Endogenous Upstream Bioelectrical Modulation of Inflammatory, Angiogenic, and Anti- Senescent Pathways in Human Dermal Fibroblasts Using the REAC-ACT Protocol

Endogenous Upstream Bioelectrical Modulation of Inflammatory, Angiogenic, and Anti- Senescent Pathways in Human Dermal Fibroblasts Using the REAC-ACT Protocol | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Endogenous Upstream Bioelectrical Modulation of Inflammatory, Angiogenic, and Anti- Senescent Pathways in Human Dermal Fibroblasts Using the REAC-ACT Protocol Sara Cruciani, Vania Fontani, Arianna Rinaldi, Salvatore Rinaldi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6701676/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chronic low-grade inflammation and cellular senescence contribute significantly to skin aging and impaired tissue repair. Fibroblasts, key regulators of extracellular matrix remodeling and cytokine activity, are strategic targets for regenerative interventions. This study evaluates the effects of the Anti-Inflammatory Cellular Treatment (ACT) protocol, based on upstream endogenous bioelectrical modulation using Radio Electric Asymmetric Conveyer (REAC) technology, on human dermal fibroblasts (HFF-1), focusing on key molecular pathways involved in inflammation, oxidative stress, angiogenesis, and cellular senescence. HFF-1 cells underwent nine 30-minute REAC-ACT sessions. Gene expression was analyzed via RT-qPCR; protein levels of cytokines and related mediators were measured using ELISA and immunofluorescence. Statistical significance was assessed with Kruskal–Wallis, ANOVA with Tukey correction, and Wilcoxon tests. REAC-ACT significantly upregulated SIRT1 and VEGF, with modest increases in Nox4. Key cytokines (IL-1α, IL-1β, IL-2, IL-8) were selectively elevated, suggesting a reparative rather than pro-inflammatory response. FOXO1 expression increased, while mTOR was downregulated, indicating activation of antioxidant and anti-senescent signaling. REAC-ACT exerts upstream regulatory effects on inflammation, vascular remodeling, and senescence-related signaling, supporting its potential as a non-invasive therapeutic strategy in regenerative and anti-aging dermatology. These findings were consistently observed across biological replicates, supporting the reproducibility and translational relevance of this upstream bioelectrical modulation approach. Biological sciences/Biotechnology Biological sciences/Biotechnology/Regenerative medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Chronic low-grade inflammation and cellular senescence are pivotal contributors to tissue degeneration 1 , impaired extracellular matrix remodeling 2 , and disrupted microcirculation 3 . These processes are especially relevant in cutaneous fibroblasts, which orchestrate structural maintenance, cytokine regulation, and vascular signaling 4 , 5 . Bioelectrical dysregulation in fibroblast function has been increasingly recognized as a central mechanism in both aging and inflammatory skin conditions 6 . The Radio Electric Asymmetric Conveyer (REAC) technology offers a non-invasive modality to restore endogenous bioelectrical activity 7 , thereby reprogramming fibroblast behavior 8 and modulating inflammatory 9 , 10 and reparative pathways 11 , 12 . This study investigates the effects of the REAC Anti-Inflammatory Cellular Treatment (ACT) protocol on human foreskin fibroblasts (HFF1), focusing on oxidative stress regulation, vascular remodeling, and cytokine expression. Degenerative alterations of the subcutaneous tissue, often underestimated due to their superficial appearance, are sustained by underlying biological dysfunctions such as chronic low-grade inflammation, impaired microcirculation, and altered fibroblast activity. These features make fibroblast-targeted upstream endogenous bioelectrical modulation a promising therapeutic strategy. Fibroblasts are central players in maintaining skin structure and function 13 , producing extracellular matrix components 14 and regulating inflammatory responses 15 . Dysregulation of fibroblast activity contributes to aging 16 , scarring 17 , and persistent low-grade inflammation of subcutaneous tissues. 18 . Therefore, targeting fibroblasts with therapies that enhance their functionality holds promise for improving skin quality. REAC-ACT treatment acts by restoring altered endogenous bioelectrical activity 19 , 20 , a mechanism that is broadly applicable to all cell types 7 , including various fibroblast subtypes 21 . REAC-ACT has been investigated in preclinical and translational contexts for tissue degeneration related to inflammation 22 . This study investigates how REAC-ACT modulates expression of key genes involved in inflammation, vascular remodeling, oxidative stress, and cellular senescence. Markers such as SIRT1 23 , VEGF 24 , Nox4 25 , and cytokines (IL-1a, IL-1b, IL-2, IL-8, TNF-α) 26 were analyzed. While often associated with pathology, inflammation also plays essential roles in repair and homeostasis 27 . Fibroblasts contribute to this process by secreting pro-angiogenic factors, including VEGF 28 , a function regulated by SIRT1 29 , which coordinates redox response and inflammation. SIRT1is in turn involved in the regulation of the mTOR signaling pathway, especially in response to nutrients and cellular stress. The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates cell growth and proliferation. SIRT1 activation has been shown to inhibit mTOR activity and cellular senescence 30 . NADPH oxidase 4 (NOX4), an enzyme that generates reactive oxygen species (ROS), is also involved in fibroblast activation and differentiation during inflammation, playing an important role in activating tissue repair processes 31 . Understanding how REAC-ACT modulates these interconnected pathways provides insight into its reparative and anti-aging effects. Results Effect of REAC-ACT treatment on key molecular markers involved in vascular function and oxidative stress regulation Cells were exposed to nine 30-minute sessions of REAC-ACT treatment, and the relative expression levels of each marker were normalized to untreated control samples. Statistical analyses revealed significant changes in the expression of several markers. The sirtuin-1 (SIRT1) gene (Fig. 1 , panel A), a critical regulator of cellular senescence and inflammation, exhibited a substantial 2.7-fold increase in expression following REAC-ACT treatment as compared to untreated controls (Ctrl = 1.0, ACT = 2.7, p < 0.05). This upregulation suggests that REAC-ACT enhances fibroblast survival by suppressing pro-inflammatory cytokine release and extending cellular lifespan through deacetylation of target substrates. At the same time, vascular endothelial growth factor (VEGF), a key mediator of angiogenesis and vascular maintenance, showed a statistically significant 1.5-fold increase in expression following REAC-ACT treatment (Ctrl = 1.0, ACT = 1.5, p < 0.05) (Fig. 1 , panel B). While this change may appear modest, it is likely physiologically relevant when considered alongside the concurrent upregulation of NADPH oxidase Nox4 (Fig. 1 , panel C), which stabilizes VEGFR2 and promotes endothelial differentiation. Nox4, which plays a crucial role in maintaining low hydrogen peroxide (H 2 O 2 ) levels and promoting endothelial differentiation, demonstrated a slight but notable increase in expression (Ctrl = 0.9, ACT = 1.1, p = 0.08). Although the fold-change is relatively small, previous studies have demonstrated that even minor alterations in Nox4 activity can significantly impact vascular function. Enhanced Nox4 expression likely contributes to the overall improvement in endothelial health induced by REAC-ACT, further supporting its role in optimizing vascular function and tissue repair. Together, these findings suggest that REAC-ACT enhances vascular function and improves blood supply to treated areas, addressing chronic inflammation-associated microcirculatory dysfunction in subcutaneous tissue. 3.2 Effect of REAC-ACT treatment on cytokine profiles The analysis of cytokine profiles revealed significantly changes in the expression levels of several inflammatory mediators, highlighting the ability of REAC-ACT to selectively modulate inflammatory responses. Interleukins IL-1a and IL-1b, traditionally associated with inflammation, exhibited significant increases (IL1a: Ctrl = 1.0, ACT = 4.2, p < 0.01; IL1b: Ctrl = 1.0, ACT = 3.7, p < 0.01) (Fig. 2 , panel A and B respectively), as compared to untreated controls. Similarly, IL-2, a pleiotropic cytokine known to stimulate T-cell proliferation and immune regulation, showed the most striking increase, with a remarkable 42-fold upregulation (Ctrl = 1.0, ACT = 42.0, p < 0.001) (Fig. 2 , panel C). Additionally, interleukin-8 (IL8), which mediates neutrophil recruitment and chemotaxis during early phases of tissue repair, exhibited a moderate 2.6-fold increase (Ctrl = 0.8, ACT = 2.6, p < 0.05). (Fig. 2 , panel D). These findings underscore the dual roles of these cytokines in both inflammation and tissue repair processes, indicating that REAC-ACT promotes a balanced inflammatory response rather than indiscriminately inducing inflammation. Effect of REAC-ACT treatment on cytokine release Tumor necrosis factor-alpha (TNF-α), a prototypical pro-inflammatory cytokine, showed minimal alteration (Ctrl = 0.95, ACT = 1.02, p > 0.05) (Fig. 3 , panel A) in terms of gene expression, while being most detectable in medium, which concentrations were measured by ELISA assay (Fig. 3 , panel B). IL-6, a key marker of early inflammation also shows an increase, albeit not significant, in ACT-treated HFF1 (Fig. 4 , panel A), while IL-10, well known as anti-inflammatory cytokine, shows a slight decrease in its concentrations (Fig. 4 , panel B), as compared to untreated controls. This finding aligns with the hypothesis that REAC-ACT promotes healing while minimizing excessive inflammation. In summary, REAC-ACT treatment significantly modulated the expression of key molecular markers in HFF1 fibroblasts, as summarized in Table 1 below: Table 1 Effects of REAC-ACT treatment on the expression of key molecular markers in HFF-1 fibroblasts. Data are expressed as fold change relative to untreated control (Ctrl) cells. REAC-ACT significantly upregulated the expression of genes involved in anti-aging (SIRT1), angiogenesis (VEGF), and inflammation (IL-1α, IL-1β, IL-2, IL-8), while showing a slight, non-significant increase on oxidative stress marker Nox4 and no significant change in TNF-α expression. Statistical significance is indicated by p-values. Marker Control (Ctrl) REAC-ACT Fold Change p-value SIRT1 1.0 2.7 2.7x < 0.01 VEGF 1.0 1.5 1.5x < 0.05 Nox4 0.9 1.1 1.2x 0.08 IL-1a 1.0 4.2 4.2x < 0.001 IL-1b 1.0 3.7 3.7x < 0.01 IL-2 1.0 42.0 42.0x < 0.01 IL-8 0.8 2.6 3.3x 0.05 These results indicate that REAC-ACT not only suppresses excessive inflammation but also enhances endothelial differentiation, promotes tissue repair processes, and modulates redox homeostasis. Furthermore, the observed upregulation of Sirt1 highlights the potential of REAC-ACT to delay cellular senescence and combat aging-related skin degeneration. Effect of REAC-ACT treatment on antioxidant signaling pathway SIRT1 is an NAD + dependent deacetylase 33 that plays a significant role in the oxidative stress response by stimulating the transcription factor FOXO 34 . FOXO activation is in turn responsible for the activation of several proteins related to the oxidative stress response 34 . The increased expression of FOXO1, as emerged by immunofluorescence analysis (Fig. 5 ), is a direct consequence of the increased expression of SIRT1 observed in ACT-treated HFF1 (Fig. 6 ), as compared to untreated controls. The mTOR pathway 34 also interacts with SIRT1, that negatively regulates mTOR in response to stress 35 . Here, we observed the decreased expression of mTOR in ACT-treated HFF1 (Fig. 6 ), as compared to untreated control cells. Discussion Sirtuin-1 (SIRT1) is a key regulator of cellular senescence and inflammation 23 , suppressing pro-inflammatory cytokine release and extending cellular lifespan through deacetylation of target substrates 32 . Our data reveals a significant 2.7-fold increase in SIRT1 expression (p < 0.05), indicating that REAC-ACT enhances fibroblast survival and reduces inflammatory signaling 33 , 34 . This effect is particularly relevant in aging skin, where increased senescence and inflammation contribute to tissue degeneration. By activating SIRT1, REAC-ACT may delay aging processes 32 and promote healthier skin. Additionally, SIRT1 activation has been linked to improved mitochondrial function 35 and metabolic health 36 , 37 . This effect is closely related to the modulation of FOXO1 and mTOR signaling pathways exerted by SIRT1, as observed by immunofluorescence 38 , suggesting that REAC-ACT could have systemic benefits beyond local skin improvements. Given the pivotal role of SIRT1 in delaying aging processes 39 , 40 and promoting healthier skin, this finding highlights the potential of REAC-ACT to combat aging-related skin degeneration. Tissue repair processes critically depend on angiogenesis. Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and vascular maintenance, promoting the formation of new blood vessels and ensuring adequate nutrient supply to tissues 41 . The statistically significant 1.5-fold increase in VEGF expression (p < 0.05) suggests that REAC-ACT enhances vascular function and improves blood supply to treated areas, addressing chronic inflammation-associated microcirculatory dysfunction in subcutaneous tissue. When combined with the upregulation of Nox4, which stabilizes VEGFR2 42 and promotes endothelial differentiation, by maintaining low hydrogen peroxide (H2O2) levels 43 , these findings support the hypothesis that REAC-ACT optimizes vascular health and tissue repair. Although the increase in NOX4 expression was relatively modest (1.1-fold, p = 0.08), this finding aligns with previous studies suggesting that even small changes in NOX4 activity can have profound effects on vascular function. Enhanced NOX4 expressions likely contribute to the overall improvement in endothelial health induced by REAC-ACT. One of the most striking findings of this study is the dramatic upregulation of certain cytokines, particularly IL-2 (42-fold, p < 0.001), alongside increases in IL-1a (4.2-fold, p < 0.01) and IL-1b (3.7-fold, p < 0.01). While these results might initially appear counterintuitive given the anti-inflammatory intent of REAC-ACT, it is important to consider the dual roles of these cytokines in inflammation and tissue repair. For example, IL-2 is known to stimulate T-cell proliferation and immune regulation 44 , 45 , potentially contributing to tissue repair processes. Similarly, IL-1a and IL-1b, although traditionally associated with inflammation, play roles in wound healing and extracellular matrix (ECM) remodeling 46 . The lack of significant change in TNF-α (1.1-fold, p > 0.05) further supports the notion that REAC-ACT selectively modulates cytokine profiles rather than indiscriminately promoting inflammation. The combined effects of SIRT1 activation, VEGF and Nox4 modulation, and cytokine regulation highlight the comprehensive nature of REAC-ACT's impact on fibroblast functionality. By enhancing vascular function, reducing oxidative stress, delaying cellular senescence, and promoting tissue repair, the REAC-ACT treatment protocols offers a holistic approach to combating aging and inflammatory skin conditions. Furthermore, its ability to modulate cytokine profiles suggests potential applications beyond chronic inflammation-associated microcirculatory dysfunction in subcutaneous tissue, including wound healing, scar prevention, and dermatological disorders characterized by chronic inflammation. This study has certain limitations that should be acknowledged. First, the sample was restricted to a single cell line (HFF1), which may not fully capture the variability of fibroblast responses across different populations or pathological conditions. Second, while qPCR provided valuable insights into gene expression changes, the inclusion of functional assays, such as analysis of oxidative stress or enzymatic activity measurements, would enhance the interpretation and robustness of these findings. Methods Cell culturing conditions Human foreskin fibroblasts (HFF1) 47 were obtained from ATCC (Manassas, VA, USA) and cultured in a Dulbecco’s modified Eagle’s Medium (DMEM) (Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS Life Technologies), 2 mM l-glutamine (Euroclone, Milano, Italy) and 1% of penicillin/streptomycin (Euroclone, Milano, Italy). The REAC-ACT treatment was conducted via the REAC BENE mod 110 device (ASMED, Scandicci, Italy), which is specifically designed to deliver asymmetrically conveyed radio electric fields for upstream endogenous bioelectrical modulation. The device parameters are pre-set and non-adjustable by the operator, ensuring standardization and reproducibility. Nine 30-minute sessions were conveyed under controlled conditions (37°C, 5% CO₂), ensuring consistent cellular viability and experimental reproducibility. The control groups were cultured under identical environmental conditions without exposure to the radio electric asymmetrically conveyed field, which served as a baseline for comparison. The device parameters, fixed by the manufacturer, preclude operator adjustments, standardizing treatment across replicates. Gene expression analysis Total RNA was extracted from HFF1 using the RNeasy Mini Kit (Qiagen, 40724 Hilden, Germany) at the end of the treatment, and quantified by the NanoDrop One/OneC Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific, Grand Island, NY, USA). Real-time quantitative PCR was performed using Luna ® Universal One-Step RT-qPCR Kit (New England Biolabs), in a CFX Thermal Cycler (Bio-Rad, Hercules, CA, USA). Target Ct values of each sample were normalized to hGAPDH, used as a reference gene and the relative values of all analyzed genes were expressed as fold of change (2 −∆∆Ct ) of mRNA levels observed in untreated control cells. The primers used were from Thermo Fisher Scientific (Grand Island, NY, USA). All experiments were conducted using at least three independent biological replicates (n ≥ 3). ELISA assays HFF1 culture supernatants were collected from untreated control cells and from treated cells at the end of REAC-ACT exposure. The concentrations of IL-6, IL-10 and TNF-α were determined using streptavidin-HRP conjugated systems Human IL-6 Mini ABTS ELISA Development kit (PeproTech EC, Ltd., London, UK), IL-10 Mini ABTS ELISA Development kit (PeproTech EC, Ltd., London, UK) and Human TNF-α Mini TMB ELISA Development kit (PeproTech EC, Ltd., London, UK), respectively. For each marker analyzed, standard curves were prepared accordingly to manufacturer’s instructions. Each sample was assayed in duplicate, and values were expressed as the mean ± SD of 2 measures per sample. Immunofluorescence analysis Immunofluorescence analysis was used to quantify the expression of FOXO1, Sirt1 and mTOR. At the end of REAC-ACT exposure, cells were fixed for 30 min at RT with 4% paraformaldehyde (Sigma Aldrich Chemie GmbH, Germany) and then permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific, Grand Island, NY, USA)-PBS for 1h at RT in gentle agitation. Blocking solution of 4% bovine serum albumin (BSA)-0. 1% Triton X-100 in PBS (Thermo Fisher Scientific, Grand Island, NY, USA) was used for 1 h with agitation at RT and then cells were incubated overnight at 4°C in agitation with primary anti-FOXO1 and double-labeled anti-Sirt1 and anti-mTor antibodies. At the end of incubation, the cells were washed three times for 5 min in PBS and incubated with fluorescence-conjugated secondary antibodies (Life Technologies, USA) at 37°C for 1 h in the dark. Nuclei were labeled with 1 µg/mL 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Grand Island, NY, USA). Fluorescence was acquired with a fluorescence microscope (Thunder Imager DMi8, Leica, Nussloch, Germany). Statistical analysis Statistical analysis was performed via GraphPad Prism 9.0 software (GraphPad, San Diego, CA, USA). For this study, the Kruskal–Wallis rank sum test, two-way analysis-of-variance (ANOVA) test with Tukey’s correction and the Wilcoxon signed-rank test were used, with a p value < 0.05 assumed to be statistically significant. We considered *p < 0.05, **p < 0.01, ***p < 0.001, and ****p ≤ 0.0001. Declarations Competing Interests S.R. and V.F. are inventors of the REAC technology patent. A.R. is the daughter of S.R. and V.F. The remaining authors declare no competing interests. Funding No Funding Author Contribution Conceptualization, SR, SC, VF. and MM.; methodology, SR, SC, AR, VF. and MM; validation, SR, SC, VF, AR and MM; formal analysis, SC, MM, AR, SR, and VF; investigation, SR, SC, AR, VF and MM; data curation, SR, SC, AR , VF and MM; writing original draft preparation, SR, SC, VF, AR. and MM; writing review and editing, SR, SC, VF, AR and MM; visualization, SC, MM.; supervision, SR, and MM; project administration, SR, VF and MM. All authors have read and agreed to the published version of the manuscript. Data Availability Data is provided within the manuscript References Garcia-Dominguez, M. Pathological and Inflammatory Consequences of Aging. Biomolecules 15 10.3390/biom15030404 (2025). Cifuentes, M. et al. Low-Grade Chronic Inflammation: a Shared Mechanism for Chronic Diseases. Physiol. (Bethesda) . 40 , 0. 10.1152/physiol.00021.2024 (2025). Mengozzi, A. et al. 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The remaining authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6701676","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":515270425,"identity":"f005a92a-17fc-45d8-9dc9-40c4a81aeb4f","order_by":0,"name":"Sara Cruciani","email":"","orcid":"","institution":"University of Sassari","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Cruciani","suffix":""},{"id":515270426,"identity":"d584303a-79da-4064-87e0-a6878a7e3125","order_by":1,"name":"Vania Fontani","email":"","orcid":"","institution":"Rinaldi Fontani 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14:14:33","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115430,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/0b40c090229dc4135b4cc62d.html"},{"id":91874468,"identity":"aa4dd9e9-8875-4743-a04e-b79a812d2f94","added_by":"auto","created_at":"2025-09-22 14:22:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15885,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of SIRT1 (panel A), VEGF (panel B) and Nox4 (panel C) was evaluated in HFF1 at the end of REAC-ACT exposure, as compared to untreated controls. The mRNA levels for each gene were normalized to Glyceraldehyde-3-Phosphate-Dehydrogenase (hGAPDH) and expressed as fold of change (2−∆∆Ct) of the mRNA levels observed in untreated controls defined as 1 (mean ±SD; n=6). Data are expressed as mean ± SD (* p ≤ 0.05; **p ≤ 0.01).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/7205cead9198c796e81a5f0d.png"},{"id":91873995,"identity":"5da7cdb1-c533-4e64-ba36-90c0264e8d28","added_by":"auto","created_at":"2025-09-22 14:14:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33157,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of IL-1a (panel A), IL-1b (panel B), IL-2 (panel C) and IL-8 (panel D) was evaluated in HFF1 at the end of REAC-ACT exposure, as compared to untreated controls. The mRNA levels for each gene were normalized to Glyceraldehyde-3-Phosphate-Dehydrogenase (hGAPDH) and expressed as fold of change (2−∆∆Ct) of the mRNA levels observed in untreated controls defined as 1 (mean ±SD; n=6). Data are expressed as mean ± SD (* p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/6900b7281efd339695bdbd79.png"},{"id":91874469,"identity":"c995889f-fff6-48f5-8540-7c9d99678cad","added_by":"auto","created_at":"2025-09-22 14:22:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15381,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of TNF-α (panel A) and the concentration of TNF-α (panel B) were evaluated in HFF1 at the end of REAC-ACT exposure, as compared to untreated controls. The mRNA levels for each gene were normalized to Glyceraldehyde-3-Phosphate-Dehydrogenase (hGAPDH) and expressed as fold of change (2−∆∆Ct) of the mRNA levels observed in untreated controls defined as 1 (mean ±SD; n=6). Data are expressed as mean ± SD (**** p ≤ 0.0001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/68478dbc316923861f9b1a6f.png"},{"id":91873998,"identity":"6b5cd02a-d945-443b-9466-63b76a56b2c9","added_by":"auto","created_at":"2025-09-22 14:14:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15391,"visible":true,"origin":"","legend":"\u003cp\u003eIL-6 and IL-10 quantification by ELISA. IL-6 (panel A) and IL-10 (panel B) were measured in HFF1 at the end of REAC-ACT exposure, as compared to untreated controls. Data are expressed as mean ± SD (** p ≤ 0.01; *** p ≤ 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/c79735b1398c7c8e502ffc7f.png"},{"id":91874018,"identity":"465df3dc-e39a-4fd6-8636-7a15d681fbc5","added_by":"auto","created_at":"2025-09-22 14:14:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":128753,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence analysis of FOXO1 expression in HFF1 at the end of REAC-ACT exposure, as compared to untreated controls. Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars: 40 µm. The figures are representative of different independent experiments. Fields with the highest yield of positively stained cells are shown.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/7e6f47ad6803b3b42dba234d.jpg"},{"id":91874470,"identity":"341bbde4-4826-4f7d-9c06-a06377f986a4","added_by":"auto","created_at":"2025-09-22 14:22:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":94583,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence analysis of SIRT1 and mTOR expression in HFF1 at the end of REAC-ACT exposure, as compared to untreated controls. Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars: 40 µm. The figures are representative of different independent experiments. Fields with the highest yield of positively stained cells are shown.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/694c66c0ec709af4de709fb0.jpg"},{"id":100788160,"identity":"cf4364b6-3f8a-4d34-bf52-c3b3a7918dfd","added_by":"auto","created_at":"2026-01-21 12:05:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":960369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6701676/v1/c931cdf3-8aeb-4494-a549-8d72944c1b11.pdf"}],"financialInterests":"Competing interest reported. S.R. and V.F. are inventors of the REAC technology patent. A.R. is the daughter of S.R. and V.F. The remaining authors declare no competing interests.","formattedTitle":"Endogenous Upstream Bioelectrical Modulation of Inflammatory, Angiogenic, and Anti- Senescent Pathways in Human Dermal Fibroblasts Using the REAC-ACT Protocol","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic low-grade inflammation and cellular senescence are pivotal contributors to tissue degeneration \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, impaired extracellular matrix remodeling \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and disrupted microcirculation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These processes are especially relevant in cutaneous fibroblasts, which orchestrate structural maintenance, cytokine regulation, and vascular signaling\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Bioelectrical dysregulation in fibroblast function has been increasingly recognized as a central mechanism in both aging and inflammatory skin conditions \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The Radio Electric Asymmetric Conveyer (REAC) technology offers a non-invasive modality to restore endogenous bioelectrical activity \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, thereby reprogramming fibroblast behavior \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and modulating inflammatory \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and reparative pathways \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This study investigates the effects of the REAC Anti-Inflammatory Cellular Treatment (ACT) protocol on human foreskin fibroblasts (HFF1), focusing on oxidative stress regulation, vascular remodeling, and cytokine expression. Degenerative alterations of the subcutaneous tissue, often underestimated due to their superficial appearance, are sustained by underlying biological dysfunctions such as chronic low-grade inflammation, impaired microcirculation, and altered fibroblast activity. These features make fibroblast-targeted upstream endogenous bioelectrical modulation a promising therapeutic strategy.\u003c/p\u003e\u003cp\u003eFibroblasts are central players in maintaining skin structure and function\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, producing extracellular matrix components \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and regulating inflammatory responses \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Dysregulation of fibroblast activity contributes to aging \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, scarring \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and persistent low-grade inflammation of subcutaneous tissues. \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, targeting fibroblasts with therapies that enhance their functionality holds promise for improving skin quality. REAC-ACT treatment acts by restoring altered endogenous bioelectrical activity \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, a mechanism that is broadly applicable to all cell types \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, including various fibroblast subtypes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eREAC-ACT has been investigated in preclinical and translational contexts for tissue degeneration related to inflammation \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This study investigates how REAC-ACT modulates expression of key genes involved in inflammation, vascular remodeling, oxidative stress, and cellular senescence. Markers such as SIRT1\u003csup\u003e23\u003c/sup\u003e, VEGF \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, Nox4 \u003csup\u003e25\u003c/sup\u003e, and cytokines (IL-1a, IL-1b, IL-2, IL-8, TNF-α) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e were analyzed. While often associated with pathology, inflammation also plays essential roles in repair and homeostasis \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Fibroblasts contribute to this process by secreting pro-angiogenic factors, including VEGF \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, a function regulated by SIRT1 \u003csup\u003e29\u003c/sup\u003e, which coordinates redox response and inflammation. SIRT1is in turn involved in the regulation of the mTOR signaling pathway, especially in response to nutrients and cellular stress. The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates cell growth and proliferation. SIRT1 activation has been shown to inhibit mTOR activity and cellular senescence \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. NADPH oxidase 4 (NOX4), an enzyme that generates reactive oxygen species (ROS), is also involved in fibroblast activation and differentiation during inflammation, playing an important role in activating tissue repair processes \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Understanding how REAC-ACT modulates these interconnected pathways provides insight into its reparative and anti-aging effects.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eEffect of REAC-ACT treatment on key molecular markers involved in vascular function and oxidative stress regulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells were exposed to nine 30-minute sessions of REAC-ACT treatment, and the relative expression levels of each marker were normalized to untreated control samples. Statistical analyses revealed significant changes in the expression of several markers. The sirtuin-1 (SIRT1) gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, panel A), a critical regulator of cellular senescence and inflammation, exhibited a substantial 2.7-fold increase in expression following REAC-ACT treatment as compared to untreated controls (Ctrl\u0026thinsp;=\u0026thinsp;1.0, ACT\u0026thinsp;=\u0026thinsp;2.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This upregulation suggests that REAC-ACT enhances fibroblast survival by suppressing pro-inflammatory cytokine release and extending cellular lifespan through deacetylation of target substrates. At the same time, vascular endothelial growth factor (VEGF), a key mediator of angiogenesis and vascular maintenance, showed a statistically significant 1.5-fold increase in expression following REAC-ACT treatment (Ctrl\u0026thinsp;=\u0026thinsp;1.0, ACT\u0026thinsp;=\u0026thinsp;1.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, panel B). While this change may appear modest, it is likely physiologically relevant when considered alongside the concurrent upregulation of NADPH oxidase Nox4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, panel C), which stabilizes VEGFR2 and promotes endothelial differentiation. Nox4, which plays a crucial role in maintaining low hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels and promoting endothelial differentiation, demonstrated a slight but notable increase in expression (Ctrl\u0026thinsp;=\u0026thinsp;0.9, ACT\u0026thinsp;=\u0026thinsp;1.1, p\u0026thinsp;=\u0026thinsp;0.08). Although the fold-change is relatively small, previous studies have demonstrated that even minor alterations in Nox4 activity can significantly impact vascular function. Enhanced Nox4 expression likely contributes to the overall improvement in endothelial health induced by REAC-ACT, further supporting its role in optimizing vascular function and tissue repair. Together, these findings suggest that REAC-ACT enhances vascular function and improves blood supply to treated areas, addressing chronic inflammation-associated microcirculatory dysfunction in subcutaneous tissue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e3.2 Effect of REAC-ACT treatment on cytokine profiles\u003c/p\u003e\u003cp\u003eThe analysis of cytokine profiles revealed significantly changes in the expression levels of several inflammatory mediators, highlighting the ability of REAC-ACT to selectively modulate inflammatory responses. Interleukins IL-1a and IL-1b, traditionally associated with inflammation, exhibited significant increases (IL1a: Ctrl\u0026thinsp;=\u0026thinsp;1.0, ACT\u0026thinsp;=\u0026thinsp;4.2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; IL1b: Ctrl\u0026thinsp;=\u0026thinsp;1.0, ACT\u0026thinsp;=\u0026thinsp;3.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel A and B respectively), as compared to untreated controls. Similarly, IL-2, a pleiotropic cytokine known to stimulate T-cell proliferation and immune regulation, showed the most striking increase, with a remarkable 42-fold upregulation (Ctrl\u0026thinsp;=\u0026thinsp;1.0, ACT\u0026thinsp;=\u0026thinsp;42.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel C). Additionally, interleukin-8 (IL8), which mediates neutrophil recruitment and chemotaxis during early phases of tissue repair, exhibited a moderate 2.6-fold increase (Ctrl\u0026thinsp;=\u0026thinsp;0.8, ACT\u0026thinsp;=\u0026thinsp;2.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, panel D).\u003c/p\u003e\u003cp\u003eThese findings underscore the dual roles of these cytokines in both inflammation and tissue repair processes, indicating that REAC-ACT promotes a balanced inflammatory response rather than indiscriminately inducing inflammation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of REAC-ACT treatment on\u003c/b\u003e cytokine release\u003c/p\u003e\u003cp\u003eTumor necrosis factor-alpha (TNF-α), a prototypical pro-inflammatory cytokine, showed minimal alteration (Ctrl\u0026thinsp;=\u0026thinsp;0.95, ACT\u0026thinsp;=\u0026thinsp;1.02, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel A) in terms of gene expression, while being most detectable in medium, which concentrations were measured by ELISA assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, panel B). IL-6, a key marker of early inflammation also shows an increase, albeit not significant, in ACT-treated HFF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, panel A), while IL-10, well known as anti-inflammatory cytokine, shows a slight decrease in its concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, panel B), as compared to untreated controls. This finding aligns with the hypothesis that REAC-ACT promotes healing while minimizing excessive inflammation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn summary, REAC-ACT treatment significantly modulated the expression of key molecular markers in HFF1 fibroblasts, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below:\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of REAC-ACT treatment on the expression of key molecular markers in HFF-1 fibroblasts. Data are expressed as fold change relative to untreated control (Ctrl) cells. REAC-ACT significantly upregulated the expression of genes involved in anti-aging (SIRT1), angiogenesis (VEGF), and inflammation (IL-1α, IL-1β, IL-2, IL-8), while showing a slight, non-significant increase on oxidative stress marker Nox4 and no significant change in TNF-α expression. Statistical significance is indicated by p-values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMarker\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl (Ctrl)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eREAC-ACT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFold Change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSIRT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.7x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVEGF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.5x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNox4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.2x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-1a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.2x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-1b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.7x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42.0x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.3x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNF-α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.1x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThese results indicate that REAC-ACT not only suppresses excessive inflammation but also enhances endothelial differentiation, promotes tissue repair processes, and modulates redox homeostasis. Furthermore, the observed upregulation of Sirt1 highlights the potential of REAC-ACT to delay cellular senescence and combat aging-related skin degeneration.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEffect of REAC-ACT treatment on antioxidant signaling pathway\u003c/h2\u003e\u003cp\u003eSIRT1 is an NAD\u0026thinsp;+\u0026thinsp;dependent deacetylase \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e that plays a significant role in the oxidative stress response by stimulating the transcription factor FOXO \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. FOXO activation is in turn responsible for the activation of several proteins related to the oxidative stress response \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The increased expression of FOXO1, as emerged by immunofluorescence analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), is a direct consequence of the increased expression of SIRT1 observed in ACT-treated HFF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), as compared to untreated controls. The mTOR pathway \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e also interacts with SIRT1, that negatively regulates mTOR in response to stress \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Here, we observed the decreased expression of mTOR in ACT-treated HFF1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), as compared to untreated control cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSirtuin-1 (SIRT1) is a key regulator of cellular senescence and inflammation \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, suppressing pro-inflammatory cytokine release and extending cellular lifespan through deacetylation of target substrates \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our data reveals a significant 2.7-fold increase in SIRT1 expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that REAC-ACT enhances fibroblast survival and reduces inflammatory signaling \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This effect is particularly relevant in aging skin, where increased senescence and inflammation contribute to tissue degeneration. By activating SIRT1, REAC-ACT may delay aging processes \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and promote healthier skin.\u003c/p\u003e\u003cp\u003eAdditionally, SIRT1 activation has been linked to improved mitochondrial function \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and metabolic health \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This effect is closely related to the modulation of FOXO1 and mTOR signaling pathways exerted by SIRT1, as observed by immunofluorescence \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, suggesting that REAC-ACT could have systemic benefits beyond local skin improvements. Given the pivotal role of SIRT1 in delaying aging processes \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and promoting healthier skin, this finding highlights the potential of REAC-ACT to combat aging-related skin degeneration. Tissue repair processes critically depend on angiogenesis. Vascular endothelial growth factor (VEGF) plays a pivotal role in angiogenesis and vascular maintenance, promoting the formation of new blood vessels and ensuring adequate nutrient supply to tissues \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The statistically significant 1.5-fold increase in VEGF expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) suggests that REAC-ACT enhances vascular function and improves blood supply to treated areas, addressing chronic inflammation-associated microcirculatory dysfunction in subcutaneous tissue. When combined with the upregulation of Nox4, which stabilizes VEGFR2 \u003csup\u003e42\u003c/sup\u003e and promotes endothelial differentiation, by maintaining low hydrogen peroxide (H2O2) levels \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, these findings support the hypothesis that REAC-ACT optimizes vascular health and tissue repair. Although the increase in NOX4 expression was relatively modest (1.1-fold, p\u0026thinsp;=\u0026thinsp;0.08), this finding aligns with previous studies suggesting that even small changes in NOX4 activity can have profound effects on vascular function. Enhanced NOX4 expressions likely contribute to the overall improvement in endothelial health induced by REAC-ACT.\u003c/p\u003e\u003cp\u003eOne of the most striking findings of this study is the dramatic upregulation of certain cytokines, particularly IL-2 (42-fold, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), alongside increases in IL-1a (4.2-fold, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and IL-1b (3.7-fold, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). While these results might initially appear counterintuitive given the anti-inflammatory intent of REAC-ACT, it is important to consider the dual roles of these cytokines in inflammation and tissue repair. For example, IL-2 is known to stimulate T-cell proliferation and immune regulation \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, potentially contributing to tissue repair processes. Similarly, IL-1a and IL-1b, although traditionally associated with inflammation, play roles in wound healing and extracellular matrix (ECM) remodeling \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The lack of significant change in TNF-α (1.1-fold, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) further supports the notion that REAC-ACT selectively modulates cytokine profiles rather than indiscriminately promoting inflammation.\u003c/p\u003e\u003cp\u003eThe combined effects of SIRT1 activation, VEGF and Nox4 modulation, and cytokine regulation highlight the comprehensive nature of REAC-ACT's impact on fibroblast functionality. By enhancing vascular function, reducing oxidative stress, delaying cellular senescence, and promoting tissue repair, the REAC-ACT treatment protocols offers a holistic approach to combating aging and inflammatory skin conditions. Furthermore, its ability to modulate cytokine profiles suggests potential applications beyond chronic inflammation-associated microcirculatory dysfunction in subcutaneous tissue, including wound healing, scar prevention, and dermatological disorders characterized by chronic inflammation.\u003c/p\u003e\u003cp\u003eThis study has certain limitations that should be acknowledged. First, the sample was restricted to a single cell line (HFF1), which may not fully capture the variability of fibroblast responses across different populations or pathological conditions. Second, while qPCR provided valuable insights into gene expression changes, the inclusion of functional assays, such as analysis of oxidative stress or enzymatic activity measurements, would enhance the interpretation and robustness of these findings.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eCell culturing conditions\u003c/h2\u003e\u003cp\u003eHuman foreskin fibroblasts (HFF1) \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e were obtained from ATCC (Manassas, VA, USA) and cultured in a Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s Medium (DMEM) (Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS Life Technologies), 2 mM l-glutamine (Euroclone, Milano, Italy) and 1% of penicillin/streptomycin (Euroclone, Milano, Italy). The REAC-ACT treatment was conducted via the REAC BENE mod 110 device (ASMED, Scandicci, Italy), which is specifically designed to deliver asymmetrically conveyed radio electric fields for upstream endogenous bioelectrical modulation. The device parameters are pre-set and non-adjustable by the operator, ensuring standardization and reproducibility. Nine 30-minute sessions were conveyed under controlled conditions (37\u0026deg;C, 5% CO₂), ensuring consistent cellular viability and experimental reproducibility. The control groups were cultured under identical environmental conditions without exposure to the radio electric asymmetrically conveyed field, which served as a baseline for comparison. The device parameters, fixed by the manufacturer, preclude operator adjustments, standardizing treatment across replicates.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGene expression analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from HFF1 using the RNeasy Mini Kit (Qiagen, 40724 Hilden, Germany) at the end of the treatment, and quantified by the NanoDrop One/OneC Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific, Grand Island, NY, USA). Real-time quantitative PCR was performed using Luna\u003csup\u003e\u0026reg;\u003c/sup\u003e Universal One-Step RT-qPCR Kit (New England Biolabs), in a CFX Thermal Cycler (Bio-Rad, Hercules, CA, USA). Target Ct values of each sample were normalized to hGAPDH, used as a reference gene and the relative values of all analyzed genes were expressed as fold of change (2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e) of mRNA levels observed in untreated control cells. The primers used were from Thermo Fisher Scientific (Grand Island, NY, USA). All experiments were conducted using at least three independent biological replicates (n\u0026thinsp;\u0026ge;\u0026thinsp;3).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eELISA assays\u003c/h2\u003e\u003cp\u003eHFF1 culture supernatants were collected from untreated control cells and from treated cells at the end of REAC-ACT exposure. The concentrations of IL-6, IL-10 and TNF-α were determined using streptavidin-HRP conjugated systems Human IL-6 Mini ABTS ELISA Development kit (PeproTech EC, Ltd., London, UK), IL-10 Mini ABTS ELISA Development kit (PeproTech EC, Ltd., London, UK) and Human TNF-α Mini TMB ELISA Development kit (PeproTech EC, Ltd., London, UK), respectively. For each marker analyzed, standard curves were prepared accordingly to manufacturer\u0026rsquo;s instructions. Each sample was assayed in duplicate, and values were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of 2 measures per sample.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence analysis\u003c/h3\u003e\n\u003cp\u003eImmunofluorescence analysis was used to quantify the expression of FOXO1, Sirt1 and mTOR. At the end of REAC-ACT exposure, cells were fixed for 30 min at RT with 4% paraformaldehyde (Sigma Aldrich Chemie GmbH, Germany) and then permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific, Grand Island, NY, USA)-PBS for 1h at RT in gentle agitation. Blocking solution of 4% bovine serum albumin (BSA)-0. 1% Triton X-100 in PBS (Thermo Fisher Scientific, Grand Island, NY, USA) was used for 1 h with agitation at RT and then cells were incubated overnight at 4\u0026deg;C in agitation with primary anti-FOXO1 and double-labeled anti-Sirt1 and anti-mTor antibodies. At the end of incubation, the cells were washed three times for 5 min in PBS and incubated with fluorescence-conjugated secondary antibodies (Life Technologies, USA) at 37\u0026deg;C for 1 h in the dark. Nuclei were labeled with 1 \u0026micro;g/mL 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Grand Island, NY, USA). Fluorescence was acquired with a fluorescence microscope (Thunder Imager DMi8, Leica, Nussloch, Germany).\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed via GraphPad Prism 9.0 software (GraphPad, San Diego, CA, USA). For this study, the Kruskal\u0026ndash;Wallis rank sum test, two-way analysis-of-variance (ANOVA) test with Tukey\u0026rsquo;s correction and the Wilcoxon signed-rank test were used, with a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 assumed to be statistically significant. We considered *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026le;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.R. and V.F. are inventors of the REAC technology patent. A.R. is the daughter of S.R. and V.F. The remaining authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNo Funding\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, SR, SC, VF. and MM.; methodology, SR, SC, AR, VF. and MM; validation, SR, SC, VF, AR and MM; formal analysis, SC, MM, AR, SR, and VF; investigation, SR, SC, AR, VF and MM; data curation, SR, SC, AR , VF and MM; writing original draft preparation, SR, SC, VF, AR. and MM; writing review and editing, SR, SC, VF, AR and MM; visualization, SC, MM.; supervision, SR, and MM; project administration, SR, VF and MM. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGarcia-Dominguez, M. 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Toxicol.\u003c/em\u003e \u003cb\u003e86\u003c/b\u003e, e99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cptx.99\u003c/span\u003e\u003cspan address=\"10.1002/cptx.99\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6701676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6701676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic low-grade inflammation and cellular senescence contribute significantly to skin aging and impaired tissue repair. Fibroblasts, key regulators of extracellular matrix remodeling and cytokine activity, are strategic targets for regenerative interventions. This study evaluates the effects of the Anti-Inflammatory Cellular Treatment (ACT) protocol, based on upstream endogenous bioelectrical modulation using Radio Electric Asymmetric Conveyer (REAC) technology, on human dermal fibroblasts (HFF-1), focusing on key molecular pathways involved in inflammation, oxidative stress, angiogenesis, and cellular senescence. HFF-1 cells underwent nine 30-minute REAC-ACT sessions. Gene expression was analyzed via RT-qPCR; protein levels of cytokines and related mediators were measured using ELISA and immunofluorescence. Statistical significance was assessed with Kruskal\u0026ndash;Wallis, ANOVA with Tukey correction, and Wilcoxon tests. REAC-ACT significantly upregulated SIRT1 and VEGF, with modest increases in Nox4. Key cytokines (IL-1α, IL-1β, IL-2, IL-8) were selectively elevated, suggesting a reparative rather than pro-inflammatory response. FOXO1 expression increased, while mTOR was downregulated, indicating activation of antioxidant and anti-senescent signaling. REAC-ACT exerts upstream regulatory effects on inflammation, vascular remodeling, and senescence-related signaling, supporting its potential as a non-invasive therapeutic strategy in regenerative and anti-aging dermatology. These findings were consistently observed across biological replicates, supporting the reproducibility and translational relevance of this upstream bioelectrical modulation approach.\u003c/p\u003e","manuscriptTitle":"Endogenous Upstream Bioelectrical Modulation of Inflammatory, Angiogenic, and Anti- Senescent Pathways in Human Dermal Fibroblasts Using the REAC-ACT Protocol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 14:14:28","doi":"10.21203/rs.3.rs-6701676/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5addf5d4-ee3f-4fc1-83aa-3eaca5352d21","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54732215,"name":"Biological sciences/Biotechnology"},{"id":54732216,"name":"Biological sciences/Biotechnology/Regenerative medicine"}],"tags":[],"updatedAt":"2026-01-21T11:57:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-22 14:14:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6701676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6701676","identity":"rs-6701676","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}