Comparative assessment of pulsed electromagnetic fields (PEMF) and pulsed radio frequency energy (PRFE) on an in vitro wound healing model

Abstract

Pulsed electromagnetic fields (PEMF) and pulsed radio frequency energy (PRFE) have been shown to accelerate wound healing process due to its ability to stimulate cellular proliferation. However, comparative efficacy of PEMF and PRFE on an in vitro wound healing study has yet to be studied. The present study examined the effect of PEMF and PRFE on an in vitro wound healing model developed by using 3T3 mouse fibroblasts. Cells were exposed to PEMF (75 Hz frequency, square waveform, and 1-mT magnetic field) and PRFE (27.12 MHz, Phase-shift keying (PSK), and 13-dBm amplitude signal) for 5 hours. The migration rates of 3T3 fibroblasts were determined by capturing images at time points of 0, 12, 24, 48, and 72 hours. Cell proliferation was also quantified. The results of the migration and proliferation assays showed that PEMF and PRFE applied groups had significantly greater cell proliferation and migration compared to control group. In addition, PRFE application showed significantly faster wound area closure compared to PEMF application.

Keywords

Pulsed radio frequency energy (PRFE)pulsed electromagnetic fields (PEMF)wound healing rectangular spiral applicator

1. Introduction

Chronic wounds, which directly affect the quality of life and contribute significantly to the cost of health care in the world, are major healthcare problem [1]. Chronic ulcers or wounds are defined as wounds that have failed to proceed through the overlapping phases in less than 6 weeks. Wound healing is a complex and dynamic series of biological processes which includes synthesis of the bioregulatory molecule nitric oxide (NO) and regeneration of the damaged or lost tissue [2]. All stages of the repair process are controlled by wide variety of different mediators like extracellular matrix (ECM) molecules, platelets, inflammatory cells, growth factors, cytokines, and chemokines [3]. These molecules affect promoters in the wound healing stages by activating and attracting neutrophils, macrophages, endothelial cells and fibroblasts [4]. The wound healing process can be divided into four overlapping phases: (i) homeostasis, (ii) inflammatory phase, (iii) proliferative phase or new tissue formation (neoangiogenesis, proliferation, re-epithelialization), and (iv) tissue remodeling (remodeling of ECM) [5]. Recently, there have only been a handful of technical advances that have contributed to changes in the discipline of wound management. It is reported that wound healing process could be accelerated when conventional treatments are supported by adjuvant therapies such as hyperbaric oxygen therapy [6], negative pressure wound therapy [7], ozone therapy [8], topical oxygen therapy [9], laser therapies [10], and electrical stimulation [11]. Some of these adjuvant therapies may act by cellular, chemical, biological, or biochemical processes. Despite these advancements, no therapeutic method has been defined so far due to the subjectivity and complexity of the wound healing process [12].

In the last century, electromagnetic fields (EMF) have achieved an important role as an adjuvant therapy in medicine [13]. It was reported that low-frequency EMF may affect various functions of different pathways such as signal-transduction cascades [14], gene transcription [15], cell growth [16], and Ca ${}^{2+}$ , Na ${}^{2+}$ , K ${}^{2+}$ ion transport system [17]. Additionally, it was demonstrated that low-frequency EMF stimulation increases the proliferation of fibroblasts [18] and endothelial cells [19], and induces differentiation of skin fibroblasts in culture [20]. Low-magnitude and low-frequency pulsed electromagnetic fields have been used for treating soft tissue injuries [21], breast cancer [22], skin ulcers [23], non-uniform bone fractures [24], and degenerated nerves in the past several decades [25]. Time varying electromagnetic fields comprising rectangular or arbitrary waveforms ( $<$ 100 Hz) are described as pulsed electromagnetic fields (PEMF). Pulse modulated radio frequency waveforms, in particular, those in the 15–40 MHz range, are described as pulsed radio frequency fields (PRFE). The Federal Communications Commission assigned three frequencies at the short end of the radio frequency band (40.68, 13.56, and 27.12 MHz) for medical use in 1947 [26]. Most radio frequency electromagnetic field therapy devices operate at 27.12 MHz in clinical practice [27]. Therapies using these have been shown to enhance healing when used as adjunctive therapy for a variety of wound healing cases [28]. However, the effect of PEMF and PRFE application on in vitro wound healing model have yet to be investigated. From this viewpoint, the main purpose of this study is to investigate the effects of PRFE at 27.12 MHz on the proliferation and migration of cells involved in in vitro wound healing. The other objective of this study was to compare for the first time the effect of PEMF and PRFE on an in vitro wound healing model developed with 3T3 mouse fibroblasts.

2. Materials and methods

2.1 Exposure systems

Bipolar power supply (Kikusui, PBZ40-10, Santa Clara, CA, USA), Pasco Helmholtz coils (Pasco, Roseville, CA, USA), and PASPORT 2-Axis Magnetic Field Sensor (Pasco, Roseville, CA, USA) were used to design and characterize the PEMF setup. A PEMF exposure system was used to generate uniform time varying magnetic fields in the low-frequency range, with a magnetic induction (magnetic flux density) of up to 5 mT (Fig. 1A). Pasco Helmholtz coil system was used to deliver PEMF signal and magnetic field intensity was quantified by magnetic field sensor.

Figure 1.

PEMF exposure system (A), PRFE system (B), Schematic diagram ofexposure setup and experimental timeline (C).

DDS function generator (Suin TFG8080, Shijiazhuang, China), rectangular spiral planar applicator, and spectrum analyzer (GW Instek, New Taipei City, Taiwan) were used for developing PRFE setup. A PRFE exposure system with different signal modulations (PFS, FSK, AM, etc.) was used to generate uniform time varying magnetic fields at 27.12 MHz (Fig. 1B). Our designed 27.12 MHz PRFE applicator was used to deliver PRFE signal. The applicator was simulated by Finite Integration Method (FIT) based CST Studio Suite 2015 (Darmstadt, Germany).

2.2 Characterization of PEMF/PRFE exposure systems

Schematic diagram of exposing PEMF and PRFE to cell culture plate is shown in Fig. 1C. The PEMF exposure system comprised one Helmholtz coil system measuring 20 cm in diameter, and the PFRE exposure system comprised a square patch applicator with an edge length of 12 cm. A current flow of 0.4 A passed through two 500-turn Helmholtz coils setups comprising two field coils mounted on a base to provide a uniform magnetic field between the coils. The diameter of the copper wire was 0.8909 mm. The coils were connected in series and each coil was powered by a power supply at 75 Hz frequency, 1.3 ms pulse duration with a square waveform. The system enabled exposure with low frequencies and a magnetic field strength of up to 5 mT. The magnetic field was measured using the hall effect sensor. A DDS function generator fed the applicator with a frequency of 27.12 MHz, PSK modulation, and a 13-dBm amplitude signal.

2.3 Theory-Biot-Savart law

The Biot-Savart law is used for computing the magnetic field ( $\vec{B}$ ) generated at some arbitrary point (P) by a steady current (i). It relates this incremental magnetic field, located a distance from an incremental length of wire, with the current flowing in the direction of the incremental length.

Biot-Savart law, $\Delta\vec{B}=\frac{\mu_{0}i}{4\pi}\frac{\Delta\vec{l}\sin\Theta}{r^{2}}$ or in vector form, $\Delta\vec{B}=\frac{\mu_{0}i}{4\pi}\frac{\Delta\vec{l}\times\vec{r}}{r^{3}}$ .

The vector $\Delta\vec{B}$ can be resolved into parallel and perpendicular components with respect to the central axis along x using the unit vectors $\hat{i}$ and $\hat{j}$ so that

$\displaystyle\Delta\vec{B}=\Delta B_{\parallel}\hat{i}+\Delta B_{\bot}\hat{j}$ (1)

where $\Delta\vec{B}_{\parallel}$ is a component along the x-axis and $\Delta\vec{B}_{\bot}$ is a component perpendicular to the x-axis. $\Delta\vec{B}_{\parallel}$ and $\Delta\vec{B}_{\bot}$ are related to the magnitude of $\Delta\vec{B}$ , $\Delta\vec{B}$ , by the following equations

$\displaystyle\Delta\vec{B}_{\parallel}=\Delta\vec{B}\cos\alpha$ (2)

and

$\displaystyle\Delta\vec{B}_{\bot}=\Delta\vec{B}\sin\alpha$ (3)

where $\alpha$ is the angle between the plane of the loop and the vector from the line segment $\Delta\vec{l}$ to point P.

The total magnetic field is then found by summing all the increments of $\Delta\vec{B}$ for every increment of $\Delta\vec{l}$ so that

$\displaystyle\vec{B}=\sum\Delta\vec{B}=\sum\Delta B_{\parallel}\hat{i}+\sum% \Delta B_{\bot}\hat{j}$ (4)

The magnetic field in the center of Helmholtz coil system can be calculated by

$\displaystyle\vec{B}=\frac{\mu_{0}i}{4\pi}\frac{R}{(R^{2}+x^{2})^{3/2}}2\pi R=% \frac{\mu_{0}i}{2}\frac{R^{2}}{(R^{2}+x^{2})^{3/2}}$ (5)

and

$\displaystyle\vec{B}=\frac{\mu_{0}i}{2}\frac{R^{2}}{(R^{2}+x^{2})^{3/2}}$ (6)

Both the PEMF and PRFE stimulated samples were maintained outside the incubator at room temperature. The PRFE exposure system comprised a field generator, an amplifier, and a wideband applicator. Figure 2 shows the diagram of PEMF exposure system. Cells were placed in the central part of the system that presented the highest field homogeneity. Cell behavior and morphological changes were examined under a phase contrast microscope. 3T3 fibroblasts seeded plate was kept under the same conditions without exposure and were used as control group.

Figure 2.

Simulation results of the PEMF Helmholtz coil setup; Helmhltz coil with cell culture (A), Magnetic field intensity calculation along the petri dish (A/m), inlet image shows the 3-D distribution of magnetic field (B), B-field (mT) measurement (C).

2.4 Fabrication of the PRFE applicator

The rectangular spiral structure is a variation of the Archimedes spiral structure, which has a simple structure and higher space utilization. Therefore, helical applicator in this study was designed in a planar rectangular spiral structure. A rectangular spiral structure effectively reduces aplicator size and weight. In this design, a metal pin that contacted with the first segment of the rectangular spiral is connected to the inner conductor of the coaxial cable and the outer conductor is attached to the ground.

2.5 Cell Cultures

The 3T3 fibroblast cell line (the 3T3 abbreviation comes from three-day transfer inoculum 3 $\times$ 10 ${}^{5}$ cells) was generously donated by the Ege Research Group of Animal Cell Culture and Tissue Engineering Laboratory (Department of Bioengineering, Ege University, Izmir, Turkey). All chemicals used in cell culture experiments were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cells were routinely grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS), 1% L-glutamine, and 0.1% penicillin/streptomycin. The cells were cultured in 75-cm ${}^{2}$ culture flasks at 37 ${}^{\circ}$ C in a 5% CO ${}_{2}$ atmosphere. All cultures were passaged two or three times per week at 80% confluence with a trypsin/EDTA detachment. For this study, passage P19 was used.

2.6 In vitro wound closure (scratch) assay

3T3 cells were seeded in 24-well plates at a density of 1 $\times$ 10 ${}^{5}$ cells/ml and allowed to grow for 24 h at 37 ${}^{\circ}$ C in a 5% CO ${}_{2}$ atmosphere. A small linear scratch was created in the confluent monolayer by gently scraping with sterile 200- $\mu$ l pipette tips (care was taken during the scratching process to ensure that universal size and distance was produced for all samples). Cells were rinsed with sterile PBS to remove cellular debris before adding the media. PEMF and PRFE treatments were performed for 5 hours and the closure rate of the scratch area was evaluated. During the treatment, all plates including the control group were kept at room temperature. The scratched area was then inspected microscopically at 0, 12, 24, 48, and 72 hours. As the 3T3 cells migrated to fill the scratched area, the images were acquired at 0, 12, 24, 48, and 72 hours after the magnetic field exposure by using an Olympus CKX41 inverted light microscope (Olympus, Tokyo, Japan) with a 10 $\times$ phase objective. For quantification, the enclosed area of the wounds calculated and compared to the control with the software ImageJ 1.32 Software (National Institute of Health, MD, USA) at 0 and 72 hours (Fig. 5B). The experiments were performed in triplicate.

2.7 Cell proliferation analysis

The proliferation of cells after the treatment was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After the treatments, the medium was replaced and the MTT substrate (Vybrant ${}^{\@setsize{\scriptsize}{9.5pt}{\viiipt}{\@viiipt}{\textregistered}}$ MTT Cell Proliferation Assay Kit, Invitrogen, Carlsbad, CA, USA) was added to the final concentration of 0.5 mg/ml. The cells were incubated for 2 h in culture conditions and formazan salts dissolved with DMSO. The absorbance was measured at 540 nm using a Synergy ${}^{\text{TM}}$ HTX microplate reader (BioTek, Winooski, WT, USA). Data were recorded and analyzed for the assessment of the effects of PEMF and PRFE treatments on cell proliferation and growth inhibition. The percentage of growth inhibition was calculated from the optical density (OD) obtained from the MTT assay. Measured absorbance values were correlated to the equivalent number of cells by using a calibration curve constructed with reference number of cells. The procedure was also repeated at 12, 24, 48, and 72 hours. The experiments were performed in triplicate and data were analyzed using Excel Professional Plus 2013 software.

2.8 Statistical analysis

Differences among groups were determined by one-way ANOVA, followed by Tukey’s post hoc test. $p<$ 0.05 value was considered as statistically significant. The error bars represent mean $\pm$ standard errors (SEs) for five independent experiments ( $n=$ 3); each was carried out with two replications.

3. Results and discussion

3.1 Designing and characterization of pemf/prfe exposure systems

New methods of rapid tissue regeneration are being investigated to shorten the wound healing process. In recent years, the effects of physical methods such as electric currents, laser beams, and ultrasound on the repair of tissue injury have been demonstrated in experimental studies. The effect of low-frequency PEMF and 27.12 MHz PRFE on different cells in wound healing remains to be investigated. Therefore, this study was focused on the effects of pulsed electromagnetic fields on the healing process in an in vitro wound healing model. In this study, we intended to design an applicator by examining the effects of PRFE conducted at 27.12 MHz on experimental in vitro wound healing model. PRFE and PEMF application on in vitro wound closure were also compared in order to observe superior electromagnetic field for cell proliferation and wound healing.

In vitro scratch was created in 3T3 mouse fibroblasts seeded 24 well plates and placed in PEMF Helmholtz coil system (Fig. 2A). The magnetic field distribution between two identical coils were simulated in MATLAB. The parameters of the simulation were taken as following. The z component of magnetic field intensity is sketched from $x=-$ 15 cm to $x=$ 15 cm for PEMF coils on the same axis. The simulation results showed that the dominant component of magnetic field intensity is made to be homogenous in magnitude on horizontal plane by the use of Helmholtz coils (Fig. 2B). The magnetic field intensity was measured throughout the magnetic field application on cell culture plate. The results showed that approximately 1 mT magnetic field intensity horizontally was maintained during 5 hours of PEMF application (Fig. 2C).

The PRFE applicator was initially designed for free space conditions and was later optimized for simulated biological materials. To meet the initial design requirements, various analytical approximate approaches can be used for 27 MHz. The proposed applicator has dimensions of 120 $\times$ 120 $\times$ 1.6 mm, 14 turn numbers, and a copper thickness of 0.8909 mm (Fig. 3A). The applicator was fabricated on an FR4 dielectric substrate of thickness $h=$ 1.6 mm, loss tangent 0.02 and a relative permittivity of 4.4. The magnetic field distribution on the applicator was simulated by using CST Studio Suite 2015 program. The simulation result showed that the magnetic field can be applied on the cell culture homogenously with our designed 27 MHz PRFE applicator (Fig. 3B). The delivered power to the cells was measured by a spectrum analyzer (Fig. 3C). The power transmitted by the applicator was measured around $-$ 60.8 dBm which is equal to 8.3 mW (the reference power is $-$ 40 dBm).The distributed magnetic fields by two systems were simulated and analyzed. Our results clearly showed that the magnetic field distributions on the cells were uniform in both system.

Figure 3.

Simulation results of the proposed PRFE applicator; Photograph of the fabricated rectangular 27.12 MHz spiral applicator (A), Simulation of magnetic fields (A/m) on the applicator (B), Measured power level of PRFE applicator (C).

3.2 Analysis of in vitro wound closure assay

The effect of certain PEMF and PRFE sequences on in vitro wound healing were compared by exploring the proliferation rate and wound closure area of 3T3 mouse fibroblasts. Recent studies clearly showed that both PEMF and PRFE techniques enhanced bone healing and wound repair [29, 30, 31]. PEMF treatment with range from 15–75 Hz has been shown effective results on wound [32], cartilage [33], bone [34], and ligament healing [35]. Although the exact mechanism of cellular response is still unclear, it was proven that PEMF has positive impact on wound healing process by inducing cell migration and proliferation [36], DNA synthesis [37], protein [38] and growth factors expression [39], nitric oxide signaling and cytokine modulation [40]. Similar to our observation, Seeliger et al., demonstrated that the PEMF could enhance the proliferation of human tendon fibroblast in an in vitro wound healing model [32]. Furthermore, it was also reported that human skin keratinocyte [41] and endothelial cells [42] migration and proliferation can also be enhanced by EMF application. In another study, Štolfa et al., examined the effect of EMF on proliferation of human chondrocytes. It was reported that PEMF with a frequency of 21.2 MHz enhanced proliferation of chondrocytes [43]. Similarly, our results demonstrated that PEMF application significantly increased proliferation of 3T3 fibroblasts. PEMF exposure systems were also assessed on in vivo studies. For instance, Goudarzi et al. studied the effect of PEMF exposure for 1 h per day fro 10 days and investigated the wound healing in diabetic rats. It was demonstrated that PEMF can enhance wound healing in diabetic rats with parameter of 20 Hz frequency, 4 ms pulse width, and 8 mT magnetic field intensity [44].

The images of in vitro scratch assay were acquired at 0, 12, 24, 48, and 72 hours after the PEMF and PRFE exposure and were shown in Fig. 4. The in vitro wounds were uniformly formed as it is presented at 0 h images of control, PEMF, and PRFE exposed groups. It was observed that compared to control group, the ability of wound repair was higher in PEMF and PRFE for each time point. Especially, PRFE application substantially increased the wound healing process compared to control and PEMF group. The percentage of healed wound area on PEMF, PRFE and control groups was measured and analyzed quantitatively by using the acquired images at 0 and 72 h time points. The ability of wound closure of PRFE treated groups was significantly higher ( $p<$ 0.05) compared to that of the control group (Fig. 5B). For instance, at 72 h, PRFE exposed group occupied the 83.7 $\pm$ 1.13 wound area while control and PEMF exposed group occupied 67.4 $\pm$ 21 and 78.4 $\pm$ 2.4, respectively. These results are consistent with the MTT assay results where PRFE applied groups had significantly high cell number. PRFE exposed group showed significantly larger wound area healing compared to PEMF. Our results are consistent with the previous statement by Sunkari et al. where they demonstrated that low-intensity EMF significantly increased human dermal fibroblast proliferation. They related that increased proliferation rate with the expression of human fibroblast growth factor 1 after EMF exposure [45]. In another study, Seeliger et al. reported that low-frequency PEMF exposure program consisted of 10 minutes at a fundamental frequency of 33 Hz and 20 minutes at 7.8 Hz. accelerated the in vitro wound healing of patellar tendon fibroblast [32].

Figure 4.

Acquired images of PEMF and PRFE exposed and non-exposed (control) in vitro scratch assay at 0, 12, 24, 48, and 72 hours (Scale bars represent 200 $\mu$ m).

Figure 5.

MTT assay results of 3T3 fibroblast proliferation on control, PEMF and PRFE exposed groups at 0, 12, 24, 48, and 72 hours (A). Percentage of wound closure of PEMF and PRFE treated groups (B).

Cell proliferation on PEMF, PRFE and control groups were assessed by an MTT assay at 0, 12, 24, 48 and 72 hours (Fig. 5A). MTT results indicated that PEMF and PRFE induce significant increase in metabolic activity of 3T3 fibroblast which leads to higher cell number. For instance, cell numbers for PRFE group at 12, 48 and 72 hours were significantly higher compared to control group ( $p<$ 0.05). For PEMF group, significant difference on cell numbers were only observed at 48 and 72 hours of MTT assay. PRFE applied groups showed significant difference on the cell numbers compared to PEMF applied groups at only 72 hours. These results were in agreement with Vianale et al. who studied the effect of low-frequency EMF on keratinocyte proliferation and production of chemokines that have vital role in wound healing mechanism. They reported that EMF exposure significantly increased keratinocyte growth through inhibition of inflammatory process [16].

PRFE has also been used as soft tissue repair and pain relief treatment with 13–27.12 MHz carrier frequency range [31]. Studies indicated that PRFE treatment could trigger signal transduction cascades that have ability to transition gene expression, which results in increasing growth factors, protein modification, cell adhesion-related proteins, cyclins and DNA replication factors [31]. For instance, Moffett et al. used a flat spiral antenna with six turns which transmit 591 V/m electric and 6.7 A/m magnetic field intensities as PRFE applicator [46]. They indicated that the PRFE application increase endogenous opionid expression and proliferation of endothelial cells. Similarly in our study, we designed a PRFE applicator which can transmit 4400 V/m electric and 18 A/m magnetic field intensities. Although the applicator geometries differ in our study it was observed that 3T3 fibroblast proliferation enhanced.In another study, Moffett et al. demonstrated in vitro wound model that PRFE therapy have effect on proliferation of human keratinocyte and fibroblast cell lines [47, 48]. It has been shown previously both PEMF and PRFE treatments had an effect on in vitro wound healing models but there is no literature that shows the comparison of the two methods of therapy. In this study, we compared two methods to investigate the effects on cell proliferation and wound closure time on same cell line. Although PEMF and PREF therapies could activate different pathways on cells, rapid and synchronous increases on cell number can be seen. Recently, along with comprehension, the mechanisms of PEMF signals has allowed technologic advances on improvement of PRFE devices [28]. With advantages of opportunity to design portable, disposable and even availability on clothes, PRFE devices may provide effective treatment options with the patient comfort.

4. Conclusion

In conclusion, our findings indicated that both PEMF at 75 Hz and 27.12-MHz carrier frequency PRFE application accalarated cell proliferation and in vitro wound clousure compared to control group. Also, PRFE applied groups showed significantly increased cell numbers compared to PEMF applied groups. Similarly, significantly improved wound area closure rate was observed under the influence of PRFE. Thus, these results may be corralated with the complex wound healing phenomena and PRFE application could be an supportive tratment option for wound healing process. The promising in vitro wound healing results that show the superior effect of PRFE application should be strengthed by in vivo and clinical studies. Our findings may be helpful in the field of wound healing and may support the development of new strategies for tissue regeneration.

Footnotes

Acknowledgments

This work was supported by the Scientific and Technical Research Council of Turkey (TÜBİTAK) under Grant No 114E490. Authors would like to acknowledge Ege University Research Group of Animal Cell Culture and Tissue Engineering (EgeREACT) for donating 3T3 mouse fibroblasts. Finally, authors would like to thank Bonegraft Biological Materials Co. (Izmir, Turkey) for providing technical assistance in cell culture studies.

Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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