Wang Yingnan, Zhao Miaomiao, Li Wen, Yang Yuzhi, Zhang Zhenliang, Ma Ruijie, Wu Mengjie
The Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou, China.
Front Cell Dev Biol. 2021 Apr 1;9:656153. doi: 10.3389/fcell.2021.656153. eCollection 2021.
Temporomandibular joint osteoarthritis (TMJOA) seriously affects the health of patients, and the current treatments are invasive and only used for advanced cases. Bone marrow mesenchymal stem cell (BMSC)-derived small extracellular vesicles (BMSC-sEVs) may represent a safer and more effective treatment, but their role in TMJOA has not been elucidated. This study attempted to analyze the cartilage reconstruction effect of BMSC-sEVs on TMJOA and the mechanism underlying this effect. BMSC-sEVs were isolated and purified by microfiltration and ultrafiltration and were subsequently characterized by nanoparticle tracking analysis, electron microscopy, and immunoblotting. TMJOA models were established and , and hematoxylin-eosin staining, immunohistochemistry, and histological scoring were performed to analyze the histological changes in TMJOA cartilage tissues treated with BMSC-sEVs. The proliferation, migratory capacity, and cell cycle distribution of TMJOA cartilage cells treated with BMSC-sEVs were detected. Furthermore, the related mechanisms were studied by bioinformatic analysis, immunoblotting, and quantitative PCR, and they were further analyzed by knockdown and inhibitor techniques. The acquisition and identification of BMSC-sEVs were efficient and satisfactory. Compared with the osteoarthritis (OA) group, the condylar tissue of the OA group treated with BMSC-sEV (OA) showed an increase in cartilage lacuna and hypertrophic cartilage cells in the deep area of the bone under the cartilage. Significantly upregulated expression of proliferating cell nuclear antigen and cartilage-forming factors and downregulated expression of cartilage inflammation-related factors in OA were observed. In addition, we found higher rates of cell proliferation and migratory activity and alleviated G1 stagnation of the cell cycle of OA. Autotaxin was found in the BMSC-sEVs, and key factors of the Hippo pathway, Yes-associated protein (YAP), phosphorylated Yes-associated protein (p-YAP), etc. were upregulated in the OA group. Treatment with BMSC-sEVs after autotaxin knockdown or inhibition no longer resulted in expression changes in cartilage-forming and inflammation-related factors and key factors of the Hippo pathway. These results suggest that the autotaxin-YAP signaling axis plays an important role in the mechanism by which BMSC-sEVs promote cartilage reconstruction in TMJOA, which may provide guidance regarding their therapeutic applications as early and minimally invasive therapies for TMJOA, and provide insight into the internal mechanisms of TMJOA.
颞下颌关节骨关节炎（TMJOA）严重影响患者健康，目前的治疗具有侵入性，且仅用于晚期病例。骨髓间充质干细胞（BMSC）衍生的小细胞外囊泡（BMSC-sEVs）可能是一种更安全、更有效的治疗方法，但其在TMJOA中的作用尚未阐明。本研究试图分析BMSC-sEVs对TMJOA的软骨重建作用及其潜在机制。通过微滤和超滤分离并纯化BMSC-sEVs，随后通过纳米颗粒跟踪分析、电子显微镜和免疫印迹对其进行表征。建立TMJOA模型，并进行苏木精-伊红染色、免疫组织化学和组织学评分，以分析用BMSC-sEVs处理的TMJOA软骨组织的组织学变化。检测用BMSC-sEVs处理的TMJOA软骨细胞的增殖、迁移能力和细胞周期分布。此外，通过生物信息学分析、免疫印迹和定量PCR研究相关机制，并通过敲低和抑制剂技术进行进一步分析。BMSC-sEVs的获取和鉴定高效且令人满意。与骨关节炎（OA）组相比，用BMSC-sEV（OA）处理的OA组髁突组织软骨下骨深部区域的软骨陷窝和肥大软骨细胞增多。观察到OA中增殖细胞核抗原和软骨形成因子的表达显著上调，软骨炎症相关因子的表达下调。此外，我们发现OA的细胞增殖率和迁移活性更高，且细胞周期的G1停滞得到缓解。在BMSC-sEVs中发现了自分泌运动因子，OA组中Hippo通路的关键因子，如Yes相关蛋白（YAP）、磷酸化Yes相关蛋白（p-YAP）等上调。自分泌运动因子敲低或抑制后用BMSC-sEVs处理不再导致软骨形成和炎症相关因子以及Hippo通路关键因子的表达变化。这些结果表明，自分泌运动因子-YAP信号轴在BMSC-sEVs促进TMJOA软骨重建的机制中起重要作用，这可能为其作为TMJOA的早期和微创治疗的治疗应用提供指导，并深入了解TMJOA的内在机制。
