The Synergistic Effect of Photobiomodulation, Methylglyoxal, and Complex Magnetic Fields on Human Dermal Fibroblasts: Potential Applications for Chronic Wound Treatments.

The Synergistic Effect of Photobiomodulation, Methylglyoxal, and Complex Magnetic Fields on Human Dermal Fibroblasts: Potential Applications for Chronic Wound Treatments. | 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 Research Article The Synergistic Effect of Photobiomodulation, Methylglyoxal, and Complex Magnetic Fields on Human Dermal Fibroblasts: Potential Applications for Chronic Wound Treatments. Emira D'Amico, Tania Vanessa Pierfelice, Loredana D'Ercole, Paola Di Fermo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7599081/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Lasers in Medical Science → Version 1 posted 14 You are reading this latest preprint version Abstract Purpose This paper aimed to verify how a new protocol, recently proposed for treating chronic wounds due to its excellent antimicrobial properties, affects human dermal fibroblasts (NHDFs). Methods Single and combined action of light-emitting diodes (LED), complex magnetic fields (CMFs), and methylglyoxal (MGO) on cell viability and activity of dermal fibroblasts (NHDFs) were investigated. Our first objective was to exclude any toxicity of this combined treatment on these cells. NHDFs were exposed to LED light for 17 min, CMFs for 22 min, MGO, MGO + LED, and MGO + CMFs, and then were assessed for cell viability, morphology, cytoskeletal integrity, collagen type I production, and migration capacity. Results of combined treatments were compared with those of single treatments and unexposed controls. Results NHDFs exposed to both single and combined treatments maintained viability, morphology, and cytoskeletal integrity, showing no signs of cytotoxicity. MGO at low concentrations was non-toxic and, combined with other technologies, was able to confer beneficial effects on cell adhesion. LED stimulated collagen type I synthesis, and the production increased in samples subjected to the combined action of MGO + LED. CMFs notably accelerated fibroblasts' migration in scratch assays, and when combined with MGO, they further enhanced this effect. Conclusions The combined use of MGO + LED and MGO + CMFs produced more significant effects than separate treatments, probably because magnetic fields and light therapy enhanced cellular uptake and receptor sensitivity. The tested protocols were not only non-toxic but also promoted beneficial effects on the vitality and activity of dermal fibroblasts, confirming their potential in treating chronic wounds. photobiomodulation magnetic fields MGO regeneration derma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Chronic wounds are lesions of the skin that fail to progress through the physiological phases of healing in an orderly and timely manner. These wounds represent a significant clinical challenge due to their persistent inflammation, high susceptibility to infections, and impaired tissue regeneration [ 1 ]. Conventional treatments for chronic wounds include debridement, infection control, moisture-balancing dressings, compression therapy, and supportive therapies like growth factors, negative pressure wound therapy, and bioengineered skin substitutes. However, these treatments present various limitations, including high cost, limited effectiveness in complex cases, pain, prolonged sessions, invasiveness, and inability to fully address underlying systemic issues or cellular dysfunctions such as fibroblast senescence [ 1 ]. In addition to canonical treatments, innovative and effective strategies have been tested. Among potential therapeutic options, novel technologies and natural compounds have been proposed. Photobiomodulation (PBM) is a non-invasive treatment that utilizes low-dose light irradiation to promote tissue repair, reduce inflammation, and alleviate pain [ 2 , 3 ]. Numerous research investigations have reported that PBM accelerates wound healing [ 4 , 5 ]. Light-emitting diodes (LEDs) have been shown to be an effective additional treatment method for chronic wounds in people with diabetes in various in vivo studies [ 2 , 6 ]. In particular, red-light photobiomodulation has been studied for its potential to enhance cell proliferation, migration, and collagen synthesis, which are essential for wound repair [ 7 , 8 ]. Several studies have demonstrated the antibacterial effects of red-light irradiation at 630 nm against both gram-positive and negative bacteria [ 9 – 13 ]. Among the other potential alternatives for chronic wounds, cellular models exposed to electromagnetic fields (EMFs) showed various biological processes, including the induction of anti-inflammatory pathways and the reduction of reactive oxygen species (ROS)[ 14 ]. Complex magnetic fields (CMFs) are composed of EMFs signals of different frequencies, intensities, pulses, and waveforms. The CMFs device is characterized by different programs consisting of a sequence of small, single steps of magnetic fields (3–5 min each), with frequencies ranging between 6 and 70 Hz, intensities between 6 and 95 microT, and complex waveforms with multiple harmonics[ 15 ]. The magnets are formed by a winding of 650 turns of enameled copper wire with a 0.35 mm section. External dimensions of the coil 110 mm, internal dimensions of the coil 12 mm, thickness of the coil 8 mm. Some of these programs have been tested in different conditions [ 15 – 17 ]. These studies have shown a relevant anti-virulence action against C. albicans and no cytotoxicity effects on human gingival fibroblasts [ 15 – 17 ]. Zanotti et al. demonstrated the antioxidant, anti-inflammatory, and wound-healing potential of CMFs [ 18 ]. Among natural molecules, methylglyoxal (MGO), contained in Manuka honey, has been recently investigated for its anti-inflammatory and antimicrobial properties [ 19 ]. MGO is effective against various microorganisms, including some strains of gram-positive bacteria and certain gram-negative bacteria, and C. albicans [ 19 , 20 ]. However, the effects of MGO on mammalian cells require careful evaluation due to its possible cytotoxicity. In literature, studies have shown that MGO affects cell viability, proliferation, and apoptosis in a dose-dependent manner [ 21 , 22 ]. Lee JH. et al, observed that at high concentrations (0.6–1 mM) MGO can induce apoptosis by triggering oxidative stress, mitochondrial dysfunction, and DNA damage in various endothelial cells [ 22 ]. Zhang X. et al. described a cytotoxic effect in human umbilical vein endothelial cells (HUVECs) exposed to 0.8 mM of MGO for 5 hours. MGO treatment resulted in p53 phosphorylation, cell cycle arrest, and induction of autophagy [ 21 ]. Although these innovative strategies are potentially promising, they are still far from completely solving the problem of chronic wounds. Indeed, PBM has been used in medicine for the past three decades, it is still a developing form of therapy, and exploring the combination of PBM with molecules like MGO might be a promising and potentially impactful direction for research and therapy development. Despite encouraging results, none of these therapies alone is universally recognised for the treatment of chronic wounds. The ideal treatment should promote antibacterial effects while also inducing tissue regeneration, thereby minimizing cytotoxic effects. A recent study demonstrated that the combination of MGO with red PBM and CMFs yielded a more potent antimicrobial effect against Staphylococcus aureus, Pseudomonas aeruginosa , and Candida albicans compared to single treatments alone. These approaches may exhibit considerable potential as a treatment for chronic wounds; however, infection control and the progression of wound healing are equally paramount in the management of these lesions. [ 20 ]. Beyond its antimicrobial properties, a treatment should not exhibit undesirable effects on the cells responsible for tissue healing and turnover. Thus, this study aimed to investigate the effects of single and combined MGO with red PBM and CMFs on normal human dermal fibroblasts (NHDFs). 2. METHODS 2.1. Cell culture Normal human dermal fibroblasts (NHDFs) were purchased by Sigma Aldrich (Darmstadt, Germany) and were cultured with low glucose Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% of Foetal Bovine Serum (FBS), 1% of penicillin-streptomycin, 1% of L-glutamine (Corning, New York, USA) and 1 ng/mL Fibroblast Growth Factor (FGF) (Sigma Aldrich) at 5% CO 2 and 37°C. 2.2. Treatment conditions MGO Methylglyoxal solution 40 wt.% in H 2 O (Sigma-Aldrich, Milan, Italy) at the concentration of 16 µg/mL. The MGO concentration was chosen based on the results of a previous study [ 20 ]. For the cytotoxicity test, MGO was added to the cell culture media at increasing concentrations, ranging from 16 µg/mL to 11000 µg/mL, to determine at which concentration this molecule exhibits cytotoxicity. CMFs The cells were exposed to the ANTIBACTERIAL PROGRAM, 22 min, of the C.M.F. device, Slim version (Medicina Fisica Integrata, M.F.I., Rome, Italy). CMFs were applied at a 90-degree angle to the sample under treatment [ 20 ]. PBM Irradiation with red light was applied using an AlGaAs power LED device (TL-01; ALPHAStrumenti s.r.l., Pero (MI), Italy) characterized by a wavelength of 630 nm, an intensity of 380 mW/cm², and a light dose of 23 J/cm², as previously described [ 9 ]. The time of irradiation was established at 17 min based on previous studies [ 13 , 20 , 23 ]. The summary of the material and methods and the principal findings was shown in Fig. 1 . 2.3. Evaluation of cytotoxicity and cell viability 10 4 cells/well were seeded in 96-well plate. Then, NHDFs were treated as explained in the experimental design section, and the viability was determined using the MTS assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. 2.4 Evaluation of cell density and shape Cell density and shape of NHDFs were evaluated by toluidine-blue staining. 2×10 4 cells/well were cultured in a 24-well plate and treated as described in the experimental design section. After 24h, NDHFs were fixed with 70% cold ethanol and stained with toluidine blue. Then, cells were observed using an optical microscope (Leica, Wild Heerbrugg, Wetzlar, Germany) at 3x and 25x magnification. 2.5 Immunofluorescence NHDFs were cultured in 8-well culture glass slides (Corning, Glendale, AZ, USA) at a density of 1.3×10 4 /well and treated according to the experimental design for 24h. Cells were fixed with 4% paraformaldehyde (PFA) (BioOptica, Milan, Italy) in 0.1 M PBS (Lonza, Basel, Switzerland). Then, the cells were washed three times with PBS and permeabilized with 0.1% Triton X-100 (BioOptica) in PBS for 5–6 minutes. The cytoskeleton actin and the nuclei have been stained, respectively, with rhodamine-phalloidin (Invitrogen) and DAPI (4’, 6-Diamidino-2-phenylindole dihydrochloride; Sigma), both prepared 1:1000 in PBS and maintained for 1h at 37°C. The images were acquired through the Zeiss LSM800 confocal system (Carl Zeiss, Jena, Germany). 2.6 Cells count, surface area determination, and nuclei to plasma ratio Cell surface area (SA) was determined to characterize how cells spread on the surface under given conditions. This value describes the average surface area occupied by cells. Images of fluorescently stained cells were binarised using ImageJ's tools. From these images, the surface area (µm 2 ) occupied by cells was determined with ImageJ software. Next, cell nuclei were counted to obtain the number of cells (n°) using ImageJ software[ 24 ]. This software was used to create a binary mask of the nuclei or cytoplasm, to measure the Nucleus to cytoplasm (N/C) ratio. Next, the effective area (%) of the total nuclei was divided by the effective area (%) of the total cytoplasm of cells[ 24 ]. Five images, acquired during three repetitive experiments, were analyzed per condition. 2.7 SEM observation The adhesion capability of cells was tested using scanning electron microscopy (SEM). 10 4 cells/well were seeded on titanium surfaces (Implacil, DeBortoli, São Paulo, Brazil) as a support [ 15 ]and treated as described in the experimental design. After 24h, samples were fixed with 2.5% glutaraldehyde for 1 h, dehydrated using increasing concentrations of ethanol and sputtered with gold. A SEM (Phenom-World B.V., Eindhoven, The Netherlands) was used to observe the samples at 245x and 1000x magnification. 2.8 Wound healing assay 3.5∙10 4 cells/well were cultured in 24 well-plates until the confluence was reached. Then, a scratch was made in each well using a 200µl pipette tip, and NDHFs were subjected to the treatment according to the experimental design. The wound areas were acquired with a camera connected to an inverted optical microscope (Leica) at 4x magnification at 0, 24, and 48h. The wound areas, expressed as a percentage, and the migration rate (µm/h) were measured using the software ImageJ 1.52q (National Institutes of Health, Bethesda, MD, USA). 2.9 Picrosirius red staining and spectrophotometric analysis NHDF cells were cultured in 24-well plates at a density of 5∙10 4 cells/well and treated in accordance with the experimental design. After 7 days, cells were fixed with 2.5% glutaraldehyde for 2h, incubated with the picrosirius red staining (Sigma Aldrich) at room temperature for 1h, and images were captured using a stereomicroscope (Leica) at 25×. Then, cells were subjected to three rounds of 0.1% acetic acid washing and to 0.1 N sodium hydroxide. The spectrophotometrical analysis was performed by reading the optical density (OD) at 540 nm. 2.10 Statistics All experiments were performed in triplicate, so for each experimental group/condition the biological sample size was 3. Statistical analysis was performed using GraphPad 5 (GraphPad, San Diego, CA, USA) software. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to evaluate the differences between groups and intragroup analysis at different time-points. For the quantitative analysis of the scratch assay, two-way ANOVA with Tukey’s post hoc test was applied. A p-value of less than 0.05 was considered statistically significant. 3. RESULTS 3.1 Evaluation of MGO cytotoxicity A variable percentage of viability, ranging from 95% to 130%, was observed after treating NHDFs with MGO at concentrations from 16 µg/mL to 9500 µg/mL (Fig. 2 A). Only at the concentrations of 10000 µg/mL and 11000 µg/mL, a severe decrease in the viability was observed, obtaining values of ~ 50%. These values appeared significant compared to CTR ( p < 0.0001). 3.2 Viability of dermal fibroblasts No significant differences were found in the comparison of the unexposed CTR versus the tested groups. The viability of NHDFs exposed to single treatments of MGO 16 µg/mL and LED showed a cell growth comparable to CTR. NHDFs exposed to CMFs showed a slight enhancement of proliferation, but not statistically significant with respect to controls. Also, the combination of MGO + LED as well as MGO + CMFs showed similar values to CTR (Fig. 2 B). 3.3 Cell density, morphology, and adhesion Cells exposed to the treatments were very similar to those of untreated CTR in their morphological features, density, and surface adherence. All groups maintained high cell viability, and cell density appeared preserved (Fig. 3 A), together with the structure of nuclei and cytoskeletal filaments (Fig. 3 B). The number of cells in all treated groups was higher than in the controls, although the data were not statistically significant (Fig. 3 C). Also, at the SEM, all exposed cells maintained their original shape. Numerous interconnections and filopodia among cells were observed at 1000x (Fig. 3 D). The combined action of MGO + LED and MGO + CMFs promoted a slight increase in cell adherence to the titanium surfaces, respect single treatments and unexposed controls. The most notable increase in cell number was observed in cells treated with CMFs, particularly in the combined MGO + CMFs. Treated cells displayed different surface areas, indicating varying abilities to spread on the surface (Fig. 3 E-F). A larger surface area corresponds to greater adhesion. The most marked change was observed in MGO and in MGO + CMFs. N/C ratio decreased in all treated cells, except for those treated with CMFs, whose values were similar to those of the control group. 3.4 Wound healing potential All treatments significantly induced cell migration and wound reduction over time (Fig. 4 A). The graph quantifying wound area (µm²) over time (Fig. 4 B) showed that at 24 hours, CTR had the smallest wound area (0.64 ± 0.02%). All exposed groups showed a statistically significantly larger wound area at 24 hours compared to CTR ( p < 0.001). On the contrary, MGO + CMFs showed a significantly larger area than the single actions of MGO and CMF alone (p < 0.001). At 48 hours, all groups showed a decrease in wound area; however, unexposed controls demonstrated the best wound closure performance (Fig. 4 B). A statistically significant difference (p < 0.001) was confirmed at 48 hours for the wound area in the comparison between CTR (0.59±0.03%) and the exposed groups. Surprisingly, the LED group was not the worst, as it reduced the wound area to 5.37%. On the contrary, MGO + CMFs was the group that at 48h was characterized by the highest wound area (8.75±0.16%), followed by LED, MGO (4.08±0.09%), MGO + LED (3.61±0.80%), and CMFs (3.26±0.09%). Comparing the single and combined actions of the tested technologies, at 48 MGO + LED showed a significantly smaller area compared to LED alone ( p < 0.001), but no significant difference compared to MGO. Conversely, the combined action of MGO + CMFs showed a larger area compared to CMFs and MGO alone ( p < 0.001). The rate of cells moving towards the scratched area revealed that untreated cells and cells treated with CMFs, MGO + LED, and MGO + CMFs migrated faster to close the gap of a scratch than MGO and LED-treated cells (Fig. 4 C). CMFs showed a significantly higher rate compared to MGO + LED ( p < 0.001), MGO + CMFs ( p < 0.001), MGO ( p < 0.001), and LED ( p < 0.001). 3.5 Collagen production Picrosirius red staining revealed more intense red deposits after irradiation with LED and MGO + LED compared to other treatments (Fig. 5 A). The spectrophotometric analysis confirmed the qualitative evaluation with quantitative measurements. In detail, the MGO + LED condition exhibited the highest collagen deposition, with a significant increase in OD values compared to CTR ( p < 0.001), MGO ( p < 0.001), CMFs (p < 0.005), and MGO + CMFs (p < 0.05). LED also showed moderate increases in collagen deposition, and no significant differences were found respect MGO + LED. On the contrary, significant differences (p < 0.001) were found at the intergroup analysis between LED and CMFs, MGO, MGO + CMFs, and CTR. Listing the groups in descending order of collagen production, the greater values were found for MGO + LED and LED, followed by CMFs, MGO, MGO + CMFs, and CTR. No significant differences were found between CMFs, MGO, MGO + CMFs, and CTR (Fig. 5 B). 4. DISCUSSION The combined action of MGO + LED, and MGO + CMFs demonstrated significant potential against chronic wounds due to their antimicrobial effects. However, there is a gap in the literature about the potential effects of these combined strategies on NHDFs. Considering the impact of the single treatments, MGO at the dose used by Diban et al [ 20 ], 16 µg/mL, showed no cytotoxicity against NHDF. In particular, this molecule has shown no cytotoxicity up to a concentration of 10,000 µg/mL, indicating that it remains safe at or below this dosage. In the literature, the effects of MGO remain somewhat controversial. Several in vitro studies have demonstrated that MGO, at concentrations between 0.6–1 mM, can inhibit cell growth by inducing apoptosis [ 25 , 26 ]. Conversely, an in vivo study found that MGO did not produce harmful effects on the vital organs of various animal models [ 27 ]. Additionally, other single treatments, such as CMFs and LED, did not affect the viability of NHDFs. These results were in line with previous literature, which indicates that pulsed electromagnetic fields (PEMF) enhance early wound healing and promote myofibroblast proliferation in diabetic rats [ 28 ]. Vinck et al. reported positive effects of PBM, using various wavelengths (950 nm, 660 nm, and 570 nm), on fibroblast proliferation [ 29 ]. Jere et al. recently reported a significant increase in migration and proliferation of normal, wounded, diabetic, and diabetic-wounded WS1 fibroblast cells when irradiated with a 660 nm diode laser at a fluence of 5 J/cm² [ 30 ]. Surprisingly, the combination of 16 µg/mL MGO + CMFs, and MGO + LED in the study of Diban et al. was characterized by a remarkable antimicrobial activity, in terms of viable cell count, motility, and cellular membrane permeability and fluidity, against Candida albicans and Pseudomonas aeruginosa , and did not affect NHDFs' proliferation [ 20 ]. The absence of cytotoxicity of this protocol was tested in this study and confirmed through optical, confocal, and scanning electron microscope observations, which showed the typical spindle-shaped morphology and cytoskeleton of normal fibroblasts in both exposed and unexposed cells The observed increase in density of the actin filaments in MGO-treated cells suggested a response to a possible stress induced by this molecule. This might reflect cytoskeletal remodeling in response to the glycation stress induced by MGO[ 31 ]. Interestingly, this effect was not accompanied by significant morphological alterations in actin filament organization, although an increase in filament thickness. The combined treatments (MGO + LED and MGO + CMFs) appeared to mitigate MGO-induced effects, maintaining actin architecture and spreading areas similar to control cells. This may suggest a protective or modulatory role of LED or CMFs against MGO-induced cytoskeletal stress, potentially by influencing signaling pathways involved in cytoskeletal dynamics or oxidative stress responses. N/C ratio decreased in treated cells, except for those treated with CMFs alone, which maintained control-like values. A lower N/C ratio typically indicates increased cytoplasmic volume, potentially reflecting enhanced cell spreading or cytoplasmic reorganization[ 24 , 32 ]. This finding aligns with the surface area measurements and supports the hypothesis that CMFs may promote or preserve cytoplasmic integrity and cell adhesion capacity under stress conditions. Indeed, in the initial stages of wound healing, preserving cytoskeletal integrity enables fibroblasts to migrate to the wound site and deposit extracellular matrix, thereby reducing treatment-related stress [ 33 ]. Photobiomodulation seemed to delay wound closure, as evidenced by partial closure at 24 h. There is insufficient evidence of the effects of red LED irradiation on scratch assay closure on NHDFs after 24 hours. Theodoro et al. showed that red LED irradiation at 635 nm, 1.45 J/cm² of NIH/3T3 fibroblasts had no significant differences compared to untreated controls regarding the proliferation and migration rate during the first 4 days of observation [ 34 ]. However, after 48 hours, the wound closure in the LED group increased significantly and was nearly complete, confirming that LED irradiation promoted wound closure, even though the migration rate was lower than that of untreated cells. This finding was in agreement with a recent study indicating that the cell migration was unaffected until 12h upon 630 nm LED treatment and started to accelerate after 24h [ 35 ]. MGO at the concentration used in this study did not seem to affect wound closure, although other concentrations between 7.5 and 10 mM MGO led to enlarged scratch areas in NHDF after 26 hours, indicating a significant decline in cell migration and viability [ 36 ]. The study of the wound area showed that at 24 h, the combined action of MGO + LED produced better results than LED alone, but worse results than MGO alone. Conversely, at 48 h, the combined action showed better results than the single treatments, but with significant results only compared to LED. On the contrary, the combined action of MGO + CMF showed worse results compared to the single treatments, both at 24 and 48 hours.Migration rate, quantified by dividing the change in wound width by the time spent in migration, was highest in CMFs-treated cells. The application of CMFs seemed to accelerate the migration rate of MGO-treated cells. Indeed, the combined action of MGO + CMFs showed a significantly higher migration rate compared to MGO alone, at 24h. The contradictory findings of various studies on MFs suggest that combinations of intensity and treatment period may produce different effects on extracellular matrix synthesis and remodelling, cell proliferation, and migration [ 37 , 38 ]. The combination of MGO + LED stimulated a better wound closure than LED and MGO alone, at 24h. At 48 h, no significant differences were found between single and combined action for the migration rate. The remodelling phase of chronic wound healing involves collagen synthesis [ 39 ]. In the present study, LED-irradiated cells produced significantly more collagen than untreated cells. In particular, MGO + LED improved the effects of MGO in the synthesis of collagen by fibroblasts. On the contrary, the combination of MGO + CMFs did not increase the collagen deposition, with respect to CTR, MGO, and CMFs alone. Literature describes the beneficial effects of light therapy on collagen production. A histology study reported differences in collagen content in an animal model. It showed that collagen fibers were more organized in diabetic and non-diabetic rats after low-level laser therapy (904nm) compared to untreated ones [ 40 ]. The effects of LED on both migration and collagen synthesis are highly dependent on wavelength, fluence, and treatment protocol, and the effect on fibroblasts can persist up to 21 days after irradiation [ 41 ]. In this study, LED irradiation seems to decrease wound closure and migration rate with respect to controls at 24 h, but at 48 h, the same cells increased their values, reducing the differences with other groups. Moreover, at 7 days, the collagen production was higher in the LED group, confirming that this treatment stimulated cell activity and the effects persisted for more days after irradiation. In this study, each combined treatment exerted beneficial effects on cells. MGO at low concentrations was not cytotoxic for dermal fibroblasts; for this reason and due to its antibacterial properties, it might be used as MGO-based dressings to treat many types of wounds, including chronic wounds. The eco-friendly technologies CMFs seemed to be a promoter of the proliferation and cell migration that characterize the initial phase of the healing process. In contrast, LED appeared to stimulate the deposition of collagen type I, which is crucial in the late stage of the healing process. Dang et al. observed that a wavelength of 800 nm and a fluence of 40 J/cm 2 increased skin collagen synthesis via the Smad pathway [ 42 ]. At a wavelength of 670 nm, Otterco et al. observed an improved wound healing process on wounded rats compared to the non-irradiated control group. The authors noted a significant decrease in the inflammatory cytokine TNF-α, and an increase in collagen type I [ 43 ]. Fibroblasts are responsible for generating the majority of the extracellular matrix (ECM) during tissue repair, which is crucial for tissue remodeling and the complete closure of wounds. Collagen, a key element of the ECM, is produced and regulated through a balance between its synthesis and degradation by enzymes like matrix metalloproteinases (MMPs). The findings of this study indicate that combining technologies such as CMFs and PBM with molecules like MGO yields greater benefits than using either treatment individually. A possible explanation might be that LED, and CMFs can affect drug transport and cellular receptor sensitivity. It has been reported that MFs can influence ion channels and signaling pathways, thereby altering cell responses to bioactive molecules [ 44 ]. LED light can increase cell membrane permeability, microcirculation, and cellular metabolism, which can enhance penetration and cellular uptake of molecules [ 45 ]. Further research, especially in vivo studies to validate their clinical relevance, is needed. In the future, the combination of PBM and CMFs may not only be feasible but could also represent a promising and innovative direction in biomedical research. Both PBM and CMFs therapies are highly adaptable and well-suited to personalized medicine, offering the potential for individualized treatment strategies. By adjusting key parameters, such as wavelength, fluence, field strength, and duration, these therapies can be tailored to the specific needs and biological responses of each patient. With further research and clinical validation, the integration of PBM and CMFs could become a powerful and non-invasive approach to precision therapy. 5. CONCLUSIONS This study showed that all devices tested, at the parameters described, were not toxic and are safe for dermal fibroblasts, but produce beneficial effects on the cellular activity of dermal fibroblasts, which are mainly responsible for skin wound healing. Declarations Funding Declaration Funded by European Union-Next Generation EU, program “MUR-Fondo Promozione e Sviluppo-DM737/2021, SCIAMI”, “Eco-friendly antimicrobial Strategies to fight Chronic-wound Infections Associated with Multidrug resistant pathogens for the development of Innovative medical systems”. Author Contribution Author contributionsConceptualisation S.D., M.P., L.D., G.I.; Data curation: E.D., M.P; Investigation: E.D., T.V.P., M.P., S.D.; Methodology: S.D., M.P., L.D., G.I.; Project administration: S.D., M.P., L.D., P. D.F.; Resources: S.D., L.D.; P.D.F.; Supervision: S.D., M.P., L.D., G.I.; Validation: S.D., M.P., L.D., G.I.; Visualisation: S.D., M.P., L.D. G.I.; Writing – original draft: E.D.; T.V.P.; M.P.; Writing – review and editing: S.D., G.I. P.D.F, L.D. ORCIDEmira D’Amico https://orcid.org/0000-0003-2414-7130Tania Vanessa Pierfelice https://orcid.org/0000-0002-5886-2186Loredana D’Ercole https://orcid.org/0000-0002-4466-3987Paola Di Fermo https://orcid.org/0000-0001-6025-1133Giovanna Iezzi https://orcid.org/0000-0002-2391-6594Simonetta D’Ercole https://orcid.org/0000-0002-4797-4019Morena Petrini https://orcid.org/0000-0002-3849-4304DECLARATIONSEthical Approval Not applicable.Competing interests: The authors declare no conflicts of interest concerning this project. The funders were not involved in the study design; in the collection, analysis, and interpretation of data; in the writing of this article; or in the decision to submit it for publication.Clinical trial number Not applicable. Acknowledgement AcknowledgmentsThe authors would like to thank Dr. Stefania Lepore for her assistance with imaging editing. The authors would like to express their gratitude to Marco Mantarro and M.F.I. Medicina Fisica Integrata, Italy, for providing the free use of the CMFs device, and to Alphastrumenti for the free use of the red light device.Data availability statementThe data that support the findings of this study are available from the corresponding author upon reasonable request. Data Availability Data is provided within the manuscript, but if other data are necessary, you can contact the corresponding authors References Frykberg RG, Banks J (2015) Challenges in the Treatment of Chronic Wounds. 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1","display":"","copyAsset":false,"role":"figure","size":208320,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the material and methods and the principal findings (realized by BioRender.com).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7599081/v1/88c1bb7b3be79c3f59576d8f.jpg"},{"id":93599838,"identity":"b516d1f1-5caf-4a91-aa6a-ba4e766124f1","added_by":"auto","created_at":"2025-10-15 14:38:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eBoxplots illustrating MGO cytotoxicity by MTS assay. Results were expressed in the form of optical density (OD) at 490 nm. (*p\u0026lt; 0.05; ***p \u0026lt; 0.0001). \u003cstrong\u003e(B)\u003c/strong\u003e Boxplots illustrating the viability of NHDFs exposed to MGO, LED, and CMFs as single treatments and combined. Results were expressed in the form of optical density (OD) at 490 nm. No statistically significant differences were found with respect to unexposed CTR.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7599081/v1/d18727c83ce62dcfb39ce9e4.jpg"},{"id":93599841,"identity":"3012fbca-5854-41a1-a615-2c8645a9b7da","added_by":"auto","created_at":"2025-10-15 14:38:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":553136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eToluidine blue staining observations at the optical microscope at 24h. Magnification: 3x (scale bar: 500µm), 25x (scale bar: 300µm). \u003cstrong\u003e(B) \u003c/strong\u003eCSLM observations of NHDFs unexposed and exposed to single and combined treatments after 24h. DAPI staining (blue fluorescence) highlights the nuclei of cells; Phalloidin/ rhodamine staining (green fluorescence) shows the cytoskeleton. Magnification: 63x, Scale bar: 20μm. Changes in filament thickness were noted, with filaments appearing slightly thicker in MGO-treated cells. \u003cstrong\u003e(C)\u003c/strong\u003e SEM observations of NHDFs unexposed and exposed to single and combined treatments after 24h. Magnification: 245x (scale bar: 300µm), 1000x (scale bar: 80µm). \u003cstrong\u003e(D)\u003c/strong\u003e Count (n°) of cells (Box plot denotes an average number of cells in the images). \u003cstrong\u003e(E)\u003c/strong\u003e Spreading area (SA) of cells. Box plot denotes an average surface area of cells in the images. (\u003cstrong\u003eF)\u003c/strong\u003eNucleus to cytoplasm (N/C) ratio of cells. (Box plot denotes an average surface area of cells in the images). Boxplots D,E,F represent basic statistical parameters (mean, median, standard deviation, and 25% and 75% percentiles from n=5 fluorescent images). Statistical significance: not statistically significant (p\u0026gt;0.05).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7599081/v1/538a03b3eebdb499efe130f6.jpg"},{"id":93599950,"identity":"da0b2377-e6a0-4eee-a187-cb3a8230a0ab","added_by":"auto","created_at":"2025-10-15 14:40:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":311280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eScratch assay of unexposed and exposed groups at different timings: 0, 24, and 48h. The unclosed area has been highlighted in blue (magnification 4x, scale bar: 300 µm). The comparisons are shown with respect CTR at t0. \u003cstrong\u003e(B) \u003c/strong\u003ePercentage of wound area and \u003cstrong\u003e(C)\u003c/strong\u003e migration rate calculated on the scratch assay. (** p\u0026lt;0.001; ***p\u0026lt;0.0001). The comparisons are shown with respect CTR at t0.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7599081/v1/b7b0d90a824128b1f7663f44.jpg"},{"id":93599922,"identity":"e2cecd46-4237-45e1-80b0-b1821790b4db","added_by":"auto","created_at":"2025-10-15 14:40:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":240799,"visible":true,"origin":"","legend":"\u003cp\u003ePicrosirius red staining of CTR and exposed groups observed at the optical microscope, magnification: 25x; scale bar 300μm \u003cstrong\u003e(A).\u003c/strong\u003eSirius-Red staining quantification \u003cstrong\u003e(B)\u003c/strong\u003e at spectrophotometric analysis, expressed as optical density (OD) (*p\u0026lt;0.05; **p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7599081/v1/fcbf4c5e6775581d5a2c7f37.jpg"},{"id":98243904,"identity":"0fdc46d1-970c-4140-b6da-23e808521dc8","added_by":"auto","created_at":"2025-12-15 16:11:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2142156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7599081/v1/7510c2b8-ace5-4046-bcc1-9bced93bdbe3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Synergistic Effect of Photobiomodulation, Methylglyoxal, and Complex Magnetic Fields on Human Dermal Fibroblasts: Potential Applications for Chronic Wound Treatments.\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eChronic wounds are lesions of the skin that fail to progress through the physiological phases of healing in an orderly and timely manner. These wounds represent a significant clinical challenge due to their persistent inflammation, high susceptibility to infections, and impaired tissue regeneration [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConventional treatments for chronic wounds include debridement, infection control, moisture-balancing dressings, compression therapy, and supportive therapies like growth factors, negative pressure wound therapy, and bioengineered skin substitutes. However, these treatments present various limitations, including high cost, limited effectiveness in complex cases, pain, prolonged sessions, invasiveness, and inability to fully address underlying systemic issues or cellular dysfunctions such as fibroblast senescence [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition to canonical treatments, innovative and effective strategies have been tested. Among potential therapeutic options, novel technologies and natural compounds have been proposed.\u003c/p\u003e\u003cp\u003ePhotobiomodulation (PBM) is a non-invasive treatment that utilizes low-dose light irradiation to promote tissue repair, reduce inflammation, and alleviate pain [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Numerous research investigations have reported that PBM accelerates wound healing [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Light-emitting diodes (LEDs) have been shown to be an effective additional treatment method for chronic wounds in people with diabetes in various \u003cem\u003ein vivo\u003c/em\u003e studies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In particular, red-light photobiomodulation has been studied for its potential to enhance cell proliferation, migration, and collagen synthesis, which are essential for wound repair [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Several studies have demonstrated the antibacterial effects of red-light irradiation at 630 nm against both gram-positive and negative bacteria [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Among the other potential alternatives for chronic wounds, cellular models exposed to electromagnetic fields (EMFs) showed various biological processes, including the induction of anti-inflammatory pathways and the reduction of reactive oxygen species (ROS)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Complex magnetic fields (CMFs) are composed of EMFs signals of different frequencies, intensities, pulses, and waveforms. The CMFs device is characterized by different programs consisting of a sequence of small, single steps of magnetic fields (3\u0026ndash;5 min each), with frequencies ranging between 6 and 70 Hz, intensities between 6 and 95 microT, and complex waveforms with multiple harmonics[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The magnets are formed by a winding of 650 turns of enameled copper wire with a 0.35 mm section. External dimensions of the coil 110 mm, internal dimensions of the coil 12 mm, thickness of the coil 8 mm.\u003c/p\u003e\u003cp\u003eSome of these programs have been tested in different conditions [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These studies have shown a relevant anti-virulence action against \u003cem\u003eC. albicans\u003c/em\u003e and no cytotoxicity effects on human gingival fibroblasts [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Zanotti et al. demonstrated the antioxidant, anti-inflammatory, and wound-healing potential of CMFs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong natural molecules, methylglyoxal (MGO), contained in Manuka honey, has been recently investigated for its anti-inflammatory and antimicrobial properties [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. MGO is effective against various microorganisms, including some strains of gram-positive bacteria and certain gram-negative bacteria, and \u003cem\u003eC. albicans\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the effects of MGO on mammalian cells require careful evaluation due to its possible cytotoxicity. In literature, studies have shown that MGO affects cell viability, proliferation, and apoptosis in a dose-dependent manner [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Lee JH. et al, observed that at high concentrations (0.6\u0026ndash;1 mM) MGO can induce apoptosis by triggering oxidative stress, mitochondrial dysfunction, and DNA damage in various endothelial cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Zhang X. et al. described a cytotoxic effect in human umbilical vein endothelial cells (HUVECs) exposed to 0.8 mM of MGO for 5 hours. MGO treatment resulted in p53 phosphorylation, cell cycle arrest, and induction of autophagy [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough these innovative strategies are potentially promising, they are still far from completely solving the problem of chronic wounds. Indeed, PBM has been used in medicine for the past three decades, it is still a developing form of therapy, and exploring the combination of PBM with molecules like MGO might be a promising and potentially impactful direction for research and therapy development.\u003c/p\u003e\u003cp\u003eDespite encouraging results, none of these therapies alone is universally recognised for the treatment of chronic wounds. The ideal treatment should promote antibacterial effects while also inducing tissue regeneration, thereby minimizing cytotoxic effects. A recent study demonstrated that the combination of MGO with red PBM and CMFs yielded a more potent antimicrobial effect against \u003cem\u003eStaphylococcus aureus, Pseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eCandida albicans\u003c/em\u003e compared to single treatments alone. These approaches may exhibit considerable potential as a treatment for chronic wounds; however, infection control and the progression of wound healing are equally paramount in the management of these lesions. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Beyond its antimicrobial properties, a treatment should not exhibit undesirable effects on the cells responsible for tissue healing and turnover. Thus, this study aimed to investigate the effects of single and combined MGO with red PBM and CMFs on normal human dermal fibroblasts (NHDFs).\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell culture\u003c/h2\u003e\u003cp\u003eNormal human dermal fibroblasts (NHDFs) were purchased by Sigma Aldrich (Darmstadt, Germany) and were cultured with low glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) enriched with 10% of Foetal Bovine Serum (FBS), 1% of penicillin-streptomycin, 1% of L-glutamine (Corning, New York, USA) and 1 ng/mL Fibroblast Growth Factor (FGF) (Sigma Aldrich) at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Treatment conditions\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eMGO\u003c/strong\u003e\u003cp\u003eMethylglyoxal solution 40 wt.% in H\u003csub\u003e2\u003c/sub\u003eO (Sigma-Aldrich, Milan, Italy) at the concentration of 16 \u0026micro;g/mL. The MGO concentration was chosen based on the results of a previous study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For the cytotoxicity test, MGO was added to the cell culture media at increasing concentrations, ranging from 16 \u0026micro;g/mL to 11000 \u0026micro;g/mL, to determine at which concentration this molecule exhibits cytotoxicity.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCMFs\u003c/strong\u003e\u003cp\u003eThe cells were exposed to the ANTIBACTERIAL PROGRAM, 22 min, of the C.M.F. device, Slim version (Medicina Fisica Integrata, M.F.I., Rome, Italy). CMFs were applied at a 90-degree angle to the sample under treatment [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePBM\u003c/strong\u003e\u003cp\u003eIrradiation with red light was applied using an AlGaAs power LED device (TL-01; ALPHAStrumenti s.r.l., Pero (MI), Italy) characterized by a wavelength of 630 nm, an intensity of 380 mW/cm\u0026sup2;, and a light dose of 23 J/cm\u0026sup2;, as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The time of irradiation was established at 17 min based on previous studies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThe summary of the material and methods and the principal findings was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Evaluation of cytotoxicity and cell viability\u003c/h2\u003e\u003cp\u003e10\u003csup\u003e4\u003c/sup\u003e cells/well were seeded in 96-well plate. Then, NHDFs were treated as explained in the experimental design section, and the viability was determined using the MTS assay (Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Evaluation of cell density and shape\u003c/h2\u003e\u003cp\u003eCell density and shape of NHDFs were evaluated by toluidine-blue staining. 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well were cultured in a 24-well plate and treated as described in the experimental design section. After 24h, NDHFs were fixed with 70% cold ethanol and stained with toluidine blue. Then, cells were observed using an optical microscope (Leica, Wild Heerbrugg, Wetzlar, Germany) at 3x and 25x magnification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Immunofluorescence\u003c/h2\u003e\u003cp\u003eNHDFs were cultured in 8-well culture glass slides (Corning, Glendale, AZ, USA) at a density of 1.3\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well and treated according to the experimental design for 24h. Cells were fixed with 4% paraformaldehyde (PFA) (BioOptica, Milan, Italy) in 0.1 M PBS (Lonza, Basel, Switzerland). Then, the cells were washed three times with PBS and permeabilized with 0.1% Triton X-100 (BioOptica) in PBS for 5\u0026ndash;6 minutes. The cytoskeleton actin and the nuclei have been stained, respectively, with rhodamine-phalloidin (Invitrogen) and DAPI (4\u0026rsquo;, 6-Diamidino-2-phenylindole dihydrochloride; Sigma), both prepared 1:1000 in PBS and maintained for 1h at 37\u0026deg;C. The images were acquired through the Zeiss LSM800 confocal system (Carl Zeiss, Jena, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Cells count, surface area determination, and nuclei to plasma ratio\u003c/h2\u003e\u003cp\u003eCell surface area (SA) was determined to characterize how cells spread on the surface under given conditions. This value describes the average surface area occupied by cells. Images of fluorescently stained cells were binarised using ImageJ's tools. From these images, the surface area (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e) occupied by cells was determined with ImageJ software. Next, cell nuclei were counted to obtain the number of cells (n\u0026deg;) using ImageJ software[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This software was used to create a binary mask of the nuclei or cytoplasm, to measure the Nucleus to cytoplasm (N/C) ratio. Next, the effective area (%) of the total nuclei was divided by the effective area (%) of the total cytoplasm of cells[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Five images, acquired during three repetitive experiments, were analyzed per condition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 SEM observation\u003c/h2\u003e\u003cp\u003eThe adhesion capability of cells was tested using scanning electron microscopy (SEM). 10\u003csup\u003e4\u003c/sup\u003e cells/well were seeded on titanium surfaces (Implacil, DeBortoli, S\u0026atilde;o Paulo, Brazil) as a support [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]and treated as described in the experimental design. After 24h, samples were fixed with 2.5% glutaraldehyde for 1 h, dehydrated using increasing concentrations of ethanol and sputtered with gold. A SEM (Phenom-World B.V., Eindhoven, The Netherlands) was used to observe the samples at 245x and 1000x magnification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Wound healing assay\u003c/h2\u003e\u003cp\u003e3.5∙10\u003csup\u003e4\u003c/sup\u003e cells/well were cultured in 24 well-plates until the confluence was reached. Then, a scratch was made in each well using a 200\u0026micro;l pipette tip, and NDHFs were subjected to the treatment according to the experimental design. The wound areas were acquired with a camera connected to an inverted optical microscope (Leica) at 4x magnification at 0, 24, and 48h. The wound areas, expressed as a percentage, and the migration rate (\u0026micro;m/h) were measured using the software ImageJ 1.52q (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Picrosirius red staining and spectrophotometric analysis\u003c/h2\u003e\u003cp\u003eNHDF cells were cultured in 24-well plates at a density of 5∙10\u003csup\u003e4\u003c/sup\u003e cells/well and treated in accordance with the experimental design. After 7 days, cells were fixed with 2.5% glutaraldehyde for 2h, incubated with the picrosirius red staining (Sigma Aldrich) at room temperature for 1h, and images were captured using a stereomicroscope (Leica) at 25\u0026times;. Then, cells were subjected to three rounds of 0.1% acetic acid washing and to 0.1 N sodium hydroxide. The spectrophotometrical analysis was performed by reading the optical density (OD) at 540 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Statistics\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate, so for each experimental group/condition the biological sample size was 3. Statistical analysis was performed using GraphPad 5 (GraphPad, San Diego, CA, USA) software. One-way analysis of variance (ANOVA) and Tukey\u0026rsquo;s post hoc test were used to evaluate the differences between groups and intragroup analysis at different time-points. For the quantitative analysis of the scratch assay, two-way ANOVA with Tukey\u0026rsquo;s post hoc test was applied. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Evaluation of MGO cytotoxicity\u003c/h2\u003e\u003cp\u003eA variable percentage of viability, ranging from 95% to 130%, was observed after treating NHDFs with MGO at concentrations from 16 \u0026micro;g/mL to 9500 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Only at the concentrations of 10000 \u0026micro;g/mL and 11000 \u0026micro;g/mL, a severe decrease in the viability was observed, obtaining values of ~\u0026thinsp;50%. These values appeared significant compared to CTR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Viability of dermal fibroblasts\u003c/h2\u003e\u003cp\u003eNo significant differences were found in the comparison of the unexposed CTR versus the tested groups. The viability of NHDFs exposed to single treatments of MGO 16 \u0026micro;g/mL and LED showed a cell growth comparable to CTR. NHDFs exposed to CMFs showed a slight enhancement of proliferation, but not statistically significant with respect to controls. Also, the combination of MGO\u0026thinsp;+\u0026thinsp;LED as well as MGO\u0026thinsp;+\u0026thinsp;CMFs showed similar values to CTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Cell density, morphology, and adhesion\u003c/h2\u003e\u003cp\u003eCells exposed to the treatments were very similar to those of untreated CTR in their morphological features, density, and surface adherence. All groups maintained high cell viability, and cell density appeared preserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), together with the structure of nuclei and cytoskeletal filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The number of cells in all treated groups was higher than in the controls, although the data were not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Also, at the SEM, all exposed cells maintained their original shape. Numerous interconnections and filopodia among cells were observed at 1000x (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The combined action of MGO\u0026thinsp;+\u0026thinsp;LED and MGO\u0026thinsp;+\u0026thinsp;CMFs promoted a slight increase in cell adherence to the titanium surfaces, respect single treatments and unexposed controls. The most notable increase in cell number was observed in cells treated with CMFs, particularly in the combined MGO\u0026thinsp;+\u0026thinsp;CMFs. Treated cells displayed different surface areas, indicating varying abilities to spread on the surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). A larger surface area corresponds to greater adhesion. The most marked change was observed in MGO and in MGO\u0026thinsp;+\u0026thinsp;CMFs. N/C ratio decreased in all treated cells, except for those treated with CMFs, whose values were similar to those of the control group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Wound healing potential\u003c/h2\u003e\u003cp\u003eAll treatments significantly induced cell migration and wound reduction over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The graph quantifying wound area (\u0026micro;m\u0026sup2;) over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) showed that at 24 hours, CTR had the smallest wound area (0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02%). All exposed groups showed a statistically significantly larger wound area at 24 hours compared to CTR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the contrary, MGO\u0026thinsp;+\u0026thinsp;CMFs showed a significantly larger area than the single actions of MGO and CMF alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eAt 48 hours, all groups showed a decrease in wound area; however, unexposed controls demonstrated the best wound closure performance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was confirmed at 48 hours for the wound area in the comparison between CTR (0.59\u0026plusmn;0.03%) and the exposed groups. Surprisingly, the LED group was not the worst, as it reduced the wound area to 5.37%. On the contrary, MGO\u0026thinsp;+\u0026thinsp;CMFs was the group that at 48h was characterized by the highest wound area (8.75\u0026plusmn;0.16%), followed by LED, MGO (4.08\u0026plusmn;0.09%), MGO\u0026thinsp;+\u0026thinsp;LED (3.61\u0026plusmn;0.80%), and CMFs (3.26\u0026plusmn;0.09%). Comparing the single and combined actions of the tested technologies, at 48 MGO\u0026thinsp;+\u0026thinsp;LED showed a significantly smaller area compared to LED alone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but no significant difference compared to MGO. Conversely, the combined action of MGO\u0026thinsp;+\u0026thinsp;CMFs showed a larger area compared to CMFs and MGO alone (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eThe rate of cells moving towards the scratched area revealed that untreated cells and cells treated with CMFs, MGO\u0026thinsp;+\u0026thinsp;LED, and MGO\u0026thinsp;+\u0026thinsp;CMFs migrated faster to close the gap of a scratch than MGO and LED-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). CMFs showed a significantly higher rate compared to MGO\u0026thinsp;+\u0026thinsp;LED (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), MGO\u0026thinsp;+\u0026thinsp;CMFs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), MGO (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and LED (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Collagen production\u003c/h2\u003e\u003cp\u003ePicrosirius red staining revealed more intense red deposits after irradiation with LED and MGO\u0026thinsp;+\u0026thinsp;LED compared to other treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The spectrophotometric analysis confirmed the qualitative evaluation with quantitative measurements. In detail, the MGO\u0026thinsp;+\u0026thinsp;LED condition exhibited the highest collagen deposition, with a significant increase in OD values compared to CTR (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), MGO (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), CMFs \u003cem\u003e(p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005), and MGO\u0026thinsp;+\u0026thinsp;CMFs \u003cem\u003e(p\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eLED also showed moderate increases in collagen deposition, and no significant differences were found respect MGO\u0026thinsp;+\u0026thinsp;LED. On the contrary, significant differences \u003cem\u003e(p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were found at the intergroup analysis between LED and CMFs, MGO, MGO\u0026thinsp;+\u0026thinsp;CMFs, and CTR. Listing the groups in descending order of collagen production, the greater values were found for MGO\u0026thinsp;+\u0026thinsp;LED and LED, followed by CMFs, MGO, MGO\u0026thinsp;+\u0026thinsp;CMFs, and CTR. No significant differences were found between CMFs, MGO, MGO\u0026thinsp;+\u0026thinsp;CMFs, and CTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe combined action of MGO\u0026thinsp;+\u0026thinsp;LED, and MGO\u0026thinsp;+\u0026thinsp;CMFs demonstrated significant potential against chronic wounds due to their antimicrobial effects. However, there is a gap in the literature about the potential effects of these combined strategies on NHDFs. Considering the impact of the single treatments, MGO at the dose used by Diban et al [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], 16 \u0026micro;g/mL, showed no cytotoxicity against NHDF. In particular, this molecule has shown no cytotoxicity up to a concentration of 10,000 \u0026micro;g/mL, indicating that it remains safe at or below this dosage. In the literature, the effects of MGO remain somewhat controversial. Several \u003cem\u003ein vitro\u003c/em\u003e studies have demonstrated that MGO, at concentrations between 0.6\u0026ndash;1 mM, can inhibit cell growth by inducing apoptosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Conversely, an \u003cem\u003ein vivo\u003c/em\u003e study found that MGO did not produce harmful effects on the vital organs of various animal models [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, other single treatments, such as CMFs and LED, did not affect the viability of NHDFs. These results were in line with previous literature, which indicates that pulsed electromagnetic fields (PEMF) enhance early wound healing and promote myofibroblast proliferation in diabetic rats [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Vinck et al. reported positive effects of PBM, using various wavelengths (950 nm, 660 nm, and 570 nm), on fibroblast proliferation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Jere et al. recently reported a significant increase in migration and proliferation of normal, wounded, diabetic, and diabetic-wounded WS1 fibroblast cells when irradiated with a 660 nm diode laser at a fluence of 5 J/cm\u0026sup2; [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Surprisingly, the combination of 16 \u0026micro;g/mL MGO\u0026thinsp;+\u0026thinsp;CMFs, and MGO\u0026thinsp;+\u0026thinsp;LED in the study of Diban et al. was characterized by a remarkable antimicrobial activity, in terms of viable cell count, motility, and cellular membrane permeability and fluidity, against \u003cem\u003eCandida albicans\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and did not affect NHDFs' proliferation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The absence of cytotoxicity of this protocol was tested in this study and confirmed through optical, confocal, and scanning electron microscope observations, which showed the typical spindle-shaped morphology and cytoskeleton of normal fibroblasts in both exposed and unexposed cells\u003c/p\u003e\u003cp\u003eThe observed increase in density of the actin filaments in MGO-treated cells suggested a response to a possible stress induced by this molecule. This might reflect cytoskeletal remodeling in response to the glycation stress induced by MGO[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Interestingly, this effect was not accompanied by significant morphological alterations in actin filament organization, although an increase in filament thickness. The combined treatments (MGO\u0026thinsp;+\u0026thinsp;LED and MGO\u0026thinsp;+\u0026thinsp;CMFs) appeared to mitigate MGO-induced effects, maintaining actin architecture and spreading areas similar to control cells. This may suggest a protective or modulatory role of LED or CMFs against MGO-induced cytoskeletal stress, potentially by influencing signaling pathways involved in cytoskeletal dynamics or oxidative stress responses. N/C ratio decreased in treated cells, except for those treated with CMFs alone, which maintained control-like values. A lower N/C ratio typically indicates increased cytoplasmic volume, potentially reflecting enhanced cell spreading or cytoplasmic reorganization[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This finding aligns with the surface area measurements and supports the hypothesis that CMFs may promote or preserve cytoplasmic integrity and cell adhesion capacity under stress conditions. Indeed, in the initial stages of wound healing, preserving cytoskeletal integrity enables fibroblasts to migrate to the wound site and deposit extracellular matrix, thereby reducing treatment-related stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Photobiomodulation seemed to delay wound closure, as evidenced by partial closure at 24 h. There is insufficient evidence of the effects of red LED irradiation on scratch assay closure on NHDFs after 24 hours. Theodoro et al. showed that red LED irradiation at 635 nm, 1.45 J/cm\u0026sup2; of NIH/3T3 fibroblasts had no significant differences compared to untreated controls regarding the proliferation and migration rate during the first 4 days of observation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, after 48 hours, the wound closure in the LED group increased significantly and was nearly complete, confirming that LED irradiation promoted wound closure, even though the migration rate was lower than that of untreated cells. This finding was in agreement with a recent study indicating that the cell migration was unaffected until 12h upon 630 nm LED treatment and started to accelerate after 24h [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. MGO at the concentration used in this study did not seem to affect wound closure, although other concentrations between 7.5 and 10 mM MGO led to enlarged scratch areas in NHDF after 26 hours, indicating a significant decline in cell migration and viability [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The study of the wound area showed that at 24 h, the combined action of MGO\u0026thinsp;+\u0026thinsp;LED produced better results than LED alone, but worse results than MGO alone. Conversely, at 48 h, the combined action showed better results than the single treatments, but with significant results only compared to LED. On the contrary, the combined action of MGO\u0026thinsp;+\u0026thinsp;CMF showed worse results compared to the single treatments, both at 24 and 48 hours.Migration rate, quantified by dividing the change in wound width by the time spent in migration, was highest in CMFs-treated cells. The application of CMFs seemed to accelerate the migration rate of MGO-treated cells. Indeed, the combined action of MGO\u0026thinsp;+\u0026thinsp;CMFs showed a significantly higher migration rate compared to MGO alone, at 24h. The contradictory findings of various studies on MFs suggest that combinations of intensity and treatment period may produce different effects on extracellular matrix synthesis and remodelling, cell proliferation, and migration [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The combination of MGO\u0026thinsp;+\u0026thinsp;LED stimulated a better wound closure than LED and MGO alone, at 24h. At 48 h, no significant differences were found between single and combined action for the migration rate. The remodelling phase of chronic wound healing involves collagen synthesis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the present study, LED-irradiated cells produced significantly more collagen than untreated cells. In particular, MGO\u0026thinsp;+\u0026thinsp;LED improved the effects of MGO in the synthesis of collagen by fibroblasts. On the contrary, the combination of MGO\u0026thinsp;+\u0026thinsp;CMFs did not increase the collagen deposition, with respect to CTR, MGO, and CMFs alone. Literature describes the beneficial effects of light therapy on collagen production. A histology study reported differences in collagen content in an animal model. It showed that collagen fibers were more organized in diabetic and non-diabetic rats after low-level laser therapy (904nm) compared to untreated ones [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The effects of LED on both migration and collagen synthesis are highly dependent on wavelength, fluence, and treatment protocol, and the effect on fibroblasts can persist up to 21 days after irradiation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, LED irradiation seems to decrease wound closure and migration rate with respect to controls at 24 h, but at 48 h, the same cells increased their values, reducing the differences with other groups. Moreover, at 7 days, the collagen production was higher in the LED group, confirming that this treatment stimulated cell activity and the effects persisted for more days after irradiation. In this study, each combined treatment exerted beneficial effects on cells. MGO at low concentrations was not cytotoxic for dermal fibroblasts; for this reason and due to its antibacterial properties, it might be used as MGO-based dressings to treat many types of wounds, including chronic wounds. The eco-friendly technologies CMFs seemed to be a promoter of the proliferation and cell migration that characterize the initial phase of the healing process. In contrast, LED appeared to stimulate the deposition of collagen type I, which is crucial in the late stage of the healing process. Dang et al. observed that a wavelength of 800 nm and a fluence of 40 J/cm\u003csup\u003e2\u003c/sup\u003e increased skin collagen synthesis via the Smad pathway [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. At a wavelength of 670 nm, Otterco et al. observed an improved wound healing process on wounded rats compared to the non-irradiated control group. The authors noted a significant decrease in the inflammatory cytokine TNF-α, and an increase in collagen type I [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Fibroblasts are responsible for generating the majority of the extracellular matrix (ECM) during tissue repair, which is crucial for tissue remodeling and the complete closure of wounds. Collagen, a key element of the ECM, is produced and regulated through a balance between its synthesis and degradation by enzymes like matrix metalloproteinases (MMPs). The findings of this study indicate that combining technologies such as CMFs and PBM with molecules like MGO yields greater benefits than using either treatment individually. A possible explanation might be that LED, and CMFs can affect drug transport and cellular receptor sensitivity. It has been reported that MFs can influence ion channels and signaling pathways, thereby altering cell responses to bioactive molecules [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. LED light can increase cell membrane permeability, microcirculation, and cellular metabolism, which can enhance penetration and cellular uptake of molecules [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Further research, especially \u003cem\u003ein vivo\u003c/em\u003e studies to validate their clinical relevance, is needed. In the future, the combination of PBM and CMFs may not only be feasible but could also represent a promising and innovative direction in biomedical research. Both PBM and CMFs therapies are highly adaptable and well-suited to personalized medicine, offering the potential for individualized treatment strategies. By adjusting key parameters, such as wavelength, fluence, field strength, and duration, these therapies can be tailored to the specific needs and biological responses of each patient. With further research and clinical validation, the integration of PBM and CMFs could become a powerful and non-invasive approach to precision therapy.\u003c/p\u003e"},{"header":"5. CONCLUSIONS","content":"\u003cp\u003eThis study showed that all devices tested, at the parameters described, were not toxic and are safe for dermal fibroblasts, but produce beneficial effects on the cellular activity of dermal fibroblasts, which are mainly responsible for skin wound healing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eDeclaration\u003c/p\u003e\u003cp\u003eFunded by European Union-Next Generation EU, program \u0026ldquo;MUR-Fondo Promozione e Sviluppo-DM737/2021, SCIAMI\u0026rdquo;, \u0026ldquo;Eco-friendly antimicrobial Strategies to fight Chronic-wound Infections Associated with Multidrug resistant pathogens for the development of Innovative medical systems\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributionsConceptualisation S.D., M.P., L.D., G.I.; Data curation: E.D., M.P; Investigation: E.D., T.V.P., M.P., S.D.; Methodology: S.D., M.P., L.D., G.I.; Project administration: S.D., M.P., L.D., P. D.F.; Resources: S.D., L.D.; P.D.F.; Supervision: S.D., M.P., L.D., G.I.; Validation: S.D., M.P., L.D., G.I.; Visualisation: S.D., M.P., L.D. G.I.; Writing \u0026ndash; original draft: E.D.; T.V.P.; M.P.; Writing \u0026ndash; review and editing: S.D., G.I. P.D.F, L.D. ORCIDEmira D\u0026rsquo;Amico https://orcid.org/0000-0003-2414-7130Tania Vanessa Pierfelice https://orcid.org/0000-0002-5886-2186Loredana D\u0026rsquo;Ercole https://orcid.org/0000-0002-4466-3987Paola Di Fermo https://orcid.org/0000-0001-6025-1133Giovanna Iezzi https://orcid.org/0000-0002-2391-6594Simonetta D\u0026rsquo;Ercole https://orcid.org/0000-0002-4797-4019Morena Petrini https://orcid.org/0000-0002-3849-4304DECLARATIONSEthical Approval Not applicable.Competing interests: The authors declare no conflicts of interest concerning this project. The funders were not involved in the study design; in the collection, analysis, and interpretation of data; in the writing of this article; or in the decision to submit it for publication.Clinical trial number Not applicable.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgmentsThe authors would like to thank Dr. Stefania Lepore for her assistance with imaging editing. The authors would like to express their gratitude to Marco Mantarro and M.F.I. Medicina Fisica Integrata, Italy, for providing the free use of the CMFs device, and to Alphastrumenti for the free use of the red light device.Data availability statementThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript, but if other data are necessary, you can contact the corresponding authors\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFrykberg RG, Banks J (2015) Challenges in the Treatment of Chronic Wounds. 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Front Photonics 3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphot.2022.1018773\u003c/span\u003e\u003cspan address=\"10.3389/fphot.2022.1018773\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"lasers-in-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lims","sideBox":"Learn more about [Lasers in Medical Science](https://link.springer.com/journal/10103)","snPcode":"10103","submissionUrl":"https://submission.springernature.com/new-submission/10103/3","title":"Lasers in Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"photobiomodulation, magnetic fields, MGO, regeneration, derma","lastPublishedDoi":"10.21203/rs.3.rs-7599081/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7599081/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eThis paper aimed to verify how a new protocol, recently proposed for treating chronic wounds due to its excellent antimicrobial properties, affects human dermal fibroblasts (NHDFs).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSingle and combined action of light-emitting diodes (LED), complex magnetic fields (CMFs), and methylglyoxal (MGO) on cell viability and activity of dermal fibroblasts (NHDFs) were investigated. Our first objective was to exclude any toxicity of this combined treatment on these cells. NHDFs were exposed to LED light for 17 min, CMFs for 22 min, MGO, MGO\u0026thinsp;+\u0026thinsp;LED, and MGO\u0026thinsp;+\u0026thinsp;CMFs, and then were assessed for cell viability, morphology, cytoskeletal integrity, collagen type I production, and migration capacity. Results of combined treatments were compared with those of single treatments and unexposed controls.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eNHDFs exposed to both single and combined treatments maintained viability, morphology, and cytoskeletal integrity, showing no signs of cytotoxicity. MGO at low concentrations was non-toxic and, combined with other technologies, was able to confer beneficial effects on cell adhesion. LED stimulated collagen type I synthesis, and the production increased in samples subjected to the combined action of MGO\u0026thinsp;+\u0026thinsp;LED. CMFs notably accelerated fibroblasts' migration in scratch assays, and when combined with MGO, they further enhanced this effect.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe combined use of MGO\u0026thinsp;+\u0026thinsp;LED and MGO\u0026thinsp;+\u0026thinsp;CMFs produced more significant effects than separate treatments, probably because magnetic fields and light therapy enhanced cellular uptake and receptor sensitivity. The tested protocols were not only non-toxic but also promoted beneficial effects on the vitality and activity of dermal fibroblasts, confirming their potential in treating chronic wounds.\u003c/p\u003e","manuscriptTitle":"The Synergistic Effect of Photobiomodulation, Methylglyoxal, and Complex Magnetic Fields on Human Dermal Fibroblasts: Potential Applications for Chronic Wound Treatments.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 14:23:06","doi":"10.21203/rs.3.rs-7599081/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-03T20:23:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T14:02:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T16:37:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T18:21:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118818296460171227009375267201517639038","date":"2025-10-14T07:07:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333468958323509187111068325686981635674","date":"2025-10-13T21:11:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124329209154351015191834657709133816065","date":"2025-10-02T15:06:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T13:26:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"471520712371502848954639432771888518","date":"2025-09-30T06:45:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105919563396035614538670663484096128167","date":"2025-09-30T06:41:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-29T18:26:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-29T18:01:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-18T16:02:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Lasers in Medical Science","date":"2025-09-12T09:02:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"lasers-in-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lims","sideBox":"Learn more about [Lasers in Medical Science](https://link.springer.com/journal/10103)","snPcode":"10103","submissionUrl":"https://submission.springernature.com/new-submission/10103/3","title":"Lasers in Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1191233f-ac58-4c2e-9cb6-459392286164","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:03:53+00:00","versionOfRecord":{"articleIdentity":"rs-7599081","link":"https://doi.org/10.1007/s10103-025-04775-3","journal":{"identity":"lasers-in-medical-science","isVorOnly":false,"title":"Lasers in Medical Science"},"publishedOn":"2025-12-11 15:58:38","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-10-15 14:23:06","video":"","vorDoi":"10.1007/s10103-025-04775-3","vorDoiUrl":"https://doi.org/10.1007/s10103-025-04775-3","workflowStages":[]},"version":"v1","identity":"rs-7599081","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7599081","identity":"rs-7599081","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}