Aim: Cartilage tissue has limited capacity for spontaneous repair and self-renew due to the absence of vascularization and progenitor cells, thus requiring a therapeutic approach to repair tissue damage. Recently, extracellular vesicles (EVs) have attracted attentions because of their major roles in cell communication and tissue repair by regulating cellular processes such as cell growth, differentiation, and proliferation. Therefore, it is necessary to isolate extracellular vesicles with chondrogenic potential from an appropriate cellular source for cartilage regeneration. This study aims to compare the chondrogenic ability of extracellular vesicles derived from cocultured -chondrocytes / mesenchymal stem cells (CHO / MSC) at ratios of 1/2 and 1/4.
Material and Methods: Towards this goal, chondrocytes and mesenchymal stem cells were isolated from rabbit articular cartilage and bone marrow, respectively. Mesenchymal phenotype of isolated MSCs were characterized based on their surface markers and by differentiation into mesenchymal lineages. Chondrocytes and mesenchymal stem cells were co-cultured with defined ratios, their conditioned media were collected and extracellular vesicles were extracted with an ultracentrifuge. Concentrations of extracellular vesicles were determined using BCA Protein Assay Kit, then EVs were characterized in terms of size, morphology, and expression of surface markers by dynamic light scattering (DLS), scanning electron microscope (SEM) and western blotting, respectively. Mesenchymal stem cells were treated with different concentrations of extracellular vesicles (50, 100, and 150 ug/ml) for 21 days. Quantitative real time-PCR (qRT-PCR) and histological analysis were subsequently performed to assess the quality of chondrogenic differentiation among experimental groups. The differentiation of MSCs to osteogenic and adipogenic lineages  was demonstrated by Oil Red and Alizarin Red staining after 21 days.
Results: The results of flow cytometry showed that CD90 was expressed by 88.6% of the cell population as a positive marker and CD34 as a negative marker was only expressed in 5.48% of the cell population. SEM micrographs confirmed the spherical shape of CHO/ MSC-EV 1/2 and CHO/MSC-EV 1/4 and the mean particle size of isolated EVs were 51.66 ± 8.15 and 19.11 ± 6.03 nm, respectively. Special surface markers for extracellular vesicles containing CD9 and CD81 were expressed in both groups. At concentrations of 100 and 150 ug/ml of CHO/MSC-EV 1/2, higher cartilage - specific markers, including Col II were observed compared to CHO/MSC-EV 1/4. Similarly, safranin O and toluidine blue staining revealed the more deposition of aminoglycan and proteoglycan at concentrations of 100 and 150 ug/ml of CHO/ MSC-EV 1/2, compared to CHO/ MSC-EV 1/4. This study demonstrated the chondrogenesis potential of extracellular vesicles, especially in CHO /MSC-EV 1/2 and extracellular vesicles derived from co-culturing chondrocytes/MSCs improve matrix production and chondrogenesis.
Conclusion: Our research shows that chondrocyte-MSCs proximity is essential for the response as improved viability, chondrogenesis, and matrix formation in recipient MSCs. It is also proposed that co-cultured derived EVs with the ability to promote chondrogenesis have the potential to be utilized in cartilage regeneration.  Due to their immunogenicity and their low tumorigenic potential, extracellular vesicles can be used to help repair cartilage tissue.