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Ma H et al. Laser therapy for wound healing accelerate cutaneous wound healing [5]. Clinical benefits of using light irradiation through LLLT on skin wounds are also reported in the literature [6]. Furthermore, it has been re- ported that LLLT enhances cell proliferation of several cell types including fibroblasts, endothelial cells [7]. However, some studies have suggested that LLLT might have no effect on fibroblast proliferation and activity [8,9]. Inhibitory effect on fibroblast proliferation and activity using red light or infra- red light were also reported in literature [10,11]. Many stud- ies have demonstrated that the effect of light irradiation on human skin fibroblasts is dose and/or wavelength dependent [7-11]. Final purpose of our project is to determine effect of LLLT on diabetic wound healing, which is one of representing chronic wounds. Moreover, this pilot study was designed to confirm effect of LLLT on activity of healthy human fibro- blasts. In particular, we focused on cell proliferation and col- lagen synthesis, which are main contributing factors in would healing. Methods Human dermal fibroblast culture Dermal fibroblasts were obtained from cryopreserved cells derived from the dermis of 10 healthy adults who had pro- vided informed consent for their cells to be used for research purposes. Fibroblasts were cultured with Dulbecco’s modified Eagle medium/Ham’s F-12 nutrient (DMEM/F-12; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 25 mg/mL of gentamycin) in 100-mm tis- sue culture dishes (Corning Life Sciences, Corning, NY, USA) at 37°C. All cultures in this study were performed at 37°C in a 5% CO2–95% air atmosphere. Once the culture reached approximately 80% confluent, fibroblasts were dissociated by trypsinization, diluted 2.7-fold in phosphate-buffered saline without Mg2+ and Ca2+ (PBS; Gibco), and collected by centrif- ugation at 450 g for 17 minutes. Cell density was determined by trypan blue dye exclusion test. To determine the effect of lasers on cell proliferation, 3× 103 cells in 100 μL DMEM/F-12 with 5% FBS were seeded into each well of sterile 96-well culture plates (Fisher Scien- tific, Pittsburgh, PA, USA). For Immunohistochemistry (IHC) study to evaluate cell proliferation and collagen synthesis, 5 × 103 cells in 200 μL DMEM/F-12 with 5% FBS were seeded into each well of sterile 8 well cell culture slides (SPL Life Science, Pocheon, Korea). These 96-well plates and 8-well slides were then incubated at 37°C overnight to allow human fibroblasts to attach. Laser irradiation Human fibroblasts were irradiated using Light-emitting diode (Smartlux, MEDMIX, Incheon, Korea) in a light room. Three different wavelengths were treated; red light (RED; 635 ± 6 nm), infrared light (IR; 830 ± 5 nm), and a dual-wavelength output light (FX; RED+IR). Cells were irradiated with continu- ouswaveatafluenceof60J/cm2 onday1andday3(48 hours interval between irradiations). The distance from light source to fibroblasts was 15 cm [12]. Human fibroblasts without any laser exposure were used as controls. Control group plates were taken out of the incubator at the same time and placed in the same ambient environment as their matched treatment plates. On day 5 (48 hours after laser ir- radiation), cell proliferation and collagen synthesis were mea- sured. Cell proliferation assay On day 5, 96-well plates were subjected to cell proliferation analysis using EZ-Cytox Enhanced Cell Viability Assay Kit (Do- Gen, DAEILLAB, Seoul, Korea). EZ-Cytox assay measures cell mitochondrial activity based on the conversion of water-solu- ble tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4- nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate) to in- soluble formazan. Briefly, cells were incubated with WST-1 reagent for 3 hours. Absorbance was then measured at wavelength of 450 nm using a 96-well plate reader with refer- ence wavelength of 600 nm. In addition, 8-well slides were subjected to cell proliferation analysis using IHC staining. Each group of human fibroblasts was washed twice with ice-cold PBS, fixed with 4% parafor- maldehyde (pH 7.4) in 8-well slides for 30 minutes on ice. Removed the fixative and wash 3 times for 5 minutes each with PBS. Then incubated fibroblasts with 0.5% Triton X-100 in PBS for 10 minutes at room temperature to permeabilize the membranes, and rinsed three times with PBS. Endoge- nous peroxidase was then blocked by incubating with 3% H2O2 in PBS at room temperature for 30 minutes. After rinse with PBS 3 times, the 8-well slides were then incubated with primary antibody at 4°C overnight. After washing with PBS 3 times, the slides were incubated with corresponding fluores- cent dye-conjugated secondary antibody at 37°C for 1hour (protected from light). After fixation, sections were rinsed 3 times with buffer and mounted in 25 μL Vector shield with 2 www.jwmr.org https://doi.org/10.22467/jwmr.2018.00283PDF Image | Low-Level Laser Therapy on Proliferation and Collagen Synthesis of Human Fibroblasts
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