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. 2019 May 1:7:92.
doi: 10.3389/fbioe.2019.00092. eCollection 2019.

Osteoblast-Derived Vesicle Protein Content Is Temporally Regulated During Osteogenesis: Implications for Regenerative Therapies

Owen G Davies  1 , Sophie C Cox  2 , Ioannis Azoidis  2 , Adam J A McGuinness  3 , Megan Cooke  2   3 , Liam M Heaney  1 , Edward T Davis  4 , Simon W Jones  5 , Liam M Grover  2
Affiliations

Osteoblast-Derived Vesicle Protein Content Is Temporally Regulated During Osteogenesis: Implications for Regenerative Therapies

Owen G Davies et al. Front Bioeng Biotechnol. .

Erratum in

Abstract

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.

Keywords: annexin; collagen; mineralization; nano; osteoblast; vesicle.

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Figures

Figure 1
Figure 1
Characterization and differentiation of human primary osteoblasts derived from three separate donors toward a mineralizing phenotype over a period of 21 days. (A) Gene expression profiling of primary human osteoblasts showed upregulation of four osteogenic markers. (B) Differentiation of osteoblasts cultures in the presence of growth medium (GM) and mineralization medium (MM) containing 10 nM β-glycerophosphate and 50 μg/mL L-ascorbic acid over a period of 7, 14, and 21 days induced extracellular matrix mineralization, which was visualized using Alizarin red calcium stain. (C) Visual differences in mineralization observed using Alizarin red were confirmed semi-quantitatively. (D) The concentration of Alkaline phosphatase (ALP) was calculated per cell. Significant increases in ALP concentration were observed at days 7 and 14. Scale bars = 15 mm, *p ≤ 0.05. N = 3.
Figure 2
Figure 2
Comparison of sEVs released by mineralising primary osteoblasts derived from three separate donors into culture medium over a period of 21 days. Three repeats were performed for each donor. EVs were isolated during the first, second, and third week of differentiation and (A) total protein and (B) particle number quantified. (C) sEVs were visualized using TEM. Changes in sEV morphology were apparent, with visual indications of rupture evident during week 3. (D) Relative sEV diameter was assessed using dynamic light scattering (DLS) (E) Median peak intensities and weighted averages for sEVs isolated at each time point. Scale bars = 50 nm, *p ≤ 0.05. N = 3.
Figure 3
Figure 3
Analysis of differentially expressed proteins localized to osteoblast sEVs identified using LC-MS/MS. sEVs were isolated from three separate donors with three repeats performed for each donor. (A) Schematic representation of the relationship between sEVs in relation to the maturity and organization of the mineralized matrix. (B) Venn diagram depicting specific and non-specific proteins localized to sEVs isolated during 1, 2- and 3-weeks mineralization. (C) MS peak intensity plots comparing -Log10 MS-peak intensities of proteins associated with sEVs during weeks 1 (i), 2 (ii), and 3 (iii) of differentiation and their associated Pearson's correlation values. N = 3.
Figure 4
Figure 4
Comparative analysis of proteins localized to osteoblast sEVs during matrix mineralization. (A) Principal component analysis of total protein intensity. (B) Multiple orthogonal partial least squares-discriminant (OPLS-DA) analysis of between-group differences in MS protein intensity to identify differentially expressed proteins between sEVs isolated from the culture medium of mineralising osteoblasts at weeks 1, 2, and 3. Discriminatory proteins identified between (Bi) weeks 1 and 2 included 1, plectin; 2, galectin; CD59 glycoprotein; 4, aminopeptidase N; 5, chitinase-3-like protein 1; 6, collagen alpha-1 XVI chain; CD81 antigen; (Bii) weeks 2 and 3: 1, glia-derived nexin; 2, annexin A6; 3, ApoE; 4, protein transport protein Sec23A; 5, AP-2 complex subunit alpha-1/2; and (Biii) weeks 1 and 3: 1, plectin; 2, translational endoplasmic reticulum ATPase; 3, Annexin A6; 4, inactive serine protease PAMR1; 5, protein transport protein Sec23A; 6, reticulocalbin-3; 7, endoplasmin. (C) Volcano plots displaying Log2 values for protein fold-change against Log10 false discovery rate (FDR). Only proteins with a Log2 fold change of >1 and a p < 0.05 were considered to be statistically significant.
Figure 5
Figure 5
Gene ontology (GO) analysis of proteins identified as being differentially upregulated in sEVs isolated during the third week of osteoblast differentiation relative to those isolated at weeks 1 (dashed line) and 2 (block color). A total of 116 proteins were identified, with 87 and 99 proteins found to be differentially upregulated when compared with sEVs isolated at weeks 1 and 2, respectively. (A) Biological processes, (B) molecular mechanisms and (C) cellular component predicted for proteins identified at week 3.
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