PEMF Stimulation Mitochondrial Function Osteogenic Cells

Page | 003

www.nature.com/scientificreports/
Scientific Reports |
Figure 2. Electromagnetic field stimulation promotes bone-forming activity of osteogenic cells in vitro. Mouse osteogenic DM5 cells (a, b) or human BMSCs (c, d) were exposed to an electromagnetic field at 10 Gauss (G) or left untreated (0G control) in the absence or presence of antimycin A (AntA) at 0.1 mM. On day 4, cells were stained with Alizarin Red (ARed) to assess calcium deposition or with Crystal Violet (CrV) to assess proliferation (a, c). Plates were scanned and ARed and CrV staining intensities were measured with ImageJ software. ARed signal was normalized to CrV signal. Cells were also collected for RNA extraction and real- time RT-PCR analysis was performed for the indicated genes of interest normalized to B2m (b, d). Images are representatives of 4 (DM5) or 3 (human BMSC). Plots show actual data points and calculated means. P value was determined via the t-test.
Altogether, these results support the ability of EM field stimulation to upregulate mitochondrial function and OxPhos activity in osteogenic cells in vitro.
EM field stimulation promotes bone-forming activity of osteogenic cells in vitro. Previ- ous studies have established that upregulated mitochondrial function increases osteogenic potential in osteoprogenitors28,29,34,35. After determining in the previous experiment (Fig. 1) that EM field stimulation can upregulate mitochondrial OxPhos function, we next investigated whether EM field exposure enhances osteo- genesis and osteoblast function. Using Alizarin Red (ARed) staining that detects calcium deposition due to oste- oblast activity, we found that both DM5 cells and human BMSCs exposed to an EM field at 10G had significantly increased in vitro calcium deposition as compared to the nonexposed cells (Fig. 2a,c). Cellular proliferation for both cell types were not affected by EM field exposure, measured by crystal violet (CrV) staining. To confirm that the effect of EM field stimulation on osteogenic function was mediated via mitochondrial OxPhos and com- plex I activity induction, we first exposed cells in the presence of mitochondrial respiratory complex I inhibitor rotenone. However, rotenone, even at low concentrations, was toxic to both cell types (not shown). Therefore, we modified our approach and used a compound that affects the mitochondrial respiratory chain downstream from complex I, in particular, complex III inhibitor antimycin A (AntA). We observed that AntA reversed the osteoinductive effect of EM field exposure (Fig. 2a,c). In DM5 cells, mRNA expression of early osteoblast marker Ibsp was not significantly changed after 4 days of EM field treatment, while mRNA expression of late osteoblast
(2021) 11:19114 | https://doi.org/10.1038/s41598-021-98625-1 3 Vol.:(0123456789)