Osteoblast-Derived Matrix Vesicles Exhibit Exosomal Traits and a Unique Subset of microRNA: Their Caveolae-Dependent Endocytosis Results in Reduced Osteogenic Differentiation

Abstract

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.

Do Media Extracellular Vesicles and Extracellular Vesicles Bound to the Extracellular Matrix Represent Distinct Types of Vesicles?

“…In contrast collagenase and trypsin digestion, which maintained a part of surface collagens on matrix vesicles, preserved the calcium uptake [91] (Table 1). The process of trypsin digestion resulted in the release of matrix vesicles from the extracellular matrix, exhibiting an alkaline phosphatase activity six times greater than that of the plasma membrane [93] (Table 1). Liberase and blendzyme1, which is gentle digestion, was the less efficient method to release matrix vesicles than the other digestion methods, as indicated by the lower ratio of alkaline phosphatase in matrix vesicles as compared to that in media vesicles [92] (Table 1).…”

“…Initially, CD9 was considered an exosomal marker but later it was proposed that CD9 could be linked to budding vesicles [145]. CD9 was found by proteomic analysis in matrix vesicles from MG-63 osteoblasts [93] and in matrix vesicles from the growth plate cartilage [88], while it was undetected in matrix vesicles from MC3T3-E1 cells [92] and Saos-2 cells [129]. Exosomal marker CD81 was also present in matrix vesicles from MG-63 osteoblasts [93] and Saos-2 cells [129] but it remained undetected in matrix vesicles from MC3T3-E1 [92] and the growth plate cartilage [94].…”

“…CD9 was found by proteomic analysis in matrix vesicles from MG-63 osteoblasts [93] and in matrix vesicles from the growth plate cartilage [88], while it was undetected in matrix vesicles from MC3T3-E1 cells [92] and Saos-2 cells [129]. Exosomal marker CD81 was also present in matrix vesicles from MG-63 osteoblasts [93] and Saos-2 cells [129] but it remained undetected in matrix vesicles from MC3T3-E1 [92] and the growth plate cartilage [94]. The apparent contradiction lies from the fact that while various types of extracellular vesicles contain several common markers, including tetraspanins, their relative proportions vary in the different vesicle types [146].…”

“…Alternatively, the development of more selective methods to fully characterize the subpopulations of media vesicles and of matrix vesicles are needed. Nevertheless, matrix vesicles appear to be distinct from media vesicles due to their distinct TNAP specific activity and different lipid and enzyme content [93]. Matrix vesicles and media vesicles serve a different role in bones due to their location within mature bone, as they are anchored to the extracellular matrix versus free-floating [93].…”

“…Nevertheless, matrix vesicles appear to be distinct from media vesicles due to their distinct TNAP specific activity and different lipid and enzyme content [93]. Matrix vesicles and media vesicles serve a different role in bones due to their location within mature bone, as they are anchored to the extracellular matrix versus free-floating [93]. Media vesicles can communicate with distant cells, while matrix vesicles remain close to the parent cells [93].…”