Diabetic Wounded Cells Research Articles

Photobiomodulation at 660 nm Modifies Apoptosis Regulators Bcl-2 and Caspase-3 in Diabetic Wounded Cell Models in Vitro

Photobiomodulation at 660 nm Modifies Apoptosis Regulators Bcl-2 and Caspase-3 in Diabetic Wounded Cell Models in Vitro

Effect of photobiomodulation at 830 nm on gene expression correlated with JAK/STAT signalling in wounded and diabetic wounded fibroblasts in vitro.

Treatment of chronic diabetic wounds is an ongoing socio-economic challenge. Dysregulated signalling pathways characterise cells from chronic diabetic wounds. Photobiomodulation (PBM) stimulates healing by eliciting photochemical effects that affect gene regulation. JAK/STAT signalling is a primary signal transduction pathway involved in wound healing. This in vitro study aimed to determine if PBM at 830 nm and a fluence of 5 J/cm2 regulates genes related to JAK/STAT signalling in wounded and diabetic wounded fibroblast cells. A continuous wave diode laser (12.53 mW/cm2 ) was used to irradiate cells. Forty-eight hours post-PBM, RT-qPCR was used to analyse 84 genes related to JAK/STAT signalling. Five genes were upregulated and four downregulated in wounded cell models, while six genes were downregulated in diabetic wounded models. The results show drastic gene expression differences between wounded and diabetic wounded cell models in response to PBM using 830 nm.

Photobiomodulation at 660 nm Stimulates In Vitro Diabetic Wound Healing via the Ras/MAPK Pathway

Diabetic foot ulcers (DFUs) are open chronic wounds that affect diabetic patients due to hyperglycaemia. DFUs are known for their poor response to treatment and frequently require amputation, which may result in premature death. The present study evaluated the effect of photobiomodulation (PBM) at 660 nm on wound healing via activation of Ras/MAPK signalling in diabetic wounded cells in vitro. This study used four human skin fibroblast cell (WS1) models, namely normal (N), wounded (W), diabetic (D), and diabetic wounded (DW). Cells were irradiated at 660 nm with 5 J/cm2. Non-irradiated cells (0 J/cm2) served as controls. Cells were incubated for 24 and 48 h post-irradiation, and the effect of PBM on cellular morphology and migration rate, viability, and proliferation was assessed. Basic fibroblast growth factor (bFGF), its phosphorylated (activated) receptor FGFR, and phosphorylated target proteins (Ras, MEK1/2 and MAPK) were determined by enzyme-linked immunosorbent assay (ELISA) and Western blotting; nuclear translocation of p-MAPK was determined by immunofluorescence. PBM resulted in an increase in bFGF and a subsequent increase in FGFR activation. There was also an increase in downstream proteins, p-Ras, p-MEK1/2 and p-MAPK. PBM at 660 nm led to increased viability, proliferation, and migration as a result of increased bFGF and subsequent activation of the Ras/MAPK signalling pathway. Therefore, this study can conclude that PBM at 660 nm stimulates in vitro diabetic wound healing via the bFGF-activated Ras/MAPK pathway.

Synergistic Effects of Photobiomodulation Therapy with Combined Wavelength on Diabetic Wound Healing In Vitro and In Vivo.

Objective: The difficulty in chronic diabetic wound healing remains the focus of clinical research. Photobiomodulation therapy (PBMT) with different wavelengths could exert different effects on wound healing, but the effects of combined red and blue light (BL) remained unclear. Methods: Diabetic rat wound model and diabetic wounded endothelial cell model were established to observe possible effects of PBMT using combined wavelengths for wound healing. Cells and animals were separated into four groups exposed to red and/or BL. Cell viability, apoptosis, and migration, as well as the expression level of nitric oxide (NO), vascular endothelial growth factor, interleukin-6, and tumor necrosis factor-α were measured in vitro. Diabetic rats were evaluated for wound closure rates, collagen deposition, inflammation intensity, and density of neovascularization after light irradiation. Results: PBMT using combined wavelengths significantly sped up the healing process with increasing angiogenesis density, collagen deposition, and alleviating inflammation in vivo. Moreover, combined wavelength irradiation promoted cell proliferation and migration, and NO production, as well as reduced reactive oxygen species and inflammation in vitro. Conclusions: PBMT using combined wavelengths performed a synergistic effect for promoting diabetic wound healing and would be helpful to explore a more efficient pattern toward chronic wound healing.

Homeopathy and Photobiomodulation for Healing Diabetic Wounds in vitro

Photobiomodulation (PBM), as well as plant extracts of Calendula officinalis (Calen), Hypericum perforatum (Hyper), and Echinacea purpurea (Echi-p), have been used to accelerate wound healing. However, the use of homeopathic preparations of these medicinal plants, in combination with PBM, is unknown. The objective of this study was to investigate the combined wound healing potential of these therapies in vitro. Various cell models were created in vitro in commercially available human skin fibroblasts (WS1). PBM was administered using a diode laser at a wavelength of 660 nm and an energy density of 5 J/cm2. For homeopathic treatment, a 5% ethanolic complex containing Calen, Hyper, and Echi-p in 3cH potency was added to the culture medium. For combination therapy, cells were first treated with the homeopathic remedy and then irradiated. The controls included non-treated/irradiated cells and an alcohol control group. Cellular morphology was visualized 0, 24, and 48 h post-treatment. Post-treatment, cells were incubated for 48 h, and the levels of pro-inflammatory cytokines, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were determined by ELISA, cellular viability was determined by the Trypan Blue exclusion assay and adenosine triphosphate (ATP) was determined by the luminescence assay, and cytotoxicity was determined by the lactate dehydrogenase (LDH) membrane integrity assay. Morphological changes showed that, individually, PBM and the homeopathic complex hastened cellular migration in diabetic wounded cells, and combining these therapies had an even greater effect. Cellular viability remained unchanged in both treatments, but the combination therapy increased ATP (p = 0.05). Cytotoxicity was decreased by the homeopathic complex (p = 0.01) and the combination therapy (p = 0.004). TNF-α levels remained unchanged with PBM and the homeopathic complex as independent treatment interventions but combining them increased the TNF-α levels in diabetic models (p = 0.01). PBM decreased IL-6 levels, indicating an anti-inflammatory effect (p = 0.007). These cellular changes indicated that combining PBM and a 3cH homeopathic solution of Calen, Hyper, and Echi-p is promising for treating diabetic foot ulcers.

In Vitro Wound Healing Potential of Photobiomodulation Is Possibly Mediated by Its Stimulatory Effect on AKT Expression in Adipose-Derived Stem Cells.

Increasing evidence suggests that adipose-derived stem cells (ADSCs) serve as a therapeutic approach for wound healing. The aim of this study was to determine the effect of photobiomodulation (PBM) on antioxidant enzymes in ADSCs. Four ADSC cell models, namely, normal, wounded, diabetic, and diabetic wounded, were irradiated with 660 nm (fluence of 5 J/cm2 and power density of 11.2 mW/cm2) or 830 nm (fluence of 5 J/cm2 and power density of 10.3 mW/cm2). Nonirradiated cells served as controls. Cell morphology and wound migration were determined using light microscopy. Cell viability was determined by the trypan blue exclusion assay. The enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of antioxidants (superoxide dismutase (SOD), catalase (CAT), and heme oxygenase (HMOX1)). AKT activation and FOXO1 levels were determined by immunofluorescence and western blotting. The gaps (wound) in PBM-treated wounded and diabetic wounded cell models closed faster than the controls. PBM treatment significantly increased antioxidant levels in all cell models. This reflects that oxidative stress is reduced on the counterpart of increased antioxidant levels. This might be due to the activation of the AKT signaling pathway as evidenced by the increased AKT signals via western blotting and immunofluorescence. This data suggests that PBM promotes wound healing by increasing antioxidant levels by activating AKT signaling.

Selective Laser Efficiency of Green-Synthesized Silver Nanoparticles by Aloe arborescens and Its Wound Healing Activities in Normal Wounded and Diabetic Wounded Fibroblast Cells: In vitro Studies.

IntroductionSilver nanoparticles (AgNPs) have been extensively used in wound healing applications owing to their valuable physicochemical and biological properties. The main objective of this study was to evaluate the combined effects of green-synthesized silver nanoparticles (G-AgNPs) and photobiomodulation (PBM; laser irradiation at 830 nm with 5 J/cm2) in normal wounded and diabetic wounded fibroblast cells (WS1).MethodsThe combined effect of G-AgNPs and PBM was studied by various in vitro wound healing studies including cell morphology, cell migration rate and percentage wound closure, cell viability, cell proliferation, and filamentous (F)-actin and nuclear morphology staining.ResultsCell viability results revealed good cellular compatibility of G-AgNPs to WS1 cells. The combined therapy of G-AgNPs and PBM demonstrated promising results to achieve progressive migration and wound closure in both normal wounded and diabetic wounded cell models. G-AgNPs alone and in combination with PBM had no negative effect on cell viability and proliferation, and there was an increase in cell migration.ConclusionOverall, these findings demonstrate that the combined treatment of G-AgNPs and PBM does not display any adverse effects on wound healing processes in both normal wounded and diabetic wounded cell models.

Photobiomodulation and the expression of genes related to the JAK/STAT signalling pathway in wounded and diabetic wounded cells

Photobiomodulation therapy (PBMT) is a curative technique that uses low intensity light to relegate pain and inflammation, and accelerate tissue repair. At a molecular level, the effects of photobiomodulation (PBM) are not fully established. The present study aimed to assess the impact of PBM on the alteration of genes linked to Janus kinase-Signal transducer and activator of transcription (JAK-STAT) signalling in wounded and diabetic wounded cells in vitro. Cells were irradiated using a diode laser at a wavelength of 660 nm and an energy density of 5 J/cm2. RNA was extracted from cells 48 h post-irradiation, and was used to synthesise complementary deoxyribonucleic acid (cDNA) that was used in PCR arrays to profile for 84 JAK/STAT signalling related genes. Irradiation at a wavelength of 660 nm and an energy density of 5 J/cm2 significantly regulated genes related to the JAK/STAT signalling pathway in wounded and diabetic wounded cells. In irradiated wounded cells, 19 genes were significantly regulated, of which two were up-regulated and 17 were down-regulated, while 73 genes were significantly regulated in irradiated diabetic wounded cells of which 46 were up-regulated and 27 were down-regulated. This data suggests that PBM modulates gene transcription for protein synthesis and activates cellular signalling, and may indeed be helpful in enhancing diabetic wound repair.

Photobiomodulation at 660 nm Stimulates Fibroblast Differentiation.

Among many of the different complications that diabetic patients suffer, foot ulcers are the most challenging, and in many cases result in non-traumatic lower limb amputation and permanent disability. To alleviate this burden, new interventions such as photobiomodulation (PBM) have been utilized. However, the cellular pathways affected by PBM have not yet been fully recognized. The differentiation of fibroblasts into myofibroblasts forms a vital part of wound healing and is often impaired under diabetic conditions. Therefore, this study sought to investigate the effects of PBM at 660 nm on the transforming growth factor-β1 (TGF-β1)/Smad pathway and the differentiation of fibroblasts into myofibroblasts. WS1 fibroblasts were treated with PBM using a wavelength of 660 nm at a fluence of 5 J/cm2 in normal, normal wounded, diabetic, and diabetic wounded models. Post-irradiation cellular responses were observed at 24, 48, and 72 hours to ascertain morphological changes and cell viability, and the expression of fibroblast differentiation markers (Thy-1 or CD90, extra domain A fibronectin or EDA-FN and α-smooth muscle actin or α-SMA), TGF-β1, phosphorylated (p)TGF-β receptor 1 (R1), and p-Smad2/3. There was a significant increase in cell viability in all irradiated cell models, and no real significant changes in TGF-β1, pTGF-β1R1, and p-Smad2/3. As incubation time post-irradiation increased, Thy-1 (CD90) decreased, while EDA-FN and α-SMA increased in wounded models. PBM at 660 nm with 5 J/cm2 was successful in stimulating the differentiation of fibroblasts into myofibroblasts in diabetic wounded cells, which was independent of the TGF-β1/Smad pathway. Fibroblast transition into myofibroblasts is vital to wound healing, failure of which results in impaired healing; PBM is able to foster such a transition. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Cell Adhesion Molecules are Mediated by Photobiomodulation at 660 nm in Diabetic Wounded Fibroblast Cells.

Diabetes affects extracellular matrix (ECM) metabolism, contributing to delayed wound healing and lower limb amputation. Application of light (photobiomodulation, PBM) has been shown to improve wound healing. This study aimed to evaluate the influence of PBM on cell adhesion molecules (CAMs) in diabetic wound healing. Isolated human skin fibroblasts were grouped into a diabetic wounded model. A diode laser at 660 nm with a fluence of 5 J/cm2 was used for irradiation and cells were analysed 48 h post-irradiation. Controls consisted of sham-irradiated (0 J/cm2) cells. Real-time reverse transcription (RT) quantitative polymerase chain reaction (qPCR) was used to determine the expression of CAM-related genes. Ten genes were up-regulated in diabetic wounded cells, while 25 genes were down-regulated. Genes were related to transmembrane molecules, cell–cell adhesion, and cell–matrix adhesion, and also included genes related to other CAM molecules. PBM at 660 nm modulated gene expression of various CAMs contributing to the increased healing seen in clinical practice. There is a need for new therapies to improve diabetic wound healing. The application of PBM alongside other clinical therapies may be very beneficial in treatment.

Photobiomodulation at 660 nm stimulates proliferation and migration of diabetic wounded cells via the expression of epidermal growth factor and the JAK/STAT pathway

Photobiomodulation (PBM) modulates cellular processes to enhance diabetic wound healing. The photon energy activates wounded cells to proliferate and migrate. However, the signalling pathways responsible for these observations remain unknown. This study aimed to determine if PBM stimulates cellular proliferation and migration via the expression of epidermal growth factor (EGF) and activation of the Janus kinase/Signal transducer and activators of transcription (JAK/STAT) signalling pathway. Normal, wounded, diabetic and diabetic wounded cell models were exposed to PBM at a wavelength of 660nm and fluence of 5J/cm2 and incubated for 48h. Non-irradiated cells (0J/cm2) and cells exposed to exogenous EGF (rh EGF) served as controls. Cellular migration was determined microscopically at 0, 24 and 48h. Flow cytometry (BrdU) was used to determine cell proliferation, while the Trypan blue exclusion assay and adenosine triphosphate (ATP) luminescence was used to determine cell viability. The enzyme linked immunosorbent assay (ELISA) was used to analyse EGF expressed in the culture media, and phosphorylated (p-) EGF receptor (p-EGFR), p-JAK2, p-STAT1 and p-STAT5 in cells. Irradiated diabetic wounded cells showed a significant increase in EGF, and activation of its receptor (p-EGFR) and JAK/STAT (p-JAK2, p-STAT1 and p-STAT5). PBM at 660nm and 5J/cm2 is able to modulate cellular autocrine signalling, particularly the EGF/EGFR loop leading to activation of the JAK/STAT pathway which in turn stimulates cell proliferation and migration.

The role of photobiomodulation on gene expression of cell adhesion molecules in diabetic wounded fibroblasts in vitro

Cell adhesion molecules (CAMs) are cell surface glycoproteins that facilitate cell-cell contacts and adhesion with the extracellular matrix (ECM). Cellular adhesion is affected by various disease conditions, such as diabetes mellitus (DM) and inflammation. Photobiomodulation (PBM) stimulates biological processes and expression of these cellular molecules. The aim of this experimental work was to demonstrate the role of PBM at 830nm on CAMs in diabetic wounded fibroblast cells. Isolated human skin fibroblast cells were used. Normal (N-) and diabetic wounded (DW-) cells were irradiated with a continuous wave diode laser at 830nm with an energy density of 5J/cm2. Real time reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the relative gene expression of 39 CAMs 48h post-irradiation. Normalized expression levels from irradiated cells were calculated relative to non-irradiated control cells according to the 2^(−ΔΔCt) method. Thirty-one genes were significantly regulated in N-cells (28 were genes up-regulated and three genes down-regulated), and 22 genes in DW-cells (five genes were up-regulated and 17 genes down-regulated). PBM induced a stimulatory effect on various CAMs namely cadherins, integrins, selectins and immunoglobulins, and hence may be used as a complementary therapy in advancing treatment of non-healing diabetic ulcers. The regulation of CAMs as well as evaluating the role of PBM on the molecular effects of these genes may expand knowledge and prompt further research into the cellular mechanisms in diabetic wound healing that may lead to valuable clinical outcomes.

Laser Irradiation Alters the Expression Profile of Genes Involved in the Extracellular MatrixIn Vitro

The extracellular matrix (ECM) forms the basis of every phase in wound healing. Healing may be impaired if some of these components are destroyed. Photobiostimulation has demonstrated a stimulatory response in biological processes. This study aimed to evaluate various genes involved in the ECM, in response to laser irradiation. Isolated human skin fibroblasts were used in three different cell models, namely, normal, normal wounded, and diabetic wounded. Cells were irradiated with 5 J/cm2using a continuous wave diode laser emitting at a wavelength of 660 nm and incubated for 48 h. Nonirradiated (0 J/cm2) normal and diabetic wounded cells served as the control. Real-time reverse transcription (RT) quantitative polymerase chain reaction (qPCR) was used to determine the expression of 84 genes in a PCR array. There was a significant upregulation of 29 genes in the normal cells, 32 genes in the normal wounded cells, and 18 genes in the diabetic wounded cells as well as a downregulation of 19 genes (normal), 6 genes (normal wounded), and 31 genes (diabetic wounded). Low intensity laser irradiation (LILI) stimulates gene expression in various cell adhesion molecules (CAMs) and extracellular proteins at 660 nm in wounded fibroblastsin vitro.

Low-Intensity Laser Irradiation at 660 nm Stimulates Transcription of Genes Involved in the Electron Transport Chain

Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

Irradiation at 636 nm Positively Affects Diabetic Wounded and Hypoxic Cellsin Vitro

This study investigated the effect of low-intensity laser irradiation (LILI) on pro-inflammatory cytokines involved in wound healing processes in diabetes and hypoxia. Diabetes is associated with impaired wound healing and a prolonged inflammatory phase. Pro-inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 are elevated in diabetes. LILI has been reported to accelerate wound healing and decrease inflammatory cytokines. A human skin fibroblast cell line (WS1) was used in vitro. Cells were exposed to various insults, namely, wounding, and a diabetic or hypoxic environment. Experimental cells were exposed to an energy density of 5 J/cm(2) using a continuous wave 636-nm diode laser at an average power of 95 mW, an illuminated area of 9.05 cm(2), and an irradiance of 11 mW/cm(2) (irradiation time, 476 sec). The effect of laser irradiation on cytokine expression was examined at 1 or 24 h post-irradiation. Cellular morphology, viability, proliferation, and cytokine expression (IL-1β, IL-6, and TNF-α) were investigated. Translocation of nuclear factor-kappa B (NF-κB) was also determined. There was a higher rate of migration in irradiated wounded cultures, and irradiated hypoxic cells showed an improvement in cellular morphology. All cell models showed an increase in proliferation. Normal wounded cells showed a decrease in apoptosis, TNF-α, and IL-1β. Diabetic wounded cells showed an increase in viability and a decrease in apoptosis and IL-1β, whereas hypoxic cells showed an increase in viability and IL-6, and a decrease in apoptosis and TNF-α. NF-κB was translocated into the nucleus post-irradiation. Phototherapy resulted in hastened wound closure, increased proliferation, and normalization of cellular function. The decrease in the different pro-inflammatory cytokines and NF-κB translocation was model and time dependent. Overall, laser irradiation resulted in a reduction in inflammatory cytokines and directed cells into the cell survival pathway.

Low-Intensity Laser Irradiation Stimulates Wound Healing in Diabetic Wounded Fibroblast Cells (WS1)

Patients with diabetes suffer from slow-to-heal wounds, which often necessitate amputation. Low-intensity laser irradiation (LILI) has been shown to reduce the healing time in such patients. This study aimed to determine the effect of different wavelengths of LILI on cellular migration, viability, and proliferation in a wounded diabetic cell model. Diabetic wounded and unwounded human skin fibroblast cells (WS1) were irradiated at 632.8, 830, or 1,064 nm with 5 J/cm(2). Cellular morphology and migration were determined microscopically, while cellular viability was determined by ATP luminescence, and proliferation was determined by basic fibroblast growth factor expression and alkaline phosphatase activity. Diabetic wounded cells irradiated at 1,064 nm showed a lesser degree of migration, viability, and proliferation compared to cells irradiated at 632.8 or 830 nm. Cells irradiated at 632.8 nm showed a higher degree of haptotaxis and migration as well as ATP luminescence compared to cells irradiated at 830 nm. This study showed that LILI of diabetic wounded cells in the visible range (632.8 nm) was more beneficial to wound healing than irradiating the same cells to wavelengths in the infrared range. Cells irradiated at a longer wavelength of 1,064 nm performed worse.

Irradiation at 830 nm stimulates nitric oxide production and inhibits pro‐inflammatory cytokines in diabetic wounded fibroblast cells

Wound healing in diabetic patients remains a chief problem in the clinical setting and there is a strong need for the development of new, safe, reliable therapies. This study aimed to establish the effect of irradiating diabetic wounded fibroblast cells (WS1) in vitro on pro-inflammatory cytokines and the production of nitric oxide (NO). Normal, wounded and diabetic wounded WS1 cells were exposed to an 830 nm laser with 5 J/cm(2) and incubated for a pre-determined amount of time. Changes in cellular viability, proliferation and apoptosis were evaluated by the Trypan blue assay, VisionBlue fluorescence assay and caspase 3/7 activity respectively. Changes in cytokines (interleukin--IL-6, IL-1 beta and tumour necrosis factor-alpha, TNF-alpha) were determined by ELISA. NO was determined spectrophotometrically and reactive oxygen species (ROS) was evaluated by immunofluorescent staining. Diabetic wounded WS1 cells showed no significant change in viability, a significant increase in proliferation at 24 and 48 hours (P<0.001 and P<0.01 respectively) and a decrease in apoptosis 24 hours post-irradiation (P<0.01). TNF-alpha levels were significantly decreased at both 1 and 24 hours (P<0.05), while IL-1 beta was only decreased at 24 hours (P<0.05). There was no significant change in IL-6. There was an increase in ROS and NO (P<0.01) 15 minutes post-irradiation. Results show that irradiation of diabetic wounded fibroblast cells at 830 nm with 5 J/cm(2) has a positive effect on wound healing in vitro. There was a decrease in pro-inflammatory cytokines (IL-1 beta and TNF-alpha) and irradiation stimulated the release of ROS and NO due to what appears to be direct photochemical processes.

Irradiation with a 632.8 nm Helium-Neon Laser with 5 J/cm2 Stimulates Proliferation and Expression of Interleukin-6 in Diabetic Wounded Fibroblast Cells

The use of lasers has been shown to stimulate wound healing in vivo and in vitro. There is an increase in wound closure, cell viability, proliferation, and cytokine expression. If laser parameters can be optimized and standardized, and the underlying mechanisms better understood, this phototherapy can become an alternative safe treatment to slow-to-heal wounds, such as in patients with diabetes. This study aimed to determine the effect on cellular proliferation, migration, and cytokine [interleukin-6 (IL-6)] expression in diabetic and diabetic wounded fibroblast cells (WS1) post-laser irradiation. Diabetic and diabetic wounded WS1 cells were irradiated at 632.8 nm (23 mW) with 5 J/cm(2) or 16 J/cm(2). IL-6 level, cellular proliferation (neutral red assay), and morphology were then determined. Diabetic cells irradiated with 5 J/cm(2) showed no significant change, while diabetic wounded cells showed an increase in IL-6 level, proliferation, and migration. On the other hand, diabetic and diabetic wounded cells irradiated with 16 J/cm(2) showed a significant decrease in proliferation and evidence of cellular damage, and wounded cells showed no migration. This study showed that phototherapy at the correct fluence stimulates IL-6 expression, proliferation, and cellular migration in diabetic wounded cells. A fluence of 5 J/cm(2) stimulates diabetic wound healing in vitro, while 16 J/cm(2) is inhibitive.

Cellular Damage in Diabetic Wounded Fibroblast Cells following Phototherapy at 632.8, 830, and 1064 nm

Objective. This study aimed to establish if laser irradiation induces cellular and genetic damage. Background. Phototherapy has been shown to induce wound healing in diabetic wounds, however little information is known regarding light‐induced damage. Methods. Diabetic wounded fibroblasts were irradiated with 5 or 16 J/cm2 at 632.8, 830, and 1064 nm. Damage was assessed by measuring membrane and DNA damages. Cellular migration was determined by microscopy. Results. Cells irradiated with 5 J/cm2 at 632.8 and 830 nm showed a significant decrease in DNA damage while all cells irradiated with a fluence of 16 J/cm2 showed an increase in membrane and DNA damages. Conclusion. This study showed that the comet assay and LDH release were sensitive enough to pick up changes in laser‐irradiated cells. This study also showed that cellular and genetic damage inflicted on diabetic wounded cells was dependent on dose and wavelength and that cells are able to recover and respond.