Bone marrow-derived mesenchymal stem cell (BMSC) transplantation is considered a promising therapeutic approach for bone defect repair. However, during the transplantation procedure, the functions and viability of BMSCs may be impaired due to extended durations of in vitro culture, aging, and disease conditions of patients. Inspired by spontaneous intercellular mitochondria transfer that naturally occurs within injured tissues to rescue cellular or tissue function, we investigated whether artificial mitochondria transfer into pre-transplant BMSCs in vitro could improve cellular function and enhance their therapeutic effects on bone defect repair in situ. Mitochondria were isolated from donor BMSCs and transferred into recipient BMSCs of the same batch and passage. Subsequently, changes in proliferative capacity and cell senescence were evaluated by live cell imaging, Cell Counting Kit-8 assay, cell cycle analysis, Ki67 staining, qPCR and Western blot analysis of c-Myc expression, and β-galactosidase staining. Migration ability was evaluated by the transwell migration assay, wound scratch healing, and cell motility tests. Alkaline phosphatase (ALP) staining, Alizarin Red staining, and combined with qPCR and Western blot analyses of Runx2 and BMP2 were performed to elucidate the effects of mitochondria transfer on the osteogenic potential of BMSCs in vitro. After that, in vivo experiments were performed by transplanting mitochondria-recipient BMSCs into a rat cranial critical-size bone defect model. Micro CT scanning and histological analysis were conducted at 4 and 8 weeks after transplantation to evaluate osteogenesis in situ. Finally, in order to establish the correlation between cellular behavioral changes and aerobic metabolism, OXPHOS (oxidative phosphorylation) and ATP production were assessed and inhibition of aerobic respiration by oligomycin was performed. Mitochondria-recipient BMSCs exhibited significantly enhanced proliferation and migration, and increased osteogenesis upon osteogenic induction. The in vivo results showed more new bone formation after transplantation of mitochondria-recipient BMSCs in situ. Increased OXPHOS activity and ATP production were observed, which upon inhibition by oligomycin attenuated the enhancement of proliferation, migration, and osteogenic differentiation induced by mitochondria transfer. Mitochondria transfer is a feasible technique to enhance BMSC function in vitro and promote bone defect repair in situ through the upregulation of aerobic metabolism. The results indicated that mitochondria transfer may be a novel promising technique for optimizing stem cell therapeutic function.

中文翻译：

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线粒体转移增强了骨髓间充质干细胞的增殖，迁移和成骨分化，并促进了骨缺损的愈合。

骨髓来源的间充质干细胞（BMSC）移植被认为是修复骨缺损的有前途的治疗方法。然而，在移植过程中，由于延长的体外培养持续时间，衰老和患者的疾病状况，BMSC的功能和生存能力可能受到损害。受自然发生在受伤组织中的自发细胞间线粒体转移的启发，以抢救细胞或组织功能，我们研究了人工线粒体在体外能否转移到移植前的BMSCs中，可以改善细胞功能并增强其对骨缺损修复的治疗效果。从供体BMSC中分离出线粒体，并转移到相同批次和传代的受体BMSC中。后来，通过活细胞成像，Cell Counting Kit-8分析，细胞周期分析，Ki67染色，c-Myc表达的qPCR和Western印迹分析以及β-半乳糖苷酶染色来评估增殖能力和细胞衰老的变化。通过transwell迁移测定，伤口划痕愈合和细胞运动测试评估迁移能力。进行碱性磷酸酶（ALP）染色，茜素红染色以及Runx2和BMP2的qPCR和Western印迹分析，以阐明线粒体转移对BMSCs体外成骨潜能的影响。此后，通过将线粒体受体BMSC移植到大鼠颅骨临界大小的骨缺损模型中，进行了体内实验。移植后第4和第8周进行Micro CT扫描和组织学分析，以评估原位成骨性。最后，为了建立细胞行为变化与有氧代谢之间的相关性，评估了OXPHOS（氧化磷酸化）和ATP的产生，并进行了寡霉素对有氧呼吸的抑制作用。线粒体受体BMSCs表现出明显增强的增殖和迁移，并在成骨诱导时增加了成骨作用。体内结果显示，线粒体受体BMSCs原位移植后，更多的新骨形成。观察到增加的OXPHOS活性和ATP产生，这在寡霉素抑制后减弱了线粒体转移诱导的增殖，迁移和成骨分化的增强。线粒体转移是一种可行的技术，可通过上调需氧代谢来增强体外BMSC功能并促进原位骨缺损修复。结果表明线粒体转移可能是优化干细胞治疗功能的一种新的有希望的技术。