Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA.
Department of Biomedical Engineering, College of Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA.
Int J Mol Sci. 2023 Aug 14;24(16):12770. doi: 10.3390/ijms241612770.
Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-β-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.
基质小泡 (MVs) 是锚定在细胞外基质 (ECM) 中的纳米级细胞外囊泡。除了在生物矿化中发挥作用外，最近还提出成骨细胞衍生的 MVs 具有调节作用。本研究的目的是确定成骨细胞衍生的 MVs 在细胞外囊泡（如外泌体）中的特征，评估其在调节成骨细胞分化中的作用，并研究其摄取机制。MVs 是从 MG63 人成骨样细胞培养物的 ECM 中分离出来的，并通过酶活性、透射电子显微镜、纳米颗粒跟踪分析、Western blot 和小 RNA 测序进行了表征。用两种不同培养条件（生长培养基 [GM]；成骨培养基 [OM]）的 MV 处理成骨细胞，以评估它们对分化和炎症标志物产生的影响，以及对巨噬细胞极化的影响。使用亲脂性荧光染料和共焦显微镜评估 MV 内吞作用，并使用甲基-β-环糊精确定 caveolae 的作用。与质膜相比，MVs 碱性磷酸酶特异性活性增加了四倍；直径为 50-150nm；具有外泌体标记物 CD63、CD81 和 CD9 以及内体标记物 ALIX、TSG101 和 HSP70；并选择性富集与抗成骨作用和 M2 巨噬细胞极化相关的 microRNA。用 GM 或 OM 的 MV 处理会降低成骨细胞的分化。成骨细胞通过涉及 caveolae 的机制内化 MV。这些结果支持了成骨细胞产生参与调节成骨作用的 MV 的假说。
