivo experiments using a rabbit cranial defect model demonstrated that FPHA0.25 promoted early bone formation and angiogenesis in the defect area, enhanced VEGF secretion, and increased PDGFR-β expression in endothelial and mesenchymal cells. These findings suggest that fluorine-modified biogenic hydroxyapatite with an optimal fluorine concentration (FPHA0.25) may offer a promising strategy to enhance the body’s innate bone-healing potential by accelerating vascularization.

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.

Biogenic collagen membranes (BCM) have been widely used in guided bone regeneration (GBR) owing to their biodegradability during tissue integration. However, their relatively high degradation rate and lack of pro-osteogenic properties limit their clinical outcomes. It is of great importance to endow BCM with tailored degradation as well as pro-osteogenic properties. In this study, a fluoride-modified polymer-induced liquid precursor (PILP) based biomineralization strategy was used to convert the collagen membrane from an organic phase to an apatite-based inorganic phase, thus achieving enhanced anti-degradation performance as well as osteogenesis. As a result, three phases of collagen membranes were prepared. The original BCM in the organic phase induced the mildest inflammatory response and was mostly degraded after 4 weeks. The organic-inorganic mixture phase of the collagen membrane evoked a prominent inflammatory response owing to the fluoride-containing amorphous calcium phosphate (F-ACP) nanoparticles, resulting in active angiogenesis and fibrous encapsulation, whereas the inorganic phase induced a mild inflammatory response and degraded the least owing to the transition of F-ACP particles into calcium phosphate with high crystallinity. Effective control of ACP is key to building novel apatite-based barrier membranes. The current results may pave the way for the development of advanced apatite-based membranes with enhanced barrier performances. GRAPHICAL ABSTRACT

Biologically-derived hydroxyapatite is a widely used biomaterial in various clinical applications including bone augmentation. However, the osteogenic application of biological hydroxyapatite is limited by inflammatory responses, and the underlying mechanism remains unknown. The current study aimed to elucidate the molecular mechanisms underlying the inflammatory response to biological hydroxyapatite. Porcine-derived hydroxyapatite (PHA) with two sintering temperatures (800 and 1600 °C), PHA800 and PHA1600, respectively, were prepared. A PHA/macrophage co-culture model was established. Transcriptome, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) analyses were used to determine the inflammatory effects and the main pathways activated by PHA800 and PHA1600. Intracellular calcium level, PHA-induced calcium enrichment, and related biological effects were used to determine the molecular mechanism at the PHA-cell interface. PHA800 significantly upregulated a TLR4 mediated inflammatory pathway in a calcium influx-dependent manner, and the calcium enrichment activity on the surface of PHA800 promoted calcium influx. In contrast, the calcium enrichment activity on the surfaces of the PHA1600 and PHA800 pretreated groups was attenuated, resulting in decreased calcium influx and mild inflammatory effects. Our results provide a fundamental basis for the development of novel bone substitutes that elicit low levels of inflammation response.

Collagen sponges are widely used for oral tissue regeneration, due to their extracellular matrix-mimetic architecture and excellent biocompatibility. However, in practical biomedical applications, collagen sponges may exhibit hydration-induced structural instability, and there can be associated inflammatory responses under physiological conditions, potentially compromising their regenerative performance. In this study, we investigated how two cross-linking strategies—transglutaminase (TG) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS)—modulate the structural stability and inflammatory profiles of collagen sponges. TG-cross-linked sponges exhibited microstructural collapse, associated with macrophage activation and engagement of the Itgαvβ3/5–Src–RhoC–NF-κB signaling axis. In contrast, EDC/NHS-cross-linked sponges preserved a stable porous architecture, effectively suppressing this signaling cascade and establishing a low-inflammatory microenvironment. These findings elucidate a key mechanism by which cross-linking regulates the microstructural integrity of collagen scaffolds and provides in vitro-derived preliminary design principles for developing next-generation collagen biomaterials with low-inflammatory properties.