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

## TL;DR

NsPEFs-treated stem cell extracellular vesicles help treat osteoarthritis by promoting cartilage repair and immune modulation through RSPO3.

## Contribution

Discovery of RSPO3's dual role in cartilage regeneration and M2 macrophage polarization via NsPEFs-engineered extracellular vesicles.

## Key findings

- NsPEFs-ADSCs-EVs reduce cartilage degradation and shift macrophages to a pro-reparative M2 phenotype.
- RSPO3 acts both in an autocrine and paracrine manner to enhance cartilage anabolism and macrophage polarization.
- The LGR4/LRP6/β-catenin axis mediates RSPO3's pro-M2 effect in macrophages.

## Abstract

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.

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•NsPEFs-ADSCs-EVs as novel bio-active materials, exhibit high yield, homogeneity and enhanced bio-activity for OA treatment.•NsPEFs-ADSCs-EVs instruct chondrocytes to transform into active signaling hubs by ITGA4-mediated inter-cellular communication.•Chondrocyte-derived RSPO3 as a pivotal effector mediates the dual functionality, cartilage anabolism and M2 transition.•The pro-M2 effect of RSPO3 is mechanistically operated through the LGR4/LRP6/β-catenin signaling axis in macrophages.•A synergistic therapeutic strategy is proposed for OA by offering both the engineered EV platform and RSPO3 protein.

NsPEFs-ADSCs-EVs as novel bio-active materials, exhibit high yield, homogeneity and enhanced bio-activity for OA treatment.

NsPEFs-ADSCs-EVs instruct chondrocytes to transform into active signaling hubs by ITGA4-mediated inter-cellular communication.

Chondrocyte-derived RSPO3 as a pivotal effector mediates the dual functionality, cartilage anabolism and M2 transition.

The pro-M2 effect of RSPO3 is mechanistically operated through the LGR4/LRP6/β-catenin signaling axis in macrophages.

A synergistic therapeutic strategy is proposed for OA by offering both the engineered EV platform and RSPO3 protein.