A Review of Cellular Mechanism of Pulsed Electromagnetic Field (PEMF) in Inhibiting Adipogenesis of Mesenchymal Stem Cells

: PEMF is a method that utilizes electric current using a coil that is powered by dynamic electric current, it will cause a dynamic magnetic field in the coil. In the last few decades, it is known that PEMF has the potential to be used as a modality in the treatment of obesity. For this reason, PEMFs have been studied in vitro as their effect in inhibiting the adipogenesis of Mesenchymal Stem Cells (MSC). But the use of PEMF therapy in inhibiting adipogenesis still has many challenges. Understanding of the mechanisms that occur in cells that are exposed to PEMF is also still very slight. This article reviews the studies that investigated in vitro effect of PEMF in inhibiting adipogenesis studies and the underlying mechanism. In the future, it is hoped that this review can be used as a basic reference for in vivo research and clinical trials related to inhibiting adipogenesis using PEMF


Introduction
Studies have confirmed that obesity has become a serious health problem in the world (Djalalinia et al., 2015;Ng et al., 2014;Kelishadi, 2007).The prevalence of obesity from year to year continues to increase dramatically in most countries (Hruby and Hu, 2015).Data recorded by the World Health Organization (WHO) in 2016, shows that 1.9 billion adults in the world are overweight and at least 650 million people experiencing obesity (WHO, 2016).
Obesity occurs when there is excessive deposition of fat into adipose tissue (Ofei, 2005).Abnormal accumulation can cause various negative effects on the body, including cancers, type 2 diabetes, hypertension, stroke, coronary artery disease, congestive heart failure, asthma, chronic back pain, osteoarthritis, pulmonary embolism, gallbladder disease, and also an increased risk of disability (Djalalinia et al., 2015).Studies also found a consistent association between the high risk of premature morbidity or mortality particularly and cardio metabolic morbidity with overweight and obesity (Reilly and Kelly, 2011).
Until now, treatment for obese patients is carried out through strict dietary control, physical exercise, changing lifestyle habits, and consuming appetite suppressant drugs (Wyatt, 2013).Managing weight in a conventional manner requires at least one year and sometimes requires more time length (Benton and Young, 2017).One of the potential modalities that can be used for obesity therapy is the use of used Electro Magnetic Field (PEMF).
PEMF is a form of biophysical stimulus that utilizes an electric current indirectly.The PEMF method is a method of utilizing electric current using a coil that is powered by dynamic electric current, it will cause a dynamic magnetic field in the coil (Yadollahpour and Rashidi, 2015).The effect of PEMF has been studied further related to its ability to inhibit adipogenesis.Adipogenesis produces adipocyte cells that function for energy storage, an imbalance in the process of storing and expending energy can cause obesity (Longo et al., 2019).
Adipogenesis can be done in vivo and in vitro.One type of cell that can be used to observe adipogenesis in vitro is the Mesenchymal Stem Cell (MSC), which has the ability to differentiate into adipocyte cells (Kozlowska et al., 2019).At the present, the research related to PEMF and adipogenesis is still limited.It is because of the limited information related to the mechanism that underlies the effects of PEMF exposure.The lack of standardization of the PEMF variables used in the experiment also becomes a problem.This review discusses in vitro studies related to PEMF exposure in inhibiting adipogenesis and its underlying cellular mechanisms.

Adipogenesis of Mesenchymal Stem Cells
Stem cells are known as cells that do not have a specialization, can renew themselves continuously, and can differentiate into several types of cells in certain physiological or experimental conditions (Wei et al., 2013;Chagastelles and Nardi, 2011).In general, stem cells can be categorized into several types, namely Embryonic Stem Cells (ESC), induced Pluripotent Stem Cells (iPSC), and adult stem cells (Zakrzewski et al., 2019).One type of adult stem cell that is widely used in the medical world is mesenchymal stem cells or human Mesenchymal Stem Cells (hMSC), this type of stem cell is multipotent and non-hematopoietic (Parekkadan and Milwid, 2010).Like other adult stem cells, mesenchymal stem cells can proliferate over a long period of selfrenewal.Mesenchymal Stem Cells (MSC) have the capacity to differentiate into mesodermal, ectodermal, and endodermal lineages (Wakao et al., 2012).
MSC is present in almost every tissue.25 MSC, which were first investigated, were obtained from bone marrow sources (Ullah et al., 2015;Orbay et al., 2012).Until now, MSC has been isolated from several other parts of the body, including adipose tissue (Bunnell et al., 2008), umbilical cord (Hassan et al., 2017), amnion (Araújo et al., 2018), chorion (Araújo et al., 2018), placenta decidua (Shaer et al., 2014), dental tissue (Huang et al., 2009), peripheral blood (Trivanović et al., 2013), synovial fluid (Harvanová et al., 2011), skin (Orciani and Di Primio, 2013) and Wharton's jelly (Varaa et al., 2019).As a result of MSC being isolated from various sources and by various means of isolation and propagation, the determination of the characteristics of MSC is inconsistent and differs from one laboratory to another (Dominici et al., 2006).Therefore, the International Society for Cellular Therapy (ISCT) determines standard criteria for MSC characteristics.The first criterion, MSC must be able to stick to plastic (plastic adherent) when in a standard culture using culture containers.Second, 95% of the MSC population must positively express CD105, CD73, and CD90, which are measured using flow cytometry.MSC must also have 2-5% negative expressions of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR.Third, MSC must be able to differentiate into osteoblasts, adipocytes, and chondroblasts under standard in vitro differentiation conditions (Dominici et al., 2006).
Adipogenesis refers to the process of adipose tissue formation, this process consists of many stages that begin with the expansion of MSC clones, MSC differentiation into preadipocytes, and the formation of mature adipocytes (Fink and Zachar, 2011;Chen et al., 2016;Kwan et al., 2015).MSC cultured in vitro with adipogenic induction media are able to complete their adipogenesis in various time frames, ranging from 7 to 21 days (Lee and Fried, 2014).Adipogenic induced cells will undergo morphological changes and have vacuoles or lipid "droplets" as a sign of differentiated cells (Lee and Fried, 2014) These cells can be retained in culture for more than 40 days, the longer the time culture and induction of adipogenic media, the size of lipid droplets will grow even greater (Kawai and Rosen, 2012).Understanding the process of differentiation can help design potential antiobesity strategies (Lee and Fried, 2014).
Adipogenesis is a complex process, this process is integrated with other processes, which involve many transcription factors that have a specific role (Shaer et al., 2014;Takada et al., 2009).Molecular and cellular mechanisms of adipocyte differentiation have been studied in depth through in vitro and in vivo studies (Takada et al., 2009).After the expansion of preadipocytes, MSC will differentiate into mature adipocytes under strict control of various transcription factors, including C/EBPβ/δ and PPARγ (Takada et al., 2009;Moseti et al., 2016).PPARγ is a nuclear receptor as well as a transcription factor that plays a key role in adipogenesis (Takada et al., 2009).Activation of PPARγ is the initiation of adipogenesis because PPARγ is a transcription factor that activates adipogenic genes that play a role in adipogenesis (Tontonoz et al., 1994).Excessive PPARγ expression in fibroblast cell lines is known to initiate adipogenesis (Barak et al., 1999).In contrast, PPARγ defects in ESC and mouse embryonic fibroblasts are known to inhibit the occurrence of adipogenesis in fibroblast cell lines known to initiate adipogenesis (Lefterova et al., 2014).
PPARγ is a nuclear receptor and a ligand dependent transcription factor that play a role in the differentiation and regulation of adipose cells (Ma et al., 2018;Cariou et al., 2012).PPARγ also plays a role in the process of storing lipids, thermogenesis, active modulators of lipid metabolism, and insulin sensitivity (Tontonoz et al., 1994).Some lipid metabolites have been known to be involved as adipocytes.PPARγ activators, including unsaturated fatty acids or poly-unsaturated fatty acids, eicosanoids, prostaglandins, 15-deoxy-Δ12,14 prostaglandin J2 (Ma et al., 2018).However, these molecules are known to have a low affinity for PPARγ and their numbers are also small in adipocyte cells (Ma et al., 2018).This makes the physiological relevance of these molecules as endogenous PPARγ ligands (Ma et al., 2018).At this time, physiologically relevant PPARγ ligands remain unknown (Ma et al., 2018).The known exogenous PPARγ ligands are Thiazolidinediones (TZD), which is an anti-diabetes drug (Dave et al., 2012).

Correlation Between Adipogenesis and Obesity
Adipose cells resulting from adipogenesis have the main function of energy storage and lipid homeostasis (Choe et al., 2016).In mammals, adipose tissue consists of at least two types of adipose tissue, namely white adipose tissue or White Adipose Tissue (WAT) and brown adipose tissue or Brown Adipose Tissue (BAT) (Gómez-Hernández et al., 2016;Marcelin and Chua Jr, 2010).WAT is the dominant type of adipose tissue, WAT is located in the subcutaneous and visceral parts that surround internal organs, such as the heart, intestine, kidney, and gonad (Betz and Enerbäck, 2015).The primary function of WAT is as a storage of energy in the form of triglycerides (Betz and Enerbäck, 2015).In humans, the development of WAT occurs mostly after birth and continues throughout life.45 the BAT has a primary role as a temperature regulator.64BAT has the ability to change chemical energy and instantly becomes hot when activated by the nervous system sympathetic (Ljungberg, 2006).BAT allows small mammals to maintain a constant body temperature at cold (Ljungberg, 2006).
Adipose tissue function in general is closely related to the process of lipid metabolism.The process of lipid metabolism begins with the process of digestion of fat consumed, this fat will go through the process of digestion and break down into fatty acids and glycerol.Glycerol is a component that dissolves in water so that glycerol will easily enter the circulatory system to the liver.Conversely, fatty acids have water insoluble properties, so fatty acids will be emulsified before going to the small intestine to be absorbed.In small intestinal epithelial cells, fatty acids are converted to triglycerides.Triglycerides are the main energy source in the body, because triglycerides have hydrophobic properties, triglycerides are transported through the circulation in a complex form called lipoprotein (Desai et al., 2013).In the body, lipoprotein is metabolized through two pathways, namely the exogenous and endogenous pathways (Desai et al., 2013).
The exogenous pathway is the pathway for transporting lipids from the small intestine to the tissues, in which the triglycerides are circulated in the form of chylomicrons (Desai et al., 2013).A large number of triglycerides carried in the chylomicron form are then hydrolyzed by Lipoprotein Lipase (LPL).The bond between chylomicrons and LPL causes triglycerides to be hydrolyzed into Free Fatty Acids (FFA).Most of this FFA will be used as energy in the muscles and heart or directed for re-esterification and storage in adipose tissue.Meanwhile, the remnants of the hydrolyzed chylomicron will be cleansed from the circulation by absorption by the liver through LDL Receptor (LDLR) or LDL-Receptor Related Protein (LRP) (Desai et al., 2013).
Endogenous lipoprotein pathways are pathways for the distribution of lipids from the liver to peripheral tissue (Desai et al., 2013).These pathways begin with the secretion of Very Low-Density Lipoprotein (VLDL) particles that are rich in triglycerides from liver hepatocyte cells.These particles are then hydrolyzed by LPL and become FFA which will be stored in muscle and adipose tissue.Another result of this hydrolysis is Intermediate Density Lipoprotein (IDL) particles, some of the IDL produced will go to the liver and be further metabolized by Hepatic Lipase (HL).Some LDL will carry out its role in circulation, which is to transport cholesterol to peripheral tissues.In contrast, High Density Lipoprotein (HDL) plays a role in returning excess cholesterol in the peripheral tissues to the liver (Desai et al., 2013).
An imbalance between energy storage and expense in adipose tissue will cause obesity (Choe et al., 2016).In addition, obesity can also be caused by an increase in the process of adipogenesis (Camp et al., 2002).An increase in the process of adipogenesis also impacts an increase in the mass of the adipose tissue followed by an increase in the number of cells fat (hyperplasia) and increased fat cell size (hypertrophy) (Wade, 2013).In this context, a decrease in adipose tissue mass can be directly related to the healing process of obesity.A decrease in adipose tissue mass is known to be able to cause loss of lipids through lipolysis and loss of mature fat cells through apoptosis (Choe et al., 2016).

Pulsed Electromagnetic Field
Electromagnetic modalities include all types of modalities that use electricity to produce electric and magnetic fields (Malmivuo and Plonsey, 1995).Generally, this stimulation is carried out using electronic devices outside biological networks to cause electrical effects in the network (Lu et al., 2015).The large electric fields that will appear on the network depend on tissue type and exposure characteristics.A magnetic field of 0.1-1 mT can produce an inductive electric field of 1-100 mV/cm in the target network.One of the electromagnetic based stimulation methods that are widely used in the health field is PEMF.
PEMF is known as a form of biophysical stimulus that utilizes an electric current indirectly.PEMF allows a dynamic electric current to flow through the Helmholtz coil so as to produce a dynamic magnetic field effect with pulsed intervals that can stimulate the microenvironment to influence physiological activity in organisms during pulsed (Haghnegahdar et al., 2014) These events are in accordance with Maxwell's equations expressed by the formula ∇  E = dB/dt.
Electromagnetic waves are electric currents and paired magnetic fields produced by the movement of electrons (Malmivuo and Plonsey, 1995).This radiation produces energy that moves and spreads over time (Touitou and Selmaoui, 2022).Electromagnetic radiation has a spectrum that contains a series of electromagnetic waves in different frequencies, which cannot be seen by humans with the eye naked (Touitou and Selmaoui, 2022).These electromagnetic waves can be generated, transmitted, and distributed according to three frequency ranges, namely: The Extremely Low Frequency range (ELF) covers frequencies around the number 50-60 Hz, the intermediate frequency i.e., the frequencies produced by electrical lines and electronic electrical equipment range from 300 Hz to <10 MHz and radio frequencies (10 MHz to 300 GHz) including radar, radio broadcast, television and telecommunications (Yadollahpour and Rashidi, 2015).
Electromagnetic waves have ionization properties.Based on its characteristic ionization properties, the spectrum of electromagnetic radiation can be divided into two main categories, ionizing and non-ionizing radiation (Malmivuo and Plonsey, 1995).Waves with ionizing frequencies have enough energy to remove electrons from atoms or molecules so that they are harmful to cells (Malmivuo and Plonsey, 1995).Examples of ionizing radiation are ultraviolet, X-rays, and gamma radiation.
The use of electromagnetic waves in the health field has been known for thousands of years (Gómez-Hernández et al., 2016;Fukada and Yasuda, 1957).The first written document related to the application of electric waves for medical purposes was known to have existed in (Ullah et al., 2015;Fink and Zachar, 2011) CE when Scribonius Largus recommended the use of torpedo fish to cure headaches and gouty arthritis (Huegel et al., 2018).
Research into electromagnetic waves and their biological effects continues to grow.In the early 1800s, researchers in the fields of physics and biology for the first time discovered the relationship between physical forces including mechanical force, electricity, magnetism, and ultrasonic waves to the biological state of bones (Fukada and Yasuda, 1957).Departing from these findings, the use of electromagnetic waves in the field of bone healing began known.This is supported by research conducted by Fukada and Yasuda (1957) Who found an endogenous electrical effect on bone biology processes, particularly in fracture healing (Huegel et al., 2018).
The utilization of electromagnetic waves in the health field does not stop at the process of healing bones.Electromagnetic waves are also known to have the potential to help heal wounds in the skin and underlying tissues (Francisco et al., 2013), reduce postoperative pain and swelling (Janicak and Dokucu, 2015) and other forms of electromagnetic exposure can also be used to cure depression (Janicak and Dokucum 2015).

Pulsed Electromagnetic Field as a Potential Bioelectromagnetic Therapy to Inhibit Adipogenesis of Mesenchymal Stem Cells
The use of PEMF in medical therapy becomes larger.Several studies related to this problem, in more detail, are described in Table 1.
Unlike the PEMF studies on bone healing and another therapy, studies related to PEMF and their effects on cell differentiation, especially adipogenesis, have not been done much, so the response mechanism that occurs in cells due to PEMF exposure is not well known.In recent times, there have been several studies focusing on the effects of electromagnetic waves on cell differentiation, specifically in the inhibition of adipogenesis for the treatment of obesity.In vitro research conducted by Du et al. (2014) Concluded that electromagnetic wave exposure can induce osteogenesis and inhibit the differentiation of mesenchymal stem cells to become adipocyte cells (Du et al., 2014).Other studies conducted by Lu et al. (2015) Also support the same thing, namely exposure to electromagnetic waves can suppress the expression of transcription factors that support adipogenesis (Lu et al., 2015).
The inhibition of adipogenesis due to PEMF exposure is supported by research conducted by Du et al. (2014) in this study, the results were obtained that PEMF exposure with a frequency of 7.5 Hz, magnetic field magnitude 0.4 T and exposure time of 2 h/day for 15 days of adipogenesis can inhibit the expression of adipogenic genes.However, in this study, checking of gene expression was only carried out on the last day of exposure, i.e., on the 15 th day so it could not be seen how the pattern of gene expression occurred during the 15 days of the exposure time.The results also showed that on the 4 th and 7 th day after exposure, no statistically significant differences were found in PPARγ gene expression.Although seen in the PEMF group, PPARγ expression had a lower tendency than the two control groups.There were no significant differences found on days 4 and 7 can be caused by several factors, including the micro conditions that are applied to cells.It is known that the microenvironment contributes to cell differentiation.Several studies related to PEMF and adipogenesis of MSC in more detail are described in Table 2.

The Mechanism of Adipogenesis Inhibition in Mesenchymal
Stem Cells by Pulsed Electromagnetic Field Changes in the magnetic field produced by electromagnetic exposure stimulus will produce an electric field induced in the network.Cellular and biophysical mechanisms of electromagnetic waves toward cells are still the focus of research.The high complexity of the molecular mechanisms underlying cell responses to exposure and the many types of exposure are one of the obstacles to the difficulty of elaborating on the focus of the research (Francisco et al., 2013).
The main theory that can explain the molecular mechanism of cell response to electromagnetic waves is the theory of changes in plasma membrane potential.Electric fields exposed to cells can act as extracellular signals to cells and will affect ion transport at the cellular level (Ross et al., 2015).It is known that the magnetic gradient of electromagnetic waves can penetrate deeper tissues and work directly on cells by changing the potential of the plasma membrane.Ross et al. (2015) this is thought to have an impact on the movements of molecules that will pass through the plasma membrane (Ross et al., 2015), so as to be able to influence cell activity.In plasma membranes, there are voltage gated ion channels that can open and close in response to changes in membrane potential.When the channel is open, the ions will easily pass through the membrane, but when closed, then the flow of ions will be prevented from passing through the membrane (Fig. 1).Research on cellular mechanisms and cell response to electromagnetic field stimulation has been investigated using mesenchymal stem cells (Zhang et al., 2016).It is known that electromagnetic field stimulation can increase cytosolic Ca2+ concentrations via the calmodulin pathway, which then triggers cell proliferation (Zhang et al., 2016;Brighton et al., 2001).High levels of Ca2+ in cells are believed to be the effect of activating voltage-gated Ca2+ channels on the plasma membrane due to external electromagnetic exposure (Zhang et al., 2016).Mesenchymal stem cells also have other voltage-gated ion channels, such as voltage gated Na + channels and voltage gated K + channels, but to the extent that There is currently no research examining whether other voltage gated ion channels are also activated due to electromagnetic exposure.However, if you see the effect of electromagnetic exposure on the voltage-gated Ca2+ channel, then it is possible that other voltage gated ion channels can also be activated by electromagnetic stimuli Zhang et al., 2016.Electromagnetic field stimulation is able to change the plasma membrane potential thereby activating voltage gated Ca2+ channels and increasing cytosolic Ca2+ concentrations (Araújo et al., 2018;Desai et al., 2013;Camp et al., 2002).Increased Ca2+ concentrations are known to have an important role in regulating intracellular signaling activation, cell proliferation, and cell differentiation, especially in the induction process of osteogenesis and adipogenesis inhibition (Zhang et al., 2016;Bae et al., 2018).
Inhibition of adipogenesis caused by an increase in intracellular Ca2+ can occur through the calcineurin activation pathway (Meldolesi, 2008).Through this pathway, it is known that intracellular Ca2+ increase modulates adipocyte lipid metabolism and inhibits the early stages of adipogenesis in mice and mice (Shi et al., 2000).The further evaluation also concluded that an increase in intracellular Ca2+ could inhibit the metabolism of adipocyte lipids and inhibit the initial stages of adipogenesis in mice and mice.Shi et al. (2000) direct adipogenesis in humans, which is characterized by decreased PPAR expression (Shi et al., 2000).
Calreticulin protein (CRT) is a protein that keeps the Ca2+ concentration in the cytosol high.In this situation, Ca2+ will bind to the Calmodulin subunit (CaM) so that it activates Calcineurin (CaN).This activation will give a specific signal to the nucleus compartment to induce the transcription Factor (F1) and block the activation of the RXR-PPARγ complex.As a result, gene transcription involved in adipogenesis is blocked and adipogenesis cannot take place (Meldolesi, 2008).Conversely, if Ca2+ concentrations are low in the cytoplasm, the calcium/calmodulin dependent protein kinase II (CaMKII) enzyme will be activated via the c-Srk pathway.A possible consequence of CAMKII phosphorylation is the activation of another transcription Factor (F2) which promotes activation of the RXR-PPAR kompleks complex thereby inducing adipogenesis.
Inhibition of adipogenesis is antagonistic to the process of osteogenesis.If adipogenesis is inhibited, the osteogenesis pathway is induced, and vice versa.Previous studies related to the effect of PEMF exposure on osteogenesis showed that PEMF exposure was able to increase Wnt5a expression (Umiatin et al., 2019).Increased Wnt5a expression was known to inhibit adipogenesis by suppressing PPAR expression (Takada et al., 2009).The induction of osteogenesis can be triggered by the activation of the Wnt5a/β-catenin signaling pathway, this activation is followed by the inhibition of adipogenesis which is characterized by a decrease in adipogenesis markers namely PPARγ and leptin (Bae et al., 2018).The relationship between activating osteogenesis through the Wnt5a signaling pathway that causes adipogenesis inhibition (Takada et al., 2009).Activation of the non-canonical Wnt signaling pathway can directly suppress the PPARγ function in the mesenchymal stem cell differentiation process.Umiatin et al. (2019) this will bind to RXR-PPARγ and inhibit the process of adipogenesis (Ayala et al., 2018).
Research conducted by Ayala et al. (2018) analyzed the effects of dynamic magnetic field exposure of 17-70 mT on the potential changes in the skeletal muscle cell plasma membrane.In this study, a mathematical equation was obtained that produced a change in membrane potential of 8 mV for 1 sec.To reach the threshold for changes in membrane potential, a value of 10-15 mV is required (Ayala et al., 2018).Increased cytosolic Ca2 + leads to the activation of calmodulin thereby inducing the potential for MSC differentiation in the chondrogenic pathway (Uzieliene et al., 2018).Increased cytosolic Ca2 + is chained by the activation of Voltage Operated Calcium Channels (VOCC) and Transient Receptor Potential (TRP) due to electromagnetic stimulus (Uzieliene et al., 2018).

Conclusion
In conclusion, after reviewing the recent scientific literature, PEMF therapy can be one of the potential methods to inhibit adipogenesis.It is proven by in vivo experiments with positive results.But there is no standard for adipogenesis inhibition therapy using PEMF.So, it is necessary to carry out further research by paying attention to variations in physical parameters including frequency, magnetic intensity, and duration of exposure to find the optimal parameters for adipogenesis inhibition using a specific PEMF device design.
Unfortunately, to date, there have been no in vivo experiments related to the inhibition of adipogenesis.For further research, the in vivo study is highly recommended on this topic to find out whether PEMF exposure can have an effect at the organism level.

Table 1 :
The use of PEMF as a potential therapeutic tool