Both ALP and TRAP activity increase upon exposure to more than 10 mT of ELF-EMFs in the goldfish scale. (A) Schematic diagram of the experimental method. Extracted goldfish scales were unexposed (u.e.) or exposed to ELF-EMFs at varying intensities (3, 5, 8, 10, 20, or 30 mT) for 24 h, followed by measurement of ALP or TRAP activity in each goldfish scale. (B,C) Boxplots show the relative values of ALP (B) and TRAP activity (C) with the mean value in the u.e. scale as 1. “+” within the boxplot denotes the mean value. A total of 56–100 goldfish scales from 7 to 10 goldfish were used in each condition. Asterisks indicate the p-value in one-way ANOVA with Dunnett’s test vs. u.e. group. **p < 0.01, ***p < 0.001.

Both osteoblast and osteoclast activity increase upon exposure to 10 mT of ELF-EMFs in the zebrafish fractured scale. (A) Schematic diagram of the experimental method. After fracture stimulation, zebrafish were placed in a ring-shaped tank and unexposed (u.e.) or exposed to ELF-EMFs with varying intensities (3, 10, or 30 mT) for 4 h, followed by imaging of zebrafish scales by confocal microscopy. (B) Representative images of fractured scales in osterix:mCherry; trap:GFP double-transgenic zebrafish unexposed or exposed to ELF-EMFs at 24 h.p.fr. White dotted lines and solid lines indicate the fracture site and contour of the zebrafish scale, respectively. Lower panels of trap:GFP show a high magnification view of the fracture site (boxed region). Bars, 200 μm (osterix:mCherry and upper panels of trap:GFP); 100 μm (lower panels of trap:GFP). (C,D) Mean fluorescent intensity of osterix:mCherry per zebrafish scale and percentage of trap:GFP⁺ area (coverage area) per zebrafish scale in fractured scales from zebrafish unexposed or exposed to ELF-EMFs. A total of 17–20 scales from 3 zebrafish were used in each condition. Asterisks indicate the p-value in one-way ANOVA with Dunnett’s test. Error bars, s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001.

Eddy currents generated outside the body have no effect on osteoblast or osteoclast activity. (A,B) Representative images of fractured scales in osterix:mCherry; trap:GFP double-transgenic zebrafish exposed to 10 or 30 mT ELF-EMFs in 0.5% NaCl at 24 h.p.fr. White dotted lines and solid lines indicate the fracture site and contour of the zebrafish scale, respectively. Lower panels of trap:GFP show a high magnification view of the fracture site (boxed region). Bars, 200 μm (osterix:mCherry and upper panels of trap:GFP); 100 μm (lower panels of trap:GFP). (C,D) Mean fluorescent intensity of osterix:mCherry per scale and percentage of trap:GFP⁺ area (coverage area) per zebrafish scale in fractured scales from zebrafish unexposed or exposed to 10 or 30 mT ELF-EMFs in freshwater (“Fresh”) or 0.5% NaCl (“NaCl”). The data in fresh water are used from Figures 2C,D. A total of 12–18 scales from 3 zebrafish were used in each condition. Asterisks indicate the p-value in one-way ANOVA with Dunnett’s test. Error bars, s.e.m.; **p < 0.01, ***p < 0.001, n.s., not significant.

Both osteoblast and osteoclast numbers increase upon exposure to 10 mT of ELF-EMFs in the fractured scale. (A) Representative results of flow cytometric analysis of cells from intact scales (left panel), unexposed (u.e.) fractured scales (middle panel), or fractured scales exposed to 10 mT ELF-EMFs (right panel) at 24 h.p.fr. After fracture stimulation, zebrafish were placed in the ring-shaped tank and were unexposed or exposed to 10 mT ELF-EMFs for 4 h. Zebrafish scales were extracted at 24 h.p.fr., and cell samples were prepared for flow cytometric analysis. Red and green gates show trap:GFP^–osterix:mCherry⁺ (“mCh⁺”) and trap:GFP^high (“GFP^high”) cells, respectively. (B,C) Percentage (B) and absolute number of mCh⁺ and GFP^high cells per scale (C) in intact scales, unexposed fractured scales (u.e. fractured), or fractured scales exposed to 10 mT ELF-EMFs (10 mT fractured). A total of 12 samples from 6 zebrafish were used in each condition. Asterisks indicate the p-value in one-way ANOVA with Dunnett’s test. Error bars, s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001.

Exposure to 10 mT of ELF-EMFs enhances Wnt and Notch signaling in the fractured scale. (A,B) Relative gene expression of osteoblast markers (alpl, col1a1a, and rankl) and osteoclast markers (trap, nfatc, and rank) in intact scales, unexposed fractured scales (u.e. fractured), or fractured scales exposed 10 mT ELF-EMFs (10 mT fractured) at 24 h.p.fr. (A) and signaling molecules for osteoblasts and/or osteoclasts in intact scales, u.e. fractured scales, or 10 mT fractured scales at 4 h.p.fr. (B). Relative expression levels were calculated by ΔΔCt method with the reference gene of ef1a. Asterisks indicate the p-value in one-way ANOVA with Dunnett’s test. Error bars, s.e.m. (n = 5 for each). *p < 0.05, **p < 0.01, ***p < 0.001. (C) Gene set enrichment analysis (GSEA) of Wnt target genes (upper panel) and Notch target genes (lower panel) in fractured scales unexposed or exposed to 10 mT ELF-EMFs at 24 h.p.fr. (n = 3 for each) (D) Hierarchical clustering of selected Wnt target genes in intact scales (Int_1–3), u.e. fractured scales (u.e._1–3), or 10 mT fractured scales (10 mT_1–3) at 24 h.p.fr. (n = 3 for each).

Expression of β-catenin is increased at the fracture site upon exposure to 10 mT of ELF-EMFs. (A) Expression of β-catenin in the fractured scale unexposed (u.e.) or exposed to 10 mT ELF-EMFs at 4 h.p.fr. After fracture stimulation, zebrafish were placed in the ring-shaped tank and were unexposed or exposed to 10 mT ELF-EMFs for 4 h. Zebrafish scales were then stained with rabbit anti-β-catenin antibody, followed by anti-rabbit IgG Alexa Fluor 647-conjugated secondary antibody and Hoechst 33342. White dotted lines and solid lines indicate the fracture site and contour of the zebrafish scale, respectively. Right panels show a high magnification view of the fracture site. Bars, 200 μm (left panels) and 40 μm (right panels). (B) Expression of β-catenin in cells from fractured scales unexposed or exposed to 10 mT ELF-EMFs at 4 h.p.fr. After fracture stimulation, zebrafish were placed in the ring-shaped tank and were unexposed or exposed to 10 mT ELF-EMFs for 4 h. Cells were then collected from zebrafish scales, smeared, and stained with rabbit anti-β-catenin antibody, followed by anti-rabbit IgG Alexa Fluor 647-conjugated secondary antibody and Hoechst 33342. Bottom panels show Hoechst (nuclei), β-catenin expression, and merged images of the dotted region. Arrowheads indicate the nucleus. Bars, 10 μm. (C,D) Percentage of β-catenin (+) cells within total cells and percentage of nuclear β-catenin (+) cells within total β-catenin (+) cells in the u.e. fractured scale or the fractured scale exposed to 10 mT ELF-EMFs at 4 h.p.fr. A total of 6 samples from 3 zebrafish were used in each condition. Asterisks indicate the p-value in unpaired two-tailed Student’s t-test. Error bars, s.e.m.; *p < 0.05.; ***p < 0.001.

Inhibition of Wnt/β-catenin signaling reduces both osteoblasts and osteoclasts in the ELF-EMF exposed scale. (A) Schematic diagram of the experimental method. After fracture stimulation, zebrafish scales were extracted, placed in a tube containing conditioned medium supplemented with DMSO or IWR-1-endo (20 μM), and unexposed or exposed to 10 mT ELF-EMFs for 4 h. Cells were then collected from the zebrafish scales at 24 h.p.fr. and analyzed by flow cytometry. (B) Representative results of flow cytometric analysis of cells from fractured scales unexposed or exposed to 10 mT ELF-EMFs in the presence of DMSO or IWR-1-endo at 24 h.p.fr. Red and green gates show trap:GFP^–osterix:mCherry⁺ (“mCh⁺”) and trap:GFP^high (“GFP^high”) cells, respectively. (C,D) Percentage (C) and absolute number of mCh⁺ and GFP^high cells per zebrafish scale (D) in fractured scales unexposed or exposed to 10 mT ELF-EMFs in the presence of DMSO or IWR-1-endo. A total of 10 samples from 5 zebrafish were used in each condition. Asterisks indicate the p-value in one-way ANOVA with Dunnett’s test. Error bars, s.e.m. (E) Representative images of fractured scales in osterix:mCherry; trap:GFP double-transgenic zebrafish exposed to 10 mT ELF-EMFs in the presence of DMSO or IWR-1-endo at 24 h.p.fr. White dotted lines and solid lines indicate the fracture site and contour of the zebrafish scale, respectively. Insets of trap:GFP show a high magnification view of the fracture site (boxed region). Bars, 200 μm (osterix:mCherry and trap:GFP); 100 μm (insets of trap:GFP). (F) Relative gene expression of an osteoblast marker (col1a1a), osteoclast marker (nfatc), and Wnt target genes (fos1b, ccn1, and mmp9) in the intact DMSO-treated, unexposed fractured DMSO-treated, 10 mT fractured DMSO-treated, and 10 mT fractured IWR-1-endo-treated scale at 24 h.p.fr. Asterisks indicate the p-value in one-way ANOVA with Dunnett’s test. Error bars, s.e.m. (n = 6 for each). *p < 0.05, **p < 0.01, ***p < 0.001.