Low-level laser therapy (LLLT) is a nonthermal technology that can be used to modulate cellular activity through light irradiation at specific pulse sequences.
Healthy human fibroblasts obtained from cryopreserved cells (n=10) were irradiated at wavelength of 635 nm (RED), 830 nm (IR), and 635 nm+830 nm (FX) with the same fluence of 60 J/cm2 , after seeding into 96-well plates. A group in which no laser exposure was applied was assigned as control. Fibroblast proliferation was examined by EZ-Cytox enhanced cell viability assay and immunohistochemistry (IHC). Collagen synthesis was measured by IHC. IHC pictures were analyzed to identify the intensity values of collagen type I as quantity results.
Irradiation at FX and IR groups showed a significant increase in fibroblast proliferation and collagen synthesis compared to control and RED groups. There was no significant difference in fibroblast proliferation and collagen synthesis between FX group and IR group.
Healthy human fibroblasts showed better cell proliferation and collagen synthesis when they were irradiated at wavelength of 635 nm+830 nm or 830 nm.
The prevalence of chronic wounds is increasing dramatically, as the populations of industrialized countries age and become more sedentary. Chronic wounds that respond poorly to conventional treatment, making them very difficult to manage [
Low-level laser therapy (LLLT) is a nonthermal technology that can be used to modulate cellular activity through light irradiation at specific pulse sequences [
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 fibroblasts. In particular, we focused on cell proliferation and collagen synthesis, which are main contributing factors in would healing.
Dermal fibroblasts were obtained from cryopreserved cells derived from the dermis of 10 healthy adults who had provided 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 tissue 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 centrifugation 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 Scientific, 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.
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 continuous wave at a fluence of 60 J/cm2 on day 1 and day 3 (48 hours interval between irradiations). The distance from light source to fibroblasts was 15 cm [
On day 5, 96-well plates were subjected to cell proliferation analysis using EZ-Cytox Enhanced Cell Viability Assay Kit (DoGen, DAEILLAB, Seoul, Korea). EZ-Cytox assay measures cell mitochondrial activity based on the conversion of water-soluble tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate) to insoluble 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 reference 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% paraformaldehyde (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. Endogenous 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 fluorescent dye-conjugated secondary antibody at 37°C for 1 hour (protected from light). After fixation, sections were rinsed 3 times with buffer and mounted in 25 μL Vector shield with DAPI (4´-6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA) for staining nuclei.
Collagen type I synthesis assay was performed using IHC staining. The detailed procedure has been described in cell proliferation subsection. These 8-well slides were incubated with primary antibody (anti-collagen type 1; Abcam, Cambridge, MA, USA) and secondary antibody (anti-rabbit-FITC; 1:200, Abcam) for staining collagen type I.
All IHC pictures were taken at the exact center of 8-well slides (200×magnification). Images were analyzed to identify the intensity values of blue color pixel and green color pixel as quantity results, for the reason that nuclei stained by DAPI showed blue color, while collagen type I stained by FITC showed green color in IHC images. Whole slide image analysis was performed using positive pixel count using Adobe Photoshop CS6 (Adobe Systems, San José, CA, USA).
All experiments were performed in triplicates and average value was used as each data. Results are expressed as means±standard deviations. Statistical comparisons were performed with Wilcoxon signed ranks test and paired t-test. Statistical significance was considered at P<0.05. Data were analyzed using Statistical Product and Service Solutions version 20 (SPSS; Systat Software, IBM, Armonk, NY, USA).
In cell proliferation analysis using EZ-Cytox Kit, FX group (635 nm+830 nm) demonstrated highest cell number, followed by IR and RED groups. Cell proliferation in the FX and IR groups showed statistically significant differences compared with RED and control group. However, there was no statistically significant difference between FX and IR groups (
Collagen synthesis was increased in all irradiated groups compared to that in the control group. However, statistically significant differences were detected in IR group and FX group (
According to previous research, LLLT has effect on energy and absorption levels of cells relevant to their respiratory chains [
Another optimal parameter for LLLT is fluence. Exposure to lower-fluence laser light (from 0.09 to 20 J/cm2) [
Our findings indicated that LLLT at wavelength of 830 nm or 635 nm+830 nm with fluence of 60 J/cm2 could modulate fibroblast proliferation and collagen synthesis, with cells irradiated at 635 nm+830 nm possibly giving better results. It is difficult to compare our results with observations of other studies in the literature, because most studies have reported only one or two different single wavelength and treatment design varies depending on individual studies [
Previous studies have demonstrated action mechanisms of visible to near-infrared radiation on cells. It has been suggested that we should pay an attention to energy values relevant to the respiratory chain of cells. The effect of electromagnetic wavelength on cellular energy transfer has become evident [
As a conclusion, our study demonstrated that dual-wavelength light (635 nm+830 nm) or infrared light (830 nm) has stimulating effect on proliferation and collagen synthesis of human fibroblasts
No potential conflict of interest relevant to this article was reported.
This article was presented as a oral presentation at the Internarional 75th Congress of the Korean Society of Plastic and Reconstructive Surgeons on Nov 10–12, 2017.
Result of cell number using EZ-Cytox kit.
Representative samples of cell number study using immunohistochemistry.
Result of cell number using immunohistochemistry.
Representative samples of collagen synthesis study using immunohistochemistry.
Result of collagen synthesis using immunohistochemistry.
Statistical significances in cell proliferation analysis using EZ-Cytox
Control | 635 nm | 830 nm | 635 nm+830 nm | |
---|---|---|---|---|
Control | - | 0.391 | ||
635 nm | 0.391 | - | ||
830 nm | - | 0.113 | ||
635 nm+830 nm | 0.113 | - |
P<0.001,
P<0.01.
Statistical significances in cell proliferation analysis using IHC staining
Control | 635 nm | 830 nm | 635 nm+830 nm | |
---|---|---|---|---|
Control | - | 0.264 | ||
635 nm | 0.264 | - | ||
830 nm | - | 0.348 | ||
635 nm+830 nm | 0.348 | - |
P<0.001,
P<0.01,
P<0.05.
Statistical significances in collagen synthesis analysis using IHC staining
Control | 635 nm | 830 nm | 635 nm+830 nm | |
---|---|---|---|---|
Control | - | 0.221 | ||
635 nm | 0.221 | - | ||
830 nm | - | 0.123 | ||
635 nm+830 nm | 0.123 | - |
P<0.001,
P<0.01.