Extracellular vesicles from TNF-α and TGF-β1-treated ADSCs promote tissue-engineered cartilage regeneration by modulating macrophages via the miR-378a-3p/SIRPα axis.

BACKGROUND: Mesenchymal stem cells (MSCs) possess immunomodulatory properties and are concurrently influenced by the local inflammatory microenvironment. Extracellular vesicles (EVs) derived from MSCs (MSCs-EVs) replicate the therapeutic effects of their parent cells while avoiding the limitations of cell therapy. Elucidating the impact of diverse inflammatory factors on the composition and functionality of MSCs-EVs is crucial for their optimal use, though this remains incompletely understood. The aim of this study was to explore the immunomodulatory effects and underlying mechanisms of EVs derived from adipose-derived stem cells (ADSCs) pretreated with TNF-α and TGF-β1 (α-EVs and β-EVs) on macrophages and tissue-engineered cartilage regeneration. METHODS: Isolation and identification of EVs from ADSCs cultured in 3D spheres. The effects on chondrocyte and macrophage proliferation, migration, and polarization were evaluated in vitro. In vivo, chondrocytes-laden porous GelMA hydrogel with EVs were implanted into mice to assess chondrocyte regeneration and macrophage infiltration. Small RNA sequencing revealed distinct EVs-miRNA expression profiles, and the target validation confirmed the molecular mechanism. RESULTS: In vitro, both α-EVs and β-EVs demonstrated the capacity to modulate macrophage phenotypes. α-EVs more effectively reduced M1 macrophage markers and enhanced M2 polarization. Besides, β-EVs exhibited a stronger inhibitory effect on macrophage proliferation and migration, while also promoting chondrocyte proliferation and extracellular matrix (ECM) formation. In vivo, β-EVs significantly improved ECM deposition and chondrocyte maintenance, while both EVs groups reduced M1 infiltration and increased M2 presence. Small RNA sequencing identified miR-378a-3p upregulation in both α-EVs and β-EVs, targeting Signal-regulatory protein alpha (SIRPα) to modulate the immune status of macrophages. CONCLUSIONS: Both TNF-α and TGF-β1 enhanced the immunomodulatory effects of EVs, with TGF-β1 showing a stronger capacity to promote chondrocyte proliferation and ECM synthesis. The miR-378a-3p/SIRPα axis was identified as a key mechanism underlying the protective effects of both α-EVs and β-EVs. This study provides valuable insights into optimizing EVs-based regenerative strategies to regulate the local inflammatory microenvironment and promote the regeneration of engineered tissues.