Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study

Abstract

Background: Although pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation.

Methods: BMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05.

Results: Biochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control.Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation.

Conclusions: PEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo.

Figure 1

Effect of PEMF stimulation on DNA amounts in two different cell types. A) human bone marrow derived stromal cells, obtained from 4 different donors (n = 3 per donor), B) human fetal pre-osteoblasts (n = 3). Cells were cultured in osteogenic medium to full mineralization. Significant differences due to PEMF are marked * (p < 0.05, Wilcoxon signed rank test).

Figure 2

Effect of PEMF stimulation on osteogenic differentiation of human BMSCs. Figure shows alkaline phosphatase activity (A) and calcium deposition in the extracellular matrix (B) of these cells. Cells were obtained from 4 differenent donors (n = 3 per donor). Significant differences due to PEMF are marked * (p < 0.05, Wilcoxon signed rank test).

Figure 3

Effect of PEMF on osteoblast-relavant genes in human BMSCs. Gene expression ratios of selected genes plotted as expression in PEMF-treated condition relative to non-treated control. Targeted genes are A) bone morphogenetic protein 2 (BMP-2); B) transforming growth factor beta 1 (TGF-β1); C) osteoprotegrin (OPG); D) receptor activator of NF-kappa-B ligand (RANKL); E) matrix metalloproteinase 1 (MMP-1); F) matrix metalloproteinase 3 (MMP-3). Significant differences due to PEMF are marked * (p < 0.05), n = 4.

Figure 4

Effect of PEMF on osteoblast-relavant genes in human BMSCs. Gene expression ratios of selected genes plotted as expression in PEMF-treated condition relative to non-treated control. Targeted genes are G) collagen 1 (COL1); H) osteocalcin (OC); I) bone sialoprotein 1 (IBSP); J) osteonectin (SPARC); K) osteopontin (SPP1). Significant differences due to PEMF are marked * (p < 0.05), n = 4.

Figure 5

Effect of PEMF on OPG/RANKL expression ratio in human BMSCs. Gene expression ratios of OPG/RANKL plotted as expression in PEMF-treated condition relative to non-treated control. Significant differences due to PEMF are marked * (p < 0.05), n = 4.

Figure 6

Western blot showing activated, phosphorylated ERK (ERK1/2-P) next to total ERK (ERK1/2) specific signals as loading control. Human fetal pre-osteoblasts (SV-HFO) and bone marrow stromal cells (BMSC) were cultured up to day 14 prior to PEMF treatment. PEMF exposure of 15 minutes was compared with control (0 minutes of PEMF).