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
4
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.
6
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.
9
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.
12
control 415nm 632nm 830nm
0 20 40 60 80 100
Migration rate (%)
Normal Keloid
*
A
13
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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REFERENCES
1. Almeida-Lopes L, Rigau J, Zangaro RA, Guidugli-Neto J, Jaeger MM:
Comparison of the low level laser therapy effects on cultured human gingival fibroblasts proliferation using different irradiance and same fluence. Lasers Surg Med 29: 179-184, 2001
2. Ayuk SM, Houreld NN, Abrahamse H: Collagen production in diabetic wounded fibroblasts in response to low-intensity laser irradiation at 660 nm. Diabetes Technol Ther 14: 1110-1117, 2012
3. Barolet D, Boucher A: Prophylactic low-level light therapy for the treatment of hypertrophic scars and keloids: a case series. Lasers Surg Med 42: 597-601, 2010
4. Basso FG, Oliveira CF, Kurachi C, Hebling J, Costa CA: Biostimulatory effect of low-level laser therapy on keratinocytes in vitro. Lasers Med Sci 28: 367-374, 2013
5. Basso FG, Pansani TN, Turrioni AP, Bagnato VS, Hebling J, de Souza Costa CA:
In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts. Int J Dent 2012: 719452, 2012
6. Bonatti S, Hochman B, Tucci-Viegas VM, Furtado F, Pinfildi CE, Pedro AC, Ferreira LM: In vitro effect of 470 nm LED (Light Emitting Diode) in keloid fibroblasts. Acta Cir Bras 26: 25-30, 2011
7. Carvalho RL, Alcantara PS, Kamamoto F, Cressoni MD, Casarotto RA: Effects of low-level laser therapy on pain and scar formation after inguinal herniation surgery: a randomized controlled single-blind study. Photomed Laser Surg 28:
417-422, 2010
19
8. Crisan B, Soritau O, Baciut M, Campian R, Crisan L, Baciut G: Influence of three laser wavelengths on human fibroblasts cell culture. Lasers Med Sci 28:
457-463, 2013
9. Danno K, Mori N, Toda K, Kobayashi T, Utani A: Near-infrared irradiation stimulates cutaneous wound repair: laboratory experiments on possible mechanisms. Photodermatol Photoimmunol Photomed 17: 261-265, 2001
10. Evans DH, Abrahamse H: Efficacy of three different laser wavelengths for in vitro wound healing. Photodermatol Photoimmunol Photomed 24: 199-210, 2008
11. Frigo L, Favero GM, Lima HJ, Maria DA, Bjordal JM, Joensen J, Iversen VV, Marcos RL, Parizzoto NA, Lopes-Martins RA: Low-level laser irradiation (InGaAlP-660 nm) increases fibroblast cell proliferation and reduces cell death in a dose-dependent manner. Photomed Laser Surg 28 Suppl 1: S151-156, 2010
12. Fushimi T, Inui S, Nakajima T, Ogasawara M, Hosokawa K, Itami S: Green light emitting diodes accelerate wound healing: characterization of the effect and its molecular basis in vitro and in vivo. Wound Repair Regen 20: 226-235, 2012
13. Hawkins D, Abrahamse H: Effect of multiple exposures of low-level laser therapy on the cellular responses of wounded human skin fibroblasts. Photomed Laser Surg 24: 705-714, 2006
14. Houreld N, Abrahamse H: Low-intensity laser irradiation stimulates wound healing in diabetic wounded fibroblast cells (WS1). Diabetes Technol Ther 12:
971-978, 2010
15. Houreld NN, Abrahamse H: Laser light influences cellular viability and
20
proliferation in diabetic-wounded fibroblast cells in a dose- and wavelength-dependent manner. Lasers Med Sci 23: 11-18, 2008
16. Houreld NN, Sekhejane PR, Abrahamse H: Irradiation at 830 nm stimulates nitric oxide production and inhibits pro-inflammatory cytokines in diabetic wounded fibroblast cells. Lasers Surg Med 42: 494-502, 2010
17. Kwon HH, Lee JB, Yoon JY, Park SY, Ryu HH, Park BM, Kim YJ, Suh DH: The clinical and histological effect of home-use, combination blue-red LED phototherapy for mild-to-moderate acne vulgaris in Korean patients: a double-blind, randomized controlled trial. Br J Dermatol 168: 1088-1094, 2013
18. Mamalis AD, Lev-Tov H, Nguyen DH, Jagdeo JR: Laser and light-based treatment of Keloids - a review. J Eur Acad Dermatol Venereol, 2013
19. Oplander C, Hidding S, Werners FB, Born M, Pallua N, Suschek CV: Effects of blue light irradiation on human dermal fibroblasts. J Photochem Photobiol B 103:
118-125, 2011
20. Peplow PV, Chung TY, Ryan B, Baxter GD: Laser photobiomodulation of gene expression and release of growth factors and cytokines from cells in culture: a review of human and animal studies. Photomed Laser Surg 29: 285-304, 2011
21. Posten W, Wrone DA, Dover JS, Arndt KA, Silapunt S, Alam M: Low-level laser therapy for wound healing: mechanism and efficacy. Dermatol Surg 31: 334-340, 2005
22. Profyris C, Tziotzios C, Do Vale I: Cutaneous scarring: Pathophysiology, molecular mechanisms, and scar reduction therapeutics Part I. The molecular basis of scar formation. J Am Acad Dermatol 66: 1-10; quiz 11-12, 2012
21
23. Saygun I, Karacay S, Serdar M, Ural AU, Sencimen M, Kurtis B: Effects of laser irradiation on the release of basic fibroblast growth factor (bFGF), insulin like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3) from gingival fibroblasts. Lasers Med Sci 23: 211-215, 2008
24. Schroeder P, Lademann J, Darvin ME, Stege H, Marks C, Bruhnke S, Krutmann J: Infrared radiation-induced matrix metalloproteinase in human skin:
implications for protection. J Invest Dermatol 128: 2491-2497, 2008
25. Sekhejane PR, Houreld NN, Abrahamse H: Irradiation at 636 nm positively affects diabetic wounded and hypoxic cells in vitro. Photomed Laser Surg 29:
521-530, 2011
26. Silveira PC, Streck EL, Pinho RA: Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy. J Photochem Photobiol B 86: 279-282, 2007
27. Webb C, Dyson M: The effect of 880 nm low level laser energy on human fibroblast cell numbers: a possible role in hypertrophic wound healing. Journal of Photochemistry and Photobiology B: Biology 70: 39-44, 2003
28. Webb C, Dyson M, Lewis WH: Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts: possible mechanisms for increase in cell counts. Lasers Surg Med 22: 294-301, 1998
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연구결과: 3, 10, 30 J/cm2 의 광선 치료 시에 정상 및 켈로이드 섬유아세포 의 생존률에 차이가 없었다. 3, 10, 30 J/cm2 의 용량으로 광선을 조사 후 증
식률 비교한 예비실험에서 정상 섬유아세포는 3 J/cm2 의 용량을 조사 시에
증식률이 높아졌으나 켈로이드 섬유아세포의 경우 3 J/cm2 의 용량을 조사하
였을 때 증식률이 대조군에 비해 더 높았다. 따라서 정상 섬유아세포는 3 J/cm2 의 용량으로, 켈로이드 섬유아세포에서는 10 J/cm2 의 용량으로 광선 을 조사하였다. 632 nm 에서 통계학적으로 유의하게 정상 섬유아세포의 증식 률이 증가되었으며 켈로이드 섬유아세포의 증식률은 감소되었다. 830 nm 에 서 정상 섬유아세포의 이동률이 통계학적으로 유의하게 증가하였으나 켈로이 드 섬유아세포의 이동률은 파장별로 유의한 차이를 보이지 않았다.
결론: 632 nm 의 저출력 광선치료는 정상 섬유아세포의 증식을 촉진시키며
결론: 632 nm 의 저출력 광선치료는 정상 섬유아세포의 증식을 촉진시키며