M2 Macrophage‐Derived Exosomes Promote Tendon‐to‐Bone Healing by Alleviating Cellular Senescence in Aged Rats

PURPOSE: To explore the potential of M2 macrophage-derived exosomes (M2-Exos) in enhancing tendon-to-bone healing in aged rats by mitigating cellular senescence of bone marrow-derived stem cells (BMSCs). METHODS: In vitro, the effects of M2-Exos on alleviating cellular senescence and improving chondrogenic potential of senescent BMSCs were evaluated. Rats (24 young and 48 aged) with chronic rotator cuff tear (RCT) were repaired and assigned into 3 groups: young group (young rats injected with fibrin at the enthesis), aged group (aged rats injected with fibrin at the enthesis), and aged + M2-Exos group (aged rats injected with fibrin containing M2-Exos at the enthesis). At 6 and 12 weeks after repair, enthesis regeneration was evaluated. Proteomic analysis was conducted to explore the mechanism through which M2-Exos mitigated cellular senescence. RESULTS: In senescent BMSCs treated with M2-Exos, there was a reduction in senescence biomarkers including senescence-associated β-galactosidase, p53, p21, and senescence-associated secretory phenotype (P < .001). M2-Exos also enhanced chondrogenic potential of senescent BMSCs, reflected in greater Bern score (P < .001) and increased expression of Sox9 (P = .013), Col2a1 (P < .001), and Acan (P < .001). Histologically, aged rats treated with M2-Exos demonstrated significantly greater histologic scores (P < .001 at both 6 and 12 weeks) and increased fibrocartilage regeneration at the enthesis. Biomechanically, these rats exhibited greater failure load, stiffness, and stress (all P < .001) at 12 weeks. Mechanistically, proteomic analysis suggested that M2-Exos might alleviate cellular senescence by potentially regulating DNA replication and repair. CONCLUSIONS: M2-Exos can significantly alleviate BMSC senescence and thereby enhance tendon-to-bone healing in an aged rat RCT model. CLINICAL RELEVANCE: This study suggests the potential utility of M2-Exos as a therapy for RCT in the older population.