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Introduction

Bone defects are major problems in trauma orthopedics, leading to non-union and limb disability, making the prognosis of the patient unsatisfactory. It causes severe damage to the physical and mental health of the patient, with a huge economic burden on both patients and society (1). Fracture healing is divided into four major overlapping phases: Inflammation, bridging osseous callus formation, callus mineralization and bone remodeling (2). Functional reduction of osteoblasts is an important cause of bone defects. Therefore, pharmacological or physical interventions that induce osteoblast differentiation and proliferation, thereby promoting bone formation, are potential strategies for treating bone defects.

Low-frequency-pulsed electromagnetic field (LPEMF) is a noninvasive and inexpensive method that has shown efficacy against a wide range of diseases of the skeletal muscle system (3,4). LPEMF may inhibit osteoclast differentiation in vitro (5,6), accelerate bone defect repair and shorten the healing time of bone defects by promoting osteogenic differentiation of bone marrow stromal cells (BMSCs) (2). BMSCs have multidirectional differentiation potential and are the main source of osteoblasts (7). Osteogenic differentiation of BMSCs is the physiological basis of bone defect repair and promoting osteogenic differentiation of BMSCs can substantially shorten the time required for bone defect repair (8). Therefore, investigating the underlying mechanisms of LPEMF in osteoblasts is essential for understanding its efficacy in treating osteoblast-related diseases.

Connexin 43 (Cx43) is the most abundant gap junction protein in bone (9). Cx43 is the most widely expressed connexin among bone cell types, encoded by Gja1 gene (10) and expressed at the cell surface, where it mediates cell communication and allows small molecules and ions to pass between cells (9,11). Cx43 plays important roles in bone formation and homeostasis. Osteoblasts regulate cell proliferation, differentiation and metabolic functions via intercellular communication (9,10,12-14). Cx43 is also expressed in cartilage and is necessary for mineralization and osteoclast formation, suggesting that Cx43 may play an important role in multiple stages of fracture healing (15-17). In three-dimensional culture, overexpression of Cx43 in BMSCs enhances the size and spatial distribution of gap junctions, intercellular communication and expression of osteogenic markers (18).

LPEMF has the ability to promote the osteogenic differentiation of BMSCs, accelerate the process of bone defect repair and shorten the healing time of bone defects. However, the mechanism of LPEMF on Cx43 expression in promoting the osteogenic differentiation of BMSCs remains to be elucidated. The present study provided a comprehensive and systematic annotation of the role of Cx43 in the regulation of the osteogenic differentiation of BMSCs by LPEMF through in vitro experiments at the molecular and cellular levels to explore the mediation of Cx43 and provide a target for the treatment of bone defects.

Materials and methods

Isolation and culture of BMSCs

Primary rat BMSCs were isolated from specific pathogen free (SPF) four to six-week-old rats weighing 100-150 g Sprague-Dawley, which were provided by the SPF grade animal house of Shantou University Medical College (Guangdong, China). A total of 30 rats, 50% male and 50% female, were libitum fed in the same environment were maintained at an indoor temperature of 22˚C, relative humidity of 18-22%, and a 12-h light/dark cycle. All animal experimental procedures were approved by the Institutional Ethics Committee for Animal Experimentation of Ganzhou People's Hospital (Ganzhou, China). Rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30 mg/kg) and euthanized by cervical dislocation. The two lower limbs were rapidly dissociated and disinfected by immersion in 75% ethanol for 5-10 min, under aseptic conditions. Subsequently, the femoral bone marrow was lavaged and the lavage fluid was inoculated into a 10-cm dish using the whole bone marrow adherent culture method (19). Briefly, the cells were seeded onto flasks for culturing in a CO2 cell incubator. The culture medium was maintained at 37˚C and 5% CO2 in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 0.5 mg/ml penicillin/streptomycin. Cell morphology and growth were observed daily using an inverted microscope. When the cell confluence reached 70-90%, a subculture was carried out and the cells were cultured to the third generation for subsequent experiments.

LPEMF exposure

To evaluate the proliferation and osteogenic differentiation behavior of BSMCs under different intensity gradients of LPEMF, the cells were continuously exposed to sinusoidal LPEMF (20, 40, 60, 80 and 100 Hz) of five different gradient signals with a density of 1 mT in the incubator. A pulsed electromagnetic field in vitro cell intervention system (DG1022U; Rigol Technologies, Inc.) generated electromagnetic field signals at a predetermined frequency. The magnetic field was generated using a pair of 60-turn Helmholtz coils. The coil diameter was 300 mm and the spacing was 122.5 mm. A Petri dish was placed at the center of the coil. The control cells were placed in another incubator under the same conditions, but without LPEMF.

BMSCs osteogenic differentiation culture

When P3 cells were cell confluence reached 70-90%, they were subcultured and inoculated at 5,000-10,000 cells/cm2. The cells were incubated overnight in a humidified incubator at 37˚C, with 5% CO2 and 95% air and then replaced with an osteogenic induction medium (alpha-MEM; (Gibco; Thermo Fisher Scientific, Inc.; cat. no. C12571500BT) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.; cat. no. 12664025), 50 µM vitamin C (MilliporeSigma; cat. no. A4544), 10 mM beta-sodium glycerophosphate (MilliporeSigma; cat. no. G9422) and 100 nM dexamethasone (MilliporeSigma; cat. no. D1756) for further culture. The osteogenic induction medium was replaced once every 2-3 days and cell growth was observed under a light microscope every day. The cells were cultured until they were terminated at 9 days and tested for ALP activity. After 14 days of cell culture, total RNA and total protein were extracted for PCR and Western blotting experiments. Cell cultures were terminated at 14 days and Alizarin red S staining was performed.

Cell Counting Kit-8 (CCK-8) assay

To evaluate cell proliferation, BMSCs were inoculated into 96-well plates at a density of 1x104 cells per well and incubated in a CO2 cell incubator (37˚C, 5% CO2) for 4 h. Cells were cultured 24 h after electromagnetic field treatment for 5 days and 10 µl CCK8 (Dojindo Laboratories, Inc.) dye solution was added to each well. After 2 h, the absorbance of each well was measured at 450 nm according to the manufacturer's instructions. Each data point was obtained as the mean of five wells.

ALP activity detection

ALP activity is an early marker of bone formation (20). After cells were incubated for the indicated times, cells were washed once with PBS at 4˚C, lysed with 1% Triton X-100 and the lysates collected. After centrifugation for 5 min at 4˚C and 1,174 x g, the supernatant was collected. Color substrate (50 µl) and 50 µl of the group sample or sample buffer (blank control) was added to the 96-well plate, then placed on a shaker for mixing. After incubation at 37˚C for 10 min, 100 µl of the reaction stop solution was added to each well. The absorbance of each well was measured at 405 nm by using a microplate reader.

Alizarin red S staining and quantification

After 14 days of cell culture under different conditions, the medium was removed and 1 ml PBS was added to each well. The cells were gently washed once, fixed with 2 ml anhydrous ethanol for 30 min and cleaned once with ultra-pure water. Alizarin red S solution (1 ml, 0.1%) was added after the cell surface dried. The bed was shaken at 22˚C room temperature for 15 min, the Alizarin red S solution was removed, the cells were rinsed again with ultra-pure water three times and the staining of calcium nodules was observed under a light microscope after the cells were dried naturally. After Alizarin Red S staining was completed, 2 ml of 10% CPC sodium phosphate buffer solution was added to each well and incubated at 22˚C room temperature for 15 min to elute Alizarin Red S. The Alizarin red S eluent was diluted 10 times with ultra-pure water and 100 µl diluent was absorbed into 96-well plates with five wells per group. The absorbance of each well was measured at 540 nm using a microplate analyzer.

RNA extraction and reverse transcription-quantitative (RT-q) PCR

After treatment, total RNA was extracted from each group of cells using TRIzol® reagent (Thermo Fisher Scientific, Inc.) and the RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Next, mRNA was reverse-transcribed into cDNA using a reverse transcription system (Toyobo Life Science) according to the manufacturer's protocol. Then, cDNA was used as a template to perform qPCR (BeyoFast SYBR Green qPCR Mix; Beyotime Institute of Biotechnology) under the following thermal cycler conditions: 95˚C for 5 sec, followed by 45 cycles, including denaturation at 95˚C for 30 sec in an ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.); further it was annealed at 60˚C for 30 sec and extended at 72˚C for 30 sec. GAPDH was used as an internal control, and the relative expression level of mRNA was calculated using the 2-ΔΔCq method (21). Primers were designed and synthesized by BGI Biological Co. and the sequences are listed in Table I.

Table I Primers used for quantitative PCR.

Table I

Primers used for quantitative PCR.

Target gene	Oligonucleotide sequence
Runx2	5'-GGGACCGTCCACTGTCACTTTAA-3' (Forward)
	5'-TACAAGTGGCCAGGTTCAACGA-3' (Reverse)
BSP	5'-GCTATGAAGGCTACGAGGGTCAGGATTAT-3' (Forward)
	5'-GGGTATGTTAGGGTGGTTAGCAATGGTGT-3' (Reverse)
Bglap	5'-AGGTGGTGAATAGACTCCG-3' (Forward)
	5'-GGCTGTGCCGTCCATACTT-3' (Reverse)
Osx	5'-GGAGGCACAAAGAAGCCATA-3' (Forward)
	5'-GGGAAAGGGTGGGTAGTCAT-3' (Reverse)
GAPDH	5'-TCCTGCACCACCAACTGCTTAG-3' (Forward)
	5'-AGTGGCAGTGATGGCATGGACT-3' (Reverse)

Western blotting

Western blotting was performed according to standard procedures. Briefly, proteins were extracted from cells or tissues using RIPA lysis buffer (Beyotime Institute of Biotechnology; cat. no. P0013B). Protein concentrations were determined using the BCA Protein Assay Kit (Solarbio, Beijing, China). After denaturation ~20 µg of protein per lane was separated by 10% sodium dodecyl sulfate polyacrylamide gel, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (0.45 µm; MilliporeSigma). The PVDF membranes were blocked with Tris-buffered saline (TBS) containing 5% nonfat milk for 1 h at room temperature. Subsequently, it was incubated with primary antibodies [rabbit anti-Cx43 (1:1,000; Cell Signaling Technology; cat. no. 3512), rabbit anti-GAPDH (1:1,000; Panera; cat. no. SF-PA005)] overnight at 4˚C, then washed with TBST (0.1% Tween20) and incubated with the corresponding secondary antibody (Jackson ImmunoResearch; cat. no. 111-035-003) for 2 h. A chemiluminescence system (ECL Kit; Biosharp Life Sciences) was used to observe the protein bands and digital images were captured, which were then analyzed using ImageJ v6.0 (National Institutes of Health).

Statistical analysis

All data were analyzed using SPSS 19.0 software (IBM Corp.) and the results were expressed as mean ± standard deviation. The t-test was used for comparisons between two groups and one-way ANOVA followed by Tukey's post hoc test was used for comparisons between multiple groups. P<0.05 was considered to indicate a statistically significant difference. All experiments were independently repeated at least three times.

Results

Effect of different frequencies of LPEMF treatment on the proliferation ability of BMSCs

Evaluation of the effect of LPEMF on BMSCs proliferation in vitro: BMSCs were treated with different intensity gradients (0, 20, 40, 60, 80 and 100 Hz) of LPEMF for 24 h and the viability of BMSCs treated with LPEMF for 24 h was determined using CCK-8. The results showed that the proliferation viability of BMSCs increased with increasing magnetic field strength from 0 to 80 Hz peaking at 80 Hz (P<0.05). However, when the intensity reached 100 Hz, the proliferation viability of BMSCs was significantly inhibited and exhibited toxic side effects (Fig. 1). Therefore, the 100 Hz magnetic field intensity was removed in subsequent experiments as the research condition and 80 Hz magnetic field stimulation was used as the optimal stimulation intensity for the LPEMF treatment of BMSCs.

Figure 1 Effect of different frequencies of LPEMF treatment on rat BMSCs. The proliferation activity of BMSCs increased with the increase of magnetic field intensity at 0-80Hz and reached the peak value at 80 Hz. When the intensity reached 100 Hz, the proliferation activity of BMSC was significantly inhibited. Data is expressed as mean ± standard deviation and the experiments were performed in quadruplicate (n=4). *P<0.05 vs. controls at the same time point; #P<0.01 vs. controls at the same time point, controls were anterior frequency gradient at the same time point-. LPEMF, low-frequency-pulsed electromagnetic field; BMSC, bone marrow-derived mesenchymal stem cell.

Effect of LPEMF treatment on ALP activity during osteogenic differentiation of BMSCs

To test whether LPEMF can promote the osteogenic differentiation of BMSCs, the ALP activity of each group was measured. The results showed that, compared with the control group, the ALP activity of BMSCs pretreated with LPEMF at different frequencies was enhanced on day 9 after osteogenic induction culture and the intensity showed a dose-dependent trend, with significant differences among all groups (Fig. 2).

Figure 2 Effect of different frequency LPEMF treatment on the ALP activities of BMSCs. ALP activities were measured by conventional methods and the data represent the mean ± standard deviation of quadruplicate samples (n=4). #P<0.05 vs. the 0 Hz group; *P<0.01 vs. the 20 Hz group; **P<0.01 vs. the 40 Hz group; ##P<0.05 vs. the 60 Hz group. LPEMF, low-frequency-pulsed electromagnetic field; ALP, alkaline phosphatase; BMSC, bone marrow-derived mesenchymal stem cell.

Effect of LPEMF treatment on the expression of osteogenic marker genes during osteogenic differentiation of BMSCs

The present study examined the effect of LPEMF treatment on the expression of osteogenic marker genes during osteogenic differentiation of BMSCs. Following LPEMF treatment at different frequencies, BMSC osteogenic differentiation was induced and cultured for 7 and 14 days and the transcription levels of the osteogenic marker genes Runx2, BSP, Bglap and Osx in each group were measured. The results showed that the transcription levels of osteogenic marker genes on day 7 (Fig. 3A) and 14 (Fig. 3B) were enhanced with an increase in the LPEMF frequency and there was a significant difference between the groups with different frequencies (P<0.05). After cells were treated with LPEMF at the same frequency, the transcription levels of osteogenic marker genes on day 14 were higher than those on day 7 (P<0.05).

Figure 3 Expression of osteogenic gene expression was measured using RT-qPCR after (A) 7 and (B) 14 days of osteogenic induction and different frequency LPEMF treatment. Expression of Runx2, BSP, Bglap and Osx was measured using real-time RT-qPCR after 7 and 14 days of induction, respectively. LPEMF, low-frequency-pulsed electromagnetic field. Data are expressed as the mean ± standard deviation of the values in 0 Hz. Values represent analysis from three separate experiments. *P<0.05 vs. the 0 Hz group; #P<0.05 vs. the 20 Hz group; **P<0.05 vs. the 40 Hz group; ##P<0.05 vs. the 60 Hz group. RT-qPCR, reverse transcription-quantitative PCR; LPEMF, low-frequency-pulsed electromagnetic field.

Effect of LPEMF treatment on osteogenic mineralization of BMSCs

The effect of LPEMF treatment on osteogenic mineralization of BMSCs was investigated. BMSCs from each group were terminated after being cultured under osteogenic inductive conditions for 14 days after treatment with LPEMF at different frequencies and mineralization was assessed using Alizarin Red S staining. The gross specimens are shown (Fig. 4A). The formation of calcium nodules was lowest in the control group and numerous calcium nodules were observed in the other groups. The amount of calcium nodule deposition increased with an increase in LPEMF frequency. Alizarin Red S quantitative results showed that the number of calcium nodules significantly increased with an increase in frequency in each group (P<0.05; Fig. 4B).

Figure 4 Mineralization level as assessed by alizarin red S staining after 14 days of differentiation. (A) Alizarin red S staining with (B) quantitative results of calcium deposition after 14 days of osteogenic induction and different frequency LPEMF treatment. Data represent the mean ± standard deviation of five independent experiments (n=5). *P<0.05 vs. the 0 Hz group; #P<0.05 vs. the 20 Hz group; **P<0.05 vs. the 40 Hz group; ##P<0.05 vs. the 60 Hz group. LPEMF, low-frequency-pulsed electromagnetic field.

Effect of LPEMF treatment on Cx43 expression during osteogenic differentiation of BMSCs

To detect the expression of Cx43 following LPEMF treatment during the process of osteogenic differentiation of BMSCs, Cx43 expression levels in BMSCs treated with LPEMF at 0 and 80 Hz for 14 days after osteogenic induction culture were determined using western blotting. The results showed that Cx43 expression was significantly enhanced in the 80 Hz LPEMF-treated group compared with that of the 0 Hz LPEMF-treated group (P<0.05; Fig. 5).

Figure 5 Cx43 expression after 14 days of osteogenic induction and 80 Hz LPEMF treatment. (A and B) Cx43 expression after 14 days of osteogenic induction and 80 Hz LPEMF treatment. Data represent the mean ± standard deviation of five independent experiments (n=5). *P<0.05 vs. the 0 Hz group. Cx43, connexin 43; LPEMF, low-frequency-pulsed electromagnetic field.

Inhibition of Cx43 expression attenuates the role of LPEMF in promoting osteogenic differentiation of BMSCs

To verify the role of Cx43 in promoting the osteogenic differentiation of BMSCs by LPEMF, BMSCs were transfected with a CX43-specific lentivirus vector to inhibit the expression of Cx43. Transfection efficiency was detected using western blotting and the results showed that Cx43 expression was significantly inhibited in BMSCs after transfection with the Cx43 specific lentivirus vector. Following Cx43 inhibition, BMSCs were treated with an 80 Hz LPEMF. The indicators of osteogenic differentiation of BMSCs were significantly lower than those of Cx43 without inhibition (P<0.05; Fig. 6).

Figure 6 Osteogenic differentiation level of BMSCs while inhibiting Cx43 expression. (A and B) Cx43 expression after 14 days of osteogenic induction, 80 Hz LPEMF treatment and Cx43 inhibition. (C) Effect of different LPEMF treatment frequencies on the ALP activities of BMSC expression of osteogenic gene expression was measured using reverse transcription-quantitative PCR after (D) 7 and (E) 14 days of osteogenic induction. (F) Alizarin red S staining and (G) quantitative results of calcium deposition after 14 days of osteogenic induction and different LPEMF treatment frequencies. Data represent the mean ± standard deviation of four independent experiments (n=4). *P<0.05 vs. the 0 Hz group; #P<0.05 vs. the 80 Hz group. Cx43, connexin 43; LPEMF, low-frequency-pulsed electromagnetic field; ALP, alkaline phosphatase; BMSC, bone marrow-derived mesenchymal stem cell.

Discussion

The present study established an in vitro osteogenic differentiation model for LPEMF-induced BMSCs. It showed that the proliferation, osteogenic differentiation and mineralization of BMSCs were enhanced in a magnetic field intensity-dependent manner by LPEMF intervention.

BMSCs are the main cell source of osteoblasts in fracture healing. They are adult stem cells with self-renewal, proliferation and multidirectional differentiation potential and can differentiate into osteoblasts, adipocytes, chondrocytes, nerve cells and myoblasts (22,23). The osteogenic differentiation of BMSCs is a continuous and dynamic process. Generally, osteogenic differentiation of BMSCs is divided into three stages: Osteoblast precursor cells, immature osteoblasts and osteoblasts. Massive deposition of mineralized matrix marks the formation of osteoblasts and the main component of the mineralized matrix is calcium salt (24). Therefore, the present study investigated the mechanism of LPEMF healing from a cytological perspective and used BMSCs to study the process of LPEMF-promoting BMSC osteogenic differentiation from multiple aspects and at different stages. LPEMF has been used to promote fracture healing for more than half a century and Wang et al (25) showed that it plays a positive role in promoting bone regeneration. However, the mechanism through which LPEMF promotes bone regeneration remains to be elucidated. Currently, there are theories, such as ‘non-thermal effect’ (26), ‘window effect’ hypothesis (27), local blood flow increase theory, calcium ion deposition theory (28), mediating mechanism of second messenger molecule (29) and endocrinoid effect of pulsed electromagnetic field (30).

ALP is an enzyme secreted by BMSCs during the early stages of osteogenic differentiation and its activity reaches its peak before the mineralization stage. Through the hydrolysis of pyrophosphate, ALP provides phosphate ions and hydroxyl radicals for the deposition of hydroxyapatite crystals, thus forming a mineralized bone matrix (31). The proliferation of osteoblasts isolated from ALP knockout mice was normal, but there was no normal mineral salt deposition following culture. The quantitative measurement of ALP activity is often used as an indicator of changes in the strength of early osteogenic differentiation (20). Runx2 activation controls the initiation of the osteogenic differentiation switch in BMSCs and promotes osteoblast maturation (32). Runx2 gene defects cause BMSCs to have no osteogenic differentiation ability and endometrial and endochondral osteogenic functions are lost in Runx2 knockout mice, suggesting osteoblast maturation disorder (33). Runx2 binds to osteoblastic heterozygous elements to regulate the expression of osteoblastic extracellular matrix protein genes Bglap, osteopontin, BSP and Col1α1 and participate in the synthesis of extracellular matrix (34). Osx is a key gene that controls BMSCs during osteoblast differentiation. Osx was first discovered during the process of osteogenic differentiation induced by BMP-2 in C2C12 myoblasts and regulates the expression of Bglap, osteopontin, bone salivariin, type I collagen and other genes (35). BSP and Bglap are secreted by mature osteoblasts, which together with osteopontin and type I collagen participate in the synthesis of osteoids and deposition of calcium salts and other minerals (36). The increased secretion of BSP and osteocalcin signals the transformation of immature osteoblasts into mature osteoblasts and can be used as indicators of middle and late osteogenic differentiation of BMSCs (36-38). Alizarin red S forms a complex with calcium ion chelation and is an orange-red deposition. Alizarin red S staining is commonly used to detect the maturity of BMSCs during the late stage of osteogenic differentiation (39). In the present study, ALP activity and Runx2, Osx, BSP and Bglap were used as markers of osteogenic differentiation of BMSCs. Alizarin Red S staining was used to detect calcium nodules and reflect the level of osteogenic differentiation in BMSCs. The results of the present study showed that LPEMF stimulated the proliferation of BMSCs in a frequency-dependent manner in the range of 0-80 Hz, whereas at 100 Hz, LPEMF inhibited the growth of BMSCs, presenting a toxic side effect. Therefore, it can be concluded that a magnetic field with a frequency <80 Hz is safe for stimulating BMSCs. Following LPEMF treatment, the activity of ALP and expression of the osteogenic marker genes Runx2, BSP, Bglap and Osx notably increased during BMSCs osteogenic differentiation. The mineralization level of BMSCs was also notably increased, suggesting that LPEMFs can promote bone formation.

Cx43 is a connexin encoded by Gja1 that is widely expressed in bone tissue cells (40). Cx43 is an essential gene for the survival, proliferation, differentiation and other physiological processes of osteoblasts and interference with Cx43 expression can significantly inhibit the differentiation of osteoblasts (40-42). In a three-dimensional osteogenic induction culture of BMSCs, the overexpression of Cx43 increased the number and spatial distribution of GJIC and enhanced the transcription level of osteogenic marker genes (43). Osteoblast differentiation of BMSCs is regulated by various signaling pathways. The present study hypothesized that LPEMF may promote BMSC osteoblast differentiation by regulating Cx43 gene expression. The results showed that Cx43 expression was significantly increased in BMSCs treated with 80 Hz LPEMF for 14 days after osteogenic induction. According to the present study, LPEMF may promote osteogenic differentiation of BMSCs by upregulating ALP activity, expression of osteogenic marker genes and calcium salt formation and Cx43 upregulation may be involved in LPEMF promoting the osteogenic differentiation of BMSCs; however, the specific mechanism is still not completely clear and needs to be further verified by more cell and animal experiments. In tissue engineering, BMSCs have the characteristic of directional differentiation into osteoblasts and are often used as the seed cells of bone cells. Using a pulsed electromagnetic field to stimulate osteogenesis has the advantages of being non-invasive, having no complications and suitable for popularization and application. The application of LPEMF to promote the osteogenic differentiation of BMSCs will provide a new treatment for clinical bone nonunion and bone defects.

In conclusion, the present study concluded that when the LPEMF frequency was <80 Hz, the proliferation, osteogenic differentiation and mineralization of BMSCs were enhanced in a magnetic field intensity-dependent manner. LPEMF enhanced the osteogenic differentiation of BMSCs by regulating ALP activity and osteogenic marker gene expression. The upregulation of Cx43 expression may be involved in the process by which LPEMF promotes the osteogenic differentiation of BMSCs. This provided an experimental basis for the treatment of non-unions and osteoporosis caused by bone defects. These findings suggested that LPEMF may be an effective strategy for the treatment of osteoblast-related diseases.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the funding of the 2023 Technology + Medical joint plan project-Key research and development plan-Ganzhou People's Hospital (grant nos. 2023LNS17521 and 2023LNS17517) and the Science and Technology planning project of Jiangxi Provincial Administration of Traditional Chinese Medicine (grant no. 2019A430).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

FL, HG and ZLu conceived the study. FL performed the experiments. ZLu, HG and ZLi analyzed the data. FL wrote the manuscript. FL, HG and ZLu confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ganzhou People's Hospital Animal Care and Use Committee (Ganzhou, China; approval no. 20211210038).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References