NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties.

Osteoarthritis (OA) remains a debilitating joint disorder due to the lack of disease-modifying therapies that can simultaneously halt cartilage degradation and modulate the aberrant immune microenvironment. This study demonstrated the therapeutic potential of extracellular vesicles derived from adipose-derived stem cells preconditioned with nanosecond pulsed electric fields (NsPEFs-ADSCs-EVs). Administration of NsPEFs-ADSCs-EVs significantly attenuated OA progression, as indicated by alleviated cartilage degradation, and a marked shift in synovial macrophage from the pro-inflammatory M1 to the pro-reparative M2 phenotype. Mechanistically, we discovered that NsPEFs-ADSCs-EVs, via surface-enriched ITGA4, activated the PI3K/Akt pathway to instruct the increased secretion of R-spondin 3 (RSPO3). We further unveiled a novel dual function of chondrocyte-derived RSPO3. It acted in an autocrine manner to enhance chondrocyte anabolism and in a paracrine manner to directly drive M2 macrophage polarization. The pro-M2 effect was specifically mediated through the activation of the LGR4/LRP6/β-catenin signaling axis in macrophages. Collectively, this work elucidates a previously unrecognized paracrine axis wherein NsPEFs-engineered EVs deploy RSPO3 as a significant coordinator to synchronously promote cartilage regeneration and immune resolution. Our findings not only reveal RSPO3 as a promising therapeutic target but also establish the NsPEFs platform as a efficient strategy for generating functionally enhanced EVs, offering a novel cell-free strategy for OA therapy.