Menstrual blood-derived mesenchymal stromal cell extracellular vesicles stimulate chondrocytes and cartilage extracellular matrix synthesis in vitro

Human mesenchymal stromal cells are frequently studied for the development of novel technologies for preventing osteoarthritis development or repairing cartilage tissue after traumas. Menstrual blood-derived mesenchymal stromal cells (MenSCs) are less studied, however, they possess a strong therapeutic potential due to their secretome, including extracellular vesicles (EVs). The aim of this study was to evaluate the potential of MenSC-EVs in stimulating chondrocyte functions, as well as cartilage tissue repair. Chondrocyte proliferation, morphology (holomonitor), sex hormone receptor expression (ELISA, RT-qPCR), and chondrogenic capacity (RT-qPCR, histology) were evaluated after 3 days of MenSC-EV treatment. Cartilage explants were isolated and treated with EVs for 3 days and cultured for 3 and 7 days under inflammatory (IL-1β) or regenerative (TGF-β3) conditions. Cartilage oligomeric matrix protein (COMP) secretion and glycosaminoglycan (GAG) release were assessed by ELISA and spectrophotometry, with extracellular matrix (ECM) deposition evaluated by histology/immunohistochemistry, infrared absorption spectroscopy and gene expression by RT-qPCR. Cytokine and growth factor secretion were quantified in explant and chondrocyte culture supernatants using multiplexed Luminex assays. MenSC-EVs did not affect chondrocyte migration/motility, speed/perimeter and proliferation after 7 days. However, EVs increased progesterone receptor expression, as well as ECM production together with collagen type II and TGF-β3 receptor gene expression in chondrocytes after 21 days with TGF-β3. Cartilage degradation was diminished after treatment with MenSC-EVs according to lower levels of COMP and GAGs released under conventional or inflammatory conditions. Moreover, MenSC-EVs did not significantly affect inflammatory cytokine secretion in cartilage explants stimulated or not with IL-1β and in chondrocyte monolayer, except for secretion of IL-6. In addition, cartilage ECM components (collagen II and aggrecan), as well as TGF-β3 receptor gene expression were significantly higher in MenSC-EV treated cartilage explant samples, as compared to non-treated. Infrared absorption spectroscopy of MenSC-EV treated cartilage explants corroborated ECM restoration, with samples exhibiting higher amide I/II intensities and a stronger carbohydrate-associated band near 1030 cm^{- 1}, consistent with increased collagen II and proteoglycan content. This study demonstrates the potential of MenSC-EVs stimulating ECM synthesis in chondrocytes, which may turn out to be a promising cell-free therapy in the future.