Extracellular vesicles (EVs) may have a key therapeutic role and offer an innovative treatment for osteoarthritis (OA). Studies have shown that ratio of MSC/chondrocyte could affect their therapeutic outcomes. Here, we investigate the chondrogenic potential and therapeutic effect of EVs derived from MSCs and chondrocytes in the naïve, chondrogenically primed, and co-culture states to treat OA. EVs are isolated from naïve MSCs (M-EV), chondrogenically primed MSCs (cpM-EV), chondrocytes (C-EV), and co-cultures of chondrocytes plus MSCs at ratios of 1:1 (C/M-EV), 2:1 (2C/M-EV), and 4:1 (4C/M-EV). We characterized the isolated EVs in terms of surface markers, morphology, size, and zeta potential, and evaluated their chondrogenic potential in vitro by qRT-PCR and histological analyses. Next, these EVs were intra-articularly injected into osteoarthritic cartilage of a rat model and assessed by radiography, gait parameters, and histological and immunohistochemical analyses. EVs obtained from chondrocytes co-cultured with MSCs resulted in improved matrix production and functional differentiation. Our research showed that close proximity between the two cell types was essential for this response, and improved chondrogenesis and matrix formation were the outcomes of this interaction in vitro. Furthermore, in the in vivo rat OA model induced by a monoiodoacetate (MIA), we observed recovery from OA by increasing ratio of the C/M-derived EV group compared to the other groups. Our findings show that the increasing chondrocyte ratio to MSC leads to high chondrogenic induction and the therapeutic effect of harvested EVs for cartilage repair.

细胞外囊泡（EV）可能具有关键的治疗作用，并为骨关节炎（OA）提供创新的治疗方法。研究表明间充质干细胞/软骨细胞的比例可能会影响其治疗结果。在这里，我们研究了源自 MSC 和软骨细胞的 EV 在幼稚、软骨形成引发和共培养状态下治疗 OA 的软骨形成潜力和治疗效果。EV 分离自幼稚 MSC (M-EV)、软骨引发的 MSC (cpM-EV)、软骨细胞 (C-EV) 以及软骨细胞与 MSC 以 1:1 的比例共培养 (C/M-EV)， 2:1 (2C/M-EV) 和 4:1 (4C/M-EV)。我们从表面标记、形态、大小和 zeta 电位方面对分离的 EV 进行了表征，并通过 qRT-PCR 和组织学分析评估了它们的体外软骨形成潜力。接下来，将这些 EV 关节内注射到大鼠模型的骨关节炎软骨中，并通过放射线照相、步态参数以及组织学和免疫组织化学分析进行评估。从与 MSC 共培养的软骨细胞中获得的 EV 可改善基质生产和功能分化。我们的研究表明，两种细胞类型之间的紧密接近对于这种反应至关重要，并且软骨形成和基质形成的改善是这种体外相互作用的结果。此外，在由单碘乙酸（MIA）诱导的体内大鼠 OA 模型中，我们观察到通过与其他组相比增加 C/M 衍生 EV 组的比例来从 OA 中恢复。我们的研究结果表明，软骨细胞与 MSC 的比例增加会导致高软骨形成诱导和收获的 EV 对软骨修复的治疗效果。