Table_2_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.

成骨细胞来源的细胞外囊泡（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可以直接从成骨细胞培养介质中分离出来，其在再生医学领域的应用引起了越来越多的关注。然而，目前的治疗进展受到对它们在基质矿化过程中精确时间贡献缺乏理解的制约。本研究通过将sEV的谱系和蛋白质含量与矿化状态进行时间对齐，推动了当前知识的进步。sEVs在1周、2周和3周期间从矿化初级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 nm和164 nm；第2周：59 nm和220 nm），表明具有类似于外泌体（44 nm和59 nm）和微囊泡（164 nm和220 nm）粒子尺寸的异质群体。通过液相色谱串联质谱（LC-MS/MS）进行蛋白质组学分析揭示了随着矿化过程的进展，EV定位蛋白质与矿化之间的相关性逐渐下降，分别对应的皮尔逊相关系数为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）进一步突出了随着矿化过程的推进，蛋白质含量的时间依赖性变化。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，共116种囊泡定位蛋白质显著上调（其中56%非特异性，19%与第1周相比，25%与第2周相比）。对这些蛋白质进行的基因本体富集分析突出了与基质组织相关的基因过度表达。值得注意的是，磷脂结合和钙通道蛋白（A2、A5和A6）的存在增加，这些蛋白指示囊泡的成核能力随时间推移而逐渐变化，以及与周围细胞外基质（ECM）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的发展变化产生影响。这些发现对于EV的选择和应用在再生医学领域具有潜在意义。