Abstract
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.