Low level light therapy (LLLT) is using photobiomodulation effect of visible to near infrared spectrum light (Posten et al., 2005). It is thought that photon absorbs at the cytochrome c oxidase enzyme which participates of the oxidative respiration of mitochondria resulting in making a cascade of gene transcription and alteration of various cellular properties (Silveira et al., 2007). Its photobiomodulatory effect is applied in clinically for reducing pain, inflammation and edema, promoting healing of wound, and preventing tissue damage. Nowadays, some clinical observations and small studies are published that LLLT is effective to preventing hypertrophic scar or keloid and stimulating wound healing without any additional pain or discomfort (Barolet and Boucher, 2010; Carvalho et al., 2010; Mamalis et al., 2013).
Wound healing is divided inflammatory phase, proliferation phase, and remodeling phase. After disruption of skin, hemostasis is occurred immediately and inflammatory cells are recruited. Neutrophils kill microbes and macrophages are engulfing the tissue debris. Growth factors, cytokines, and chemokines secreted by inflammatory cells make a complex process of interaction between many cells. During proliferation phase, macrophages secrete platelet-derived growth factors (PDGF), and transforming growth factor (TGF)-β1 and endothelial cells make new vessels. These processes make fibroblasts increase their proliferation and product type III collagen. During remodeling phase, type III collagen is degraded and replaced with type I collagen result in reconstruction of dermis and wound undergoes a contractile response (Profyris et al., 2012). There are no effective modalities accelerating wound healing yet. However, above mentioned, some studies are proved that LLLT is effective to promoting wound healing and preventing hypertrophic scar. LLLT could effect on keratinocytes, inflammatory cells and fibroblasts, and that is resulting in alteration of their motility, proliferation and secretion properties of cytokines (Posten et al., 2005; Peplow et al., 2011). Among these various cells, because fibroblasts play a crucial role in wound healing, LLLT at certain fluences and wavelengths can enhance the release growth factors from fibroblasts and stimulate cell proliferation or migration. Therefore, we planned the study that reveals
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which wavelength and fluence could enhance fibroblast proliferation and migration.
Keloid is contributed by several factors: proliferation of fibroblast, excessive production of collagen synthesis by TGF-β1 and reduction of collagen degeneration (Profyris et al., 2012). Considering excessive production of collagen generation and reduction of collagen degeneration are closely related with fibroblast activity and proliferation, fibroblast is a key cell in keloid and hypertrophic scar. LLLT could mortify keloid fibroblast view in clinical use of LLLT in preventing of recurring keloid after surgical excision or laser ablation of keloid (Barolet and Boucher, 2010). However, studies about the effect of LLLT on fibroblasts from keloid or hypertrophic scar are relatively fewer than those from normal or wounded fibroblasts.
Thus, this study aimed to investigate the effect of blue (415 nm), red (632 nm) and infrared (830 nm) light emitting diodes (LED) to wound healing, especially in normal and keloid dermal fibroblasts.
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II. MATERIALS AND METHODS
A. Isolation and culture of normal and keloid fibroblasts
Normal fibroblasts were from human foreskin after phimosiectomy and keloid fibroblasts were from keloid-revision surgery. After washing with phosphate buffered saline (PBS) and removing fat, the dermal fibroblasts extracted from skin. Normal and keloid dermal fibroblasts cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum and 1% streptomycin at 37°C under a humidified atmosphere of 5% carbon dioxide. Fibroblasts were grown as monolayers on plastic Petri dishes and used 10th and 30th passages.
B. Irradiation source
APL® (Medro medical division, Seoul, Korea) was used for this study. This device produced blue light (415 nm), red light (632 nm), and infrared light (830 nm). The power fluence of each wavelength is 172 mW/cm2 for blue, 205 mW/cm2 for red, 50 mW/cm2 for infrared. During irradiation, cells were maintained in 0.5ml PBS and the distance from light source is 1 cm. After irradiation, PBS was replaced by DMEM.
C. Viability assay
To find out whether LLLT could affect to cell viability, EZ-CyTox cell viability assay kit (Daeillab, Korea) was used. Normal and keloid fibroblasts were seeded at 6 x104 cells/well in 24-well plate and were irradiated with 3, 10, and 30 J/cm2 energy fluence of LED. After irradiation, the cells were incubated for 24 hours at the atmosphere above mentioned. EZ-CyTox solution (40 μl) was added to each well and the mixtures were incubated for 3 hours at 37°C. Absorbance was measured for 3 hours using an ELISA reader (Molecular Device, Menlo Park, CA, USA) at 450nm.
D. Proliferation assay
Trypan blue exclusion assay was done for measure proliferation rate. Many previous studies about effect of LLLT used very variable fluences of light. As preliminary study to
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determine the most suitable fluence of irradiation, we conducted 3, 10, and 30 J/cm2 fluence of LED to normal and keloid fibroblasts. Normal fibroblasts were seeded at 1 x 105 cells/well and keloid fibroblasts were seeded at 8.5 x 104 cells/well in 24-well plate.
Because cells were filled up about 80% of plate to giving uniformly irradiation to each cells, the count of fibroblasts per well is different between normal and keloid fibroblasts.
After irradiation, the cells were incubated for 24 hours at the atmosphere above mentioned. The DMEM in each well was aspirated and replaced by 0.12% trypsin to detach cells from well. Then, 50 μL of this cell suspension were added to 50 μL of 0.04%
trypan blue dye. To counting live cells, 10 μL of the solution were taken to a hemocytometer and examined the microscope (Olympus, Tokyo, Japan). Proliferation rate was defined the ratio of live cell count of irradiation group after 24 hour per that of non-irradiated control. After choosing the most suitable light fluence for both fibroblasts, we repeated the proliferation assay at the determinate light fluence.
D. Migration assay
Normal fibroblasts were seeded at 3 x 105 cells/well and keloid fibroblasts were seeded at 2.5 x 105 cells/well in 6-well plate for cell migration assay. To make a cell-free area, each dish was scratched straight on the center by sterile 2 mL yellow pipette tip.
After wash-out with PBS, each wavelength of LEDs irradiated to the fibroblasts. After LED irradiation, the cells were incubated and we took photos at the 0, 12, 24 hours at the same point of each well using microscope (Olympus, Tokyo, Japan). We analyzed with the photos, and calculated cell migration area into the hollow space using computer software (Image-Pro Plus, MEDIA CYBERNETICSA, Silver Spring, MD, USA).
Migration rate (%) is defined the ratio of migration area into the hollow space after 24 hours per hollow space induced by pipette tip at the initial.
G. Statistical analysis
Data are expressed as mean and standard deviation (SD). A one-way analysis of variance with Dunnett’s multiple compression test using SPSS Statistics Desktop 20.0.0 (IBM, Armonk, NY, USA) was performed. A P value <.05 was considered statistically
5 significant.
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III. RESULTS
A. Viability assay
Three times of experiments are conducted to find out whether LLLT could affect to cell viability. Normal fibroblasts irradiated with 3 J/cm2 in all wavelengths showed slightly higher viability and normal fibroblasts irradiated with 10 J/cm2 in all wavelengths showed slight lower viability than nonirradiated normal control. Keloid fibroblasts were shown higher viability than normal fibroblasts, regardless of the light fluences and wavelengths. Keloid fibroblasts irradiated with any fluence and any wavelength showed slight lower viability rate than nonirradiated keloid control. However, there was no statistically significant effect of LLLT on both normal and keloid fibroblasts in viability assay (Fig. 1.and Table 1).
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Fig. 1. Viability assay of normal and keloid fibroblasts after 24 hours of irradiation with 415, 632, and 830 nm, at fluence of 3, 10, and 30 J/cm2. Normal fibroblasts irradiated with 3 J/cm2 in all wavelengths showed slightly higher viability and normal fibroblasts irradiated with 10 J/cm2 in all wavelengths showed slight lower viability than nonirradiated normal control. Keloid fibroblasts irradiated with any fluence and any wavelength showed slight lower viability rate than nonirradiated keloid control.
8 B. Proliferation assay
After performed twice experiences, proliferation rate of normal fibroblasts has a tendency to increase after irradiation in all fluences and all wavelengths. However, there were not dose-dependent results in proliferation rate of normal fibroblast among 3, 10 and 30 J/cm2 irradiation group (Fig. 2.). If irradiation at 3, 10 and 30 J/cm2 made similar effect on cells, using lowest dose is more suitable and convenient for executing study.
Proliferation rate of keloid fibroblasts at 3 J/cm2 in all three wavelengths was higher than nonirradiated control, while proliferation rate at 10 and 30 J/cm2 is lower than nonirradiated control (Fig. 2.). If irradiation at 10 and 30 J/cm2 showed similar effect on cells, using lower dose is more suitable and convenient for executing study.
Thus, normal fibroblasts were irradiated at 3 J/cm2 and keloid fibroblasts were irradiated at 10 J/cm2 in further repeated study.
Fig 2. Proliferation rate (%) of normal and keloid fibroblasts after 24 hours of irradiation with 415, 632, and 830 nm, at the 3, 10, and 30 J/cm2. Proliferation rate of normal fibroblasts has a tendency to increase after irradiation in all three fluences and all wavelengths. Proliferation rate of keloid fibroblasts is increased at the fluence of 3 J/cm2, but decreased at the fluence 10 and 30 J/cm2.
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Normal fibroblasts were irradiated by fluence of 3 J/cm2 and results calculated as mean and standard deviation were from 5 times of experience. Comparing with nonirradiated control, proliferation rate of normal fibroblasts irradiated with 415 nm was 113.25 ± 8.13, that with 632 nm was 112.8 ± 5.87, and that with 830 nm was 105.61 ± 3.82 (Table 2).
Only at the 632 nm, there was significantly increase the proliferation rate after LLLT (P=0.034, Fig. 3.). Keloid fibroblasts were irradiated by fluence of 10 J/cm2 and results were from 6 times of experiments. Comparing to nonirradiated control, proliferation rate irradiated with 415 nm was 85.02 ± 10.24, that with 632 nm was 82.42 ± 7.33, and that with 830 nm was 89.88 ± 11.84 (Table 2). Only at the 632 nm, there was significant decreased the proliferation rate of keloid fibroblasts after LLLT (p=0.010, Fig. 3.).
Table 2. Proliferation rate after LLLT at 3 J/cm2 in normal fibroblasts and at 10 J/cm2 in keloid fibroblasts.
Normal fibroblasts Keloid fibroblasts Mean, SD (%) P value Mean, SD (%) P value
Control 100 ± 0 100 ± 0
415 nm 113.25 ± 8.13 0.089 85.02 ± 10.24 0.070
632 nm 112.8 ± 5.87 0.034 82.42 ± 7.33 0.010
830 nm 105.61 ± 3.82 0.122 89.88 ± 11.84 0.344
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Fig. 3. Proliferation rate after LLLT at 3 J/cm2 in normal fibroblasts and at 10 J/cm2 in keloid fibroblasts. Normal fibroblasts irradiated with the 632 nm significantly increased the proliferation rate than control. Keloid fibroblasts irradiated with the 632 nm were significantly decreased the proliferation rate than control.
11 C. Migration assay
Normal fibroblasts were irradiated by fluence of 3 J/cm2 and the results calculated as mean and standard deviation were from 8 times of experiments. Cell migration rate (%) in control was 66.81 ± 11.57, that in 415 nm was 71.16 ± 11.27, that in 632 nm was 74.64 ± 10.60, and that in 830 nm was 85.44 ± 6.83 (Table 3). Although normal fibroblasts irradiated in any wavelength showed more migration rate than in nonirradiated control, only when irradiated with 830 nm were significant (p=0.013, Fig.
4a. and 4b.). Keloid fibroblasts were irradiated by fluence of 10 J/cm2 and results were from 20 times of experiments. Cell migration area (%) in control was 75.02 ± 14.64, that in 415 nm was 79.15 ± 11.78, that in 632 nm was 81.74 ± 7.33, and that in 830 nm was 82.86 ± 5.77 (Table 3). Although cells irradiated in each wavelength show more migration than in nonirradiated control, there is no significant difference (Fig. 4a. and 4c.).
Table 3. Migration rate after LLLT at 3 J/cm2 in normal fibroblasts and at 10 J/cm2 in keloid fibroblasts.
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control 415nm 632nm 830nm
0 20 40 60 80 100
Migration rate (%)
Normal Keloid
*
A
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Fig. 4. Migration assay after LLLT at 3 J/cm2 in normal fibroblasts and at 10 J/cm2 in keloid fibroblasts. Migration rate (%) of normal fibroblasts was significant increased only after irradiation with 830 nm. That of keloid fibroblasts irradiated was increased slightly than nonirradiated control, but it was no statistically significant (A). Photos of normal fibroblasts irradiated with 3 J/cm2 were taken after 0, 6, 12, and 24 hours (B).
Photos of keloid fibroblasts irradiated with 10 J/cm2 were taken after 0, 6, 12, and 24 hours (C).
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IV. DISCUSSION
According to a definition of LLLT, various wavelength and energy dose are used in each study. Whereas blue light had no effect to normal fibroblast in our study, other study revealed daily irradiation at 410 nm or 420 nm inhibit proliferation of dermal fibroblast at 5-10 J/cm2 (Oplander et al., 2011). As it is reveals that the blue light has anti-inflammatory effect and phototoxic effect on Propionibacterium acne, LLLT with blue light is used in acne treatment in clinically rather than wound healing (Kwon et al., 2013).
Red light appears to be the most frequently investigated wavelength at the cellular level.
Single irradiation of 5 J/cm2 or irradiation of 2.5 J/cm2 on two consecutive days using 632 nm light stimulated migration and proliferation of fibroblasts. Furthermore, higher doses (10 and 16 J/cm2) decreased in cell viability and cell proliferation with a significant amount of cell damage (Hawkins and Abrahamse, 2006). When normal wounded skin fibroblast cells were irradiated at 632, 830, or 1,064 nm, cells irradiated at 632 nm with 5 J/cm2 showed highest migration rate (Evans and Abrahamse, 2008; Houreld and Abrahamse, 2008; Houreld and Abrahamse, 2010), while cells irradiated at 830 nm showed higher migration rate in our study. The previous reported study revealed that fibroblasts in diabetic condition increase cell migration, proliferation and collagen production after irradiation with 660 nm diode laser at 5 J/cm2 (Ayuk et al., 2012). The studies executed with human gingival fibroblasts revealed that irradiation with each 670, 685, 692, 780, or 786 nm light at 2 or 3 J/cm2 accelerated proliferation and migration of gingival fibroblasts (Almeida-Lopes et al., 2001; Saygun et al., 2008; Basso et al., 2012).
On the near-infrared wavelength region, irradiation of 830 and 980 nm influences fibroblasts mitochondrial activity compared to the 2,940 nm wavelength which produces apoptosis (Crisan et al., 2013). Thought in a diabetic condition, wounded fibroblasts showed a significant increase in proliferation after 830 nm diode laser irradiation with a fluence of 5 J/cm2 (Houreld et al., 2010). These different results of proliferation and migration could be related to the cell type and/or the wavelength used in each study. Also in this study, there is no significant effect of LLLT in EZ-CyTox proliferation assay, contrast to in trypan blue proliferation assay.
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Although some studies reveal the mechanism of effect of LLLT, most part of mechanism of migration or proliferation of fibroblasts after LLLT are unrevealed. It is thought that photon from LLLT absorbs in cytocrome c enzyme in mitochondria, and makes an intracellular response, such as activating transcription factors, increasing reactive oxygen species, and nitric oxide. Irradiation of red (632 nm, 636 nm, and 638 nm) light is significantly increased keratinocyte growth factor, hepatocyte growth factor, basic fibroblast growth factor, and interlukin (IL)-6 and decreased tumor necrosis factor (TNF)-α, and IL-1β (Evans and Abrahamse, 2008; Sekhejane et al., 2011; Fushimi et al., 2012). Normal wounded fibroblasts increased their proliferation and decreased TNF-α and IL-1β after irradiation at 830 nm with 5 J/cm2 (Houreld et al., 2010). Exposing fibroblasts to near infrared radiation was shown to induce reactive oxygen species formation and lead to the subsequent increased expression of matrix metalloproteinase (Danno et al., 2001; Schroeder et al., 2008). However, further studies are needed to investigate how these alterations of growth factors and cytokines induce proliferation and migration of fibroblasts.
Studies about LLLT affect on fibroblasts derived from keloid or hypertrophic scar are only a few. Irradiation with 660 nm reduced keloid fibroblast death significantly (Frigo et al., 2010) and stimulated proliferation of fibroblasts derived from hypertrophic scar (Webb et al., 1998). While most studies show LLLT effect to fibroblast to increase proliferation, one study which is used light with 880 nm at 2.4 and 4 J/cm2 revealed that both normal and hypertrophic scar derived fibroblasts showed mild decrease of cell number (Webb and Dyson, 2003). Furthermore, irradiation with 470 nm LED with 60, 122 and 183 J/cm2 did not affect in proliferation of keloid fibroblasts(Bonatti et al., 2011). Because studies about the effect of LLLT on keloid or hypertrophic scar derived fibroblasts are relatively fewer than normal fibroblasts, there is no consensus of LLLT effect and further studies are needed to investigate.
Although this study is focused on fibroblasts, LLLT could also promote biostimulatory effects on keratinocytes. LLLT promoted an increase of keratinocyte metabolism, proliferation, and type I collagen and vascular endothelial growth factor (VEGF) gene expression (Basso et al., 2013). LLLT using 638 nm or 518 nm light promoted the
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migration of keratinocytes, and only 518 nm light make increase gene expression and cytokine secretion of heparin-binding epidermal growth factor-like growth factor and VEGF (Fushimi et al., 2012). In vivo, LLLT may affect both keratinocytes and fibroblasts and it could make synergetic effect to promote wound healing.
This study has some limitations. Each of two proliferation assays was shown different results in this study. It could be explained that effect of LLLT is slight week to detect by EZ-CyTox proliferation assay or more repeated studies are needed. Light dose of normal and keloid fibroblasts is different, because these two cell lines is complete different. So, we conducted the preliminary study to determine most effective dose of each cells and chose the most suitable light dose. Although we only focused on proliferation and migration of fibroblasts in this study, wound healing is a multi-complex process participating various cells, including keratinocyte, endothelial cells and inflammatory cells. Even if specific light promotes proliferation and migration of fibroblast, this contributes a small part of wound healing processing. We cannot prove any specific cytokines, chemokines, molecules, or genes which induced proliferation or migration of fibroblasts. Another consideration is that successful in vitro results do not always translate to the in vivo application. Further studies about molecular and gene levels of LLLT effect which induced proliferation or migration of fibroblasts is needed.
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V. CONCLUSION
In conclusion, this study demonstrates LLLT effect on normal fibroblasts. Irradiation with 632 nm light could accelerate proliferation of fibroblasts, and irradiation with 830 nm light could enhance wound healing by increasing migration of fibroblasts. Although further studies are needed, when irradiating with 632 nm light, it seems inhibit proliferation of keloid fibroblasts. However, it is likely to occur on a higher dose than the dose irradiated on normal fibroblasts.
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