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.

Keywords: caveolin; differentiation; endocytosis; extracellular vesicles; matrix vesicles; microRNA; osteoblasts; osteogenesis.

Figure 1

Matrix vesicles display extracellular vesicle characteristics and demonstrate uptake by osteoblast-like cells. Quantification of alkaline phosphatase specific activity for cell pellet lysate (CP), plasma membrane (PM), and matrix vesicle (MV) isolates (A); transmission electron microscopy at 50 K magnification of MV demonstrated circular vesicles with a bilaminar membrane ((B), scale bar = 100 nm); TEM of green inset in B at 100 K magnification ((C), scale bar = 50 nm); size distribution of osteoblast-derived MVs using nanoparticle tracking analysis with median of 105 nm (green line), mean of 115 nm (red line) and standard deviation of ±85 nm (blue dashed lines); the results are mean of three independent measurements (D); Western blot analysis of protein expression in CP, PM, and MV isolates (E); osteoblasts treated with PKH26-stained MVs (red fluorescence), DRAQ5 nuclear counterstain (blue fluorescence) and plasma membrane counterstain (yellow fluorescence) using confocal microscopy ((F), scale bar = 50 µm); region of interest to highlight PKH26-stained MVs surrounding osteoblast nuclei ((G), scale bar = 10 µm); NaCl control-treated osteoblasts with DRAQ5 nuclear counterstain and plasma membrane counterstain ((H), scale bar = 50 µm); quantification of cells with a minimum of one spot of MV fluorescence between MV and NaCl control images (I). Results are expressed as the mean ± SEM of 2 independent reviewers. Groups not sharing a letter are considered statistically different at α = 0.05 by one-way ANOVA with Tukey post hoc test. Group with * is significantly different from the vehicle at an α = 0.05 using unpaired t-test.

Figure 2

Small RNA sequencing suggests selective packaging of microRNA into MG63 matrix vesicles. Principal component analysis plot of components 1 and 2 for all six samples with grouping of cell lysate (CP) samples (pink) and matrix vesicle (MV) samples (teal) (A); Venn diagram displaying overlap of unique microRNA with differential expression between CP and MV samples (B); heatmap of sample z-scores clustered with Euclidean distance measure (C); volcano plot of microRNA with Log2 fold change greater than ±2 and adjusted p-value less than 0.05 (D).

Figure 3

Differentially expressed microRNA of matrix vesicles suggest an anti-osteogenic role in osteoblasts and regenerative role in macrophages. Differentially expressed microRNA (adjusted p-value < 0.05 and absolute Log2 fold change >2) with pro-, anti-, or unclear osteogenic potential based on the literature search (A); differentially expressed microRNA with promotion of M1 or M2 macrophage polarization according to literature search (B).

Figure 4

Treatment of MG63 cells with matrix vesicles decreases markers of differentiation. Quantification of DNA (A), alkaline phosphatase (B), osteocalcin (C), osteopontin (D), vascular endothelial growth factor (E), interleukin-6 (F), and interleukin-10 (G) in MG63 cells treated for 24 or 48 h with or without 5 µg/mL matrix vesicles (MV) harvested from MG63s grown in growth media until 24 h after confluence; relative quantification of messenger RNA (mRNA) expression of BGLAP (H), RUNX2 (I), and Osterix/SP7 (J) in MG63 cells treated for 24 or 48 h with or without 5 µg/mL matrix vesicles (MV) from the same conditions. mRNA was quantified via RT-PCR and normalized to GAPDH expression. Data are from a representative experiment and are shown as mean ± SEM of n = 6 per group. Groups not sharing a letter are considered statistically different at α = 0.05 using one-way ANOVA with Tukey post-hoc test.

Figure 5

Matrix vesicle composition is sensitive to culture conditions. Quantification of DNA (A), alkaline phosphatase (B), osteocalcin (C), osteopontin (D), vascular endothelial growth factor (E), interleukin-6 (F), and interleukin-10 (G) in MG63 cells treated for 24 or 48 h with or without 5 µg/mL matrix vesicles (MV) harvested from MG63s grown in osteogenic media for 10 days. Data are from a representative experiment and are shown as mean ± SEM of n = 6 per group. Groups not sharing a letter are considered statistically different at α = 0.05 using one-way ANOVA with Tukey post-hoc test.

Figure 6

MG63 exosomes display characteristics and treatment effects different from matrix vesicles. Quantification of alkaline phosphatase specific activity for cell pellet lysate (CP) and exosome (EX) isolates (A); Size distribution of MG63-derived EXs using nanoparticle tracking analysis with median of 42 nm (green line), mean of 59 nm (red line) and standard deviation of ±44.6 nm (blue dashed lines), results are mean of three independent measurements (B); Western blot analysis of protein expression in CP and EX isolates (C); Quantification of DNA (D), alkaline phosphatase (E), and osteocalcin (F) in MG63 cells treated for 24 or 48 h with 5 µg/mL matrix vesicles (MV), EX, or saline control. MVs and EXs were harvested from the trypsinized cell layer or conditioned media, respectively, of MG63s grown in growth medium until 24 h after confluence. Data are from a representative experiment and are shown as mean ± SEM of n = 6 per group. Group with * is significantly different from the vehicle at an α = 0.05 by unpaired t-test. Groups not sharing a letter are considered statistically different at α = 0.05 using one-way ANOVA with Tukey post-hoc test.

Figure 7

Inhibition of caveolae-mediated endocytosis prevents uptake and effect of matrix vesicles on osteoblast-like cells. MG63 cells pre-treated with 10 mM vehicle (A) or methyl-β-cyclodextrin (β-CD; (B)) followed by treatment with PKH26-stained MVs (red fluorescence), DRAQ5 nuclear counterstain (blue fluorescence) and plasma membrane counterstain (yellow fluorescence) using confocal microscopy (scale bar = 50 µm); quantification of cells with a minimum of one spot of MV fluorescence between vehicle and β-CD images (C); quantification of DNA (D) and alkaline phosphatase-specific activity (E) in MG63 cells pre-treated with increasing concentrations of β-CD followed by treatment for 48 h with or without 5 µg/mL matrix vesicles (MV) harvested from MG63s grown in growth media until 24 h after confluence. Results are expressed as the mean ± SEM of 2 independent reviewers. Group with * is significantly different from the vehicle at an α = 0.05 by unpaired t-test. Groups not sharing a letter are considered statistically different at α = 0.05 by one-way ANOVA with Tukey post-hoc test.

Figure 8

Schematic representation of osteoblast-derived matrix vesicles. Matrix vesicles (MVs), with their protein and microRNA (miRNA) enrichment, are produced by osteoblasts and become embedded in the mineralized extracellular matrix. During osteoclast-mediated resorption of bone, MVs are released from the mineralized matrix to act in an autocrine and/or paracrine fashion on nearby osteoblasts. These MVs are taken up by osteoblasts via caveolin-mediated endocytosis, where they are able to release their miRNA cargo intracellularly. miRNAs interact with target messenger RNA (mRNA) to active or inhibit translation into proteins, slowing production of osteoblastic differentiation markers. Image created with BioRender.com.