Table_1_Osteoblast-Derived Vesicle Protein Content Is Temporally Regulated During Osteogenesis: Implications for Regenerative Therapies.xlsx

Osteoblast-derived extracellular vesicles (EV) are a collection of secreted (sEVs) and matrix-bound nanoparticles that function as foci for mineral nucleation and accumulation. Due to the fact sEVs can be isolated directly from the culture medium of mineralizing osteoblasts, there is growing interest their application regenerative medicine. However, at present therapeutic advancements are hindered by a lack of understanding of their precise temporal contribution to matrix mineralization. This study advances current knowledge by temporally aligning sEV profile and protein content with mineralization status. sEVs were isolated from mineralizing primary osteoblasts over a period of 1, 2, and 3 weeks. Bimodal particle distributions were observed (weeks 1 and 3: 44 and 164 nm; week 2: 59 and 220 nm), indicating a heterogeneous population with dimensions characteristic of exosome- (44 and 59 nm) and microvesicle-like (164 and 220 nm) particles. Proteomic characterization by liquid chromatography tandem-mass spectrometry (LC-MS/MS) revealed a declining correlation in EV-localized proteins as mineralization advanced, with Pearson correlation-coefficients of 0.79 (week 1 vs. 2), 0.6 (2 vs. 3) and 0.46 (1 vs. 3), respectively. Principal component analysis (PCA) further highlighted a time-dependent divergence in protein content as mineralization advanced. The most significant variations were observed at week 3, with a significant (p < 0.05) decline in particle concentration, visual evidence of EV rupture and enhanced mineralization. A total of 116 vesicle-localized proteins were significantly upregulated at week 3 (56% non-specifically, 19% relative to week 1, 25% relative to week 2). Gene ontology enrichment analysis of these proteins highlighted overrepresentation of genes associated with matrix organization. Of note, increased presence of phospholipid-binding and calcium channeling annexin proteins (A2, A5, and A6) indicative of progressive variations in the nucleational capacity of vesicles, as well as interaction with the surrounding ECM. We demonstrate sEV-mediated mineralization is dynamic process with variations in vesicle morphology and protein content having a potential influence on developmental changes matrix organization. These findings have implications for the selection and application of EVs for regenerative applications.

成骨细胞衍生的细胞外囊泡（EV）是一组分泌的（sEVs）和基质结合的纳米颗粒，它们作为矿物核化和积累的焦点。鉴于sEVs可以直接从矿化成骨细胞的培养介质中分离出来，其在再生医学中的应用日益受到关注。然而，目前治疗进展受到对它们在基质矿化中精确时间贡献缺乏理解的阻碍。本研究通过将sEV特征和蛋白质含量与矿化状态进行时间对齐，推进了当前的知识。sEVs在1周、2周和3周期间从矿化原代成骨细胞中分离出来。观察到双峰粒子分布（第1周和第3周：44和164纳米；第2周：59和220纳米），表明存在一个具有外泌体（44和59纳米）和微泡样（164和220纳米）粒子特征的异质性群体。通过液相色谱串联质谱（LC-MS/MS）进行的蛋白质组学特征分析揭示了随着矿化进程的推进，EV定位蛋白质与矿化状态的关联性逐渐下降，相应的皮尔逊相关系数分别为0.79（第1周与第2周）、0.6（第2周与第3周）和0.46（第1周与第3周）。主成分分析（PCA）进一步强调了随着矿化进程的推进，蛋白质含量随时间依赖性的差异。在第3周观察到最显著的变化，粒子浓度显著下降（p < 0.05），这是EV破裂和增强矿化的视觉证据。在第3周，总共116个囊泡定位蛋白质显著上调（其中56%非特异性，19%相对于第1周，25%相对于第2周）。对这些蛋白质的基因本体富集分析突出了与基质组织相关基因的过度表达。值得注意的是，磷脂结合和钙通道结合的附蛋白（A2、A5和A6）的增加，这表明囊泡核化能力的渐进性变化以及与周围细胞外基质的相互作用。我们证明sEV介导的矿化是一个动态过程，囊泡形态和蛋白质含量的变化可能对基质组织发育变化产生影响。这些发现对EV的选择和应用以及再生医学应用具有重要意义。