Hypoxia-preconditioned tendon stem cell-derived extracellular vesicles promote tendon regeneration by modulating macrophage heterogeneity

Tendon injuries result in poor healing outcomes and functional impairment. Extracellular vesicles (EVs) derived from tendon stem cells (TSCs) have shown therapeutic potential for tendon regeneration. Because tendon tissues exist in a relatively hypoxic microenvironment under both physiological and injured conditions, we investigated whether hypoxic preconditioning could enhance the biological activity of TSC-derived EVs. In this study, EVs were isolated from rat tendon stem cells cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions, referred to as EVs and Hypo-EVs, respectively. To explore the molecular mechanisms underlying macrophage regulation by Hypo-EVs, RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) to induce an M1 inflammatory phenotype and subsequently treated with Hypo-EVs or normoxic EVs. Total RNA was extracted and subjected to high-throughput RNA sequencing (RNA-seq). The transcriptomic profiles generated in this study provide insights into the regulatory effects of hypoxia-preconditioned TSC-derived EVs on macrophage polarization and inflammatory signaling pathways during tendon repair.