Journal of Nutritional Biochemistry Milk extracellular vesicles accelerate osteoblastogenesis but impair bone matrix formation  Marina C. Oliveira, Onno J. Arntz, Esmeralda N. Blaney Davidson, Peter L.E.M. van Lent, Marije I. Koenders, Peter M. van der Kraan, Wim B. van den Berg, Adaliene V.M. Ferreira, Fons A.J. van de Loo  Journal of Nutritional Biochemistry  Volume 30, Pages 74-84 (April 2016) DOI: 10.1016/j.jnutbio.2015.11.017 Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 1 Mice treated with BMEVs in two different concentrations (4.7×106/mL (low) or 14.3×106/mL (high)) by oral delivery in the drinking water during 7weeks. (A) The percentage of trabecular bone and (B) number of osteocytes in the proximal epiphysis and metaphysis of the tibia by hematoxylin–eosin staining. Quantification of (C) lamellar bone (mauve) and woven bone (mauve-blue) by Alcian Blue-Hematoxylin/Acid Fuchsin-Eosin staining. (D) Percentage of adipocytes area in the bone marrow. (E) Representative histology of proximal tibia in each group. Bars represent the mean±S.E.M. (n=5–7). ⁎⁎P<.01, ⁎⁎⁎P<.001 vs. PBS group. Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 2 Uptake of bovine derived-milk extracellular vesicles (BMEVs) by human mesenchymal stem cells (hMSC), dose finding of BMEVs and effect of BMEVs treatment on hMSC differentiated into osteoblast for 21days. (A) hMSCs uptake of BMEVs labeled with PKH67 dye (green) after 24h of treatment at 37°C or 4°C (control). (B) Representative pictures of cells stained with Alizarin red after BMEVs treatment (200μg/ml) (25×). Quantification of (C) mineralization by Alizarin red staining and (D) cell proliferation by crystal violet staining in hMSCs that received different concentrations of BMEVs (2, 20 and 200μg/ml). (E) XTT assay measured after 4, 8 and 24h of substrate addition. Collagen production is represented by (F) photomicrographs of Picrosirius Red/Fast green staining (100×), (G) ratio of collagen/cells determined by Picrosirius red/crystal violet staining and (H) mRNA expression of col1α1 and col1α2. (I) Alkaline phosphatase enzymatic activity. (J) mRNA expression of RUNX2, OSX, ALP, OC, OPN, RANKL, OPG, TGF-Β1, TGF-Β2, BMP-2, FGF-2, WISP-1 and AXIN2 of hMSCs in the presence of osteogenic medium and BMEVs (200μg/ml) (n=4). Bars represent the mean±S.E.M. ⁎P<.05, ⁎⁎P<.01, ⁎⁎⁎P<.001 vs. control. Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 3 Kinetic effect of bovine derived-milk extracellular vesicles (BMEVs) on osteoblast differentiation. Data represent BMEVs (200μg/ml) treatment on human mesenchymal stem cells (hMSCs) for 4, 7, 14, 21 and 28days in the presence of osteoblast differentiation medium. (A) Representative pictures (25×), (B) quantification of mineralization and (C) number of mineralized nodules/well by Alizarin red staining. (D) Quantification of cell proliferation by crystal violet staining. (E) The ratio of collagen/cells at days 21 and 28 determined by Picrosirius red/crystal violet staining. Expression of osteogenic genes (F) SOST, (G) RUNX2, (H) COL1A1, (I) COL1A2, (J) osteocalcin, (K) osteopontin, (L) FGF-2 and (M) WISP-1 in hMSCs induced to differentiate into osteoblast (n=4). Bars represent the mean±S.E.M. ⁎P<.05, ⁎⁎P<.01, ⁎⁎⁎P<.001 vs. control of the respective time. Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 4 The effect of duration by removing bovine derived-milk extracellular vesicles (BMEVs) from osteoblast differentiation medium. Data represent time combinations of treatments with BMEVs (200μg/ml) on hMSCs finishing at day 28. BMEVs were added at day 0 and replaced by only osteogenic medium at the times indicated: day 4, 7, 14 and 21. The control (osteogenic medium) and BMEVs D28 groups were maintained with the same medium composition during all period of treatment. (A) Representative pictures of cells stained by Alizarin red (4× and 25×). Quantification of (B) mineralization by Alizarin red staining and (C) cell proliferation by crystal violet staining. (D) Ratio between collagen and cells determined by Picrosirius red/crystal violet staining. (E) Cells mRNA expression of RUNX2, osteocalcin, collagen 1α1, osteopontin, FGF-2 and WISP-1 (n=4). Bars represent the mean±S.E.M. ⁎P<.05, ⁎⁎P<.01, ⁎⁎⁎P<.001 vs. control. Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 5 The effect of duration by adding bovine derived-milk extracellular vesicles (BMEVs) on osteoblast differentiation medium. Data represent time combinations of treatments with BMEVs (200μg/ml) on hMSCs finishing at day 28. Osteogenic medium was initiated at day 0, and BMEVs were added at the times indicated: day 4, 7, 14 and 21. The control (osteogenic medium) and BMEVs D0 groups were maintained with the same medium composition during all period of treatment. (A) Representative pictures of cells stained with Alizarin red (4× and 25×). Quantification of (B) mineralized by Alizarin red staining and (C) cell proliferation by crystal violet staining. (D) Ratio between collagen and cells determined by Picrosirius red/crystal violet staining. (E) Cells mRNA expression of RUNX2, osteocalcin, collagen 1α1, osteopontin, FGF-2 and WISP-1 (n=4). Bars represent the mean±S.E.M. ⁎P<.05, ⁎⁎P<.01, ⁎⁎⁎P<.001 vs. control. Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 6 Effect of bovine derived-milk extracellular vesicles (BMEVs) on osteoblast differentiation. BMEVs contributed with the increase in FGF-2 and WISP-1 expression since the beginning of treatment that may maintain the higher expression of RUNX2 in the cells. This effect leads to the predominance of characteristics related to immature osteoblast phase at day 21. Immature osteoblasts have a higher proliferation rate and may express osteopontin and RANKL, leading to immature bone formation and increase in bone turnover. At day 28 is observed an increase in the expression of osteocalcin, which is a marker for mature osteoblast and mineralization, but a reduction in collagen type I production and osteonectin expression. In addition, the increased expression of sclerostin by BMEVs at the same time point may be indicative for osteocytes' presence that appears to be differentiated faster than the control group. The sclerostin may contribute to Wnt signaling inhibition, subsequent osteoclast activation and cessation of mineralization. Altogether, these processes may contribute to the less quality of bone matrix formation. Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Supplementary Fig. 1. Human fetal bone marrow-derived mesenchymal stem cells (hMSCs) treated with bovine milk-derived extracellular vesicles (BMEVs), boiled BMEVs (15min at 105°C) or latex beads during 4days. All of treated groups received the particle concentration of 200μg/ml. mRNA expression by qRT-PCR in the cells for (A) Runx2, (B) FGF-2 and (C) Collagen1α1 genes. Bars represent the mean±S.E.M. ⁎⁎P<.01, ⁎⁎⁎P<.001 vs. control; #P<.05, ##P<.01, ###P<.001 vs. BMEVs. Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Supplementary Fig. 2. miRNA content on bovine derived-milk extracellular vesicles (BMEVs). Detection of miRNA content on BMEVs for miR-29a, −29b, −29c and miR-92a (control) by StepOne RT-PCR system (A) or visualized on an agarose ethidium-bromide gel (B). Journal of Nutritional Biochemistry 2016 30, 74-84DOI: (10.1016/j.jnutbio.2015.11.017) Copyright © 2016 Elsevier Inc. Terms and Conditions