Electric Stimulation at 448 kHz Modulates Proliferation and Differentiation of Follicle Dermal Papilla Cells

Abstract

Dermal papilla cells (DPCs) regulate the hair cycle and play important roles in hair growth and regeneration. Alopecia is a pathology caused by a deregulation in the hair cycle phases. Currently, the use of physical therapies such as radiofrequency (RF) as an alternative to pharmacological treatment is increasing. Electrical stimulation by capacitive resistive electrical transfer (CRET) is one of these therapies. The objective of the present study was to analyze the effect of RF-CRET currents on DPCs. Cells were treated with subthermal 448 kHz CRET currents with two different types of signals: standard (CRET-STD) or modulated (CRET-MOD). Viability (XTT Assay), proliferation (Ki67 and ERK1/2), apoptosis (p53 and caspase 3), differentiation (β-catenin and α-SMA), and anagen markers (versican and PPARγ) were analyzed by immunofluorescence and immunoblot. CRET caused effects on the proliferation and survival of DPCs associated with increases in the expression of p-MAPK-ERK1/2, cyclin D1, and decreases in the expression of p53 and caspase 3. Also, CRET caused significant transient increases in the expression of β-catenin, involved in hair growth, and in the expression of anagen phase markers such as versican and PPARγ related to hair follicle maintenance. The present study highlights the ability of treatment with CRET therapy to cause molecular alterations in DPC involved in hair regeneration.

1. Introduction

Early hair loss is medically called alopecia, and the number of people suffering from it is increasing, approaching 10 million worldwide. Hair not only protects from solar radiation and exposure to heat/cold but also contributes to personal appearance. For this reason, its progressive decline has both a cosmetic and social impact [1].

Hair goes through three stages in the hair cycle: the anagen, catagen, and telogen phases. Thanks to this hair cycle of hair loss and new hair growth, their number remains relatively constant. Changes in the growth cycle that lead to alopecia may be due to shortening of the anagen phase, premature entry into the catagen phase, or prolongation of the telogen phase. There is a wide variety of factors that can affect this hair growth, such as the hormonal component, nutritional status, exposure to radiation, environmental toxins, and certain medications [2,3]. Recently, the development of new therapeutic agents for the prevention of hair loss is receiving more and more attention. Pharmacological therapy of alopecia includes the use of minoxidil, finasteride, dutasteride, Janus kinase (JAK) inhibitors, and valproic acid.

Minoxidil is the first-line treatment for alopecia and is a topically applied vasodilator. Its main mechanism is to increase blood flow to the scalp. Although the exact mechanism of action of minoxidil has not yet been elucidated, available research results suggest that its effects are mediated by increased nutrient delivery to hair follicles through vasodilation, potassium channel opening, and activation of extracellular signals, which may stimulate hair follicles and prolong the anagen phase of the hair cycle. It has also been observed that minoxidil can increase the size of hair follicles, improving hair density. Numerous studies have shown that minoxidil is effective in promoting hair growth in men and women with androgenic alopecia [4].

On the other hand, finasteride and dutasteride prevent hair loss by inhibiting the enzyme 5α-reductase type I and type II, which converts testosterone into dihydrotestosterone (DHT), a hormone that contributes to the miniaturization of hair follicles in androgenic alopecia. By reducing DHT levels, finasteride can help prevent the progression of hair loss and, in some cases, promote hair growth. Finasteride has proven highly effective in clinical studies, with a significant response rate in men with androgenic alopecia [5].

Other drugs such as valproic acid induce hair shaft elongation by expressing specific differentiation proteins. JAK inhibitors (JAKi) are small molecules that inhibit the kinase activity of JAKs and effectively decrease the intracellular transduction of the JAK/STAT pathway, which promotes the transcription of a wide variety of proinflammatory genes in the hair follicle (HF) [6].

Despite their pharmacological efficacy, some of these medications have adverse effects, such as allergic contact dermatitis, erythema, and itching. Additionally, minoxidil has limited efficacy that varies from person to person and tends to be more effective in the early stages of hair loss. In addition, both minoxidil and finasteride require continued use, as discontinuation of treatment often results in loss of the recovered hair. In addition, long-term use of finasteride causes male sexual dysfunction and is considered one of the main causes of infertility and teratogenicity in women [5,6,7]. These undesirable effects have increased interest in the development of other therapies with fewer adverse effects. Thus, the use of physical stimuli such as LEDs, lasers, and electrical stimulation has emerged as a novel alternative for the treatment of hair loss. Among these non-surgical therapies, low-level laser therapy (LLLT) has shown promising results [8]. This type of device exerts its effect through photobiomodulation with red light, which can stimulate hair growth by accelerating the mitosis of keratinocytes and fibroblasts, inhibiting nitric oxide, and reducing inflammation [9,10]. The use of laser and energy-assisted drug delivery (LEADD) for the treatment of alopecia is also currently used. In a very recent review [11], all the studies described observed a positive effect of LEADD treatment. The strongest evidence for LEADD in alopecia is its use with topical corticosteroids. Non-ablative fractionated lasers (NAFL) and fractionated radiofrequency are other current non-pharmacological therapeutic options [12].

Radiofrequency (ablative or non-ablative) is another physical therapy used in alopecia treatment and has been linked to an increase in HF growth [12]. The application of RF induces an increase in temperature caused by the resistance of the tissues to the passage of the current. Among the effects induced by the non-ablative treatment, changes in cell proliferation, neoangiogenesis, neocollagenesis, or increase in dermal thickness have been described [13,14,15,16]. Non-invasive or minimally invasive procedures are used in RF treatment and can be applied to any type of skin with a minimum risk of complications or side effects [17]. RF has also been used in cosmetic treatments that include skin sagging, wrinkles, body/skin rejuvenation, treatments for facial expression lines, recent and late fibrosis, scars and adhesions, cellulite, and localized fat [18]. Studies on the actual efficacy of radiofrequency in hair loss are still limited, although some preliminary results suggest that it may be beneficial, especially in combination with other treatments. Improvement is usually gradual and depends on multiple sessions. However, its long-term efficacy and ability to reverse androgenetic alopecia are still under investigation [13].

One of the RF therapies that applies this type of electrical stimulation is CRET. This therapy applies 448 kHz currents to induce electrical or electrothermal stimulation of target tissues. CRET is a non-invasive technology that has been successfully used in the regeneration of muscle tissue, bone as well as tendons and ligaments [14,15]. Recent studies have shown that this therapy generates neocollagenogenesis when applied in thermal conditions, and its application in subthermal conditions induces anti-inflammatory and antiedematous effects [16]. In alopecia, in a preliminary study by our group, 20 patients with female pattern hair loss (FPHL) were treated with CRET therapy. Trichoscopic data revealed widespread and statistically significant hair redensification (10–15% over pre-treatment values) in all treated scalp areas [17]. Previous studies have shown that the cellular response to CRET varies depending on the type of signal applied. Thus, in a study carried out on hair follicles of patients with androgenic alopecia (AGA-HF) treated with CRET, the standard unmodulated signal was more efficient in increasing proliferation and inhibiting death in the different AGA-HF populations than the modulated signal, as well as in decreasing the expression of metalloproteinases [18].

The objective of the present study was to analyze the effect of RF-CRET currents on human DPC. The dermal papilla is considered the control center for hair growth. Its cells control the development and growth of hair follicles and induce cells in the surrounding matrix to proliferate, migrate, and differentiate [19]. Given the relevance of this cell type in hair growth, this study analyzed the expression of different factors involved in its proliferation and differentiation of this cell type in the absence and presence of two types of CRET signal, standard and modulated.

2. Materials and Methods

2.1. Cell Culture

Human follicle dermal papilla cells (DPC; 67 age, Caucasian) were obtained from PromoCell (C-12072, Heidelberg, Germany) through the local distributor. Cells were routinely grown in DPC growth medium (PromoCell, cat. No. C-26501) with supplement mix (PromoCell) and 1% penicillin-streptomycin (10,000 U/mL; Gibco; Grand Island, NE, USA). Cultures were maintained and grown in an incubator (Forma Scientific, Thermo Fisher, Waltham, MA, USA) cells at 37 °C in a humidified atmosphere at 5% CO₂. Cells from passage numbers 3 to 6 were used for this study. DPCs were passaged every 7 days, detached with Detachkit (PromoCell, cat. No. C-41220), and trypsin neutralizing solution (PromoCell).

In each experimental run, cells were plated either directly on the bottom of 60 mm plastic Petri dishes or on glass coverslips placed inside the dishes (Nunc, Roskilde, Denmark) for immunofluorescence assays. Depending on the aim of the corresponding experiment, a total of 4 to 10 Petri dishes were plated per experimental replicate.

2.2. CRET Treatments

The procedure and materials for in vitro capacitive resistive electrical therapy (CRET) treatments have been described in previous studies [20,21]. Briefly, the RF currents were delivered through pairs of sterile stainless-steel electrodes designed ad hoc for in vitro stimulation. A total of 3 or 4 days after seeding, depending on the experiment, pairs of sterile electrode ions were inserted in all Petri dishes and connected in series. Only the electrodes corresponding to plates intended for electrical stimulation were energized using a signal generator (INDIBA Activ HCR 902, INDIBA^®, Barcelona, Spain) adapted to supply a current identical to that applied in the treatment of patients. The remaining un-energized plates were sham-exposed simultaneously inside an identical, separate CO₂ incubator.

DPCs were treated with two different types of signals: a standard 448 kHz sinusoidal, non-modulated signal (CRET-STD), or 20 kHz, 40% amplitude modulation of the 448 kHz signal (CRET-MOD). In both cases, the stimulation pattern consisted of 5 min pulses of the 448 kHz current applied intermittently and delivered at a subthermal density of 100 μA/mm², separated by 235 min interpulse lapses and administered for a total of 4, 8, 12, 24, 48 or 72 h. Previous studies revealed that the same exposure parameters used in this study were the most effective in obtaining proliferative and cell differentiation responses in different cell types, such as human stem cells, keratinocytes, and fibroblasts. Therefore, the same density and stimulation pattern was used in the present study [20,22,23].

2.3. XTT Viability Assay

One of the objectives of this study was the analysis of cell viability. For this purpose, the XTT cell proliferation assay kit (2,3-bis-2-methoxy-4-nitro-5-sulfophenyl)-2H- tetrazolium-5-carboxanilide, Roche, Rotkreuz, Switzerland) was used. The metabolically active cells reduced XTT into colored formazan compounds were quantified with a microplate reader (TECAN, Männedorf, Switzerland) at a 450 nm wavelength. In this experiment, DPCs seeded at densities of 5500 cells/cm₂ were incubated for 3 days. After 48 h of CRET or sham treatment, the cells were incubated for 3 h with the tetrazolium salt XTT in a 37 °C and 6.5% CO² atmosphere, as recommended by the manufacturer. A microplate reader (TECAN, Männedorf, Switzerland) at a 450 nm wavelength was used to quantify colored formazan Three experimental replicates of the experiment were conducted.

2.4. Immunofluorescence for Ki67, p53, β-Catenin, α-SMA, and Collagen III

After CRET or sham exposure of the samples cultured on coverslips, the expression of Ki67, p53, β-catenin, α-SMA, or Collagen III was characterized by indirect immunofluorescence. The cells were fixed with 4% paraformaldehyde and permeabilized with 95/5 ethanol/acetic acid, or Triton X-100, 0.05% for Collagen III at the end of treatment. The cells were incubated overnight with monoclonal primary antibody anti-Ki67 [SP6] (1:250, cat. No. ab16667; Abcam; Cambridge, UK), Mouse monoclonal anti-p53 (Novocastra, NCL-L-p53-D07, Thermo Fisher Scientific Inc., Rockford, IL, USA), α-SMA monoclonal mouse (1:400, Sigma, A2547, Merck KGaA, Darmstadt, Germany), β-catenin monoclonal mouse (1:100, sc 7963, Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit polyclonal collagen III antibody (1:400, Novus, Saint Louis, MO, USA NB600-594SS) at 4 °C. Then, the secondary antibody Alexa Fluor^® 488 goat anti-rabbit IgG (1:500; cat. No. A11034; Life Technologies, Eugene, Oregon, USA) or Alexa Fluor^® 488 goat anti-mouse IgG (1:500; cat. No. A11034; Life Technologies) was added, and the samples were incubated at room temperature for 1 h to reveal the proteins of interest. The nuclei were counterstained with. ProlongTM Gold antifade reagent with DAPI (cat. No. P36941; Thermo-Fisher, Hillsboro, OR, USA). An inverted fluorescence microscope (Nikon Eclipse Ts2R, Tokyo, Japan) attached to a digital camera DS-Ri2 (Nikon, Japan) was used to study the cellular samples. In each experimental repeat, fifteen microscope fields per coverslip were photographed and analyzed. Images from at least three experimental replicates were recorded, and positive cells or fluorescence intensity and total cells were counted through NIS-Elements Br image software (version 4.40, Nikon, Japan). Positive cell identification and fluorescence intensity were based on fixed thresholds of fluorescence determined and automated at the beginning of the analysis.

2.5. Immunoblotting

DPCs were lysed after CRET or sham exposure in Pierce RIPA buffer (78440, Thermo Scientific) containing protease and phosphatase cocktail (78440, Thermo Scientific). BCA protein assay (Thermo Fisher Scientific) was used to determine the total protein content of the cell lysates. Equal protein volumes from each of the samples (50–60 μg protein aliquots) were separated in 10% sodium dodecyl sulfate polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (Amersham, Buckinghamshire, UK) using a semi-dry system (Semi-Dry blotting, TRANSBLOT-SD;, Bio-Rad, Munich, Germany). The membranes were blocked with 5% BSA in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T buffer) at room temperature for 1 h. The membrane was then washed three times with TBS-T and incubated overnight at 4 °C with primary antibodies, diluted in 10% bovine serum albumin, and then incubated with primary antibodies.

Primary antibody against cyclin D1 (1:1000, cat. No. P2D11F11; Novocastra, Newcastle, UK), ERK1/2 (1:1000; cat. No. 9102S, Thermo Fisher Scientific), p-ERK1/2 (1:1000; cat No. 44-680G, Thermo Fisher Scientific), β-catenin (1:1000, sc 7963, Santa Cruz Biotechnology, Dallas, TX, USA), PPARγ (1:1000; sc81152, Santa Cruz Biotechnology), Versican (1:1000; S351-23, Invitrogen, MA, USA), anti-Actin, α-Smooth Muscle (α-SMA, 1:1000, A2547, Sigma-Aldrich, Saint Louis, MO, USA) were used. The membranes were stripped with 25 mM glycine at pH 2.0 for 30 min when it was necessary. As a loading control, anti-human -GAPDH (1:1000; sc-47724, Santa Cruz Biotechnology) was used. After incubation with the indicated primary antibodies, the protein was detected using peroxidase-conjugated secondary antibodies (ECL donkey anti-rabbit, cat. No. NA934; or sheep anti-mouse, cat. No. NA931; IgG horseradish peroxidase-linked species-specific whole; antibody GE Healthcare, Chicago, IL, USA). The immunoblot signals were quantified by densitometry. The ChemiDoc Imaging system (Bio-Rad) was used to detect ECL chemiluminescence in the blots. For semiquantitative immunoblot analysis, the optical density of the bands was measured and quantified by densitometry through computer imaging software (Quantity-One, version 4.6.7, BioRad).

2.6. Statistical Analysis

At least three independent replicates were conducted per experiment and expressed as means ± standard error (SEM). Statistical analyses were performed with Graph-Pad Prism 6.01 software (GraphPad Software, Inc., San Diego, CA, USA). A two-tailed Student’s t-test was used when comparing two samples (control and treated), and a One-way ANOVA test and post hoc Tukey’s multiple comparisons test were used when comparing the different times and the different CRET signals. Differences of p < 0.05 were considered significant statistically.

3. Results

3.1. Effects of CRET on DPC Viability and Proliferation

The XTT assay is a colorimetric, non-radioactive assay that measures the metabolic activity of viable cells. The results of this XTT assay showed a response dependent on the type of signal used. Thus, the standard signal caused an increase in the viability of the DPC cultures that was not observed when they were treated with the modulated signal. The difference in response of the standard and the modulated signal was statistically different (p < 0.05 *, One-way ANOVA Test, post hoc Tukey’s multiple comparisons test). The proportion of viable cells in a proliferative state was assessed using the proliferation marker Ki67 by immunolabeling. Ki67 is associated with the proliferative phases of the cell cycle, as its expression appears during the G1 phase of the cycle, decreases after mitosis, and, due to its very short half-life, it does not accumulate in quiescent cells. In the CRET-STD-treated DPCs, an increase in the rate of Ki67+ cells was observed (32.22 ± 8.5% p < 0.05 *) compared to the control (Figure 1).

3.2. Effects of CRET on the Expression of ERK1/2 and Cyclin D1

To study the effect of CRET on the MAPK proliferation pathway, the expression of the active (p-ERK1/2) and inactive (ERK) forms, and cyclin D1, was analyzed at different treatment times (4, 8, 12, 24, 48 h). The results showed a transient increase in the expression of p-ERK1/2 compared to the control, which was significant after 12 h of exposure to the standard signal. The longer treatments did not reflect changes with respect to control levels (Figure 2a,c). When the cells were treated with the modulated signal, no significant changes were observed in the expression of p-ERK1/2 at any of the treatment times used compared to the control (Figure 2b,c).

Cyclin D1 is involved in the initiation of DNA synthesis, in the transition from G1 phase to S phase of the cell cycle. Therefore, it is related to cell proliferation. In our experiments, treatment with the standard signal caused significant increases after 12 h of exposure that were maintained after 24 h of treatment. After 48 h of standard CRET treatment, a significant decrease in protein expression was observed (Figure 2d,f). On the other hand, the modulated signal caused a significant transient increase in its expression after 8 h of CRET treatment, which was not observed at longer or shorter treatment times, compared to its controls (Figure 2e,f).

3.3. Effects of CRET on p53 and Caspase 3 Expression

p53 and caspase 3 play a fundamental role in the regulation of cell survival and in cell death processes by apoptosis. The expression of both proteins was analyzed by immunoblot at different times (24, 48, and 72 h) of CRET treatment (standard or modulated). The results showed that only treatment with the standard signal for 48 h significantly decreased the expression of p53 (Figure 3a,b). In addition, the expression of p53 was also studied by immunofluorescence. The number of p53+ cells decreased after 48 h of treatment, although it was not statistically significant (Figure 3c,d).

As in p53, only treatment with the standard signal for 48 h significantly decreased caspase 3 expression (Figure 4a,b).

3.4. Effects of CRET on β-Catenin Expression and Localization

In DPC, the Wingless-type integration-site (Wnt)/β-catenin (β-cat) signaling pathway plays a key role in hair follicle differentiation. In this study, the expression of β-cat was analyzed in DPCs treated with CRET, using the standard or modulated signal, and at different times (4, 8, 12, 24, 48, or 72 h). Significant increases in β -cat compared to the control were observed with both types of signal, although the response times were different. Thus, CRET-standard treatment (Figure 5a,c) caused a significant increase in β-cat after 12 h, while the modulated signal (Figure 5b,c) increased its expression significantly after 48 h of treatment, compared to controls. Furthermore, the response observed to treatment with the standard signal was statistically different from that observed when the modulated signal was used (p < 0.01 **, One-way ANOVA Test, post hoc Tukey’s multiple comparisons test).

Additionally, a count of the number of β-cat+ cells was performed using immunofluorescence in order to detect the intracellular localization of the protein. The CRET treatment at 12 h showed no changes compared to the control with any of the signals used. On the contrary, after 48 h of exposure, a significant increase in the rate of β-cat+ cells was observed in the samples treated with the standard signal. This increase did not reach statistical significance when the cells were treated with the modulated signal (Figure 6a,b). The localization of β-cat observed was mostly cytoplasmic (Figure 6c).

3.5. Effects of CRET on α-SMA Expression and Localization

α-smooth muscle actin (α-SMA) is one of the stem cell markers of the dermal papilla, although it is expressed only in vitro [24]. In the present study, DPCs were treated at different times (4, 8, 12, 24, 48, and 72 h) using the standard or modulated signal, and the expression of α-SMA was analyzed by immunoblot. The results revealed a significant increase with both signals after short treatments (12 h with standard signal and 8 h with modulated signal), compared to the control. When DPCs were treated for a longer time with CRET, a decrease in the expression of this protein was observed with both types of signal (72 h with standard signal and 48 h with modulated signal) (Figure 7).

In order to analyze whether CRET modifies the localization of α-SMA, its expression and localization were analyzed by immunofluorescence. Only times at which the immunoblot had shown significant results with both signals were investigated. The images revealed that only short exposures (12 h) of modulated signal caused a significant increase in the expression of this protein, while both signals induced significant decreases at 48 h. The response to 12 h of treatment was statistically different when a modulated or a standard signal was used (p < 0.01 **, One-way ANOVA Test, post hoc Tukey’s multiple comparisons test) (Figure 8a,b). The cytoplasmic localization of the protein did not change at any of the times or with any of the signals analyzed (Figure 8c)

3.6. Effects of CRET on Anagen Markers: Versican and PPARγ

Regarding versican, with both standard and modulated signals, significant changes were observed in its expression compared to control samples. Thus, cells treated with CRET-STD showed a significant decrease after 4 h of treatment and an increase, also significant, after 12 h, compared to their controls (Figure 9a,c). The response was similar with the modulated signal, although it occurred at different treatment times. The response to the modulated signal after 12 h of treatment was significantly different from that observed after 12 h of treatment with the standard signal (p < 0.001***, One-way ANOVA Test, post hoc Tukey’s multiple comparisons test). Thus, CRET-MOD induced a significant decrease at 12 h of treatment and a significant increase at 48 h, compared to the control (Figure 9b,c).

In PPARγ, a significant increase in its expression was observed after the application of CRET, and again the response occurred at different times depending on the type of signal used. The standard signal gradually increased the expression of PPARγ until it became statistically significant after 12 h of treatment, compared to the control. After 48 h of CRET-STD, control levels were recovered (Figure 10a,c). With the modulated signal, no significant changes were observed in the first hours of treatment and it was not until 48 h of treatment that it reached a significant increase compared to controls, recovering control levels after 72 h of CRET-MOD (Figure 10b,c).

3.7. Effects of CRET on Collagen Type III Expression

DPCs preferentially express collagen type III [25]. Therefore, the expression of this type of collagen was assessed by immunofluorescence in our control cultures or those treated with CRET (standard or modulated) for 48 h. The results did not show significant changes with any of the signals used (Figure 11).

4. Discussion

DPCs are considered the key cells in the maintenance of hair follicles due to their function as initiators and regulators of hair regeneration. They are a reservoir of multipotent stem cells that play a key role in HF morphogenesis and its growth cycle. These cells undergo asymmetric divisions to maintain self-renewal and generate progenies that renew neighboring tissues [26]. During the anagen phase, the number of DPCs increases, producing signals that regulate the proliferation and differentiation of epithelial progenitors [27]. Thus, DPCs provide a controlled release of signaling molecules, such as the transforming growth β (TGFβ), Wingless-type integration site (WNT), bone morphogenetic proteins (BMPs), and fibroblast growth factor (FGF) to promote the proliferation and differentiation of these epithelial progenitor cells [28,29]. In this way, DPCs, through differentiation processes, induce the cells of the surrounding matrix to proliferate, migrate, and differentiate in the hair shaft and the inner root sheath [19]. Some researchers have suggested that certain functional abnormalities in DPCs cause hair loss due to an imbalance in the hair follicle cycle [30].

Physical therapies have been shown to have diverse effects on DPCs. Thus, irradiation with light-emitting diodes (LED) and Low-Level Light Therapy (LLLT) induce the proliferation of DPCs in vitro [31,32]. Likewise, physical therapies based on electrical stimulation are capable of inducing increases in the proliferation and migration of DPCs [33]. In the present study, the effect of treatment with CRET currents on DPC cultures has been studied. Previous results from our group showed that treatment with a standard CRET signal causes a significant increase in the proliferation of DPCs [17] and other cell types, such as keratinocytes, fibroblasts [22], and stem cells [20]. The results of the present work confirm this increase in the proliferation of DPCs, as well as their viability, induced by CRET.

Several pharmacological treatments currently used for alopecia have demonstrated their ability to alter various molecular pathways [34,35,36], including the ERK1/2 signaling pathway. Thus, it has been described that minoxidil activates the ERK1/2 pathway, resulting in an increase in cell proliferation [34]. As in the case of pharmacological treatments such as minoxidil, electrical treatment with the standard signal was able to induce the activation of MAP kinase ERK1/2 (Figure 12). This effect has already been observed in previous studies in fibroblasts treated with the same CRET treatment [22]. However, this proliferative effect was not observed with the modulated signal. This difference in effect between both types of signals could be due to the fact that only the standard signal is able to activate the Ras/ERK pathway.

A temporal evolution of cyclin D1 expression was also observed, with increases compared to the control at 8 h after the application of the modulated signal or at 12 and 24 h with the standard signal. After 48 h of standard treatment, there was a significant decrease in its expression. Cyclin D1 is involved in the initiation of DNA synthesis in the transition from the G1 phase to the S phase of the cell cycle. In the case of the standard signal, the increase in its expression would lead to greater proliferation observed in the treated cultures (Figure 12). However, in the case of the modulated signal, such an increase did not translate into a higher proliferative rate of DPCs. Although the classic role of cyclin D1 has been associated with cell cycle progression, other functions have been identified, such as cell migration and invasion, improvement of angiogenesis, inhibition of mitochondrial metabolism, and detection of DNA damage and repair [37]. Furthermore, it has been described that the different populations that make up the hair follicle present a differential expression of cyclin D1 related to their migration [38]. In the present study, the increase in cyclin D1 induced by treatment with the modulated signal could not be related to an increase in the proliferative rate of the culture. However, given the role of cyclin D1 in DNA repair, migration, or angiogenesis, it is not ruled out that the modulated form of CRET could exert its activity on these other cellular functions.

Different studies have shown that p53 is a negative regulator of the cell growth cycle that participates in follicle apoptosis during the anagen phase [38,39,40,41,42]. Caspases are also other fundamental components in this type of death in several cell types [43]. In this study, the standard signal caused a decrease in the rate of cells expressing p53 and caspase 3. This effect of CRET on p53 was also previously described in fibroblasts [44]. Therefore, the decrease in the expression of both pro-apoptotic proteins, if it also occurs in the follicles, would result in a decrease in their apoptosis during the anagen phase. Together, these data would support an effect of survival of these follicular organs induced by the standard CRET signal, which does not seem to be observed with the modulated signal.

The Wnt/β-catenin signaling is a key pathway associated with hair growth. Wnt/β-catenin plays an important role in various aspects of hair follicle development, such as maintenance of the anagen phase, and its absence leads to the catagen phase [45,46,47,48]. This pathway is involved in the proliferation and differentiation processes of bulge stem cells, regulating the expression of differentiation markers such as K15, K19, α6-integrin, and β1-integrin in papilla cells [47]. Therefore, activation of the Wnt/β-catenin signaling pathway is critical for the maintenance of the differentiation-inducing properties of the DPCs necessary for HF regeneration and hair shaft growth. The present study shows the ability of CRET therapy to cause significant transient increases in β-cat expression. Similarly, it has been described that minoxidil [35] and other drugs, such as valproic acid [36], also activate β-catenin in DPCs, thus prolonging the anagen phase, delaying catagen progression, and promoting lengthening the growth of the human hair shaft. Thus, the effect of the modulated signal on β-cat could favor a differentiation process of DPCs, while the standard signal would induce a proliferative effect in these cultures through this same pathway and/or the ERK1/2 pathway. The combined effect of both treatments could cause an increase in the duration of the anagen phase of the hair cycle.

Additionally, in the present study, anagen phase markers such as versican and PPARγ were analyzed. Versican is an extracellular matrix proteoglycan and is a specific marker of the dermal papilla. Its gene, VCAN, is strongly expressed in DPCs during the anagen phase and hair growth phase [49], so its main role is the induction and maintenance of this phase of the hair cycle, which supports its importance in maintaining normal HF growth [50]. Its expression decreases in androgenic alopecia (AGA) [49] and is downregulated in senescent DPCs [51]. Another marker of the anagen phase in HF, PPARγ, is expressed during the active growth stage of the hair cycle in keratinocytes of the inner and outer root sheath, in keratinocytes of the hair matrix, and in inductive fibroblasts of the dermal papilla. PPARγ promotes terminal differentiation in keratinocytes [49]. In DPCs, CRET causes temporary increases in versican and PPARγ expressions. Taken together, the increases in the expression of both anagen markers expression would suggest that CRET treatment promotes the duration of the follicle growth phase.

Another of the CPD markers analyzed was α-SMA. This actin is highly expressed in stem cells of the dermal papilla and is considered a relatively specific cellular marker of DPCs in vitro related to proliferation [52]. The results of the present study have shown that treatment with the CRET signal induces early increases in its expression, both with the standard signal and the modulated signal, which would be related to the proliferative effect described above. Also, in both types of treatment, significant decreases in its expression were observed after 48 h of treatment. This absence of labeling after long treatments would be associated with a spontaneous process of differentiation of the DPC stem cells in differentiated cell types, which would prevent them from being detected with this specific marker of undifferentiated cells. This loss of α-SMA expression would, therefore, support the differentiation-promoting effect of CRET treatment in DPCs.

In a recently published study from our group performed on hair follicles from patients with AGA (AGA-HF) treated with the same CRET treatment, we showed an increase in proliferation and differentiation, as well as a reduction in death in the different AGA-HF cell types. In addition, the epidermis surrounding the hair follicle also thickened compared to non-electrically treated follicles [18]. Such effects would induce the entry and permanence of the hair follicles in anagen and in this way, electrical stimulation could promote hair growth and follicular proliferation.

On the other hand, the results of the previously conducted and published clinical trial indicated that patients showed a significant increase in hair redensification three months after the end of CRET treatment. This effect was due to increases in the average number of hairs and in the number of follicular units and was detected both in areas with poor hair density according to the diagnosis prior to treatment, and in those whose hair density had been considered normal [53]. Since the main mechanism underlying abnormal hair loss is the deregulation of dermal papilla cell proliferation, the response of DPCs to subthermal electrical stimulation described here could be involved in some of the redensifying effects observed in the trichological study. In sum, this study revealed that CRET treatment caused effects on the proliferation and survival of DPCs associated with increases in the expression of p-ERK1/2, cyclin D1, and decreases in the expression of p53 and caspase 3. Also, CRET causes significant transient increases in the expression of β-catenin, involved in hair growth, and in the expression of anagen phase markers such as versican and PPARγ related to hair follicle maintenance. Thus, CRET electrical treatment would exert a response similar in nature to that obtained by drugs currently used in clinical practice such as minoxidil and valproic acid. While it is true that CRET therapy may have limitations in terms of requiring multiple sessions to achieve optimal results, it is considered a therapy with multiple applications, being useful for the treatment of musculoskeletal injuries [54,55] as well as in aesthetic medicine [56,57]. In general, RF therapies generally have few side effects, such as mild and transient discomfort at the treatment site in some patients. Specifically, CRET therapy uses 448 kHz frequency currents to generate various tissue responses in depth without heating the tissue. Since this therapy has the advantage of being noninvasive, it could be useful as a sole or complementary therapy to pharmacological treatments, shortening their use and, therefore, minimizing the adverse effects of the drugs. Although clinical case studies indicate that CRET and other RF therapies may have undesirable effects or unknown long-term consequences, it would be of interest to conduct more in-depth and/or long-term studies to rule them out [58,59].

5. Conclusions

The results of the present study show the ability of CRET treatment to promote the expression of anagen phase markers in DPCs of the hair follicle. Both types of CRET signals studied, standard and modulated, elicited transient changes in α-SMA, versican, PPARγ, β-catenin, and cyclin D1. Furthermore, while the standard signal promoted DPC proliferation and survival through increases in p-ERK1/2 expression and decreases in p53 and caspase 3 expression, the modulated signal would be involved in the induction of processes related to cell differentiation. These changes would support the results obtained in the clinical trial carried out with this therapy, which revealed a reduction in the loss of hair follicles in patients affected by alopecia and would support the efficacy of RF-based therapies for the treatment of these dermal pathologies.

However, the results shown come from an in vitro study, which represents a limitation for direct extrapolation to patients. This in vitro study aims to be the first approach to the molecular bases involved in the possible effects of CRET on the cells of the dermal papilla, an essential cellular system in the hair growth process. It would be necessary to delve deeper into the pathways that have been shown to be sensitive to CRET currents for a better understanding of the biological mechanism by which CRET acts on hair follicle cells. Likewise, it would be of great interest to advance our knowledge of the differences observed between the two types of signals, standard and modulated, and their clinical implications. Therefore, it is necessary to carry out clinical trials with a sufficient number of subjects to clarify the real effects on the pathology and to delimit the limits of use and its long-term application. In this way, the real effectiveness of CRET therapy and other RF therapies for the treatment of alopecia could be evaluated.