Effect of Pulsed Electromagnetic Field (PEMF) on Pressure Ulcer in BALB/c and C57BL/6 Mice

Abstract

Pressure ulcers (PUs) are localized injuries caused by prolonged mechanical loading and ischemia, often leading to delayed healing and high recurrence rates. Although conventional treatments aim to support tissue repair, their efficacy remains limited, prompting interest in noninvasive therapies such as the pulsed electromagnetic field (PEMF). The PEMF has been reported to enhance cellular proliferation, re-epithelialization, and collagen remodeling, but its effects in pressure ulcer models, particularly concerning genetic background, remain unclear. This study investigated the therapeutic effects of the PEMF in a murine pressure ulcer model established by ischemia and reperfusion injury induced with externally attached magnets in two mouse strains, BALB/c and C57BL/6. The PEMF (10 Hz, 24 h per day) was used to treat PU-induced mice from day 4 to day 15 in BALB/c mice and to day 14 in C57BL/6 mice. Wound healing was assessed by gross morphological observation, histological analysis, and digital quantification of epidermal lesion length and collagen-positive area. In BALB/c mice, PEMF-treated wounds showed a modest trend toward improved re-epithelialization and collagen deposition, although the differences were not statistically significant. In contrast, C57BL/6 mice exhibited a significantly shorter length of epidermal lesion in the PEMF group on day 14, indicating enhanced epidermal regeneration. Collagen analysis showed comparable levels between treated and control groups in both strains, with no significant differences observed. To further assess the cellular response to PEMF, a scratch wound assay was conducted using HaCaT cells. Quantitative analysis demonstrated that PEMF treatment accelerated cell migration and wound closure in vitro. These findings suggest that PEMF enhances epidermal regeneration and keratinocyte mobility, with therapeutic responses potentially influenced by genetic background. This study supports the potential application of PEMF in pressure ulcer treatment and underscores the importance of strain selection in preclinical wound healing research.

1. Introduction

Pressure ulcers (PUs), also known as pressure injuries, are defined as localized damage to the skin and underlying tissues resulting from sustained mechanical pressure. These lesions most commonly develop over bony prominences such as the sacrum, heels, hips, and ankles [1]. Prolonged pressure disrupts blood flow to the affected area, leading to tissue ischemia, necrosis, and, ultimately, skin breakdown. The increasing prevalence of PUs presents a major challenge in modern healthcare systems, primarily driven by population aging and the growing incidence of chronic health conditions [2]. Comorbidities such as diabetes mellitus, obesity, cardiovascular disease, and peripheral vascular disease further exacerbate susceptibility by impairing circulation, delaying wound healing, and reducing mobility—all of which promote an environment conducive to pressure-induced skin injury [3].

Beyond the mechanical insult, PUs are associated with a wide range of clinical complications that significantly compromise patient well-being. Pain is one of the most prevalent and debilitating symptoms, frequently limiting patient movement and interfering with rest, which further worsens outcomes [4]. Furthermore, the structural disruption of the skin barrier in PUs increases vulnerability to microbial invasion, predisposing patients to localized infections that may progress to systemic involvement [5]. In severe or inadequately managed cases, PUs can lead to osteomyelitis, necessitating prolonged antibiotic therapy or surgical debridement [6]. In advanced stages, infection may escalate to sepsis and multi-organ failure, posing a substantial threat to patient survival [7]. Additionally, although rare, long-standing non-healing ulcers have been reported to undergo malignant transformation into squamous cell carcinoma [8].

The pathogenesis of PUs is multifactorial, involving extrinsic mechanical forces—such as pressure, shear, and friction—as well as intrinsic physiological factors like malnutrition and immobility [9]. Among these, oxidative stress and the associated inflammatory response triggered by ischemia–reperfusion (I/R) injury are widely recognized as principal contributors to tissue damage [10]. Consequently, animal models of pressure ulceration have primarily adopted I/R injury protocols to recapitulate clinically relevant features of PU development [11]. This experimental approach typically involves cyclic application and release of external pressure, simulating the dynamic conditions seen in patients who receive periodic repositioning. The initial ischemic phase induces localized hypoxia, while subsequent reperfusion paradoxically exacerbates tissue injury through the generation of reactive oxygen species, infiltration of inflammatory cells, and microvascular dysfunction. Devices such as magnetic compression plates or pressure chambers are commonly employed to model these alternating cycles [12,13].

While conventional management strategies for PUs are stage-specific and widely practiced, they often face significant limitations. These include the high cost and discomfort of pressure redistribution systems, challenges in achieving adequate nutritional intake in malnourished patients, and difficulties with infection control [14]. Thus, there is a growing interest in complementary therapeutic approaches that address these limitations and promote endogenous tissue repair. Among them, pulsed electromagnetic field (PEMF) therapy has emerged as a promising non-invasive modality [15]. The PEMF delivers targeted electromagnetic pulses to living tissues, modulating cellular processes that are essential for wound healing. Previous studies have reported that the PEMF facilitates re-epithelialization, enhances collagen remodeling, and promotes overall tissue regeneration [16,17,18]. To further investigate the therapeutic potential of the PEMF in PUs, the present study employed a murine model of I/R-induced ulcers using an externally attached magnetic compression system. This non-invasive model allowed for the induction of acute ulcer pathology under clinically relevant conditions while avoiding confounding effects from surgical implantation. We subsequently evaluated the effects of PEMF therapy by comparing histological parameters in two strains of mice, focusing on epidermal regeneration and collagen fiber remodeling. In addition, in vitro wound healing assays were conducted to elucidate the cellular mechanisms through which the PEMF supports tissue repair.

2. Materials and Methods

2.1. Animals

Twenty-four male BALB/c and twenty-four male C57BL/6 mice (8 weeks old) were purchased from Orient Bio (Seongnam, Republic of Korea) and housed in individually ventilated cages under a 12 h light/dark cycle, with ad libitum access to food and water. The animal housing facility is equipped with a temperature- and humidity-controlled chamber to maintain constant environmental conditions (temperature: 23 ± 1 °C; relative humidity: 50 ± 10%). Potential electromagnetic interference from PEMF exposure was controlled by placing cages at least 30 cm apart, thereby minimizing the influence of the electromagnetic effect between the animal cages. All experimental procedures were approved by the Yonsei University MIRAE Campus Institutional Animal Care and Use Committee (IACUC), in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care International (#YWCL-202503-005-01).

2.2. Pressure Ulcer (PU) Model and Magnetic Core-Type PEMF (mPEMF) Treatment

After a one-week acclimation period, the mice were utilized for the experiments. Prior to the experiments, the dorsal fur of each mouse was removed with a depilatory cream and then disinfected using 70% isopropanol. A template was employed to ensure consistent placement of the magnets. The skin was gently lifted and positioned between two circular ceramic magnets (specifications provided in the Supplementary Table S1). By using this “pinch” technique, a skin bridge of approximately 5.0 mm was formed between the two magnets. To induce PUs, an ischemia–reperfusion procedure was performed three to five times per mouse. One cycle consisted of attaching the magnets for 8 to 12 h, followed by a 12 to 16 h rest period. During magnet attachment, the mice were neither restrained nor given additional anesthesia, and they had free access to both food and water. After pressure ulcer induction, mice were randomly assigned to either the pressure ulcer (PU) group or the PU + PEMF 10 Hz (P10 Hz) group to minimize allocation bias, with 12 animals per group for each mouse strain. The sample size was determined based on practical considerations, including ethical constraints and resource availability. No a priori sample size calculation was performed. The magnetic core-type PEMF device (mPEMF), was designed to emit magnetic fields in a fountain-shaped distribution pattern. The magnetic coils were positioned 1 cm below the mouse cages. PEMF treatment for mice was performed continuously at ambient temperature for 24 h per day. At the conclusion of the experiments, the mice were euthanized using carbon dioxide, and skin tissues were collected. Humane endpoints were established prior to the study, and mice were monitored daily for clinical signs of distress, including hunched posture, piloerection, reduced mobility, severe weight loss (>20%), or self-inflicted trauma. If any of these signs were observed, animals were to be euthanized immediately using carbon dioxide inhalation. However, no animals reached the humane endpoint criteria. Furthermore, no unexpected adverse events, such as infection, excessive inflammation, or mortality, were observed during the course of the study. Mild erythema and scabbing at the wound site were anticipated outcomes of the pressure ulcer model. All animals met the inclusion criteria andcompleted the experimental protocol, and no animals were excluded from the analysis.

2.3. Mouse Histology

Skin tissues from mice, including the ulcerative region and adjacent margins, were harvested and immediately fixed in 10% neutral buffered formalin (Dana Korea, Incheon, Republic of Korea) at room temperature for 72 h to ensure complete tissue fixation. Following fixation, samples were processed through standard histological processing and were embedded in paraffin. The formalin-fixed, paraffin-embedded (FFPE) blocks were then sectioned into 4 μm thick slices using a microtome (HM340E, Thermo Fisher Scientific, Waltham, MA, USA). Serial sections were stained with hematoxylin and eosin (H&E) to examine epidermal lesion length and with Masson’s Trichrome stain to evaluate collagen content. Stained sections were scanned with a digital slide scanner (Pannoramic, 3DHistech, Budapest, Hungary), and image analysis was performed using CaseViewer 2.4 software. Epidermis lesion length and collagen quantification was conducted using ImageJ software (Version 1.53, NIH, Bethesda, MD, USA). All statistical analyses were performed using GraphPad Prism 9.3.1 software (GraphPad Software, San Diego, CA, USA). Statistical significance was analyzed by a nonparametric two-tailed Mann–Whitney U test. Group allocation was concealed from investigators responsible for conducting tissue analysis, assessing outcomes, and analyzing data.

2.4. Wound Healing Assay and Solenoid-Type PEMF (sPEMF) Treatment

Human keratinocyte HaCaT cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified incubator containing 5% CO₂. Upon reaching confluence, a linear scratch wound was generated across the cell monolayer using a sterile P200 pipette tip (SPL Life Sciences, Pocheon, Republic of Korea). Detached cells and debris were removed by washing three times with 1 mL of Dulbecco’s Phosphate-Buffered Saline (DPBS; Welgene, Gyeongsan, Republic of Korea), ensuring clear wound edges. Subsequently, the medium was replaced with 2 mL of DMEM containing 10% FBS. Cells were then subjected to sPEMF treatment at 10 Hz for 1 h, followed by incubation for the indicated periods The solenoid-type PEMF system (sPEMF), producing a magnetic field with uniform spatial distribution, was used for in vitro applications requiring consistent electromagnetic exposure. Wound areas were imaged every 12 h using Optinity (×100 magnification; Korea Lab Tech, Hwaseong, Republic of Korea), and the percentage of wound closure was quantified using ImageJ software.

3. Results

3.1. PEMF Treatment Parameters

A PEMF is generated by applying pulsed currents and voltages to a coil that magnetizes the core and produces a pulsed magnetic field (Supplementary Figure S1A). The effects of electric stimulation are negligible, and frequencies commonly used in PEMF applications range from 1 Hz to below 1000 Hz. The magnetic core-type PEMF device (mPEMF) in this study consists of six circular coils with an outer diameter of 6 cm and an inner diameter 3.5 cm, designed to emit magnetic fields in a fountain-shaped distribution pattern (Figure 1, left panel). The six magnetic coils were arranged underneath the mouse housing cage, and the mice were continuously exposed to the PEMF at room temperature for 24 h per day under conditions of 24 V, with the waveform consisting of pulses (on time, 4.29 ms; off time, 10.01 ms; period, 14.3 ms; duty ratio, 30%) repeated at 10 Hz (Supplementary Figure S1B). The magnetic flux density at the core center was 24.75 mT (Figure 1, right panel).

3.2. Macroscopic Wound Healing Response to PEMF Treatment in BALB/c Mice

To establish an optimized pressure ulcer model in BALB/c mice, we initially tested various types of magnets and parameter settings using an externally applied ischemia–reperfusion (I/R) protocol. Based on the results shown in Supplementary Figures S2–S6, the neodymium magnet (12 mm × 1 mm, 123 mT) was selected for its reproducibility in inducing consistent gross and histological tissue damage (Supplementary Figure S3). This parameter was subsequently implemented in the main experimental protocol to evaluate the therapeutic efficacy of PEMF treatment. Following a 7-day acclimation period and dorsal hair removal, neodymium magnets were externally applied to the dorsal skin of BALB/c mice for three consecutive I/R cycles for three days. Gross morphological evaluation indicated that ulcer severity peaked on day 4. Therefore, PEMF treatment (magnetic core type, 10 Hz, 24 h/day) was initiated on day 4 and continued daily until day 15. Control animals received no additional treatment. The experiment was terminated on day 15, based on prior observations indicating that spontaneous healing is typically complete by day 21 in this model (Figure 2A). To assess the therapeutic effect of the PEMF, gross comparisons of wound appearance were performed between the control and PEMF-treated groups. As shown in Figure 2B, no prominent visual differences were observed between the control and PEMF-treated groups.

3.3. Histological Analysis of Epidermal Regeneration in BALB/c Mice

To characterize the histopathological features of PUs at the peak of injury, dorsal skin samples were collected on day 4 before the initiation of PEMF treatment. As shown in Figure 3A, H&E staining revealed prominent epithelial disruption, confirming the successful induction of PUs with marked structural damage at both the epidermal and dermal levels. To assess the intermediate effects of the PEMF on wound healing, histological comparisons between control and PEMF-treated groups were performed on day 10. In the control group, H&E staining showed persistent epidermal discontinuity and exposed dermis, indicating delayed re-epithelialization. In contrast, PEMF-treated tissues exhibited improved epithelial coverage, although complete regeneration had not yet occurred. To further evaluate the effects of the PEMF at a later stage of healing, additional histological analyses were conducted on day 15. In the control group, partial re-epithelialization was observed, often accompanied by residual scabs and irregular epidermal restoration. Meanwhile, the PEMF group demonstrated more advanced epithelial recovery, despite some remaining structural irregularities. To quantify the extent of epidermal regeneration, the length of discontinuous epidermis was measured on day 15 post injury. By day 15, both groups exhibited considerable re-epithelialization, and the remaining differences between groups were minimal and not statistically meaningful (Figure 3B).

3.4. Histological Analysis of Collagen Regeneration in BALB/c Mice

As shown in Figure 3A, Masson’s trichrome (MT) staining on day 4 revealed a collapsed collagen matrix in the ulcerated dermis, confirming successful induction of PUs, characterized by pronounced structural disruption at the dermal level. On day 10, the control group exhibited loosely arranged and disorganized collagen fibers, indicating limited structural recovery. In contrast, MT staining in the PEMF-treated group showed denser and more organized collagen deposition compared to corresponding H&E images, suggesting enhanced dermal remodeling in response to PEMF stimulation. By day 15, MT staining revealed ongoing collagen remodeling in both groups. The dermal structure in the control group remained immature, while the PEMF group exhibited slightly increased collagen density. However, overall dermal architecture remained largely comparable between groups at this stage (Figure 4A). Collagen remodeling was quantitatively assessed by measuring the area of MT-stained collagen. Although the PEMF-treated group exhibited a marginally reduced collagen-positive area compared to the control group, the difference did not reach statistical significance (Figure 4B).

3.5. Macroscopic Wound Healing Response to PEMF Treatment in C57BL/6 Mice

To assess the therapeutic effect of the PEMF, gross comparisons of wound appearance were performed between control and PEMF-treated C57BL/6 mice under identical ulcer-inducing conditions. Both groups underwent the same ischemia–reperfusion protocol; however, only the treatment group received daily PEMF stimulation (10 Hz, 24 h/day) from day 4 to day 14 (Figure 5A). As shown in Figure 5B, the PEMF-treated group exhibited a trend toward improved wound healing compared to controls; however, the differences were subtle and not consistently distinguishable across individuals, making it difficult to discern a clear treatment effect based solely on gross morphology.

3.6. Histological Analysis of Epidermal Regeneration and Collagen Regeneration in C57BL/6 c Mice

To investigate the effects of PEMF during the later phase of wound healing, histological analysis was performed on day 14 in C57BL/6 mice. In the control group, H&E staining revealed incomplete re-epithelialization, characterized by residual scab formation and partial restoration of the epidermal layer. In contrast, the PEMF-treated group exhibited more advanced re-epithelialization, with only minimal surface irregularities remaining. To quantitatively assess epidermal regeneration, the length of epidermal discontinuity was measured on day 14 post injury. The PEMF-treated group showed a significantly shorter average discontinuity length compared to controls, indicating a statistically meaningful enhancement in epithelial regeneration in response to PEMF treatment. These findings suggest that PEMF treatment may accelerate re-epithelialization in pressure ulcer wounds (Figure 6A). MT staining revealed evidence of ongoing collagen remodeling in both groups. Although the PEMF-treated group exhibited a slight increase in collagen density compared to controls, the overall dermal architecture remained immature and largely comparable between groups. Collagen regeneration was further evaluated by quantifying the MT-stained positive area within the dermal layer. On day 14, both groups displayed similar levels of collagen deposition, and statistical analysis confirmed that the differences were not significant. These results suggest that, at this time point, the PEMF had no marked effect on dermal collagen remodeling in this model (Figure 6B).

3.7. Effect of PEMF on In Vitro Scratch Wound Closure in HaCaT Cells

To assess the effect of PEMF on keratinocyte migration, a scratch wound healing assay was performed using HaCaT cells. After generating linear wounds, cells were either left untreated or exposed to the sPEMF, which featured a cylindrical structure with a 65 mm inner diameter and a height of 200 mm, generating a magnetic field with uniform spatial distribution stimulation (10 Hz, solenoid type) for 1 h every 12 h over a total duration of 48 h (Figure 7A). Wound closure was monitored at 24 h intervals using phase-contrast microscopy. As shown in Figure 7B, PEMF-treated cells demonstrated a more pronounced reduction in wound width compared to untreated controls over time. Quantitative analysis of wound closure confirmed this trend, indicating that PEMF stimulation facilitated keratinocyte migration and accelerated scratch wound closure in vitro (Figure 7C).

4. Discussion

To optimize a pressure ulcer model in BALB/c mice, we tested various magnet types and I/R parameters. In Supplementary Figure S3, neodymium magnets (12 mm × 1 mm, 123 mT) were applied for five I/R cycles (12 h ischemia/12 h reperfusion), which induced the most severe ulceration by day 4, with H&E staining on day 14 revealing marked epidermal loss. In contrast, ferrite magnets (15 mm × 5 mm, 120 mT) used for three I/R cycles (Supplementary Figure S2) caused moderate ulceration, with ulcerative lesions most prominent on day 7, with less severe histological damage. Supplementary Figures S4–S6 show the results using thicker neodymium magnets (up to 5 mm, 334 mT), but none produced more severe tissue disruption than the 12 mm × 1 mm, 123 mT condition. Based on these findings, the 123 mT neodymium magnet was selected for model establishment due to its consistent gross and histological damage. However, to minimize excessive hyperkeratosis resembling callus formation observed with five cycles, only three I/R cycles (12 h compression/12 h reperfusion) were applied in the current study. This protocol reliably induced ulcerative lesions while preserving structural integrity for histological evaluation.

In this study, pressure ulcers were induced using an ischemia and reperfusion protocol designed to produce full-thickness skin injury, which ensured consistent ulcer depth across all experimental animals. This consistency allowed us to minimize variability in wound severity and to evaluate the effects of the PEMF under uniform depth conditions. PEMF treatment began on day four, when the ulcers reached their maximum severity, and was applied continuously for 24 h a day until the end of the experiment. However, we recognize that both wound depth and the timing of treatment are important factors that can affect the overall effectiveness of the PEMF. In cases of deeper or chronic wounds, longer treatment durations or higher levels of stimulation may be necessary. The ideal period for intervention may also depend on the specific stage of tissue damage and healing. To improve clinical relevance, future studies should use wound models with varying depths, such as partial-thickness and full-thickness ulcers, and compare the effects of early and delayed treatment. This approach will help clarify how the response to the PEMF varies with wound depth and healing stage, and may support the development of more tailored treatment strategies for clinical use.

This study evaluated the therapeutic efficacy of PEMF treatment in a murine pressure ulcer model induced by externally applied magnetic plates. The BALB/c strain was initially used to establish a reproducible ulcer model and assess the impact of PEMF treatment. Although PEMF-treated BALB/c mice exhibited a modest reduction in epidermal lesion length and an increase in collagen deposition relative to untreated controls, these differences did not reach statistical significance, suggesting a limited effect of the PEMF on structural tissue regeneration in this strain. In contrast, application of the same protocol to C57BL/6 mice resulted in a statistically significant reduction in epidermal lesion length in the PEMF group compared to controls, while the collagen-positive area remained comparable between groups, consistent with the findings in BALB/c mice. These results suggest that the therapeutic response to the PEMF may be influenced by genetic background, with C57BL/6 mice exhibiting a more pronounced re-epithelialization response. Collectively, these findings indicate that while the PEMF promotes cellular processes involved in tissue repair, the extent of its in vivo efficacy may be modulated by strain-specific physiological characteristics and biological variability.

The differential responses observed between BALB/c and C57BL/6 mice may be attributed to inherent differences in immune profiles, wound healing mechanisms, and regenerative capacity. A key distinction lies in their immunological polarization: BALB/c mice display a T helper 2 (Th2)-dominant immune response, characterized by elevated interleukin-4 (IL-4) and IL-13 production [19]. This Th2 bias promotes humoral immunity and may prolong inflammation following tissue injury, potentially diminishing the therapeutic benefits of the PEMF [20]. In contrast, C57BL/6 mice exhibit a Th1-skewed immune profile, with enhanced production of interferon-gamma (IFN-γ) [21]. However, the role of IFN-γ in wound healing is context-dependent; it has been shown to both promote and inhibit tissue regeneration depending on tissue type and healing phase. While some studies report that IFN-γ impairs skin wound healing by suppressing transforming growth factor-beta (TGF-β) signaling, angiogenesis, and collagen synthesis [22,23], others demonstrate that IFN-γ contributes to wound maturation by modulating neutrophil activity and matrix metalloproteinase (MMP) expression [24]. Accordingly, the PEMF may influence the Th1-mediated inflammatory environment in C57BL/6 mice, thereby enhancing re-epithelialization.

Beyond immune differences, C57BL/6 mice are known to possess superior regenerative dynamics. A prior study has reported accelerated corneal re-epithelialization and faster migration of epidermal keratinocytes in C57BL/6 mice relative to BALB/c, suggesting a more responsive regenerative environment [25]. These differences in epithelial migration speed between strains may contribute to variations in wound healing kinetics. Conversely, the relatively slower epithelial migration and larger corneal size observed in BALB/c mice may hinder tissue regeneration, potentially reducing the effectiveness of interventions such as PEMF treatment. Keratinocyte migration is a critical driver of re-epithelialization, and its impairment is associated with chronic, non-healing wounds [26,27]. Consistent with this, our in vitro results showed that PEMF treatment significantly accelerated wound closure in cultured cells, supporting the notion that PEMF enhances cellular behaviors such as migration and proliferation (Figure 7). The observed strain dependency highlights the importance of host-specific factors in therapeutic efficacy. For future clinical applications, it underscores the need to consider genetic and immunological variability among human populations. Further studies involving diverse animal models or human-derived cells may help clarify the translational potential of this therapy and guide the development of personalized or stratified treatment strategies.

In our study, histological analysis revealed that while PEMF treatment promoted re-epithelialization and reduced inflammatory cell infiltration in the epidermis and upper dermis, its effects were less pronounced in the deeper dermal and subcutaneous layers. Several factors may contribute to this differential response. First, the intensity of the PEMF may diminish with increasing tissue depth, resulting in reduced bioavailability of the therapeutic signal in deeper regions [28]. Second, cellular composition and regenerative potential differ among tissue compartments. While keratinocytes may respond readily to the PEMF, deeper fibroblast populations may exhibit delayed or attenuated responses [29]. To address these limitations, future studies could consider optimizing field intensity and delivery methods to ensure more uniform penetration. Additionally, integrating imaging-based dosimetry and molecular markers across layers would help delineate the depth-specific mechanisms of PEMF action.

In this study, total collagen content was assessed using MT staining, which, while effective in visualizing total collagen content, does not differentiate between specific collagen subtypes such as collagen I, III, or V, which are known to play distinct roles during various stages of wound healing. Type III collagen is typically produced in the early proliferative phase, contributing to the initial extracellular matrix (ECM) scaffold, while type I collagen becomes dominant during the remodeling phase, imparting tensile strength and structural stability to the newly formed tissue [30]. Without distinguishing these subtypes, it is challenging to determine whether PEMF treatment preferentially influenced early-phase collagen synthesis or later-stage matrix maturation and remodeling. This lack of specificity limits our ability to precisely interpret the biological timing and quality of the collagen deposition observed in response to the PEMF. For example, an increase in overall collagen content does not necessarily indicate enhanced functional healing if it primarily reflects immature collagen accumulation [31]. To address this, future studies could incorporate immunohistochemical or molecular analyses targeting specific collagen isoforms (e.g., Col1a1, Col3a1) to provide more detailed insight into how the PEMF modulates each phase of tissue repair. This would significantly enhance the translational relevance and mechanistic understanding of PEMF’s therapeutic effects.

The PEMF is believed to accelerate wound healing through a coordinated series of bioelectrical and biochemical mechanisms initiated at the cellular membrane level. At the site of injury, minute alterations in membrane potential—induced by the PEMF—modulate ion channel activity, particularly voltage-gated and stretch-activated calcium and sodium channels [32]. These changes can result in either depolarization or hyperpolarization depending on the cell type and exposure parameters, ultimately influencing cellular excitability and triggering downstream signaling events. A central outcome of this membrane perturbation is the influx of intracellular calcium, which occurs through mechanosensitive or voltage-gated calcium channels [33]. The rise in intracellular calcium activates calmodulin-dependent kinases and initiates key signaling cascades such as the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways [34,35]. These cascades regulate various aspects of cellular function critical for wound repair, including proliferation, migration, survival, and differentiation. These calcium-dependent processes are particularly essential for keratinocyte migration and re-epithelialization during cutaneous wound healing [36]. Based on the mechanisms described above, the PEMF has the ability to further modulate molecular pathways involved in both tissue regeneration and inflammation. Several studies have reported that the PEMF enhances the expression of signaling molecules related to cell proliferation and wound healing, including IL-6, TGF-β, and inducible nitric oxide synthase (iNOS), particularly in gingival wound models. These effects are accompanied by elevated levels of MMP-2, monocyte chemoattractant protein 1 (MCP-1), and heme oxygenase 1 (HO-1), which are known to accelerate tissue repair [37]. Furthermore, the PEMF has been shown to stimulate the synthesis of growth factors such as insulin-like growth factor (IGF), TGF-β, and prostaglandin E2 (PGE2), thereby promoting ECM production and remodeling [38]. In parallel, the PEMF also exerts notable anti-inflammatory effects. It downregulates pro-inflammatory cytokines, such as IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α), and modulates intracellular signaling pathways including suppression of MMP-9 via the Akt/extracellular signal-regulated kinase (ERK) axis, inhibition of cyclooxygenase-2 (COX-2) and PGE2 expression, and attenuation of nuclear factor (NF)-κB signaling [39]. These transcriptional and post-transcriptional modifications collectively contribute to the creation of a wound microenvironment favorable for tissue regeneration. Our findings align with these previous reports. In a murine model of septic shock, the PEMF significantly altered key biological processes in the liver, such as the “response to hypoxia,” “regulation of inflammatory response,” “defense response,” and “immune system process,” as identified by transcriptomic analysis [40]. These changes were accompanied by reduced expression of inflammatory cytokines, further supporting the notion that the PEMF promotes tissue recovery through both regenerative and immunomodulatory mechanisms. In addition, the PEMF has been shown to enhance both the expression and activity of fibroblast growth factor 2 (FGF-2), a potent mediator of angiogenesis [41]. The PEMF promotes calcium/calmodulin-dependent activation of NOS, leading to NO release and elevated cGMP. This, in turn, stimulates growth factor secretion via paracrine and autocrine pathways. PEMF treatment has also been reported to facilitate FGF binding to its receptor, enhancing endothelial proliferation, migration, and differentiation [42,43]. This cascade promotes neovascularization and improves blood perfusion, ultimately accelerating tissue repair. Modulation of FGF-2 signaling is especially relevant in metabolic conditions such as diabetes, where the PEMF may help overcome impaired wound healing. Beyond these effects, the PEMF influences cell motility through structural and adhesive modifications. By regulating membrane potential and calcium signaling, the PEMF alters cytoskeletal dynamics essential for directed cell movement. It also affects the organization of focal adhesions and integrin-mediated signaling, facilitating adhesion turnover required for efficient migration [44,45,46,47,48]. Finally, the PEMF modulates the cytokine milieu within the wound microenvironment. By influencing the temporal release of pro- and anti-inflammatory cytokines during different stages of healing, it supports resolution of inflammation and progression to regenerative phases [28,49].

Importantly, the biological effects of the PEMF appear to be confined within defined therapeutic windows, which are influenced by parameters such as stimulation frequency, intensity, timing, and duration. This underscores the importance of establishing optimized stimulation protocols. In particular, stimulation frequency plays a pivotal role in determining PEMF efficacy. In this study, the PEMF was applied at 10 Hz, a frequency previously associated with anti-inflammatory and regenerative effects [50]. Various frequency ranges have been explored in the literature and may exhibit phase-specific therapeutic effects. According to Lv et al. (2021), PEMF frequencies ranging from 15 Hz to 25 Hz have been shown to promote wound closure, FGF-2 expression, endothelial cell density, and collagen deposition in diabetic wound models. These mid-range frequencies (15–25 Hz) appear to support both the proliferative and remodeling phases by enhancing granulation tissue formation, myofibroblast activation, and matrix remodeling [51]. On the other hand, Bedja-Iacona et al. (2024) demonstrated that low-frequency PEMFs (10–12 Hz) increase proliferation markers such as proliferating cell nuclear antigen (PCNA) and Ki-67 in human dermal fibroblasts, while higher frequency PEMFs (100 Hz) facilitate actin cytoskeletal organization and fibroblast migration during the proliferative phase [52]. Both frequencies were also associated with increased myofibroblast maturation, indicating differential but complementary roles in distinct aspects of tissue regeneration. Therefore, an optimal PEMF treatment strategy may require a dynamic protocol where lower frequencies (10–12 Hz) are employed during the early inflammatory and proliferative phases to promote cell proliferation, while mid-to-higher frequencies (25–100 Hz) may be more suitable during later proliferative and remodeling stages to support tissue organization, angiogenesis, and tensile strength acquisition. Future studies employing time-resolved and phase-specific PEMF protocols are warranted to refine therapeutic timing and optimize outcomes across different stages of pressure ulcer healing.

In this study, we primarily focused on the therapeutic effects of the PEMF at a fixed frequency of 10 Hz, which has been previously associated with anti-inflammatory and regenerative outcomes. We acknowledge that other key parameters—specifically magnetic field intensity, timing, and duration of daily exposure—were not systematically varied or optimized in our experimental design. The effectiveness of the PEMF may vary considerably depending on intensity, particularly with respect to the depth and type of target tissue. For example, higher intensities may be required to penetrate deeper tissues or larger anatomical regions, while superficial conditions might respond to lower intensities. Insufficient intensity may fail to induce cellular responses, especially in deeper or denser tissues [53]. Timing includes factors such as session duration, frequency of application (e.g., times per day or week), and the time of day when treatment is administered (e.g., morning vs. evening). Many studies adopt a fixed protocol without comparing different timing strategies. However, clinical evidence suggests that longer daily exposure (i.e., a higher “dose”) is associated with enhanced therapeutic outcomes, such as accelerated bone healing [54,55]. In wound healing, a study showed that different PEMF intensities had distinct benefits depending on the healing phase. A 10 mT PEMF was more effective in promoting cell proliferation and collagen deposition during the early phase, whereas a lower-intensity field (2 mT) was beneficial in the later phase, aiding re-epithelialization. Importantly, an inappropriate duration or timing could compromise the biomechanical strength of the repaired tissue, emphasizing the need for phase-specific adjustments to the stimulation protocol [56].

In our study, the selected PEMF parameters (intensity, frequency, and exposure duration) were determined based on previously published studies reporting consistent biological efficacy in tissue repair models without adverse effects. To date, PEMF therapy has been widely regarded as a non-invasive and safe modality, with no well-documented adverse effects reported under clinically relevant conditions [38]. Therefore, while the risk of harmful side effects appears minimal, applying the PEMF outside the defined therapeutic window may lead to reduced or inconsistent efficacy rather than toxicity. Such deviations could result in insufficient stimulation of target cells or suboptimal modulation of key regenerative pathways.

Although the PEMF exhibits potential for enhancing tissue regeneration—particularly during the early stages of wound repair—its efficacy in chronic or advanced-stage ulcers remains limited, emphasizing the need for further research [57]. The limited efficacy of the PEMF in chronic or advanced ulcers likely stems from several intrinsic pathophysiological features characteristic of non-healing wounds. Chronic ulcers often exhibit persistent inflammation, cellular senescence, impaired angiogenesis, excessive oxidative stress, and elevated MMP activity, all of which contribute to a hostile wound microenvironment that resists regeneration [58]. In particular, senescent fibroblasts and macrophages with an unresolving pro-inflammatory (M1) phenotype may impair the cellular responsiveness to PEMF-induced signaling pathways [59,60]. Furthermore, chronic wounds frequently harbor bacterial biofilms, which not only sustain inflammation but also impair oxygen and nutrient diffusion, potentially reducing the bioavailability or effectiveness of biophysical therapies such as the PEMF [61]. To improve PEMF efficacy in such contexts, the following strategies may be considered: adjunctive therapies that reduce chronic inflammation (e.g., topical anti-inflammatories) or modulating redox status, thereby restoring cellular responsiveness. Biofilm-targeting interventions, including debridement or antimicrobial agents, prior to PEMF application may improve tissue receptivity. Customization of PEMF parameters (e.g., intensity, frequency, duration) based on wound chronicity and inflammatory profile to enhance biological relevance and responsiveness can also be considered. Future studies should explore such combinatorial approaches and evaluate patient-specific wound characteristics to better tailor PEMF therapy for recalcitrant ulcers.

While murine models are widely used due to their well-characterized genetics, cost-effectiveness, and reproducibility, we acknowledge that they do not fully replicate the multifactorial complexity of human pressure ulcers [62]. Human pressure ulcers often develop in the context of aging, chronic comorbidities (e.g., diabetes, vascular disease), prolonged immobility, and varied anatomical and biomechanical conditions, which are difficult to fully reproduce in mice [63]. In our study, we employed a widely accepted ischemia–reperfusion-induced murine model that mimics key pathological features of human pressure ulcers, including hypoxia, inflammation, oxidative stress, and delayed wound healing [10]. However, the model lacks certain human-specific elements such as pressure-related deep tissue injury and the influence of systemic comorbid conditions [2,11]. To enhance translational relevance in future studies, alternative models such as porcine pressure ulcer models may be considered. Pigs have skin that is more anatomically and physiologically similar to human skin, including comparable epidermal thickness, dermal composition, and immune responses [64]. Additionally, three-dimensional human skin equivalents or organ-on-a-chip platforms integrating mechanical stress and inflammatory stimuli may offer promising in vitro alternatives for mechanistic and pharmacological studies [65].

In addition to genetic background, wound depth, stimulation parameters, and timing of intervention, several other important aspects and underlying mechanisms require further investigation to better clarify the therapeutic potential of the PEMF in the management of pressure ulcers. First, cellular and molecular mechanisms such as the regulation of inflammatory signaling pathways and the expression of pro-healing cytokines should be examined to understand how the PEMF influences the wound environment. This includes its effects on macrophage polarization from the M1 to the M2 phenotype [66], angiogenesis, and ECM remodeling [36]. Second, vascular dynamics and perfusion recovery under PEMF stimulation remain underexplored. Investigating how the PEMF modulates microvascular integrity, endothelial function, and tissue oxygenation could reveal additional therapeutic mechanisms [67]. Third, it is important to investigate the roles of oxidative stress and mitochondrial function in wound healing mediated by the PEMF. The PEMF has been shown to alter the production of reactive oxygen species [68] and promote mitochondrial fission [69], both of which are closely associated with tissue regeneration. Finally, the influence of microbial communities should not be overlooked, as imbalances in skin microbiota can delay healing [70]. A recent study reported that the PEMF interferes with biofilm formation by Staphylococcus epidermidis and enhances the antibiofilm effect of antibiotics against Staphylococcus aureus, which may reduce microbial colonization and the risk of infection [71]. Future research could explore how the PEMF affects the composition of skin or wound microbiota and contributes to infection control and tissue repair.

Overall, although the PEMF demonstrates mechanistic potential in promoting wound repair, its therapeutic benefits were not consistently observed across tissue layers or mouse strains in this study. These findings demonstrate the need for further investigation to identify the specific conditions under which the PEMF is most effective. Future studies should consider key variables such as genetic background, wound depth, stimulation parameters, and timing of intervention. Moreover, the use of alternative ulcer models and expanded treatment protocols may help refine PEMF-based therapies and facilitate their clinical translation for early-stage pressure ulcer management.

5. Conclusions

This study demonstrated that PEMF treatment significantly enhances epidermal regeneration in a murine pressure ulcer model, with pronounced effects observed in C57BL/6 mice. While BALB/c mice exhibited only modest improvement, a marked reduction in epidermal lesion length was evident in the C57BL/6 strain, suggesting that the therapeutic response is influenced by genetic background. Collagen deposition remained comparable between groups in both strains. In vitro scratch assays further confirmed that the PEMF facilitates keratinocyte migration and promotes wound closure. These findings collectively highlight the therapeutic potential of the PEMF and the importance of considering genetic variability when evaluating wound healing responses. The strain-specific effects observed in this study imply that intrinsic host factors, such as immune response characteristics, inflammatory signaling, and tissue repair capacity, may critically shape the efficacy of PEMF treatment. This observation suggests broader implications for developing individualized strategies in wound care. Future studies should investigate the molecular and immunological mechanisms that contribute to inter-strain differences and explore the optimal parameters for PEMF application, including frequency, intensity, and duration of exposure. Furthermore, the use of large animal models or patient-derived skin equivalents may help translate these findings into clinical practice and advance the development of effective PEMF-based therapies.