Gelatin macromolecular microspheres constructed by microfluidics regulate the TNF/HIF-1 signaling axis to reshape the bone immune microenvironment and enhance infectious bone defect repair

<h4>Background</h4>Infected bone defects are difficult to treat due to bacterial biofilms and chronic inflammation. Systemic antibiotics often fail and cause severe side effects. A dual-drug delivery system that combines antibacterial and osteo-immunomodulatory actions may offer a synergistic solution.<h4>Methods</h4>A gelatin-based microfluidic microsphere (GM@RES@VAN) co-loading vancomycin (VAN) and resveratrol (RES) was developed. The microspheres were characterized for morphology, drug release, and elemental composition. Antibacterial and biofilm-degrading effects were assessed in vitro against MRSA. A rat infected bone defect model was used to evaluate in vivo healing via micro-CT, histology, and immunofluorescence. Network pharmacology, molecular docking, and cellular assays (qPCR, Western blot, TEM, HIF inhibition) explored the molecular mechanism.<h4>Results</h4>GM@RES@VAN microspheres showed uniform size, smooth surface, and sustained dual-drug release. In vitro, the microspheres exhibited potent bactericidal activity and biofilm degradation (∗∗∗p < 0.001). In vivo, the GM@RES@VAN group significantly reduced local bacterial load, decreased inflammatory infiltration, and promoted collagen deposition and new bone formation, with increased bone mineral density and bone volume/tissue volume (∗p < 0.05). Mechanistically, resveratrol suppressed pro-inflammatory factors (IL6, TNF, IL1B), reduced HIF1A, MMP9, and PTGS2 expression, preserved mitochondrial ultrastructure, upregulated osteogenic markers (ALP, OCN, COL I) and the anti-apoptotic protein BCL2. Vancomycin primarily cleared the infection, while resveratrol acted through the TNF/HIF-1 axis to reshape the inflammatory microenvironment and promote osteogenesis-their synergy was confirmed by HIF inhibition tests.<h4>Conclusion</h4>The GM@RES@VAN dual-drug microspheres achieve programmed antibacterial and osteo-immunomodulatory effects via vancomycin-mediated infection clearance and resveratrol-driven TNF/HIF-1 pathway regulation. This combination offers a promising strategy for treating refractory infected bone defects.