成骨人类 MSC 衍生的细胞外囊泡调节 MSC 活性和成骨分化，并在大鼠颅骨缺损模型中促进骨再生。

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

背景：越来越多的证据表明细胞外囊泡（EVs）在移植的人骨髓间充质干细胞（hMSC）的旁分泌机制中发挥着关键作用。然而，对于微环境刺激对 EVs 成骨作用的影响知之甚少。本研究旨在探讨未分化 hMSC 来源的 EVs（Naïve-EVs）和 hMSC 成骨早期（Osteo-EVs）的特性和功能。另一个目的是评估 Osteo-EVs 对大鼠颅骨缺损骨再生的成骨潜力。

方法：使用排阻色谱法分离两组 EVs，并通过粒径分布、形态、流式细胞术分析和蛋白质组谱进行表征。评估 EVs（10μg/ml）对培养的 hMSC 增殖、迁移和成骨分化的影响。将 Osteo-EVs（50μg）或无血清培养基（SFM，对照）与胶原膜支架（MEM）结合，修复雄性 Lewis 大鼠临界尺寸颅骨骨缺损，并使用 µCT、组织学和组织形态计量学评估疗效。

结果：尽管 Osteo- 和 Naïve-EVs 具有相似的特征，但蛋白质组学分析显示 Osteo-EVs 中富含与骨骼相关的蛋白质。两组均增强了培养的 hMSC 的增殖和迁移，但 Osteo-EVs 在促进体外成骨分化方面更有效，表现为成骨相关基因表达增加和钙沉积增加。在大鼠颅骨缺损中，与加载 SFM 的 MEM 相比，加载 Osteo-EVs 的 MEM 导致更大且更一致的骨再生。

结论：本研究使用不同方法揭示了来自未分化 hMSC 和 hMSC 成骨早期的 EVs 在蛋白质谱和功能效应上的差异。通过颅骨骨缺损模型进一步验证了 hMSC 成骨早期衍生的 EVs 的显著蛋白质谱和细胞功能，强调了使用分化的 MSC 来产生 EVs 进行骨治疗的重要性。