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. 2026 Oct 12;32(10):e70394. doi: 10.1111/srt.70394

Effect of Dermal Fibroblast‐Conditioned Media on Human Dermal Fibroblasts Treated With Electromagnetic Transduction Therapy

Jeongah Ko 1,#, Sang Bum Suh 2,#, Yeong Ju Oh 2, Ji Youn Suh 2, Sung Bin Cho 3,✉
PMCID: PMC13656786  PMID: 42861896

ABSTRACT

Background

Electromagnetic transduction therapy (EMTT) is a high‐intensity pulsed electromagnetic field therapy used in musculoskeletal medicine. However, its therapeutic effects on human dermal fibroblasts (HDFs) remain unclear. Secretomes from human dermal fibroblast‐conditioned medium (HDFCM) can enhance wound repair and tissue regeneration.

Methods

Young and aged HDFs were treated with EMTT (40 or 80 mT) following HDFCM pretreatment (0, 30, or 60%). Proliferation and migration were assessed using cell proliferation and wound healing assays. Senescence‐associated β‐galactosidase (SA‐β‐Gal) staining and enzyme‐linked immunosorbent assay (ELISA) were performed to evaluate the effects of EMTT and HDFCM on senescence and pro‐ and anti‐inflammatory cytokine secretion.

Results

HDFCM significantly enhanced the proliferation of young and aged HDFs, particularly aged cells, in a dose‐dependent manner. EMTT at 40 mT further enhanced these effects, with 40 mT EMTT plus 30% HDFCM yielding the greatest increase in the number of aged HDF. SA‐β‐Gal activity, minimal in young cells, was elevated in aged HDFs, but was reduced by 40 mT EMTT with or without HDFCM. Contrastingly, 80 mT EMTT increased SA‐β‐Gal activity unless counteracted by 60% HDFCM. Moreover, interleukin‐6 was upregulated and transforming growth factor‐β was downregulated by HDFCM and EMTT treatments.

Conclusion

EMTT at low magnetic field strength, particularly if combined with HDFCM, promotes proliferation and migration while reducing senescence in aged HDFs. This approach may offer a promising therapeutic strategy for skin regeneration and antiaging applications, especially for aging or damaged dermal tissues.

Keywords: cellular senescence, dermal fibroblast‐conditioned media, electromagnetic transduction therapy, human dermal fibroblast, neocollagenesis, neoelastogenesis, wound healing

1. Introduction

Electromagnetic transduction therapy (EMTT) is an electromagnetic treatment using high‐intensity electromagnetic impulses with an electromagnetic field strength of 80–150 mT and an oscillating frequency > 100 kHz of each single pulse [1, 2, 3, 4, 5]. EMTT can inhibit inflammatory reactions and stimulate cell proliferation and extracellular matrix synthesis [1, 2, 3]. Previous studies have demonstrated that electromagnetic treatments at long‐acting, low‐level, and high‐frequency or high‐energy, static, and single magnetic pulses have no biological and clinical effects [4, 5]. Nonetheless, pulsed electromagnetic field, which uses an oscillating frequency of 1–10 kHz in each single pulse, reduces prostaglandin E2 and pro‐inflammatory cytokines, particularly interleukin‐6 and ‐8, by acting as an agonist of adenosine receptor A2A and A3 [5, 6, 7].

Short biophysical stimulation of mesenchymal stem cells with high‐intensity electromagnetic impulses using EMTT induces proangiogenic effects that promote stimulatory and rehabilitating effects in the clinical fields of traumatology, orthopedic surgery, and soft tissue treatment [1]. Additionally, EMTT is considered a safe and easily applicable modality for managing acute and chronic aseptic inflammatory musculoskeletal pain [5, 8]. Therefore, high‐intensity electromagnetic impulses are noninvasively delivered through the skin to targeted musculoskeletal tissues without generating thermal side effects or discomfort. Nonetheless, the effects of EMTT on the skin of aged fibroblasts and surrounding dermal conditions remain unclear.

Human dermal fibroblasts (HDFs) maintain epidermal barrier integrity and skin homeostasis by dermal‐epidermal crosstalk, regulating immune reaction and wound healing, and producing collagen substance for the dermis [9, 10, 11]. We revealed that human dermal fibroblast‐conditioned media (HDFCM) contained 337 secretory proteins, which play significant roles in various stages of wound healing, angiogenesis, hair regeneration and skin barrier integrity, and nerve restoration, by proteomic and bioinformatic analyses [12, 13, 14]. The secretomes from HDFs contain various growth factors, and the HDF secretomes induce other types of cells, including outer root sheath cells, to secrete significantly high amounts of growth factors [15]. Additionally, the incubation of plasma‐treated HDFs with HDFCM preparation markedly enhanced the stimulatory effects of low‐energy nitrogen plasma on the replication and migration of HDFs [16].

Despite independent evidence supporting EMTT‐mediated mechanotransduction and fibroblast secretome‐based skin rejuvenation, their combined effects on aged HDFs have not been systematically characterized, and the downstream cytokine responses under such combined stimuli remain poorly defined. Accordingly, this pilot in vitro study was designed to evaluate whether EMTT combined with HDFCM could enhance proliferation and migration, attenuate cellular senescence, and modulate representative secreted cytokines in aged HDFs compared with young HDFs. HDFCM was prepared exclusively from low‐passage young HDFs because the therapeutic hypothesis was that a younger dermal secretome could rescue aged‐HDF function; keratinocyte‐conditioned media were not included because EMTT targets the dermal compartment and the candidate under evaluation is a fibroblast‐derived secretome. EMTT at low‐ and high‐energy settings was noninvasively delivered to HDFs pretreated with various concentrations of HDFCM. Cell proliferation and wound‐healing properties were evaluated using cell proliferation and wound‐healing assays, and SA‐β‐Gal staining was performed to comparatively evaluate patterns of the aging process. Additionally, ELISA was performed to investigate the effects of EMTT and HDFCM on pro‐ and anti‐inflammatory cytokines, specifically IL‐6 as a pro‐inflammatory cytokine, IL‐10 as a regulatory cytokine, and TGF‐β1 as a matrix‐modulating cytokine relevant to dermal remodeling.

2. Materials and Methods

2.1. Culture of HDFs and HDFCM Preparation

Neonatal human dermal fibroblasts (HDFs; Lonza, Walkersville, MD, USA) were purchased, and the cells were cultured with Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Rockville, MD, USA) containing 5% fetal bovine serum (FBS; Gibco BRL), 2‐mM GlutaMAX (Lonza), and 1% penicillin/streptomycin (Thermo Fisher Scientific Inc., Lithuania, MA, USA) in an incubator at 37°C and 5% CO2. Cells at passages 5 and 25 were used as young and aged cells, respectively. Moreover, neonatal HDFs at passage 5 were used for HDFCM preparation [12, 15, 16]. The cells were cultured in FBS‐free Opti‐modified Eagle's medium (Gibco BRL), supplemented with 2‐ng/mL recombinant human (rh) epidermal growth factor (Lonza), 2‐ng/mL rh fibroblast growth factor (Lonza), and 2 mM GlutaMAX [12, 15, 16]. The medium, containing secretomes from HDFs, was collected after incubating the cells at 37°C and 5% CO2 for 24 h and used as HDFCM preparation (G4PRF‐300TM; BNV biolab, Seoul, Korea) [12, 15, 16].

2.2. Electromagnetic Transduction Therapy (EMTT)

Magnetolith (Storz Medical, Tägerwilen, Switzerland) was used to perform EMTT on HDF preparations at passages of 5 and 25. Before EMTT, the medium was exchanged with 0%, 30%, and 60% HDFCM, and the cells were treated at the experimental settings for EMTT, including a magnetic field strength of 40 and 80 mT, 4000 impulses per session, and 8 Hz (Figure S1A). In each experimental setting, two sessions of the EMTT were delivered at 2‐h intervals. The cells were incubated at 37°C and 5% CO2 for 24 h. The distance of the cells to the center of the coil was maintained at 0–1.5 cm, depending on the cell plate (Figure S1B).

2.3. Cell Proliferation Assay

For the cell proliferation assay, neonatal HDFs at passages 5 and 25 were seeded into 96‐well plates at a density of 6.25 ×104 cells/well and incubated for 24 h. The cells were treated with 40‐ and 80‐mT EMTT and/or 0%, 30%, and 60% HDFCM and further incubated at 37°C in a 5% CO2 incubator for 24 h. The cells in each well were treated with 10 µL of cell proliferation reagent (Cell Counting Kit‐8; Sigma‐Aldrich Corporation, St Louis, MO, USA) and incubated for 4 h. Optical density was measured at 450 nm using a microplate reader (VersaMax Absorbance Plate Reader; Molecular Devices, San Jose, CA, USA).

2.4. Wound Healing Assay

For the wound healing assay, HDFs at passages 5 and 25 were seeded into cell culture dishes (μ‐Dish 35 mm‐low; ibidi GmbH, Gräfelfing, Germany) at a density of 1.25 ×105 cells/well with cell inserts (Culture‐Insert 2 Well, Cat. #80206; ibidi GmbH) for generating a linear wound and incubated at 37°C in a 5% CO2 incubator for 48 h. Before EMTT, the medium was exchanged with 0%, 30%, and 60% HDFCM, and the cells were treated with 40‐ and 80‐mT EMTT. Immediately after EMTT treatment, the cell inserts were removed and cultured for 0, 6, 12, and 24 h. Images of the cells were obtained at each time point under a phase‐contrast light microscope at ×40 magnification (Olympus CKX53; Olympus Life Science, Tokyo, Japan). The area of the wound region was measured using ImageJ software (NIH, Bethesda, MD, USA) [17, 18].

2.5. Senescence‐associated β‐Galactosidase Analysis

The SA‐β‐Gal activity was evaluated using the Senescence Cells Histochemical Staining Kit (Cat. #CS0030, Sigma–Aldrich) according to the manufacturer's instructions. Passages 5 and 25 HDFs were seeded in cell culture dishes (Cat. #20035; SPL Life Sciences, Pocheon, Korea) at a density of 6.25 ×104 cells/well and treated with 40‐ and 80‐mT EMTT and/or 0%, 30%, and 60% HDFCM. The plates were washed with phosphate‐buffered saline and fixed with a fixation buffer for 7 min at 20°C−25°C. After fixation, the cells were washed thrice with phosphate‐buffered saline and added to the staining mixture. The stained cells were incubated overnight at 37°C without CO2 and observed using a microscope at ×10 magnification (Olympus CKX53SF). The data were presented as % of β‐Gal‐stained cells/total cells.

2.6. Enzyme‐Linked Immunosorbent Assay

The concentrations of IL‐6, ‐10, and TGF‐β in 24 h post‐treatment HDFs supernatants, which were obtained from 75‐cm2‐T flasks at each experimental condition, were determined using commercial human ELISA kits to capture the early acute cytokine‐release kinetics after EMTT and HDFCM treatment. ELISAs were performed according to the manufacturer's instructions using human IL‐6 (Lot #BMS213‐2; Thermo Fisher Scientific, Inc.) and ‐10 (Lot #BMS215‐2; Thermo Fisher Scientific, Inc.), and TGF‐β (Lot #BMS249‐4; Thermo Fisher Scientific, Inc.).

2.7. Statistical Analysis

All experiments were performed in at least three independent biological replicates and data are presented as mean ± standard deviation (SD). All data were analyzed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA), SAS version 9.4 (SAS Inc., Cary, NC, USA), and R version 4.4.1 (The R Foundation for Statistical Computing, Vienna, Austria). To assess the effects of treatment conditions on cell proliferation and inflammatory cytokine secretion, two‐way analysis of variance (ANOVA) followed by Tukey's post hoc test was performed. SA‐β‐Gal data were analyzed using the same two‐way ANOVA with Tukey's post hoc test. Additionally, to assess the effects of the treatment conditions on wound healing, a three‐way linear mixed model was used. Significant main effects or interactions were observed, and post‐hoc analysis was performed to identify specific group differences. Statistical significance was set at p < 0.05.

3. Results

3.1. Effect of EMTT and HDFCM on the Proliferation of Young and Aged HDFs

A cell proliferation assay was performed to evaluate the effects of EMTT and/or HDFCM on the proliferation of HDFs at passages 5 and 25. In HDFs at passage 5, cells treated with HDFCM with or without EMTT showed significantly increased proliferation (Figure 1A). Moreover, a remarkable increase in HDF proliferation was observed with EMTT at 40 mT compared to EMTT at 80 mT. Nonetheless, untreated HDFs and HDFs treated with EMTT at 40 mT and 60% HDFCM had a statistically significant difference (p = 0.0488).

FIGURE 1.

FIGURE 1

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Effect of EMTT and human dermal fibroblast‐conditioned media (HDFCM) on the human dermal fibroblast (HDF) proliferation. HDFs at passages (A) 5 and (B) 25 were seeded in 96‐well plates and treated with HDFCM (0 [control], 30, 60%) and EMTT (0 [control], 40, 80 mT). Cell proliferation was determined by performing WST‐8 assay at 24 h after treatment. Experiments were performed in triplicate. Data represent the mean ± SD. Significance levels are indicated as follows: *p < 0.05, **p < 0.01.

Moreover, without EMTT, aged HDF proliferation increased by 29.9% and 49.6% at 24 h after incubation with 30 and 60% HDFCM, respectively, compared to untreated control cells in a dose‐dependent manner (Figure 1B). After delivering EMTT at 40 mT, cell proliferation increased by 58.4%, 74.3%, and 108.8% at 24 h after incubation with 0%, 30%, and 60% HDFCM, respectively, compared to untreated control cells in a dose‐dependent manner. After delivering EMTT at 80 mT, cell proliferation increased by 8.7%, 27.6%, and 41.8% at 24 h after incubation with 0%, 30%, and 60% HDFCM, respectively, compared to untreated control cells in a dose‐dependent manner. Comparing the cells under the identical conditions of HDFCM incubation, the highest cellular proliferation of HDFs was observed at the experimental settings of EMTT at 40 mT (vs. no EMTT, p = 0.0018; vs. EMTT at 80 mT, p = 0.0018).

3.2. Effect of EMTT and HDFCM on Wound Healing Properties

The wound healing assay using HDFs at passage 5 without HDFCM revealed that the % area of the wound (WA%) was reduced at 6, 12, and 24 h, regardless of the EMTT settings (Figures 2, 3A). A higher degree of WA% reduction was observed in HDFs pretreated with 30% and 60% HDFCM in a dose‐dependent manner compared with untreated controls. A wound healing assay using HDFs at passage 25 without HDFCM revealed that WA% was noticeably reduced at 6, 12, and 24 h than HDFs at passage 5, regardless of the EMTT settings (Figure 4). The degree of WA% reduction was slightly higher in aged HDFs pretreated with 0% or 30% HDFCM than in those pretreated with 60% HDFCM. A three‐way mixed model analysis revealed significant differences in the groups of HDFs at passages 5 and 25 in HDFCM concentrations and time points as group (p < 0.001), time (p < 0.001), group × HDFCM (p = 0.028), group × time (p = 0.004), and group × HDFCM × time (p = 0.035), but not HDFCM (p = 0.68) or HDFCM × time (p = 0.068).

FIGURE 2.

FIGURE 2

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Wound healing assay using young HDFs. HDFs at passage 5 were seeded into cell culture dishes with cell inserts for generating a linear wound. The medium was exchanged with 0%, 30%, and 60% HDFCM, and HDFs were treated with 80‐mT EMTT. Immediately after EMTT treatment, the cell inserts were removed and further cultured for 0, 6, 12, and 24 h. Original magnification ×40.

FIGURE 3.

FIGURE 3

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A three‐way mixed model analysis of wound healing assay. A three‐way mixed model analysis was performed using data from the wound healing assay to determine the statistical differences in the passage 5 and 25 groups, time points, and (A) 0%, 30%, and 60% HDFCM concentrations and (B) 0, 40‐, and 80‐mT EMTT. Data are presented as mean ± standard deviation (SD).

FIGURE 4.

FIGURE 4

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Wound healing assay using aged HDFs. HDFs at passages 25 were seeded into cell culture dishes with cell inserts for generating a linear wound. The medium was exchanged with 0%, 30%, and 60% HDFCM, and HDFs were treated with 40‐mT EMTT. Immediately after EMTT treatment, the cell inserts were removed and further cultured for 0, 6, 12, and 24 h. Original magnification ×40.

The wound healing assay using HDFs at passage 5 without EMTT revealed that WA% was remarkably reduced at 6, 12, and 24 h, regardless of HDFCM treatment (Figure 3B). HDFs treated with 40‐ or 80‐mT EMTT had a lesser degree of WA% reduction than the untreated controls, but the difference was not statistically significant. A wound healing assay using HDFs at passage 25 without EMTT revealed that WA% was more slowly reduced at 6, 12, and 24 h than HDFs at passage 5, regardless of HDFCM treatment. In HDFs at passage 25 treated with 40‐mT EMTT, the experimental settings with no HDFCM supplementation or 30% HDFCM incubation had a higher degree of WA% reduction than the untreated controls or 40‐mT EMTT with 60% HDFCM. Among the experimental settings of the 80‐mT EMTT, the HDFs incubated with 30% HDFCM presented enhanced WA% reduction compared to other conditions. A three‐way mixed model analysis revealed significant differences in the HDF groups at passages 5 and 25, 0, 40, and 80 mT EMTT, and the time points as group (p = 0.004), time (p < 0.001), and group × time (p = 0.006), but not EMTT (p = 0.492), group × EMTT (p = 0.521), EMTT × time (p = 0.382), and group × HDFCM × time (p = 0.88).

3.3. Effect of EMTT and HDFCM on Cellular Senescence

In HDFs at passage 5 at all experimental settings, SA‐β‐Gal‐stained cells were rarely found after EMTT at 40 and 80 mT and/or 0%, 30%, and 60% HDFCM incubation (Figure 5A). In HDFs at passage 25, a significantly higher number of SA‐β‐Gal‐stained cells were found compared with passage‐5 HDFs at all corresponding experimental settings (each setting, p < 0.0001; Figures 5B, 6). Moreover, passage‐25 HDFs with no EMTT or 40‐mT EMTT exhibited a higher number of SA‐β‐Gal‐stained cells when incubated with higher concentrations of HDFCM; however, the difference was not statistically significant. Passage‐25 HDFs with 80‐mT EMTT presented relatively low SA‐β‐Gal expression by incubating with 60% HDFCM compared with 0 and 30% HDFCM; however, the difference was not statistically significant. In passage‐25 HDFs with 0 and 30% HDFCM, a significantly higher number of SA‐β‐Gal‐stained cells were found in 80‐mT HDFs (p = 0.0157 in 0% HDFCM and p = 0.0394% in 30% HDFCM).

FIGURE 5.

FIGURE 5

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Senescence‐associated beta‐galactosidase (SA‐β‐Gal) analysis. HDFs at passages (A) 5 and (B) 25 were seeded in cell culture dishes and treated with 0%, 30%, and 60% HDFCM and/or 40‐ and 80‐mT EMTT. The data are presented as % of SA‐β‐Gal‐stained cells/total cells according to the experimental settings of (C) 0, (D) 30%, and (E) 60% HDFCM concentrations. Original magnification ×10. Significance levels are indicated as follows: *p < 0.05, ****p < 0.0001.

FIGURE 6.

FIGURE 6

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Enzyme‐linked immunosorbent assay of pro‐ and anti‐inflammatory cytokines. The concentrations of interleukin (IL)‐6 and ‐10 and transforming growth factor (TGF)‐β in HDFs supernatants, which were treated with 0%, 30%, and 60% HDFCM and/or 40‐ and 80‐mT EMTT. The data were analyzed using two‐way analysis of variance. (A, C, E) HDFs at passage 5 and (B, D, F) HDFs at passage 25 for IL‐6 (A, B), IL‐10 (C, D), and TGF‐β (E, F). Data are presented as mean ± SD.

3.4. Effect of EMTT and HDFCM on Inflammatory Cytokine Secretion

In HDFs at passage 5, IL‐6 secretion was significantly increased by treatment with HDFCM (p < 0.0001) in a dose‐dependent manner but was not affected by EMTT (p = 0.3816) or HDFCM × EMTT (p = 0.4438) (Figure 6A). The secretion of IL‐6 from HDFs at passage 25 was significantly increased following treatment with HDFCM (p < 0.0001), EMTT (p < 0.0001), or HDFCM × EMTT (p < 0.0001) (Figure 6B). However, IL‐10 secretion was not affected by HDFCM (p = 0.9002), EMTT (p = 0.1922), or HDFCM × EMTT (p = 0.5591) in HDFs at passage 5, or by HDFCM (p = 0.3926), EMTT (p = 0.1355), or HDFCM × EMTT (p = 0.1538) at passage 25 (Figures 6C, D).

In HDFs at passage 5, TGF‐β secretion was significantly decreased by treating HDFCM (p < 0.0001), EMTT (p < 0.0001), and HDFCM × EMTT (p < 0.0001) (Figure 6E). Post‐hoc analysis revealed that all experimental conditions were HDFCM × EMTT, but not 30% HDFCM without EMTT versus 30% HDFCM and 40‐mT EMTT (p = 0.3925). In HDFs at passage 25, TGF‐β secretion was significantly decreased by treating HDFCM (p < 0.0001), EMTT (p < 0.0001), and HDFCM × EMTT (p < 0.0001) (Figure 6F). Post‐hoc analysis revealed that all experimental conditions of HDFCM × EMTT interacted significantly with each other. Nonetheless, the experimental conditions included 30% versus 60% HDFCM under 40‐mT EMTT (p = 0.3117) and no EMTT versus 80‐mT EMTT at 0 (p = 0.0763), 30 (p = 0.4888), and 60% (p = 0.8667) HDFCM.

4. Discussion

Presently, we demonstrated the effects of EMTT and/or HDFCM on the proliferation and wound‐healing properties of young and aged HDFs. Previously, the incubation of HDFs with HDFCM after nitrogen plasma treatment was similar or more effective in wound repair compared to the experimental conditions of recombinant human growth factor supplementation [16]. HDFs were found to notably proliferate and migrate over 24 h [16]. Unlike the previous study, which treated human bone marrow mesenchymal cells (MSCs) with EMTT over two weeks, two sessions of EMTT were delivered to HDFs at 2‐h intervals in our study, and post‐treatment cellular reactions were observed over 24 h [1].

In this study, the cell proliferation assay demonstrated that aged HDFs were more affected by pretreatment with HDFCM in a dose‐dependent manner than young HDFs. There were stimulatory effects of EMTT on young HDFs treated with EMTT at 40 mT plus 60% HDFCM compared with no EMTT or EMTT at 80 mT. Moreover, these effects were more remarkable in aged HDFs treated with EMTT at 40 mT, resulting in marked proliferation of the cells compared with no EMTT or EMTT at 80 mT, particularly combined with HDFCM. The wound healing assay revealed that %WA reduction was most remarkable in the experimental settings of EMTT at 40 mT and 30% HDFCM pretreatment.

A study reported no toxic effects of EMTT on MSCs treated with EMTT at 80 and 150 mT, three sessions/week for two weeks [1]. However, differences in the cell proliferation effects of MSCs were not significant among the untreated control and 80‐ and 150‐mT EMTT groups [1]. Nevertheless, the significantly higher concentration of vascular endothelial growth factor was found in the 80‐mT EMTT group [1]. The effects of EMTT on the cellular proliferation and migration of aged HDFs were significantly found in the 40‐mT EMTT group. We suggest that the biostimulatory effects of EMTT can be obtained according to cell type, magnetic field strength, treatment sessions, and post‐treatment culture conditions.

SA‐β‐Gal is significantly associated with the aging process by breaking down collagen and other molecules responsible for skin aging under various stress conditions [19, 20, 21, 22]. In our study, SA‐β‐Gal activity in young HDFs was rarely detected in all experimental conditions of EMTT and HDFCM incubation. Untreated aged HDFs exhibited increased SA‐β‐Gal activity, which was mildly increased by the incubation of HDFCM without EMTT in a dose‐dependent manner and decreased by delivering EMTT at 40 mT. Moreover, SA‐β‐Gal activity remarkably increased by delivering EMTT at 80 mT with 30% or without HDFCM incubation. Nonetheless, high‐energy EMTT‐treated HDFs exhibited notably decreased SA‐β‐Gal expression by high‐dose HDFCM incubation. We suggested that numerous regulatory secretomes, including transforming growth factor‐β, in HDFCM could be pivotal in reducing senescence cells and promoting tissue regeneration [12, 15, 23]. Moreover, high‐dose HDFCM incubation in our study could have provided young HDF‐mimicking environmental conditions and down‐regulated high‐energy EMTT‐induced SA‐β‐Gal activity. We suggested that EMTT at 80 mT could have been involved in growth inhibition and stress‐induced transiently increased SA‐β‐Gal activity [23]. Those EMTT‐induced tissue reactions can be clinically applied for treating inflammatory, hyperplasic, fibrotic, or neoplastic conditions [23, 24]. A previous investigation demonstrated that low‐intensity pulsed ultrasound treatment selectively increased senescence‐associated secretory phenotype secretion and promoted the migration/phagocytic activity of M1 macrophages [25]. However, whether high‐intensity EMTT‐induced transient upregulated SA‐β‐Gal activity is directly associated with accelerated senescence changes in aged tissue requires further investigation.

Clinically, if EMTT targets the skin and subcutis to promote proliferation and tissue restoration, we suggest repetitive EMTT at a low magnetic field strength with or without skin boosters, including young HDF‐derived HDFCM preparations, particularly in patients with aged skin. If EMTT targets musculoskeletal systems and deep soft tissues, EMTT at a high magnetic field has been widely used to reduce pain and induce anti‐inflammatory effects in the clinical fields of traumatology and orthopedic surgery [1, 2, 3, 4, 5]. Therefore, according to our preliminary experimental data, the pre‐ or post‐EMTT at a high magnetic field application of skin boosters on aged skin or injured areas can be recommended to promote wound healing and prevent inflammation. However, further controlled studies should be conducted to establish optimal treatment protocols for enhancing tissue restoration and inducing anti‐inflammatory effects in all layers of the skin and subcutis, deep soft tissue, and the musculoskeletal system.

In the present study, the wound‐healing response to EMTT was modest and variable, and a definitive effect of EMTT on HDF migration could not be established from this pilot experiment. Because IL‐6 is a canonical component of the senescence‐associated secretory phenotype (SASP) [25], the early 24‐h IL‐6 elevation in aged HDFs may reflect an acute mechanotransductive wound‐response signal rather than a chronic SASP‐driven state, and the concomitant decrease in TGF‐β1 further indicates that a unidirectional rejuvenation interpretation is not supported by the cytokine data alone. Because ECM‐synthesis markers, including COL1A1, COL3A1, and elastin, were not measured, the present findings represent cellular‐level responses rather than direct evidence of dermal matrix regeneration.

Although the present study was restricted to HDFs, fibroblast‐derived paracrine factors are established regulators of keratinocyte proliferation and epidermal barrier recovery [11], and the shift toward an anti‐inflammatory and matrix‐modulating cytokine profile observed here is therefore expected to influence overlying keratinocytes; this premise will be tested in keratinocyte co‐culture and three‐dimensional full‐thickness skin models. Cellular senescence also exerts context‐dependent effects in skin carcinogenesis, acting both as a tumor‐suppressive mechanism and, through SASP signaling, as a potential promoter of tumor‐permissive inflammation [23, 24], so the EMTT‐plus‐HDFCM–induced reduction of the SA‐β‐Gal+ fraction and modulation of regulatory cytokines in aged HDFs may have implications beyond dermal rejuvenation. These observations should be interpreted within the context of donor‐, passage‐, and culture‐dependent variability of fibroblast secretomes and the broader literature characterizing dermal remodeling by advanced biophysical and imaging approaches [26, 27].

This study has several limitations. First, this is a pilot in vitro investigation using commercially available young and aged HDFs from single donors, and the findings may not fully represent the heterogeneity of human skin aging; in vivo and clinical validation is required. Second, the magnitude of observed changes was modest and statistical conclusions are based on a limited number of independent biological replicates (n ≥ 3); therefore, findings should be interpreted as preliminary. Third, although we measured representative inflammatory cytokines (IL‐6, IL‐10, and TGF‐β1), we did not perform comprehensive profiling of the HDFCM secretome; the identities and relative abundances of individual growth factors and matrix‐modulating proteins responsible for the observed effects remain to be defined, and ECM‐synthesis endpoints were not measured, so direct claims of dermal matrix regeneration await quantification in follow‐up studies. Broader multiplex cytokine and damage‐associated molecular pattern (DAMP) profiling is acknowledged as a follow‐up direction. Fourth, long‐term outcomes, dose‐response across a wider parameter space, and potential off‐target effects of combining EMTT with fibroblast‐derived secretomes on SASP or on adjacent cell populations were not addressed. In addition, apoptosis and cell‐death assessment was not performed, and an undiluted HDFCM condition was not evaluated. Finally, the concentration ranges and treatment durations used here were selected for exploratory purposes and may not correspond to clinically achievable exposures. Future studies with larger sample sizes, multi‐donor material, in vivo models, and unbiased proteomic characterization of the secretome are warranted to confirm and extend these findings.

5. Conclusion

This pilot in vitro study suggests that EMTT at a low magnetic field strength (40 mT), particularly when combined with HDFCM, enhanced the proliferation and migration of aged HDFs and reduced the expression of markers of cellular senescence. These effects were dose‐dependent and were more pronounced in aged cells than in young cells. Although high‐intensity EMTT (80 mT) induced inconsistent or even adverse effects on senescence‐associated β‐galactosidase expression, high‐dose HDFCM mitigated these effects, suggesting a modulatory role for fibroblast‐derived secretomes. These pilot findings suggest that the potential use of combining EMTT with fibroblast‐based secretomes to improve skin regeneration and counteract age‐related fibroblast dysfunction. However, reciprocal experiments with aged‐HDFCM and keratinocyte co‐culture are reserved for follow‐up studies. Moreover, further studies are needed to validate these results in vivo and develop clinically applicable treatment protocols for aging skin and soft tissues.

Funding

The authors have nothing to report.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: Supplementary Figure 1.jpg

SRT-32-e70394-s001.jpg (392.9KB, jpg)

Acknowledgments

We would like to thank Hye Sun Lee, PhD (Biostatistics Collaboration Unit, Yonsei University College of Medicine, Seoul, Korea) and Hye Guk Ryu, PhD (Clinical Development Team, Cynosure Lutronic Corporation, Goyang, Korea) for their assistance with the statistical analysis.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File: Supplementary Figure 1.jpg

SRT-32-e70394-s001.jpg (392.9KB, jpg)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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