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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