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

Abstract

ABSTRACT 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.