Tetrahedral Framework Nucleic Acid Delivery of miR-210-3p Promotes Femoral Bone Regeneration Through EFNA3/AKT/STAT3-Mediated Immunomodulation.

Persistent inflammation, insufficient vascularization and impaired osteogenesis limit the repair of critical-sized femoral defects. Although microRNA-210-3p (miR-210-3p) can regulate these processes, its therapeutic use is constrained by poor extracellular stability and inefficient cellular entry. Here, miR-210-3p was incorporated into a tetrahedral framework nucleic acid (tFNA) to create tFNAs-miR-210-3p. Dynamic light scattering, zeta-potential analysis, atomic force microscopy (AFM) and capillary electrophoresis supported formation of the negatively charged nanosystem. In RAW264.7 macrophages, tFNA delivery increased Cy5-labelled miR-210-3p-positive cells from 1.24% for free miR-210-3p to 99.93%. Under lipopolysaccharide (LPS)-induced inflammatory conditions, tFNAs-miR-210-3p reduced CD86 and inducible nitric oxide synthase, increased CD206 and arginase-1, enhanced endothelial tube formation and improved osteogenic differentiation. It suppressed M1 marker CD86/iNOS and elevated M2 marker CD206/Arg-1 to resolve the inflammatory microenvironment. Mechanistically, miR-210-3p directly binds the 3'UTR of EFNA3 mRNA to inhibit its expression, thereby activating AKT/STAT3 signalling to switch macrophages toward reparative phenotype. In a rat critical-sized femoral defect model, local tFNAs-miR-210-3p treatment improved histological repair, macrophage polarisation, microcomputed-tomography indices, vascularization and osteocalcin expression at 4 and 8 weeks. These findings identify tFNA-mediated miR-210-3p delivery as a promising immunomodulatory strategy for vascularised bone regeneration.