Diabetic Wounded Fibroblast Cells Research Articles

Modulatory Effects of 830 nm on Diabetic Wounded Fibroblast Cells: An In Vitro Study on Inflammatory Cytokines.

Background:After skin damage, a complicated set of processes occur for epidermal and dermal wound healing. This process is hindered under diabetic conditions, resulting in nonhealing diabetic ulcers. In diabetes there is an increase in inflammation and proinflammatory cytokines. Modulating cells using photobiomodulation (PBM) may have an effect on inflammation and cell viability, which are crucial for the healing of wounds. Objective: This study explored the impact of PBM in the near-infrared spectrum (830 nm; 5 J/cm2) on inflammation in diabetic wound healing. Materials and Methods: Five cell models, namely normal, wounded, diabetic, diabetic wounded, and wounded with d-galactose were used. Cell morphology and migration rate were assessed, while cellular response measures included viability (Trypan blue and adenosine triphosphate), apoptosis (annexin-V/PI), proinflammatory cytokines interleukin-6, tumor necrosis factor-alpha (TNF-α), and cyclooxygenase-2, nuclear translocation of nuclear factor kappa B (NF-κB), and gene expression of advanced glycation end product receptor (AGER). Results: PBM resulted in increased levels of TNF-α, supported by activation of NF-κB. PBM stimulated translocation of NF-κB and upregulation of AGER. Conclusions: PBM modulates diabetic wound healing in vitro at 830 nm through stimulated NF-κB signaling activated by TNF-α.

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 reduces oxidative stress in diabetic wounded fibroblast cells by inhibiting the FOXO1 signaling pathway.

This study aimed to elucidate the underlying molecular mechanism of photobiomodulation (PBM) in attenuating oxidative stress in diabetic wounded fibroblast cells. Cell models were exposed to PBM at a wavelength of 660nm (fluence of 5J/cm2, and power density of 11.2mW/cm2) or 830nm (fluence of 5J/cm2, and power density of 10.3mW/cm2). Non-irradiated cell models were used as controls. Cellular migration was determined at regular time intervals (0, 12, 24 and 48h) using inverted light microscopy. Cell viability was determined by the Trypan blue exclusion assay. The levels of enzymic antioxidants superoxide dismutase (SOD), catalase (CAT), and heme oxygenase (HMOX1) were determined by the enzyme linked immunosorbent assay (ELISA). The alteration in the levels of AKT and FOXO1 was determined by immunofluorescence and western blotting. Upon PBM treatment, elevated oxidative stress was reversed in diabetic and diabetic wounded fibroblast cells. The reduced oxidative stress was represented by decreased FOXO1 levels and increased levels of SOD, CAT and HMOX1. This might be due to the activation of the AKT signaling pathway. This study concluded that treatment with PBM progressed diabetic wound healing by attenuating oxidative stress through inhibition of the FOXO1 signaling pathway.

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.

Effect of photobiomodulation on cellular migration and survival in diabetic and hypoxic diabetic wounded fibroblast cells.

A disrupted wound repair process often leads to the development of chronic wounds, and pose a major physical, social and economic inconvenience on patients and the public health sector. Chronic wounds are a common complication seen in diabetes mellitus (DM), and often the severity necessitates amputation of the lower limbs. Recently, there has been increasing evidence that photobiomodulation (PBM) initiates wound healing, including increased protein transcription for cell proliferation, viability, migration and tissue reepithelialisation. Here, the hypothesis that PBM at a wavelength of 660nm and energy density of 5J/cm2 regulates wound repair in diabetic wounded and hypoxic diabetic wounded fibroblasts by enhancing cell migration and survival was investigated. PBM increased migration and survival in diabetic wounded and hypoxic diabetic wounded fibroblasts. Our findings suggest that PBM enhances migration and survival in diabetic wounded and hypoxic diabetic wounded fibroblasts, indicating that this therapeutic method may be beneficial against chronic wounds in diabetic patients.

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.

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.

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.

Laser light influences cellular viability and proliferation in diabetic-wounded fibroblast cells in a dose- and wavelength-dependent manner

Phototherapy stimulates metabolic processes in healing wounds. Despite worldwide interest, phototherapy is not firmly established or practiced in South Africa. This study aimed to determine which dose and wavelength would better induce healing in vitro. Diabetic-induced wounded fibroblasts were irradiated with 5 or 16 J/cm(2) at 632.8, 830, or 1,064 nm. Cellular morphology, viability (Trypan blue and apoptosis), and proliferation (basic fibroblast growth factor) were then determined. Cells irradiated with 5 J/cm(2) at 632.8 nm showed complete wound closure and an increase in viability and basic fibroblast growth factor (bFGF) expression. Cells irradiated at 830 nm showed incomplete wound closure and an increase in bFGF expression. Cells irradiated at 1,064 nm showed incomplete closure and increased apoptosis. All cells irradiated with 16 J/cm(2) at all three wavelengths showed incomplete wound closure, increased apoptosis, and decreased bFGF expression. This study showed that diabetic-wounded cells respond in a dose- and a wavelength-dependent manner to laser light. Cells responded the best when irradiated with a fluence of 5 J/cm(2) at a wavelength of 632.8 nm.

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.