BACKGROUND Chondrogenic progenitor cells (CPCs) have high self-renewal capacity and chondrogenic potential. Intra-articular delivery of purified mesenchymal stem cells (MSCs) from MRL/MpJ "superhealer" mice increased bone volume during repair and prevents post-traumatic arthritis. Recently, although extracellular vesicles released from MSCs have been used widely for treating OA, the application of extracellular vesicles secreted by CPCs from MRL/MpJ mice in OA therapy has never been reported. In this study, we evaluated the effects of extracellular vesicles secreted by CPCs from control CBA (CBA-EVs) and MRL/MpJ mice (MRL-EVs) on proliferation and migration of murine chondrocytes. We also determined here if weekly intra-articular injections of CBA-EVs and MRL-EVs would repair and regenerate surgically induced model in mice. METHODS CPC surface markers were detected by flow cytometry. CBA-EVs and MRL-EVs were isolated using an ultrafiltration method. Nanoparticle tracking analysis, transmission electron microscopy, and western blots were used to identify extracellular vesicles. CBA-EVs and MRL-EVs were injected intra-articularly in a mouse model of surgical destabilization of the medial meniscus (DMM)-induced OA, and histological and immunohistochemistry analyses were used to assess the efficacy of exosome injections. We used miRNA-seq analysis to analyze the expression profiles of exosomal miRNAs derived from CBA-EVs as well as MRL-EVs. Cell-counting and scratch assays were used to evaluate the effects of CBA-EVs and MRL-EVs on proliferation and migration of murine chondrocytes, respectively. Meanwhile, a specific RNA inhibitor assesses the roles of the candidate miRNAs in CPC-EV-induced regulation of function of chondrocytes. RESULTS Both CBA-EVs and MRL-EVs stimulated chondrocyte proliferation and migration, but MRL-EVs exerted a stronger effect than CBA-EVs. The similar result was also observed in in vivo study, which indicated that injecting either CBA-EVs or MRL-EVs attenuated OA, but MRL-EVs showed a superior therapeutic effect in comparison with CBA-EVs. The results of bioinformatics analyses revealed that the differentially expressed exosomal miRNAs participated in multiple biological processes. We identified 80 significantly upregulated and 100 downregulated miRNAs. Moreover, we found that the top 20 differentially expressed exosomal miRNAs connected OA repair to processes such as AMPK signaling, regulation of autophagy, and insulin signaling. Notably, miRNA 221-3p were highly enriched in MRL-Exos and treatment with miR 221-3p inhibitor markedly decreased chondrocyte proliferation and migration induced by CBA-EVs or MRL-EVs in vitro. CONCLUSIONS This is the first study to demonstrate MRL-EVs had a greater therapeutic effect on the treatment of OA than CBA-EVs. This study will hopefully provide new insight into the pathogenesis, prevention, and treatment of OA.

中文翻译：

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关节内递送MRL / MpJ超级治愈者小鼠软骨生成祖细胞分泌的细胞外囊泡，可在小鼠损伤模型中增强关节软骨的修复。

背景技术软骨生成祖细胞（CPC）具有高的自我更新能力和软骨生成潜能。从MRL / MpJ“超愈者”小鼠关节内递送纯化的间充质干细胞（MSC）可增加修复过程中的骨量，并预防创伤后关节炎。近年来，尽管从MSC释放的细胞外囊泡已被广泛用于治疗OA，但是从未报道过由MRC / MpJ小鼠的CPC分泌的细胞外囊泡在OA治疗中的应用。在这项研究中，我们评估了由对照CBA（CBA-EVs）和MRL / MpJ小鼠（MRL-EVs）的CPC分泌的细胞外囊泡对鼠软骨细胞增殖和迁移的影响。我们还在这里确定了每周一次的CBA-EV和MRL-EV关节腔内注射是否可以修复和再生小鼠的手术诱导模型。方法采用流式细胞仪检测CPC表面标志物。使用超滤方法分离了CBA-EV和MRL-EV。纳米粒子跟踪分析，透射电子显微镜和蛋白质印迹被用来识别细胞外囊泡。将CBA-EV和MRL-EV关节内注射入半月板内侧（DMM）引起的OA的手术不稳定的小鼠模型中，并使用组织学和免疫组化分析评估外泌体注射的功效。我们使用miRNA-seq分析来分析源自CBA-EV和MRL-EV的外泌体miRNA的表达谱。细胞计数和刮擦试验分别用于评估CBA-EV和MRL-EV对鼠软骨细胞增殖和迁移的影响。与此同时，一种特定的RNA抑制剂可评估候选miRNA在CPC-EV诱导的软骨细胞功能调节中的作用。结果CBA-EV和MRL-EV均可刺激软骨细胞增殖和迁移，但MRL-EV的作用要强于CBA-EV。在体内研究中也观察到了相似的结果，这表明注射CBA-EV或MRL-EV均可减弱OA，但与CBA-EV相比，MRL-EV显示出更好的治疗效果。生物信息学分析的结果表明，差异表达的外泌体miRNA参与了多个生物学过程。我们确定了80个显着上调的miRNA和100​​个下调的miRNA。此外，我们发现前20种差异表达的外泌体miRNA将OA修复与AMPK信号传导，自噬调控，和胰岛素信号传导。值得注意的是，miRNA 221-3p在MRL-Exos中高度富集，并且用miR 221-3p抑制剂处理可显着降低体外CBA-EV或MRL-EV诱导的软骨细胞增殖和迁移。结论这是首次证明MRL-EV比CBA-EV具有更大的OA治疗效果。这项研究有望为OA的发病机理，预防和治疗提供新的见解。