Exosomes derived from 3D-cultured MSCs alleviate knee osteoarthritis by promoting M2 macrophage polarization through miR-365a-5p and inhibiting TLR2/Myd88/NF-κB pathway

Abstract

Exosomes (EXO) derived from mesenchymal stem cells (MSCs) are recently shown to have therapeutic effects in preclinical models of knee osteoarthritis (KOA). However, their potential for conversion into a clinical therapy are limited by several shortcomings such as low yield and suboptimal efficacy. This study developed 3D-EXO to improve exosome yield and efficacy through 3D-culturing of MSCs, with the aim of achieving overall better therapeutic effects compared to traditional 2D-cultured MSC-derived EXO (2D-EXO). In a rat KOA model, 3D-EXO led to better preservation of bone structure and protection from cartilage damage compared to 2D-EXO. Interestingly, in vitro experiments showed no significant differences between 3D-EXO and 2D-EXO in their direct effects on chondrocytes. To explain these findings, we looked into the possible effects of MSC-derived EXO on the immune microenvironment, specifically in modulating phenotypic expression in macrophages. In vitro investigations showed that 3D-EXO increased the expression of M2 phenotype markers in macrophages to a greater extent than 2D-EXO. RNA-seq analysis revealed that expression of miR-365a-5p was more upregulated in 3D-EXO than 2D-EXO. Through micro-RNA mimics transfection, we found that miR-365a-5p acted directly on TLR2 mRNA to inhibit TLR2 translation, indicating that 3D-EXO promoted M2 polarization of macrophages by inhibiting the TLR2/Myd88/NF-κB axis. The immunomodulatory effects of 3D-EXO were confirmed by the observation that the metabolic status of chondrocytes was significantly improved after co-culturing with 3D-EXO-treated macrophages. In summary, 3D-EXO can effectively promote M2 macrophage polarization through increased expression of miR-365a-5p, which in turn improves the metabolic state of chondrocytes and resulting in overall better in vivo therapeutic effects on KOA. The findings in this study imply the development of new therapies for KOA based on MSC-derived EXO.