Synergistic effect of electromagnetic fields and nanomagnetic particles on osteogenesis through calcium channels and p‐ERK signaling

Abstract Electromagnetic fields (EMFs) are widely used in a number of cell therapies and bone disorder treatments, and nanomagnetic particles (NMPs) also promote cell activity. In this study, we investigated the synergistic effects of EMFs and NMPs on the osteogenesis of the human Saos‐2 osteoblast cell line and in a rat calvarial defect model. The Saos‐2 cells and critical‐size calvarial defects of the rats were exposed to EMF (1 mT, 45 Hz, 8 h/day) with or without Fe3O4 NMPs. Biocompatibility was evaluated with MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) assays. This analysis showed that NMP and EMF did not induce cell toxicity. Quantitative reverse‐transcription polymerase chain reaction indicated that the osteogenesis‐related markers were highly expressed in the NMP‐incorporated Saos‐2 cells after exposure to EMF. Also, the expression of gene‐encoding proteins involved in calcium channels was activated and the calcium concentration of the NMP‐incorporated + EMF‐exposed group was increased compared with the control group. In particular, in the NMP‐incorporated + EMF‐exposed group, all osteogenic proteins were more abundantly expressed than in the control group. This indicated that the NMP incorporation + EMF exposure induced a signaling pathway through activation of p‐ERK and calcium channels. Also, in vivo evaluation revealed that rat calvarial defects treated with EMFs and NMPs had good regeneration results with new bone formation and increased mineral density after 6 weeks. Altogether, these results suggest that NMP treatment or EMF exposure of Saos‐2 cells can increase osteogenic activity and NMP incorporation following EMF exposure which is synergistically efficient for osteogenesis.


| INTRODUCTION
Electromagnetic fields (EMFs) have been widely used in the stimulation of wound healing and for relieving pain. Numerous attempts have been made to evaluate the effects of EMFs on cellular activity and proliferation. 1,2 Especially, electromagnetic stimulation has been studied for applications in treating bone disorders and regenerating bone. Several studies have reported that EMFs increase the proliferation of human osteoblasts and osteosarcoma cell lines in vitro. [3][4][5] Also, it has been reported that exposure to various EMFs (7.5-75 Hz) plays a modulatory role in the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells with an increase in alkaline phosphatase and osteogenesis-related genes. [6][7][8] EMFs have been reported to stimulate healing in disconnected fractures of the tibia, induce bone repair, 9 and to reduce the healing time following fresh fractures. 10 This knowledge is based on the discovery of the electromechanical properties of bone, 11,12 which raised the possibility that electric energy may stimulate bone formation and modify the behavior of bone cells. 13,14 Also, nanoparticles (NPs) have been widely used in biomedical applications such as drug delivery, biological labels, and the detection of proteins, and many studies have shown that NPs promote the migration and differentiation of cells, thereby inducing stem cell differentiation and stimulating wound healing. [15][16][17] Kim et al. 18 showed that Fe 3 O 4 nanomagnetic particles (NMPs) could affect cell-substrate interactions and enhance neurite outgrowth of PC12 cells. 17 Especially, surface-modified NMPs are expected to increase their circulation time, aqueous solubility, biocompatibility, and nonspecific cellular uptake and to decrease immunogenicity. 19 Zhang et al. 20 reported that PEGylated MNPs not only facilitated cellular uptake into cancer cells but also increased the yield of cell internalization. Additionally, it has been reported that PEGylated NMP increased the wound-healing effect upon incorporation into human bone marrow-derived mesenchymal stem cells in injured rat spinal cord. 21 Recently, synergistic effect studies on cell proliferation and differentiation have been reported using combinations of magnetic NPs and magnetic fields. Researchers manufactured porous hydroxyapatite, poly(ε-caprolactone) (PCL), and polylactic acid scaffolds containing or coated with iron oxide magnetic NPs (20-40 nm). Then, various cells were inoculated on the scaffold, and exposed to a magnetic field, followed by an evaluation of osteogenesis. The results of these studies showed that osteogenesis efficacy was increased in the scaffolds with a magnetic field relative to the only-scaffolds groups. [22][23][24] Also, another study adhered osteoblasts on RGD-coated magnetic (4.5 µm ferromagnetic) particles and then exposed them to a magnetic field, thus applying direct mechanical stimulation to the cells (B max approximately 60 mT). Their Von Kossa staining was strong and their messenger RNA (mRNA) expression levels of osteopontin were high. 25 However, all of the previous studies evaluated the effect of cell adhesion on NMPs, not the phagocytosis efficacy of NP into the cytosol. We wanted to study the bone regeneration effect after the NMPs were injected directly into the bone defect area, following which the NMPs would be phagocytosed into the surrounding cells, but this method proved to be impossible. Therefore, we manufactured rapidly degradable collagen sponges and inoculated the NMPs just before transplantation because we wanted them to be taken up by the cells.
In the present study, we investigated the synergistic effects of EMF and Fe 3 O 4 NMP treatment on osteogenic activity. We applied EMFs and NMPs to the Saos-2 osteoblast cell line and a rat calvarial defect model. The Saos2 cells and the rat calvarial defect model were treated with 50 μg/ml of Fe 3 O 4 MPs or exposed to a frequency of 45 Hz at an intensity of 1-mT EMF for 8 h/day and examined whether treatment with Fe 3 O 4 NMPs in conjunction with exposure to EMFs is more effective in enhancing osteogenic activity.

| EMF exposure
In this study, continuous sinusoidal EMFs (B m = 1 mT, F = 45 Hz sinusoidal) were used for the experiments (Figure 1). All experimental groups were kept in a cell culture incubator at 37 ± 0.1°C and 5% CO 2 concentration.
The black product was redispersed in hexane in the presence of oleic acid (~0.05 ml) and oleylamine (~0.05 ml). The resulting Fe 3 O 4 NMPs dispersed in chloroform were encapsulated with a polyethylene glycol (PEG)-phospholipid shell to make them biocompatible. Typically, 2 ml of the organic dispersible 12-nm Fe 3 O 4 NMPs in chloroform (5 mg/ml) was mixed with 1 ml of chloroform solution containing 10 mg 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(PEG)-2000; mPEG-2000 PE; Avanti Polar Lipids Inc.) at a ratio of 5:1. After complete evaporation of the chloroform, the residue was incubated at 80°C in vacuum for 1 h. Then, 5 ml of water was added, which produced a clear and dark-brown suspension containing the PEG-PE micelles. In this study, 50 µg/ml of Fe 3 O 4 NMPs were added to the medium.

| Characterization of Fe 3 O 4 NMPs
The crystal structures of the obtained Fe 3 O 4 NMPs were studied by powder X-ray diffraction (XRD) measurements using Ni-

| Proliferation and activity assay of Saos-2
Cell proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assay. For the MTT assay, the cells were cultured in a six-well plate, and each well was supplemented with MTT (3 mg/ml; n = 4). The plates were then incubated in the dark at 37°C in an atmosphere containing 5% CO 2 for 2 h, and the supernatant was aspirated. Dimethyl sulfoxide was added, and the six-well plate was shaken slowly for 5 min. The absorption was measured at 570 nm.

| Lactate dehydrogenase (LDH) assay
LDH activity was measured using an LDH-LQ kit (Asan Pharmaceutical Inc.). Briefly, after 7 days of culture, 20-μl culture medium and 50 μl of working solution were mixed and incubated in darkness at room temperature for 30 min. The reaction was terminated by adding 1-N HCl, and the absorbance was measured at 570 nm.

| Reverse-transcription polymerase chain reaction (RT-PCR) analysis
The total RNA of the cells was isolated using 500-μl TRIzol (Sigma-Aldrich). Subsequently, 100 μl of chloroform was added, and the solution was mixed and incubated for 3 min. After centrifugation (12,000 rpm, 4°C for 15 min), the upper phase was transferred into a new tube, and 500 μl of isopropanol was added. After a 10-min incubation period and another centrifugation step (14,000 rpm, 4°C for 10 min), the supernatant was discarded. The pellet was washed with RT-PCR was routinely performed. The primer sequences used for the RT-PCR are listed in Table 1, and ImageJ software (National Institutes of Health) was used for the quantitative analysis of RT-PCR amplicons on the digitized gel images. was visualized with enhanced chemiluminescence reagent (Thermo Fisher Scientific) and photographed using a gel imaging system, ChemiDoc XRS+ (Bio-Rad). The results were quantified using ImageJ software (National Institutes of Health).

| Immunocytochemical analysis
The cells grown on coverslips were fixed using 4% paraformaldehyde for 20 min at 4°C and then washed with 10-mM Tris-HCl buffer. Then, they were incubated with the indicated primary antibodies: anti-osteocalcin (predilution, AM 386; BioGenex), anti-osteopontin (1:1000 dilution), and anti-osteonectin (1:500 dilution, AB 1858; Chemicon) for 24 h, followed by development using EnVision Plus Reagent (Dako), diaminobenzidine as a chromogen, and Mayer's hematoxylin as a counterstain. Microscopic images were captured with a Nikon digital camera attached to a Nikon Optiphot-2 microscope.

| Von Kossa staining
The mineralized matrix of the cells was assessed using 5% silver nitrate (Sigma-Aldrich) under ultraviolet light for 60 min, followed by 3% sodium thiosulphate (Sigma-Aldrich) for 5 min, and then counterstained with Van Gieson (Sigma-Aldrich) for 5 min. The mineral was stained black and the osteoid was stained red by this method.

| Immunofluorescence
For staining of the intracellular proteins, the cells were fixed and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min on ice. They were incubated with mouse primary antibodies against human osteopontin (1:1000) for 1 h, followed by a fluoresceincoupled anti-rabbit IgG secondary antibody for 1 h.

| Quantitative colorimetric calcium assay
Colorimetric calcium was measured using a QuantiChrom™ Calcium Assay Kit (Bioassay Systems). Briefly, samples were lysed with Proprep lysis buffer. Following preparation of the samples, they were mixed with working reagent by combining equal volumes of Reagent A and Reagent B. These reagents were equilibrated to room temperature before use. The mixtures were incubated for 3 min at room temperature. The amount of calcium was quantified by absorbance at 570 nm. Upstream primer sequence Downstream primer sequence
KIM ET AL.
The animals were monitored daily for the condition of the surgical wound, animal activity, food intake, and any signs of infection. Animals were euthanized with CO 2 inhalation at 6 weeks.

| Microcomputed tomographic (micro-CT) analysis
The rats were killed 40 days after surgery, and the specimens were fixed in 10% formalin for 1 week. Micro-CT scans were taken and analyzed as previously described. 26 In brief, micro-CT scans were taken using the Quantum FX micro-CT X-ray system (PerkinElmer).

| Statistical analysis
Data were analyzed using one-way analysis of variance and Student's t test. When the p value was <.05 or <.01, the difference between means was considered significant (*p < .05, **p < .01). Graphical representations were produced using Sigmaplot 2001. NMPs clearly remain without any distinctive change ( Figure 2G,H). 27 These results support the conclusion that the crystal structure of the were not observed in any of the experimental groups. Therefore, NMP incorporation, EMF exposure, and NMP incorporation with EMF exposure were not found to induce cytotoxicity.

| Effects on proliferation and cytotoxicity assays
Biocompatibility of the EMFs and NMPs was evaluated by MTT and LDH assays. The initial seeding cell number in each subculture was the same in each group. Saos-2 cell proliferation was measured by MTT assays at Days 3 and 7. The level of cell mitochondrial activity of the four experimental groups was similar ( Figure 4A). So, these results showed that the NMP treatment and/or the EMF exposure did not have an adverse effect on proliferation or mitochondrial activity ( † p > .05, † † p > .05).
For examination of the membrane damage of Saos-2 cells under different culture conditions, we performed LDH assays. The media was collected at Day 7 and analyzed. The NMP-incorporated, EMFexposed, and NMP-incorporated + EMF-exposed groups did not have increased levels of LDH secretion ( Figure 4B). No groups displayed a prominent difference in LDH ( † p > .05). Therefore, the LDH activity of the four experimental groups was similar, and it is believed that NMP and EMF did not induce cellular membrane damage.

| Expression of osteogenic-related genes
For evaluation of the effect of NMP treatment and EMF exposure on Saos-2 gene expression, total RNA was isolated from the cells of all groups, and RT-PCR was carried out ( Figure 5A). We examined the expression of each gene after normalizing it to that of GAPDH, and we reported the difference as the fold change ( Figure 5B).
The major osteogenesis markers, collagen I, osteocalcin, and bone morphogenetic protein 2 (BMP-2) were enhanced by more than 1.5-fold, and osteopontin and osteoprotegerin (OPG) were highly expressed at the transcript level by more than threefold in the NMPincorporated + EMF-exposed group compared with the control. The expression of the major bone matrix protein markers collagen I, collagen III, and osteocalcin were increased by 20%, and osteopontin, osteonectin. In the case of NMP-incorporated + EMF-exposed group, collagen I, osteocalcin, osteopontin, osteonectin, BMP-2, OPG were highly expressed, and the expression levels of the bone matrix protein genes were significantly increased.
F I G U R E 3 Morphology of the Saos-2 after electromagnetic field (EMF) treatment. Cultured in maintenance medium (A), magnetic nanoparticle (NMP)-incorporated (B), exposed to EMF (C), and NMP-incorporated with exposed EMF (NMP + EMF) (D) for 7 days. Runx-2 was measured. Runx-2 expression was enhanced in the NMPincorporated, EMF-exposed, and NMP-incorporated + EMF-exposed groups by over twofold compared with the control groups. As shown in Figure 5B, the expression level of the osteogenic-related genes was enhanced in the NMP-incorporated + EMF-exposed group compared with the other groups.

| Evaluation of calcium channels
Calcium activation affects bone formation and osteogenic differentiation. Thus, calcium channel activation was measured by RT-PCR.
After 3 days of osteogenesis with NMP-incorporation and/or EMF exposure, the mRNA levels of CACNA1G and CACNA1I were significantly increased ( Figure 5C,D). This showed that NMP plus EMF exposure induced the activation of calcium channels.
To examine the mineralization of the Saos-2 cells, a quantitative colorimetric calcium assay was performed on Day 7 ( Figure 5E). The EMF-exposed and NMP-incorporated + EMF-exposed groups exhibited higher calcium concentrations relative to the control group. Especially, the calcium concentration of the NMP-incorporated + EMF-exposed group increased by about 10% compared with the control group (*p < .05).

| EMF and NMPs increase osteogenic protein expression in Saos-2 cells
We evaluated the expression of osteogenesis-related proteins by Western blot analysis of Saos-2 cells after culture for 7 days, using βactin as an internal control. The results shown in Figure 6A indicate that the expression of osteogenic proteins increased after NMP treatment and EMF exposure compared with the control group. In particular, in the NMP-incorporated + EMF-exposed group, all osteogenic proteins (including osteopontin, osteonectin, osteocalcin, and versican) were more abundantly expressed than in the control group.
To assess the mechanism involved in the osteogenesis of Saos-2 cells, we evaluate the activation of p-ERK and p-p38 signaling.
Western blot analysis revealed that the levels of phosphorylated ERK and phosphorylated p38 increased in the EMF-exposed and NMP-incorporated + EMF-exposed group ( Figure 6B,C).

| Immunocytochemistry and immunofluorescence
To further evaluate the protein expression of osteogenesis-related proteins and mineralization, immunocytochemical staining was The EMF-exposed group exhibited a small amount of matrix mineralization, while the NMP-incorporated + EMF-exposed Saos-2 cells exhibited stronger matrix mineralization compared with the other groups on Day 7. Osteocalcin was expressed in the NMPincorporated, EMF-exposed, and NMP-incorporated + EMFexposed groups compared with the control group ( Figure 7E-H).
Immunofluorescence staining of the Saos-2 cells indicated the expression of the osteogenic-related protein, osteopontin ( Figure 8).

Saos-2 cells were fixed and labeled with anti-osteopontin and DAPI
(4′,6-diamidino-2-phenylindole). Very weak signals were detected in the control group, while bright signals were observed in the NMPincorporated, EMF-exposed, and NMP-incorporated + EMF-exposed group. Especially, many of the cells were strongly labeled in the NMP-incorporated + EMF-exposed group.

| Micro-CT 3D analysis
In the micro-CT 3D images, new bone was observed at the margin of the defect in all groups. However, the bone density was higher in the NMP-incorporated + EMF-exposed group than in the  NMP-incorporated and EMF-exposed groups at 6 weeks ( Figure 9A).
BV was significantly higher in the NMP-incorporated + EMF-exposed group than in the NMP-incorporated and EMF-exposed groups at 6 weeks ( Figure 9B). The BV of the NMP-incorporated + EMFexposed group at 6 weeks was 60.24 ± 4.9375%, which was higher than that of the NMP-incorporated group (39.224 ± 3.94%) and the EMF-exposed group (43.964 ± 5.50%) at 6 weeks. Also, the BMDs of the NMP-incorporated + EMF-exposed group were higher than in the NMP-incorporated and EMF-exposed groups at 6 weeks ( Figure 9C).

| DISCUSSION
The aim of this study was to investigate the osteogenic co-effect of EMF and NMPs on Saos-2 cells and a rat calvarial defects model. We evaluated the combination of these two parameters to induce synergic efficiency of osteogenesis in vitro and in vivo.
In this study, our data showed that no morphological changes of Also, we examined the effects of EMF and NMPs on the expression of specific osteogenesis markers, such as BSP, osteocalcin, osteopontin, osteonectin, and osteoprotegerin. The gene expression and protein levels of these markers were all increased in the NMPincorporated + EMF-exposed Saos-2 cells (Figures 5 and 6). The osteogenesis markers of osteocalcin, osteopontin, osteonectin, and BSP were particularly highly expressed in the NMP-incorporated + EMFexposed group. Osteocalcin and osteonectin are expressed in the earlier mineralization process, and osteopontin is an important marker of postmitotic osteoblasts. [34][35][36] Also, BSP is expressed in osteoblasts 37 and can function as a nucleator of mineralization in vitro and in vivo. 38 In addition, the expression levels of major bone formation genes, collagen I, collagen III, Runx-2, and bone morphogenetic protein 2 (BMP-2), were increased in the NMP-incorporated + EMF-exposed groups. It is well known that BMP-2 can induce the formation of bone 39,40 and it stimulates the expression of other osteogenic markers, such as osteopontin, osteocalcin, BSP, and alkaline phosphatase. 41,42 In our results, in the NMP-incorporated + EMF-exposed group, there was increased EMF decreased ALP activity and RANKL expression of mouse osteoblasts. 9 Although the exact cause is unknown, the differences in these experimental results are expected to be due to the differences in the equipment, as all of the electromagnetic equipment applied were not off-the-shelf devices.
As well, research using NPs has been carried out in all areas of bio-and medical fields, 44 | 1643 exposed to the field but not NMPs compared with cells only exposed to NMPs. Also, the expression of ALP and p-ERK increased strongly when the cells were exposed to both NMPs and EMFs. This can be interpreted to mean the osteogenic differentiation effect of EMFs is greater than that of magnetic NPs. 22 The results of the above report are similar to our findings, and together they can be interpreted to have confirmed that a combination of magnetic NPs and magnetic fields has a synergistic effect on osteogenesis.
This result is also related to calcium channels and calcium concentrations. The mRNA expression of CACNA1G and CACNA1I in the NMP-incorporated + EMF-exposed group was higher than that of the other groups ( Figure 5D). So, NMP-incorporated + EMF-exposed  [54][55][56][57][58][59] The p38 signaling pathway plays an important role in the regulation of osteogenesis and osteogenic differentiation. 46,60 Also, Runx-2 is involved in the production of bone matrix proteins, as it is able to promote the expression of major bone matrix protein genes, leading to an increase in immature osteoblasts differentiating from pluripotent stem cells; the immature osteoblasts then form immature bone. [61][62][63] A related study reported that osteogenic differentiation of hBMSCs using 30 nm of iron oxide NPs was induced by the Runx-2, ERK, and MAPK signaling pathways. 17 In our study, we detected increased levels of phosphorylated ERK and phosphorylated p38 after 7 days of osteogenesis in the NMPincorporated + EMF-exposed group.
We have confirmed the above results through immunocytochemical analysis. The NMP-incorporated + EMF-exposed group showed enhanced Von Kossa staining of Saos-2 cells. Von Kossa staining is generally used to quantify mineralization. 64,65 Additionally, the results showed that exposure to EMFs and NMP treatments has the potential to facilitate osteogenesis based on the results of immunocytochemical staining and immunofluorescence (Figures 7 and 8). Saos-2 cells more strongly expressed osteocalcin, osteopontin, and osteonectin in the NMP-incorporated + EMFexposed group than the control group by immunocytochemical staining. In addition, in the immunofluorescence staining, we detected strong signals in the NMP-incorporated, EMF-exposed, and NMP-incorporated + EMF-exposed groups and weak signals in the control group. This means that NMPs and EMFs promote the mineralization of Saos-2 cells.
To evaluate the bone regeneration efficacy of animals based on the in vitro results, the regeneration efficacy of NMPs and EMF were assessed using a rat calvarial defect model, and it was confirmed that there was a synergy effect of a combination of NMPs and EMF as shown in Figure 9.
Bone regeneration showed similar results to the in vitro analysis.
We predicted that NMPs contained in a collagen scaffold would be phagocytosed by migrating osteoblasts or MSCs after transplanta-