Treatment with a red‐laser‐based wound therapy device exerts positive effects in models of delayed keratinocyte and fibroblast wound healing

Light therapy is widely used in medicine. Specifically, photobiomodulation has been shown to exert beneficial effects in wound healing disorders, which present a major challenge in health care. The study's aim was providing information on the effect of a novel, red‐laser‐based wound therapy device (WTD) on keratinocytes and fibroblasts during wound healing under optimal and non‐optimal conditions.


| INTRODUC TI ON
Wound healing disorders present a major challenge in hospitals and health care settings.Inflammation plays a central role in wound healing, as inflammatory signals recruit neutrophils, macrophages and other immune cells towards the injured area, where their task is to kill invading microorganisms, remove destroyed tissue and dead cells as well as orchestrate the following repair processes. 1Recently, research has focused on the participation of the adaptive immune system in physiological wound healing, e.g.4][5] However, persistent inflammation is associated with the formation of chronic wounds, where the abundant activation of neutrophils, macrophages and other immune cells leads to the incessant release of cytokines and proteases that fuel degradation and inhibit repair processes. 1,6In accordance, continuous inflammatory TH1 conditions can delay wound closure 7 by preventing the transition from degrading to proliferative processes. 8[11] Photobiomodulation has been shown to exert beneficial effects on cell layers, matrix deposition [12][13][14][15] and wound healing, [16][17][18][19][20] while being well tolerated by cells and tissues.Particularly red light was found to promote migration of human epidermal stem cells 21 and fibroblasts isolated from diabetic patients 22 as well as increase keratinocyte proliferation. 23In accordance, light therapy is already widely used in medicine since decades while still being a discipline with great potential for innovations.However, novel light devices need new testing approaches to ensure safety and efficacy of the treatment in wound healing.One of the most studied in vitro models for wound healing is the mechanical damage to confluent cell layers ('scratch wound assay'). 24This method enables the direct measurement of cell migration and regeneration of the cell layer.Still, differences between the in vivo and the experimental in vitro conditions are generally not accounted for as the experimental prerequisites are mostly based on optimal cell conditions.Recently, we proposed the readjustment of specific cell milieus in this in vitro wound model, more closely mimicking different detrimental in vivo situations, explicitly chronic TH1 inflammation as well as TH2-dominant conditions.Scratch wound healing progression under all non-optimal environments was found to be significantly reduced. 25We now used the model to investigate the cellular effects of a novel and unique wound therapy device (WTD) in order to determine a possible mechanism for the treatment effects described in vivo.The WTD emits a coherent light beam of 650 nm, which is thought to stimulate cellular regulation processes in the recipient cells and tissues. 26It is an innovative and unique handheld device that could very likely be used to empower patients to use it themselves in a homecare setting.This is the very first study with the WTD to look into its stimulatory capacity for wound healing and investigate the effects on cell biology.The study's aim is to provide information on the cellular effects of WTD on keratinocytes and fibroblasts during wound healing on a molecular level.Fibroblasts are involved in the production of extracellular matrix and are therefore essential to fill the wound with new tissue. 278][29][30] HaCaT cells, primary epidermal keratinocytes and primary dermal fibroblasts have already been successfully employed in the scratch wound assay 31 under different wound milieus. 25Furthermore, gene expression analysis for pro-inflammatory cytokines (IL1A, IL6, CXCL8), growth factors (TGFB1, PDGFC), transcription factors (NFKB1, TP53) and heat shock proteins (HSP90AA1, HSPA1A, HSPD1) as well as desmogleins (DSG1, DSG3) in keratinocytes and collagen (COL1A1, COL3A1) in fibroblasts was performed to evaluate cellular responses to healing progression under optimal conditions and after priming with TNF-α/IFN-γ (chronic inflammatory TH1 milieu) as well as IL-4/IL-13 (TH2-dominant conditions).

| Cell compatibility tests
The WTD (Beosigner®, Vitarights Inovations GmbH) works with a laser wavelength of 650 nm, an operating current of 3.0 V DC and an output power of 1.0 mW.The operating mode continues, and the light beam is coherent with a dot shape and a beam diameter of 2.5 mm.This corresponds to an irradiance of 20 mW/cm 2 .After 48 h, medium was exchanged for fresh medium, and cells were treated for 2, 5 and 10 min (P1: continues treatment) as well as for 10 h (P2: 10 min treatment, 50 min break on repeat) with the WTD at an operating distance of 1 cm (Figure 1).This would resemble the following radiant exposures: 2.4, 6.1 and 12.2 J/cm 2 as well as 122 J/cm 2 for the repeated treatment.Both HaCaT cells and fibroblasts were cultivated after the treatments for 24 h.Afterwards, cell viability and LDH release were assessed as previously reported, 31 and DNA double-strand breaks were measured. 32Cells cultured in medium w/o treatment were used as negative controls.Triton X-100 was used as a positive control for a cytotoxic effect.Furthermore, hydrogen peroxide (H 2 O 2 ) was used as a positive control in the comet assay.Cell viability was determined on the basis of the luminometric ATP measurement using the ATPlite™-M Assay (PerkinElmer LAS) according to the manufacturer's instructions.The assay is based on the production of light that is created at the reaction of ATP with Luciferase and D-Luciferin.The emitted light is directly proportional to the ATP concentration.In brief, 1000 μL of the cell lysis solution were placed in each well, and plates were shaken for 5 min at 700 rpm.Subsequently, 150 μL were transferred into white 96-well-microtiter plates, and 50 μL substrate solution (Luciferin/ Luciferase) was added to each well.Plates were shaken again for 5 min at 700 rpm and incubated in the dark for another 10 min before the luminescence was measured using the LUMIstar® Omega (BMG Labtech).Cellular ATP concentrations were determined on the basis of an ATP standard curve, and cell viability in [%] was calculated in relation to the untreated control.The cytotoxic effect was determined by measuring the LDH release by the Cytotoxicity Detection Kit (Roche Diagnostics AG) according to the manufacturer's instructions.In short, 100 μL of the cell culture supernatant was transferred to a 96-well-microtiter-plate, and 100 μL of the tetrazoline dye solution was added per well.This was followed by an incubation step for 30 min at room temperature in the dark.Subsequently, the absorption at 490 nm was measured using the SPECTROstar® Omega (BMG Labtech).The cytotoxicity was determined as a multiple of the LDH release of the negative control.A possible genotoxic effect was assessed using the comet assay.The comet assay is based on the combination of DNA gel electrophoresis with fluorescence microscopy to visualize the migration of DNA strands from individual cells embedded in agarose. 32For this purpose, the cells were detached after treatment and incubation using trypsin-EDTA (Gibco Thermo Fisher Scientific) and taken up in cold phosphate-buffered saline (PBS; BioConcept).The cell suspension was then mixed with a 1% agar solution (Carl Roth GmbH) and applied to precoated slides (VWR International GmbH).The cells were then subjected to alkaline lysis to disrupt cell and nuclear membranes before electrophoresis (20 min, 15 V) was carried out.The DNA was then stained using propidium iodide (2.5 μg/mL; Merck KGaA).The ratio of cells with undamaged and damaged DNA was evaluated on the Scope A.1 fluorescence microscope using the AxioCam MRc (Carl Zeiss).

| Scratch wound assay
Before treatment with the WTD, cell monolayers were scratched with a sterile pipette tip and washed with medium to remove any loose cells.Afterwards, medium was renewed, and cells were treated for 5, 10 and 20 min (P1: continues treatment) with the WTD at an operating distance of 1 cm (Figure 1).This would resemble the following radiant exposures: 6.1, 12.2 and 24.4 J/cm 2 .Cells were incubated for 1, 4, 8, 24 and 48 h before staining with hematoxylin and eosin (Merck Millipore) for evaluation of scratch closure progression or gene expression analysis.Microscopic assessment of the scratches was carried out using the VHX 950F digital microscope (KEYENCE DEUTSCHLAND GmbH).The scratch area was determined in mm 2 using the VHX 950F software (KEYENCE DEUTSCHLAND GmbH).
From these results, scratch wound healing progression was calculated in [%] for each time point relative to the scratch area at 1 h.
To test the scratch wound healing under non-optimal conditions as previously reported, 25 cell culture media were replaced by primed media 24 h before the experiment.TH1 conditions were mimicked by addition of 10 ng/mL TNF-α (7Bioscience) and 5 ng/mL IFN-γ (7Bioscience), whereas TH2 conditions were simulated using 50 ng/ mL IL-4 (7Bioscience) and 50 ng/mL IL-13 (7Bioscience).

| Gene expression analysis
The mRNA isolation, cDNA synthesis and RT-qPCR were performed as previously reported. 25Concisely, cells were lysed by addition of RLT buffer (Qiagen) with 10 μL/mL β-mercaptoethanol for 3 min on ice and 3 min under shaking.Then lysates were loaded to QIA Shredder spin columns (Qiagen) and centrifuged at 4°C at 10.000 × g for 2 min.RNA purification was performed using the RNeasy® Mini Kit and the QIAcube (Qiagen) according to the manufacturer's instructions.Genomic DNA was removed using the Ribonuclease assay DNase I, RNase Free (Thermo Fisher Scientific).Finally, RNA concentration was determined using the SPECTROstar® Omega with an UV/Vis plate (BMG Labtech) by measuring the OD at 260 nm, with the assumption that OD of 1.0 equals 40 ng/mL RNA.The purity of the RNA sample was evaluated using the ratio of the absorbance at 260 and 280 nm with a ratio's threshold between 1.9 and 2.1.
All samples fulfilled this requirement.Absolute RNA amounts were F I G U R E 1 Schematic presentation of the treatment of the cell layers with the WTD (wound therapy device).The WTD works with a laser wavelength of 650 nm, an operating current of 3.0 V DC and an output power of 1.0 mW.The operating mode was continues and the light beam is coherent with a dot shape and a beam diameter of 2.5 mm.Cells were treated up to 10 h with the WTD at an operating distance of 1 cm.(Image was created with BioRe nder.com).
adjusted to 60-400 ng in 10 μL and the High-Capacity cDNA Reverse Transcription Kit by Applied Biosystems (Thermo Fisher Scientific) was used for reverse transcription.The PCR protocol (primer annealing: 10 min 25°C, reverse transcription: 120 min 37°C, termination: 5 m in 85°C) was run on the Mastercycler® gradient thermal cycler (Eppendorf).cDNA samples were stored at −80°C until further use.
RT-qPCR was performed for gene expression analyses using the QuantiNova™ SYBR Green PCR Kit (Qiagen).Briefly, master mix prepared on ice contained forward and reversed primer (test concentration each 0.5 μM) and cDNA (5 ng/mL) or Yellow Template Dilution Buffer as no template control.Using the qTOWER3G (Analytik Jena AG), the real-time amplification protocol was set for polymerase heat activation at 95°C for 3 min and 40 cycles with 3 steps: denaturation at 95°C for 5 s, annealing at 57°C for 10 s and elongation at 72°C for 10 s.Signals were detected at λex/λem 470 nm/520 nm.Finally, a melting curve from 65°C to 95°C served as amplicon control.All primer sequences or ordering IDs are listed in Table 1.Samples were measured in technical duplicates.Expression levels of target genes were normalized to the housekeeping gene ACTB.

| Statistical analysis
Two independent experiments were executed, and measurements were performed in duplicate.Gene expression levels are presented as log 2 change with a log 2 (2) = 1 implying a twofold upregulation and log 2 (0.5) = −1 indicating a downregulation by a factor of 2 compared to the untreated controls as indicated.All values are expressed as means ± SD (standard deviation).One-way analysis of variance was carried out to determine statistical significances (Microsoft® Excel 2010) as previously reported. 25Differences were considered statistically significant at a level of p < .05.

| Treatment with WTD is biocompatible
Treatments that can affect cells and tissues must be safe and biocompatible.The DIN standard EN ISO 10993-5 regulates the testing for in vitro cytotoxicity, which is often a qualitative analysis based on the study of cell damage and cell growth after treatment.In principle, cells should be used for testing the in vitro toxicity that correspond to the cells and tissues of a later application. 33,34In accordance, human keratinocytes (HaCaT cells) and human dermal fibroblasts were used to investigate the compatibility of the WTD treatment in vitro.The cell viability after 24 h was determined by luminometric determination of the cellular ATP content.Treatment with WTD for up to 10 h had no effect on the viability of keratinocytes (Figure 2A) or fibroblasts (Figure 2D) in contrast to the cytotoxicity control (Triton X-100), where a significant reduction in cell viability was observed.Biocompatibility can further be determined by other cellular TA B L E 1 List of primer sequences and order IDs for performing SYBR Green-based RT-qPCR.and molecular factors.The release of lactate dehydrogenase (LDH) is a sign of cell necrosis, where damage to the cell membrane releases the enzyme from the cytosol of the cells. 33,34The release of LDH was determined after 24 h by photometric determination of the enzyme concentration in the cell culture supernatant.Treatment with WTD for up to 10 h had no effect on LDH release by keratinocytes (Figure 2B) or fibroblasts (Figure 2E).The cytotoxicity control (Triton X-100) in comparison led to a significant increase in LDH.The comet assay (or single-cell gel electrophoresis) is a simple method for measuring DNA strand breaks in eukaryotic cells.It has applications in genotoxicity testing of new substances/treatments, monitoring of environmental genotoxin contamination and biomonitoring, as well as for basic DNA research on damage and repair mechanisms. 32The assay has the status of a standard test for evaluating the safety of drugs or other chemicals. 35If the negatively charged DNA contains double-strand breaks, the twisted DNA scaffold relaxes and the ends created by the strand breaks can migrate to the anode during a short electrophoresis, creating the shape of a comet microscopically.If the DNA is undamaged, the lack of free ends and the large size of the fragments prevent migration.24 h after the treatment of keratinocytes and fibroblasts with WTD (Figure 2C,F), a similar proportion of comet-positive cells was found compared to the medium control, while the positive control (hydrogen peroxide) showed clear DNA-toxic effects.

| WTD treatment supports scratch wound closure under non-optimal conditions
The effect of WTD treatment on wound healing of human epidermal keratinocytes (HaCaT cells) as an epithelial model and dermal fibroblasts as a dermal wound model in vitro was investigated.Three cell environments were assessed in the study: a physiological milieu, a TH1 milieu mimicking a chronic inflammatory response, and a TH2 milieu typically seen in patients with an atopic predisposition.
For the investigations, the cells were treated with WTD for 5, 10

| WTD treatment alleviates the inflammatory response in keratinocytes and induces early growth factor gene expression in fibroblasts under physiological conditions
During wound healing, there was initially a short-term increase in pro-inflammatory cytokine gene expression (IL1A, IL6 and CXCL8) as a driver of the inflammation (Figure S2).These transcript lev- genes (S100A7, RNASE7, DEFB1) were also increased.While treatment with WTD showed no significant effects on the macroscopic healing process in the epithelial wound model (HaCaT keratinocytes) under physiological conditions, the gene expression analysis revealed a significant inhibitory effect on the early gene expression of IL1A (Figure 5A,B).Moreover, it was observed that the resolution of CXCL8 and IL6 transcription was accelerated, showing significantly higher levels at 1 h (p < .05, Figure 5A) and a significant decrease at 4 h (p < .01)compared to the untreated HaCaT keratinocyte scratches (Figure 5B).In contrast, WTD augmented CXCL8 and IL6 transcript levels in fibroblasts while no distinct effect on IL1A gene expression was observed compared to the untreated fibroblast scratches under physiological conditions (Figure S4A).Treatment with WTD did not affect the expression profile of TGFB1, HSPD1 and HSPA1A or DSG1 and DSG3 in keratinocytes (Figure S3A).However, corresponding to the reduced transcript levels of CXCL8 and IL6, a reduced gene expression of the transcription factors TP53 and NFKB1 was observed (Figure S3A).WTD treatment led to a significant increase of TGFB1 (p < .05),PDGFC (p < .05)and VEGF (p < .05)gene expression in fibroblasts at 1 h as well as HSPD1 transcript levels (p < .05) at 4 h (Figure 5D-G).It was also found that WTD induced early expression of NFKB1 and increased TP53 transcript levels, especially after 20 min treatment (Figure S4A).In comparison, WTD lowered gene expression of MMP1 (Figure S4A).Moreover, a predominantly positive effect on the expression of AMP genes (DEFB1, RNASE7 and S100A7) by the keratinocytes was found (Figure 5C).

| WTD treatment supports wound healing under chronic inflammatory conditions in vitro by enhancing desmoglein and collagen gene expression as well as inducing early growth factor gene expression
Under TH1 conditions of chronic inflammation in keratinocytes and fibroblasts, the gene expression of the mediators involved in the inflammation was augmented and decreased significantly more slowly during healing progression while genes of the structural proteins were expressed in distinctly lesser amounts or later (Figure S2).WTD treatment was able to achieve significant, positive effects on healing augmenting effect on TGFB1 gene expression by keratinocytes was found (Figure 6C), while transcript levels of IL1A were significantly reduced right after BDT treatment (Figure 6D).In fibroblasts under chronic inflammatory conditions, a short BDT treatment significantly induced the expression of the growth factor genes PDGFC already after 1 h (p < .01; Figure 6G) and VEGFA with a maximum at 24 h (p < .001; Figure 6H).CXCL8 and IL6 gene expression was noted to be induced early and found to be enhanced compared to the untreated HaCaT keratinocytes scratches, which also led to an increased gene expression of the transcription factors TP53 and NFKB1 (Figure S3B).
IL6 transcript levels were further heightened in dermal fibroblasts after WTD treatment, while CXCL8 and IL1A gene expression was decreased (Figure S4B).BDT did not affect gene expression profiles of the heat shock proteins HSPA1A and HSPD1 compared to the control in keratinocyte (Figure S3B) or fibroblast scratches (Figure S4B) during wound healing.It had further had only slight effects on the different AMP gene transcript levels (Figure S3B) and MMP1 gene expression (Figure S4B) under these conditions.

| WTD increases HSPD1 transcript levels in keratinocytes and augments collagen expression in fibroblasts during wound healing under TH2 conditions in vitro
Under TH2 conditions, the acute inflammatory reaction was distinctly reduced as well as delayed after mechanical wounding of the cell layers.Likewise, the expression of important mediators was inhibited in keratinocytes and fibroblast (Figure S2).WTD treatment further selectively reduced inflammatory cytokine gene expression at early time points in keratinocytes (Figure 7A) and later in fibroblasts (Figure 7D).
Correspondingly, NFKB1 and TP53 transcript levels were either not F I G U R E 5 Gene expression analysis during keratinocyte (left panel) and fibroblast (right panel) scratch wound healing under physiological conditions after treatment with WTD.Log 2 change of mRNA expression of pro-inflammatory cytokine genes CXCL8, IL6 and IL1A at 1 h (A) and 4 h (B) as well as evaluation of antimicrobial peptide transcripts S100A7, RNASE7, DEFB1 (C) at 48 h in keratinocytes determined by real-time qPCR.Furthermore, log 2 change of mRNA expression of growth factor genes TGFB1 (D), PDGFC (E) and VEGFA (F) at 1 h as well as HSPD1 transcript levels (G) at 4 h in fibroblasts showed significant differences as determined by real-time qPCR.Values were normalized to 1-h results for the untreated control under physiological conditions.affected or slightly decreased in keratinocytes (Figure S3C) as well as in fibroblasts (Figure S4C).Furthermore, negative effects on TGFB1 gene expression at 8 h in keratinocytes were found (Figure 7B).Fibroblasts also featured significantly decreased transcript levels of TGFB1 (p < .001),PDGFC (p < .01)and VEGFA (p < .001)most notably at 8 h after 20 min BDT treatment (Figure 7E).However, a significant increase in HSPD1 gene expression was observed in keratinocytes compared to the untreated controls under TH2 conditions (Figure 7C).Furthermore, slight effects on AMP gene expression by keratinocytes were noted (Figure S3B).Despite the lack of induction of growth factor gene expression, a significant increase of COL1A1 and COL3A1 transcript levels (p < .05)was found for WTD treated fibroblasts compared to the untreated control (Figure 7F).

| DISCUSS ION
Light can have a positive influence or exert negative effects on living cells and tissues depending on wavelength, coherence and dose or intensity.3][14][15] For instance, significant induction of wound healing processes in acute and chronic wounds by wIRA treatment has been reported. 19,20It could recently be shown that wIRA radiation improved scratch healing under nonoptimal conditions in vitro. 25In contrast, UV-B irradiation under the same conditions resulted in severe damage to keratinocytes as well as fibroblasts and wound healing was impeded. 25Other light therapy devices might employ red light lasers (600-760 nm) at low intensity, e.g., 'low level laser light therapy' (LLLT), or light emitting diodes in the range of violet to green (380-495 nm), e.g., 'blue light phototherapy' and 'photodynamic therapy" (PDT).Recently, positive effects of a novel, red-laser-based wound therapy device (WTD) on stimulation of cell regeneration and wound healing of connective tissue cells were reported. 26The WTD emits a coherent light beam of 650 nm, which is thought to stimulate the recipient cells and tissues. 26In the present study, the cellular effects of the WTD were examined in order to determine the possible mechanism for the treatment effects previously observed.

F I G U R E 6
Gene expression analysis during keratinocyte (left panel) and fibroblast (right panel) scratch wound healing under TH1 conditions after treatment with WTD.Log 2 change of mRNA expression of desmoglein genes DSG1 (A) and DSG3 (B) at 24 h and growth factor TGFB1 at 8 h (C) as well as evaluation of pro-inflammatory cytokine IL1A transcripts at 1 h (D) in keratinocytes determined by real-time qPCR.In addition, Log 2 change of mRNA expression of collagen genes COL1A1 at 48 h (E) and COL3A1 at 24 h (F) and growth factors PDGFC at 1 h (G) and VEGFA at 24 h (H) in fibroblasts was determined by real-time qPCR.Values were normalized to 1-hour results for the untreated control under TH1 conditions.Although WTD showed no distinct stimulatory effects on healing in the wound models under physiological conditions, early induction of CXCL8 and IL6 gene expression accompanied by accelerated resolution was observed, while IL1A transcript levels were overall reduced compared to the control.Keratinocytes and fibroblasts secrete a broad spectrum of cytokines and chemokines and growth factors including IL-1α, IL-6 and IL-8 during wound healing. 28As the skin's 'first alert system', the IL-1 family of cytokines plays a critical role in alerting the body to imminent dangers and initiating the inflammatory cascade in the skin, as well as inducing gene expression and synthesis of other inflammatory mediators. 36,37Yet, the persistence of inflammation is detrimental and can cause cell damage, thereby blocking the healing process. 8 accordance, chronic inflammatory conditions induced in vitro by priming with TNF-α and IFN-γ reduced scratch healing compared to controls under optimal conditions, mainly by delaying cell layer regeneration. 25,380][41] However, the initial inflammatory response after injury is essential for healing onset and exciting regenerative processes; 42 therefore, promoting acute inflammation can enhance wound healing. 42,43WTD treatment exhibited the potential to ignite an early IL6 gene expression response while decreasing late pro-inflammatory cytokine gene expression together with IL1A transcript reduction under physiological as well as chronic inflammatory conditions.Similarly, low-level laser therapy 12 and cold atmospheric plasma 44 were able to induce IL-8 secretion and improve wound healing in vitro.In contrast, pro-inflammatory cytokine gene expression levels were even further reduced by WTD treatment under TH2 conditions despite the positive effect on wound healing progression.Hence, a reinitiation of the inflammatory response by induction of IL1A gene expression after IL-4/IL-13-dependent diminution, as it was previously observed for wIRA treatment, 25 cannot account for the beneficial influence of WTD in a TH2 milieu.IL-4 and IL-13 are pleiotropic cytokines involved in cell growth, immune system regulation, induction of anti-inflammatory processes and M2 macrophage promotion. 9Hence, they are believed to contribute to the wound healing process by stimulating the repair process. 11However, they have also been associated with the pathogenesis of fibroproliferative diseases such as hypertrophic scarring or systemic sclerosis. 9,45Furthermore,  impaired wound healing in keratinocytes treated with IL-4 10 or keratinocytes and fibroblasts stimulated with IL-4 and IL-13 25 has been demonstrated, which is consistent with lower wound healing rates found in atopic children. 46sitive effects of photobiomodulation on wound healing have been described, [47][48][49] which most likely can be explained by enhanced fibroblast migration and proliferation as well as augmented collagen synthesis. 50,514][55] Distinct effects on timing and release of growth factors and cytokines for example by LLLT have been noted. 56Especially bFGF production was found to be increased after the use of infrared lasers. 57,58It was further reported that LLLT increases angiogenesis and neovascularization 55 most likely by affecting VEGF, MMP-2 and HIF-1 secretion 59 and bFGF, VEGFA, SDF-1α gene expression. 47In accordance, an induction of early growth factor gene expression was observed after BDT treatment under TH1 conditions.The application of WTD had a time-dependent potentiating effect on the gene expression of TGFB1 by keratinocytes while shorter WTD treatments increased PDGFC as well as VEGFA transcript levels in fibroblasts significantly.TGF-β plays an important role in wound healing by stimulating granulation tissue formation [60][61][62][63] and fibroblast proliferation, mediating collagen production, ECM deposition and myofibroblast differentiation [64][65][66] as well as promoting angiogenesis. 67Therefore, increased TGF-β activity can accelerate wound healing 68,69 and positively influence the production of dermal-epidermal junction proteins. 70Induction of TGFB1 has also been implicated in promoting keratinocyte proliferation and migration. 71GF-C is a key component of the PDGFR-α signalling pathway and has a similar capacity to modulate fibroblast differentiation as TGF-β.72 However, overexpression can also contribute to abundant collagen accumulation, tissue fibrosis and pathological scarring.9,73,74 VEGF-A is one of the key regulators of angiogenesis, controlling formation of capillary structures, proliferation of endothelial cells and differentiation of progenitor cells.75,76 Increases of VEGFA gene expression by photobiomodulation therapy have been associated with acceleration of wound healing in a mouse model.47 The decreased TGFB1, PDGFC and VEGFA levels observed under TH2 conditions in keratinocytes and fibroblasts, probably accounting for the reduced scratch healing found here, could not be restored by WTD treatment.Therefore, the mechanism by which WTD optimizes wound healing in a TH2-dominant milieu differs from wIRA radiation, for which a significant increase in TGFB1 and PDGFC transcript levels of keratinocytes and fibroblasts was found after treatment.25 Interestingly, LLLT treatment of endothelial cells also resulted in a reduction of VEGF-A secretion, while simultaneously stimulating the proliferation of the HUVEC cells in vitro. 54Here it was shown that WTD induced an increase in HSPD1 transcript levels in keratinocytes during wound healing under TH2 conditions, which could confer the activating effect.HSPD1 is a chaperonin found in the cytosol and mitochondrial matrix 77 that has been implicated in wound healing.78 HSPA1 is another cytoprotective protein that plays a crucial role in guiding conformational status during protein folding and translocation.[82] HSPD1 and HSPA1A transcription have further been found to be significantly augmented by wIRA radiation in keratinocytes under TH1 conditions but not in the TH2 milieu.25 Regeneration of the keratinocyte and fibroblast layers and wound healing progression could be assessed by gene expression analysis of typical structural proteins in epidermis and dermis.DSG1 and DSG3 gene expression by keratinocytes increased over time during scratch wound healing, which is consistent with the regeneration of the epithelial layer and the reformation of cell-cell contacts.83 Transcript levels of the cell adhesion molecule genes DSG1 and DSG3 were found to be increased after WTD treatment under non-optimal conditions corresponding to the increased healing progression over time.Moreover, WTD treatment time-dependently affected expression of AMP genes in keratinocytes, which could indicate a positive effect on the skin's natural defenses.Previous in vivo studies have shown that AMPs are endogenously upregulated in all stages of wound healing suggesting a function outside just microbial defense and in the direction of immune response regulation, granulation tissue formation and re-epithelization.84,85 It was further observed that antimicrobial peptide transcript levels can significantly be elevated by wIRA radiation in vitro, 25 and LLLT was found to stimulate HBD-2 expression in an experimental setting of periodontal diseases.86 Regeneration of the dermal cell layers was evaluated by collagen gene transcription in fibroblasts.COL1A1 and COL3A1 levels were found to be significantly increased over time under TH1 and TH2 conditions corresponding to the improved reformation of the fibroblast cell layer. Afterconditioning with IL-4/IL-13 to establish the TH2 milieu, a COL1A1 and COL3A1 gene expression surge in fibroblasts was the only notable effect by WTD treatment accounting for the improved wound closure in vitro.
In conclusion, positive effects described for wound treatment with WTD could be replicated in vitro and seem to be to be conferred by a direct influence on cellular processes taking place in keratinocytes and fibroblasts during wound healing.This is the first in vitro study that shows a stimulatory effect of red-laser-based wound therapy on the regeneration of cell layers under TH1 and TH2 inflammatory conditions.Furthermore, it is the first study to examine the influence of WTD at the molecular level using gene expression analysis.It showed that WTD treatment alleviates the inflammatory response in keratinocytes and induces early growth factor gene expression in fibroblasts under physiological conditions.
In addition, WTD supported wound healing in chronic inflammatory conditions in vitro by improving desmoglein and collagen gene expression as well as inducing the early growth factor gene expression.
WTD treatment further increased HSPD1 transcript levels in keratinocytes and collagen expression in fibroblasts during wound healing under TH2 conditions in vitro.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.
and 20 min.The cells were then stained after 1, 4, 8, 24 and 48 h for visualization.The size of the in vitro wound and the progress of F I G U R E 2 Compatibility of WTD was investigated using human keratinocytes (A-C) and human dermal fibroblasts (D-F).Cell viability (in [%]) 24 h after WTD treatment was determined by luminometric determination of the cellular ATP content of keratinocytes (A) and fibroblasts (D).Treatment with WTD for up to 10 h had no effect on cell viability of keratinocytes compared to the untreated control (black line) in contrast to the cytotoxicity control (Triton X-100).Release of lactate dehydrogenase (LDH) was determined by photometric determination of the enzyme concentration in the culture supernatants of keratinocytes (B) and fibroblasts (E).Treatment with WTD for up to 10 h had no effect on LDH release, calculated as [x-times of the control], while the cytotoxicity control (Triton X-100) led to a distinct increase in LDH.Moreover, WTD treatment of keratinocytes (C) and fibroblasts (F) exhibited a similar proportion of comet-positive cells compared to the medium control.In contrast, the positive control (hydrogen peroxide) showed clear DNA-toxic effects.determined.Treatment with WTD showed no significant effects on healing in the epithelial (HaCaT keratinocytes) wound model under optimal conditions, referring to a physiological cell environment (FigureS1A,C).In contrast, WTD treatment retarded healing progression in dermal fibroblast scratches in a dosedependent manner (FigureS1B,D).Under TH1 and TH2 conditions, a delayed wound closure was observed in the untreated control compared to physiological conditions (Figures3 and 4).Treatment with WTD was able to achieve significant, positive effects on healing in the epithelial wound model in both inflammatory milieus (Figure3).Keratinocyte scratches showed an improvement in wound healing after treatment with WTD under TH1 (Figure3A,C) and TH2 conditions (Figure4A,C) with a significantly increased scratch closure at 24 h (p < .001)corresponding to levels under physiological milieu.Particularly, a significantly earlier onset of cell migration into the scratch wound was observed (p < .001)after WTD treatment.A clearly dose-dependent increase of the effect on keratinocytes was noted.Likewise, treatment with WTD had a beneficial influence on healing in the dermal wound model under TH1 and TH2 conditions (Figures3 and 4).WTD significantly improved wound healing of fibroblast scratches in vitro under TH1 (Figure3B,D) and TH2 conditions (Figure4B,D) with a significantly increased scratch closure at 24 h (p < .001).Again, a significantly earlier onset of cell migration into the scratch wound was observed (p < .01).Here, optimal WTD treatment time for fibroblasts was between 5 and 10 min.
els showed a steady decrease as the wound closed.At the same time, there was an increase in gene expression of growth factor TGFB, transcription factors (TP53, NFKB1) and heat shock proteins (HSPA1A, HSPD1).Likewise, genes of structural proteins such as desmoglein (DSG1, DSG3) and collagen (COL1A1, COL3A1) were increasingly expressed, while the MMP1 transcripts of the protease decreased.Furthermore, expression of AMP (antimicrobial peptides) F I G U R E 3 Wound healing under TH1 chronic inflammatory conditions after treatment with WTD.Keratinocytes (A) and fibroblasts (B) were stained after 1, 4, 8, 24 and 48 h with hematoxylin (keratinocytes) and eosin (fibroblasts) for visualization and the size of the in vitro wound and the progress of epithelial (C) and dermal (D) wound healing were determined.Treatment with WTD showed significant improvement of healing progression in keratinocyte and fibroblast scratches.

F I G U R E 4
Wound healing under TH2 atopic inflammatory conditions after treatment with WTD.Keratinocytes (A) and fibroblasts (B) were stained after 1, 4, 8, 24 and 48 h with hematoxylin (keratinocytes) and eosin (fibroblasts) for visualization and the size of the in vitro wound and the progress of epithelial (C) and dermal (D) wound healing were determined.Treatment with WTD showed significant improvement of healing progression in keratinocyte and fibroblast scratches.in the epithelial and dermal wound model under chronic inflammatory conditions.The observed earlier start of cell migration into the in vitro wound and increased healing progression was reflected in a significantly heightened gene expression of structural components such as DSG1 and DSG3 in HaCaT keratinocytes (Figure 6A,B) as well as COL1A1 and COL3A1 in fibroblasts (Figure 6E,F).In addition, an

F I G U R E 7
Gene expression analysis during keratinocyte (left panel) and fibroblast (right panel) scratch wound healing under TH2 conditions after treatment with WTD.Log 2 change of mRNA expression of pro-inflammatory cytokine genes CXCL8, IL6 and IL1A at 4 h (A) as well as evaluation of TGFB1 transcripts at 8 h (B) and HSPD1 at 4 h (C) in keratinocytes determined by real-time qPCR.Moreover, Log 2 change of mRNA expression of pro-inflammatory cytokine genes CXCL8, IL6 and IL1A at 24 h (D) as well as evaluation of growth factor TGFB1, PDGFC and VEGFA transcripts at 8 h (E) and collagen COL1A1/COL3A1 gene expression at 48 h (F) in fibroblasts was determined by real-time qPCR.Values were normalized to 1-h results for the untreated control under TH2 conditions.