ATG5-overexpressing MSCs improve ligament healing with an associated shift toward M2 macrophage polarization in aged rats.

Abstract

Objectives: Autophagy is crucial for maintaining mesenchymal stem cell (MSC) function, yet its role in age-related ligament healing deficits remains unclear. This study examined the relationship between MSC autophagic activity and age-related ligament healing and evaluated ATG5 overexpression effects on autophagy and regeneration.

Methods: Medial collateral ligament (MCL) injury models were established in young and aged rats. Healing was assessed using ultrasound, histology, immunohistochemistry, apoptosis assays, and biomechanical testing. Aged MSCs were engineered to overexpress ATG5 and transplanted into injured ligaments. Repair outcomes and macrophage polarization changes were evaluated.

Results: Aged rats exhibited impaired ligament healing, and aged MSCs displayed reduced autophagic activity. ATG5 overexpression enhanced autophagy in aged MSCs, and transplantation of ATG5-overexpressing MSCs was associated with improved ligament repair in aged rats, including increased proliferation, angiogenesis, collagen organization, and biomechanical strength, alongside reduced apoptosis. ATG5-overexpressing MSC transplantation was also associated with lower M1 and higher M2 macrophage markers and a more anti-inflammatory cytokine profile. Reduced MSC autophagy was associated with age-related ligament healing deficits.

Conclusions: Our findings support a potential relationship among ATG5-associated autophagy, macrophage phenotype, and improved ligament repair, while the causal dependence of therapeutic effects on autophagy and macrophage polarization remains to be established.