Evaluation of Pulsed Electromagnetic Field Effects: A Systematic Review and Meta-Analysis on Highlights of Two Decades of Research In Vitro Studies

Pulsed electromagnetic field (PEMF) therapy is a type of physical stimulation that affects biological systems by producing interfering or coherent fields. Given that cell types are significantly distinct, which represents an important factor in stimulation, and that PEMFs can have different effects in terms of frequency and intensity, time of exposure, and waveform. This study is aimed at investigating if distinct positive and negative responses would correspond to specific characteristics of cells, frequency and flux density, time of exposure, and waveform. Necessary data were abstracted from the experimental observations of cell-based in vitro models. The observations were obtained from 92 publications between the years 1999 and 2019, which are available on PubMed and Web of Science databases. From each of the included studies, type of cells, pulse frequency of exposure, exposure flux density, and assayed cell responses were extracted. According to the obtained data, most of the experiments were carried out on human cells, and out of 2421 human cell experiments, cell changes were observed only in 51.05% of the data. In addition, the results pointed out the potential effects of PEMFs on some human cell types such as MG-63 human osteosarcoma cells (p value < 0.001) and bone marrow mesenchymal stem cells. However, human osteogenic sarcoma SaOS-2 (p < 0.001) and human adipose-derived mesenchymal stem cells (AD-MSCs) showed less sensitivity to PEMFs. Nevertheless, the evidence suggests that frequencies higher than 100 Hz, flux densities between 1 and 10 mT, and chronic exposure more than 10 days would be more effective in establishing a cellular response. This study successfully reported useful information about the role of cell type and signal characteristic parameters, which were of high importance for targeted therapies using PEMFs. Our findings would provide a deeper understanding about the effect of PEMFs in vitro, which could be useful as a reference for many in vivo experiments or preclinical trials.


Introduction
Electromagnetic fields (EMFs) are composed of magnetic and electric fields that influence each other [1]. There are many EMF subtypes with varying frequency rates, and they can cause either positive or detrimental biological effects. For medical purposes, they can be used in diagnostic modality and be considered as a potential therapeutic option as well. On the other hand, EMFs can penetrate tissues without experiencing intensity decrement [2], pass through the cell membrane, and affect cell responses. Consequently, cells may experience diverse pathophysiological disorders like cancer, thus, elevating one's concern during the course of using EMFs for therapeutic purposes [3]. However, despite many findings, the carcinogenic role of EMF is still unclear.
Among subtypes of EMFs, low-frequency fields with specific amplitudes and waveforms are referred to as pulsed EMFs (PEMFs) [4]. Being a promising strategy and a type of the noninvasive and inexpensive physical approaches, PEMFs have exhibited therapeutic potential for treating various diseases [5]. It has already been shown that they can make changes to cell cycle, apoptosis, cell proliferation, and differentiation. Indeed, they are able to affect and alter the cell function by inducing forced vibration for free ions on the cell membrane surfaces due to an external oscillating field [6]. Irregular gating of ion channels triggered by this situation can certainly disturb the balance of transmembrane proteins and, consequently, disrupt cell function [7]. It has also been proposed that the effect of PEMFs may be propagated and amplified along the whole signal transduction pathway, thereby changing cell behavior [8]. In some studies, it has been reported that PEMFs can modulate both downstream signal transduction pathway and cell surface receptor expression/activation [8,9]. As a result, homeostatic cell functions such as differentiation, viability, proliferation, interaction with components of extracellular matrix (ECM), and communication with neighboring cells could be restored [10]. In addition, PEMFs could enhance both the neurogenic differentiation of mesenchymal stem cells (MSCs) and osteogenic differentiation. Because EMFs easily permeate through cells [4] and change the electric field of the inner cell membrane, they can induce biological changes. In particular, they  2017 Parate et al. [21] 2 BioMed Research International can induce changes in the Ca 2+ efflux and, consequently, modulate various biological effects such as nitric oxide signaling, growth factor secretion, and Mitogen-Activated Protein Kinase (MAPK)/Extracellular Signal-Regulated Kinase (ERK) [11]. It has been hypothesized that the production of second messengers is stimulated by the direct effect of PEMF on phospholipids within the plasma membrane, and subsequently, multiple intracellular signal transduction pathways are initiated [12]. There are many factors affecting the biological responses. To clarify PEMF impacts, studies have reported that signal characteristics play a crucial role in determining the type of biological responses including amplitude and frequency of exposure to the applied PEMF [13]. Indeed, to deliver a therapeutic PEMF, it is necessary to optimize these important parameters [6]. In addition, a large volume of evidence has revealed that some kinds of cells appear exquisitely sensitive to PEMF, while other types appear relatively unresponsive. For instance, undifferentiated PC12 cells are more sensitive to PEMF exposure, while differentiated PC12 cells are more resistant to stress [14]. Consequently, cell properties are of vital importance in establishing a biological response to PEMF in vitro.
Despite a relatively long history of using PEMFs in medicine, little is known about the biological mechanism of such therapies. To develop a reliable working principle of PEMF therapies, it is worth investigating the experimentally observed biological effects caused by these fields alone. Thus, in this study, a meta-analysis was performed using 3249 in vitro experimental observations available in 92 scientific journals (1999-2019) in order to determine the potential effects of PEMF on different cell types of both human and rat/mouse. Our analysis scrutinized the published experiments that had considered the effects of exposure to PEMFs (cytogenetic, gene, and protein expression analysis) on cell types from rats, mice, and humans to gain a more explicit and evidence-based conclusion on the association between PEMFs and cell responses.

Material and Methods
In Tables 1-15, the characteristics of experimental protocols and variables are presented. In this paper, cellular response (presence or absence) in human, mouse, or rat cells is defined as changes due to exposure to PEMFs. We analyzed the reported studies based on the different experimental readouts/endpoints which they used for their studies and the physiological variables they measured. These studies are shown in Figures 1-3, (human cells), Figure 4 (rat/mouse cells), and Figure 5 (other species), separately.

Collection of Raw
Data. An electronic literature search of databases including Web of Sciences and PubMed was conducted for publications in English from 1999 up to 2019.
The key terms introduced in the search engines included "pulsed electromagnetic fields" and "cell." The process of selecting the papers was carried out by reading the titles and abstracts of the studies as well as the full article when necessary. Upon omitting duplicate titles, full-text versions of the selected papers were obtained. We excluded those experiments that (1) targeted direct animal or human exposure followed by the analysis of individual cells and (2) applied the combination of PEMFs and other effective treatments, e.g., chemotherapy. After screening many research studies, 92 papers with different designs were eligible for meta-analysis.
For data analysis, the cell responses were classified as "presence" (PEMF exposure changed the cell response statistically significantly in comparison to the control group regardless of direction) and "absence" (no significant PEMF effect).
For each included study, the following data were extracted: type of cells, pulse frequency of exposure, exposure flux density, time of exposure, waveform, and assayed cell responses (cells, cell function, and DNA). Bibliographic details of the studies including the first author and year of publication were also retrieved.

Analysis of Raw
Data. According to the above explanations, given that the frequency and intensity of the mentioned exposure differ across studies, achieving different biological responses would not be unexpected. In this respect, we pooled the retrieved experimental data based on used pulse frequencies and flux densities. Our analysis considered the effect of several subgroups of pulse frequency and flux density as follows: (a) 0:1 < ƒ ≤ 10 Hz, (b) 10 < ƒ ≤ 100 Hz, (c) 100 < ƒ Hz, (d) I < 1 mT, (e) 1 ≤ I < 10 mT, (f) 10 ≤ I <

Statistical Analysis.
Microsoft Excel was used to organize the initial data and build a database. Meta-analysis combined the results obtained from separate studies with a similar outcome. The pooled results were obtained based on cell type, frequency, and intensity. A random-effect model was used to facilitate conducting the analysis, through which I 2 value was calculated as the indicator of heterogeneity. I 2 values greater than 50% could imply significant heterogeneity between the related studies. Also, the random-effect model could account for the above variation between studies, and thus, it achieved more conservative results than a fixedeffect model. Sensitivity analysis was performed to determine the effect of a particular study on the overall effect size. The presence of publication bias was tested using Begg's and Egger's regression asymmetry tests [9]. Statistical analyses were conducted using STATA version 14.0. A p value less than 0.05 was considered significant for all tests.

Results
A number of publications are analyzed in Figure 6, which provides an overview of the years of publication. Cellular response (presence or absence) was observed in human cells (2441 experiments in Figures 1-3), rat or mouse cells (854 experiments in Figure 4), and other species (11 experiments in Figure 5). The results indicated that most of the experiments were carried out on human cells, among which stem cells drew greater experimental attention. Of not, in case the analysis incorporated such parameters as exposure to PEMFs and individual cell types, the potential effects of PEMFs on cell types, such as bone marrow mesenchymal stem cells (BM-MSCs) (based on 559 reported experiments, p value < 0.001), would become clear. However, based on the reported evidence, no such effect was observed for human adipose-derived mesenchymal stem cells (AD-MSCs) and human osteogenic sarcoma SaOS-2 (p < 0:001). As a result, despite the higher susceptibility of cancer cells to PEMFS   We categorized different experimental techniques as follows: (a) cell structure (cell viability, cell morphology, apoptosis, cell proliferation, and cell differentiation), (b) cell functions (calcium concentration, signal transductions, enzyme activity, membrane potential, and membrane stability), and (c) DNA (gene expression, protein expression, ROS production, chromosome aberration, micronucleus assay, DNA damage, oxidative stress, DNA single-strand breaks, DNA double-strand breaks, and genotoxicity) in Figure 7. Our analysis of the reported results ( Figure 8) suggests that most of the experiments used experimental techniques for DNA including gene expression, protein expression, and ROS production for assaying the effect of PEMFs on cells.
We also considered the effects of different pulse frequencies of PEMFs and intensity. To do so, we pooled experimental data based on the frequencies (Figure 9), intensity levels ( Figure 10), time of exposure (Figure 11), and waveforms ( Figure 12) used in each experiment of the 92 publications Among subgroups of frequencies, significant effects were observed at 100 Hz < ƒ (p < 0:001). However, at frequencies smaller than or equal to 10 Hz, no statistically significant effects were observed. Among subgroups of intensities, the presence of response as a result of PEMFs was seen significantly in intensities between 1 and 10 mT (p < 0:05) Analysis of different times of exposure in the studies indicated on effectiveness of PEMFs in chronic exposure > 10 days (p < 0:001) and absence of cell response in acute exposure > 24 h (p < 0:001).
The cells exposed to PEMFs in in vitro experiments, which reported results (cellular response, either presence, or absence Table 1) under different exposure conditions, are shown as follows: (a) classification of experimental techniques in Figure 8, (b) frequency of PEMFs in Figure 13, (c) intensity levels in Figure 14, (d) time of exposure in Figure 15, and (e) waveform in Figure 16. It should be noted that our statistical test only reports the presence or absence of cellular responses in the literature, and it is not concerned with the increased or reduced effect of the mentioned responses.

Publication Bias and Sensitivity Analysis
The results of Egger's and Begg's test demonstrated no publication bias in the meta-analysis of cellular response (presence or absence) in human cells, rat or mouse cells, and other species according to different frequencies and intensity    To evaluate the effect of each single study on the pooled effect size, we removed each study, one by one. We found no significant effects of any individual study on the combined effect sizes in different meta-analysis presentation.  Diniz et al. [94] 10 BioMed Research International

Discussion
This study scrutinized the related scientific literature for the association between PEMFs and cell responses in vitro. Realizing that there were distinctions between cell types in terms of apoptosis, rate of proliferation and age, and other characteristics and that PEMFs parameters can be characterized in terms of frequency, intensity, time of exposure, and waveform, we investigated if there were distinct properties of positive and negative findings associated with these characteristics. The results showed that there was no significant difference between the presence and absence of the cell response to PEMF stimulation in human cells, rat/mouse cells, and other species (Figure 17 for each row (p > 0:05)). However, several aspects of our results are notable, which are given below.
Our findings demonstrated that in in vitro studies, nearly 50% of human cells (Figure 17) would undergo changes due to PEMFs, whereas fewer number of cells in rats/mice (44.61%) and other species (18.18%) were influenced by PEMFs. Thus, a large number of experiments on cells in rats/mice and other species pointed out the absence of any effect caused by PEMFs. Among the studies conducted on human cells, most of them were performed on stem cells. According to the results, it seems that the type of stem cell plays as an effective factor in intracellular processes affected by PEMFs. Especially, in the field of bone tissue engineering in which mesenchymal stem cells are activated by EMF, this finding would be considerable.
Another significant finding of our study was among osteoblast-like cells, MG-63 human osteosarcoma cells seem to be very sensitive to PEMFs (86.1%). The studies have shown that these fields could alter activity through changes in local factor production [4]. However, in human osteogenic sarcoma SaOS-2, the absence of cell response to PEMFs alone was greater in degree than the presence of cell response (75%). PEMFs appeared to have little effect on the phenotype and number of SaOS-2 cells [7].
The potential effects of PEMFs on tendon cells showed that these fields (87.74%), focusing on the potential applicability of this cell source for regenerative medicine purpose, could be effective in the treatment of tendon disorders. In fact, these fields could influence the proliferation, release of anti-inflammatory cytokines, tendon-specific marker expression, and angiogenic factor in healthy human TCs culture models [15].
Analysis of the results of other related studies concerning the effect of PEMFs on the cells of blood cancers like leukemia and lymphoma in human (and on basophilic leukemia cells in rats/mice) showed that these cells were not affected      Another part of this study focused on evaluating the role of intensity and frequency of PEMFs in stimulating cellular responses in the subgroups. This research was subject to some constraints; first, some of the related experimental studies did not provide sufficient descriptions of exposure signal characteristics, especially in expressing waveform, which in turn made us unable to interpret the results fully. Nevertheless, analysis of frequencies of PEMFs used in the studies showed that different frequencies corresponded to different levels of cellular response. In the subgroups, It is worth noting that we may be able to find optimal parameters of PEMF in future studies in the effective ranges obtained from the present study to achieve the most effective response, depending on the desired effect.
Basically, in vitro studies use cells to investigate the interaction mechanisms better by breaking down the complexity of a whole organism into a controllable system. Indeed, each cell with a model system of its own could be suitable for a       Figure 14: Intensity observed from 3306 experiments from 92 peer-reviewed scientific publications (1999-2019). Cells exposed to PEMFs in vitro experiments that reported results (cellular response (presence or absence)) for different exposure conditions. Intensity values are shown in mT.     17 BioMed Research International specific biological aspect. Therefore, although it cannot be expected that humans respond to PEMFs, studies of simple biological systems can advance our understanding about which systems in the body are more susceptible to PEMFs. Therefore, conducting an analysis similar to the present meta-analysis could be useful as a reference for many epidemiological studies or in vivo experiments using the whole organism animal models.

Conclusion
To the best of our knowledge, no other meta-analysis has investigated the effects of PEMF on cell responses in vitro. The findings of this study provided us insight into that which cell types could be more responsive to PEMFs. Additionally, we determined the range of frequencies and intensities which PEMFs appeared more effective. Future research would need to explore the effects of other variables on cell response in vitro and to investigate the effectiveness of PEMFs in vivo.

Data Availability
Access to data is restricted due to ethical concerns.

Conflicts of Interest
The authors declare that they have no conflicts of interest.