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. 2020 Mar 19;21(6):2104.
doi: 10.3390/ijms21062104.

Bone Morphogenetic Protein-2 Signaling in the Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Induced by Pulsed Electromagnetic Fields

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Free PMC article

Bone Morphogenetic Protein-2 Signaling in the Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Induced by Pulsed Electromagnetic Fields

Fernanda Martini et al. Int J Mol Sci. .
Free PMC article

Abstract

Pulsed electromagnetic fields (PEMFs) are clinically used with beneficial effects in the treatment of bone fracture healing. This is due to PEMF ability to favor the osteogenic differentiation of mesenchymal stem cells (MSCs). Previous studies suggest that PEMFs enhance the osteogenic activity of bone morphogenetic protein-2 (BMP2) which is used in various therapeutic interventions. This study investigated the molecular events associated to the synergistic activity of PEMFs and BMP2 on osteogenic differentiation. To this aim, human MSCs (hMSCs) were exposed to PEMFs (75 Hz, 1.5 mT) in combination with BMP2, upon detection of the minimal dose able to induce differentiation. Changes in the expression of BMP signaling pathway genes including receptors and ligands, as well as in the phosphorylation of BMP downstream signaling proteins, such as SMAD1/5/8 and MAPK, were analyzed. Results showed the synergistic activity of PEMFs and BMP2 on osteogenic differentiation transcription factors and markers. The PEMF effects were associated to the increase in BMP2, BMP6, and BMP type I receptor gene expression, as well as SMAD1/5/8 and p38 MAPK activation. These results increase knowledge concerning the molecular events involved in PEMF stimulation showing that PEMFs favor hMSCs osteogenic differentiation by the modulation of BMP signaling components.

Keywords: BMP; mesenchymal stem cells; osteogenic differentiation; pulsed electromagnetic fields.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dose-response effects of BMP2 (1, 10, 50, 100 ng/mL) on mineralization (A,B) and OC production (C) in hMSCs cultured in OM at 28 days. Matrix mineralization evaluated by alizarin red staining in cell monolayers (A) and spectrophotometrically quantified (B), OC levels evaluated by Elisa (C). *: p ≤ 0.05 vs. OM. °: p ≤ 0.05 vs. the lower dose of BMP2. Scale bar = 250 μm; Magnification = 10×.
Figure 2
Figure 2
Effects of PEMFs and BMP2 (10 ng/mL) used alone or in combination on osteogenic transcription factors and biochemical markers during hMSCs osteogenic differentiation. (A) DLX5 and (B) RUNX2 gene expression by RT-qPCR at 3 days, (C) ALP activity at 14 days, and (D) OC production at 28 days. °: p ≤ 0.05 vs. control. *: p ≤ 0.05 vs. OM. #: p ≤ 0.05 vs. OM + PEMF. §: p ≤ 0.05 vs. OM + BMP2.
Figure 3
Figure 3
Effects of PEMFs and BMP2 (10 ng/mL) used alone or in combination on BMP2, BMP6, ALK2 gene expression, evaluated by RT-qPCR at different times points (3, 7, 14, 28 days) during hMSCs osteogenic differentiation. °: p ≤ 0.05 vs. control. *: p ≤ 0.05 vs. OM. #: p ≤ 0.05 vs. OM + PEMF. §: p ≤ 0.05 vs. OM + BMP2.
Figure 4
Figure 4
Effects of PEMFs and BMP2 (10 ng/mL) used alone or in combination on SMAD1/5/8, p38 MAPK, and ERK1/2 phosphorylation. (A) Cell lysates obtained in all the experimental conditions (OM, OM + PEMFs, OM + BMP2, OM + PEMFs + BMP2), electrophoresed and immunoblotted with phospho-SMAD1/5/8, phospho-p38, and phospho-ERK1/2 antibodies. β-actin antibody used to ensure equal sample loading. (BD) Graphical representation of densitometry and Western blotting quantitative data for each protein. Results expressed as mean ± standard error of the mean of three independent experiments. *: p ≤ 0.05 vs. OM. #: p ≤ 0.05 vs. OM + PEMFs. §: p ≤ 0.05 vs. OM + BMP2.
Figure 5
Figure 5
(A) Dose-response effects of DM on SMAD1/5/8 activation and (B) hMSCs viability in the presence of BMP2 (10 ng/mL). hMSCs cultured in OM + BMP2 and treated with increasing doses of DM (0, 5, 10, 20 μM). (A) Cell lysates electrophoresed and immunoblotted with phospho SMAD1/5/8 antibody. β-actin antibody used to ensure equivalent sample loading. (B) Graphical representation of densitometry and Western blotting quantitative data. (C) Cell viability evaluated at different time points (3, 7, 14, 28 days). *: p ≤ 0.05 vs. BMP2 at the corresponding time point.
Figure 6
Figure 6
Effects of DM (5 µM) and SB203580 (10 µM) on hMSCs differentiated (A) in OM + BMP2, (B) in OM + PEMFs, (C) in OM + PEMFs + BMP2. DLX5 and RUNX2 gene expression (3 days), ALP activity (14 days), and OC production (28 days) evaluated. *: p ≤ 0.05 vs. cells differentiated in the absence of inhibitor treatment. #: p ≤ 0.05 vs. cells differentiated in the presence of DM.

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