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The Journal of Veterinary Medical Science logoLink to The Journal of Veterinary Medical Science
. 2024 Nov 13;87(1):90–96. doi: 10.1292/jvms.24-0318

Effects of low-level laser irradiation on canine fibroblasts

Melpa Susanti PURBA 1,4, Dito ANGGORO 1,4, Harumichi ITOH 2, Kazuhito ITAMOTO 2, Yuki NEMOTO 3, Munekazu NAKAICHI 3, Hiroshi SUNAHARA 4, Kenji TANI 4,*
PMCID: PMC11735224  PMID: 39537157

Abstract

Low-level laser (LLL) therapy is a well-known noninvasive treatment that stimulates fibroblasts to improve wound healing. LLL can improve fibroblast proliferation and migration without causing toxicity. The present study aimed to evaluate the effects of two laser wavelengths at different irradiation times on canine fibroblasts. Fibroblasts were isolated from canine oral mucosa. After seeding for 24 hr, the fibroblasts were irradiated using the Erchonia® EVL dual-diode laser at wavelengths of 405 nm (5 mW) and 640 nm (7.5 mW) with irradiation times of 120, 360, and 1,800 sec. The proliferating and viability cells were evaluated 24 hr after laser irradiation. Wound closure rates were calculated at 0, 24, and 48 hr after laser irradiation. Parameters, including proliferation cell, cell viability, and cell migration, tended to be higher in the 360-sec group (405 nm) and 120-sec group (640 nm) than in other groups. Our findings suggest that LLL therapy at wavelengths of 405 and 640 nm with an irradiation time of 120–360 sec (0.26–0.51 J/cm2) can stimulate the proliferation and migration of canine fibroblasts. This finding may contribute to a better understanding of the beneficial role of LLL stimulation in canine wound healing.

Keywords: blue light, canine fibroblast, red light, wound healing

INTRODUCTION

Fibroblasts play a crucial role in wound healing by contributing to tissue repair through various mechanisms. Fibroblasts produce extracellular matrix (ECM) components, promoting cell adhesion, tissue re-epithelialization, angiogenesis, and collagen formation [7, 13, 16, 42]. They are potential cells for regenerative medicine because they express high levels of intracellular fibroblast growth factors [54], enhancing cell proliferation and migration [29, 49, 53].

Low-level laser (LLL) therapy (also known as photobiomodulation) is a noninvasive treatment modality that uses low-power lasers with a specific wavelength of light to stimulate cellular processes without causing tissue heating, thereby promoting wound healing and reducing pain in various in vitro and in vivo studies [2, 3, 9, 28, 45]. LLL therapy has been applied for fibroblast biostimulation to improve wound healing [15, 17, 32, 48]. Fibroblast proliferation and migration are essential parameters to enhance wound healing processes. Previous studies reported that applying LLL therapy in human and animal model cells increases cell proliferation and migration [39, 50, 51].

LLL therapy is usually performed with visible or near-infrared laser light (390–1,100 nm) with relatively low fluences (0.04–50 J/cm2) and power densities (<100 mW/cm2) [2, 3, 37, 39]. The wavelength of visible light varies from 400 nm to 700 nm, which is divided by color, including violet/blue (400–500 nm), green (500–565 nm), yellow (565–590 nm), orange (590–625 nm), and red (625–700 nm) [43]. Previous studies have shown the beneficial effects of LLL therapy on wound healing in humans and animals using violet/blue light [10, 35, 46] and red light [8, 11, 30, 33, 36, 47, 52]. These studies reported that LLL therapy stimulates cell proliferation and migration.

LLL therapy has been practically reported to enhance wound healing in veterinary medicine. In dogs, Perego [41] reported that LLL therapy resulted in significant differences in post-surgical wound healing and decreased the amount of exudates in the surgical area. LLL therapy also exhibited positive healing effects in canine osteoarthritis [4], skin disease [40], and chronic wounds [22]. However, there is limited information regarding the effects of LLL therapy on canine fibroblasts, which play a crucial role in wound healing processes. Therefore, the present study aimed to investigate the effects of LLL irradiation at two different wavelengths (405 and 640 nm) and three different irradiation times (120, 360, and 1,800 sec) on canine fibroblast proliferation and wound closure. This study used primary oral mucosa fibroblast because it closely represents the origin tissue since the cells are taken directly from the tissue and not modified. Therefore, the results of this study are expected to be more representative of the tissues, which provide similar effects to in vivo and clinical conditions. We hypothesized that LLL therapy using 405 and 640 nm wavelengths can improve the proliferation and migration of canine oral mucosa fibroblasts. The results can provide data on canine fibroblasts to improve the application of LLL therapy in the veterinary field.

MATERIALS AND METHODS

Oral mucosa fibroblast cell culture

The protocol for collecting oral mucosa tissues was approved by the Committee on the Ethics of Animal Experiments of Yamaguchi University, Japan (Protocol Number 484). The oral mucosa tissues of three healthy beagles were collected independently using a 6-mm biopsy trepan (BIOPSY PUNCH, Kai Industries Co., Ltd., Gifu, Japan) with a thickness of tissue 3 mm. The isolation of fibroblast was performed according to the previous studies [1, 34, 55]. Briefly, the cells were isolated using 5,000 units of collagenase type I (Collagenase, Fujifilm Wako Pure Chemical Co., Ltd., Osaka, Japan) and cultured in Dulbecco’s Modified Eagle Medium (DMEM®, Life Technologies Corp., New York, NY, USA) with 10% fetal bovine serum (FBS, Thermo Fisher Scientific Group, Tokyo, Japan), and 100 U/mL penicillin and 100 μg/mL streptomycin (Penicillin-Streptomycin, Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan). The oral mucosa tissues were incubated overnight at 37°C under 5% CO2 and then collected. The cells were cultured with DMEM medium in an incubator for 1 hr. Then, the medium was aspirated, and the new medium was added. Since healthy fibroblasts rapidly adhere to the flask, this process removes the weakened fibroblast and other cells (such as mucosal epithelial cells). The cells were cultured for 3–5 days until fibroblast proliferation and adhesion were confirmed. Subsequently, primary fibroblasts were seeded in a new DMEM in an uncoated T-75 flask (FALCON®, Corning International Inc., Tokyo, Japan) and cultured until the cells were at 70–80% confluency. The second passage of fibroblasts was used in the experimental procedure.

Laser irradiation

LLL therapy was performed using the Erchonia® EVL dual-diode laser (Erchonia Corp., Melbourne, FL, USA), which provides two laser diodes (405 nm [violet/blue laser beam] with an output of 5 mW and 640 nm [red laser beam] with an output of 7.5 mW). Each diode emits its wavelength with a tolerance of ± 10 nm. Different groups were treated using two wavelengths (405 and 640 nm) with power output densities of 5 and 7.5 mW for 120, 360, and 1,800 sec (Table 1). The irradiation time was determined within the range of previous reports [2, 24, 36, 39]. The energy density (J/cm2) for all treatment groups was calculated by multiplying the exposure time (sec) by the laser’s power output (W), divided by the surface area (cm2). The surface area exposed to LLL therapy was identical to a 12-well plate (3.5 cm2). The fibroblasts in the well plate without irradiation were treated similarly and used as a control. The laser’s position was in contact with the bottom of the 12-well plate cover, with the laser beam placed at a 90° angle to the bottom of the 12-well plate, which was positioned in a stable supporting structure from Erchonia Corporation.

Table 1. Energy density of different groups.

Laser diode 120 sec 360 sec 1,800 sec
405 nm 0.17 J/cm2 0.51 J/cm2 2.57 J/cm2
640 nm 0.26 J/cm2 0.77 J/cm2 3.86 J/cm2
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Lactate dehydrogenase (LDH) activity assay

The LDH enzyme was assessed using a commercially available kit for measuring LDH (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories, Kumamoto, Japan). Canine oral mucosa fibroblasts were seeded in 12-well plates at an initial density of 1 × 105 cells/well and incubated for 12 hr. The laser irradiation protocol was performed as previously described (Table 1). The non-irradiated fibroblasts were used as a control group. After treatment for 24 hr, 100 μL of cell culture medium was transferred into a 96-well plate and added with 100 μL of LDH reaction solution, prepared according to the manufacturer’s guidelines. The plate was covered with foil and incubated at room temperature for 30 min. Subsequently, 50 μL of stop solution was added, and the optical density of the solution was directly measured at 490 nm using an Epoch spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The LDH secretion data were presented as absorbance with optical density value. All experiments were performed thrice for repeatability.

Cell Counting Kit-8 (CCK-8) assay

Canine oral mucosa fibroblasts were seeded in 12-well plates at an initial density of 1 × 105 cells/well and incubated for 12 hr before exposure to laser irradiation. After irradiation, the cells were incubated for 24 hr. For the cell proliferation assay, 100 μL of cell culture medium and 10 μL of CCK-8 (Dojindo Laboratories) solution were added to each well. After incubation at 37°C for 2 hr, the optical density of the solution was measured at 450 nm using an Epoch spectrophotometer. The proliferation cell data were presented as absorbance with optical density value. The cell viability was shown as a percentage calculated according to the manufacturer’s guidelines. The cell viability was calculated by dividing the average absorbance from each sample (sample-blank) by the control absorbance (control-blank) and multiplying by 100 (control: CCK-8 with medium and cells in a well [non-irradiated]; sample: CCK-8 with irradiated cells and medium; blank: CCK-8 and medium without cells in a well). All experiments were performed thrice for repeatability.

Scratch wound healing assay

Scratch assay was performed to assess the effect of LLL irradiation on canine oral mucosa fibroblasts in response to injury. The canine oral mucosa fibroblasts were cultured in 12-well plates (2 × 105 cells/well) and incubated for 24 hr until a confluent monolayer of cells was confirmed. The scratch was performed across the cell layer with a 200-μL sterile pipet tip. The medium was removed, and the cells were washed with phosphate-buffered saline to remove debris. Then, 300 μL of DMEM was added, and irradiation was performed using LLL following the irradiation times (Table 1). After irradiation, 700 μL DMEM medium was added, and the cells were incubated. Photos were taken at 0, 24, and 48 hr after treatment via an inverted fluorescence microscope (Olympus, Tokyo, Japan) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The wound closure rate was calculated by measuring the scratch area, which was not covered with fibroblast at 24 and 48 hr after treatment. The experiments were performed thrice for repeatability.

Statistical analysis

All data were represented as the mean ± standard deviation of three independent experiments. The data were analyzed using a one-way analysis of variance followed by the Tukey–Kramer honestly significant difference test (for multiple comparisons test). The calculations were performed using JMP software, version 9.0 (SAS Institute Japan, Tokyo, Japan). Statistical significance was considered at P<0.05.

RESULTS

Cell viability and LDH assay

The effects of LLL irradiation under different irradiation times on canine oral mucosa fibroblasts at 24 hr after irradiation are shown in Fig. 1. The fibroblast proliferation and viability tended to be higher in irradiated than in non-irradiated groups at both wavelengths of the laser diode. The highest proliferation of fibroblasts was observed in the 360-sec group (0.51 J/cm2) at a wavelength of 405 nm and in the 120-sec group (0.26 J/cm2) at a wavelength of 640 nm (Fig. 1A). Nevertheless, no significant differences were observed between the irradiated and non-irradiated groups at both wavelengths. The same results were also observed in terms of viability (Fig. 1B), wherein the highest viability was observed in the 360-sec group at a wavelength of 405 nm and in the 120-sec group at a wavelength of 640 nm. The viability of fibroblasts in the 120-sec group at a wavelength of 640 nm significantly differed from that of other groups (P<0.05). The results of the LDH cytotoxicity assay are shown in Fig. 2. No significant differences were observed in the LDH levels between the irradiated and non-irradiated groups (P>0.05) with absorbance ranging from 0.375 to 0.391.

Fig. 1.

Fig. 1.

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The proliferation (A) and viability (B) of canine oral mucosa fibroblasts (using CCK-8 assay) were measured at 24 hr after irradiation with single-dose LLL irradiation at wavelengths of 405 and 640 nm for 120, 360, and 1,800 sec. Proliferating fibroblasts are expressed as absorbance with optical density value, whereas the cell viability of fibroblasts is expressed as a percentage. The mean was compared by analysis of variance followed by Tukey’s honestly significant difference test. All experiments were performed thrice for repeatability. *P<0.05. CCK-8, Cell Counting Kit-8; LLL, low-level laser.

Fig. 2.

Fig. 2.

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Absorbance of LDH assay in canine oral mucosa fibroblasts at 24 hr after being treated with single-dose LLL irradiation at wavelengths of 405 and 640 nm for 120, 360, and 1,800 sec. LDH is expressed as absorbance with optical density value. The mean was compared by analysis of variance followed by Tukey’s honestly significant difference test. All experiments were performed thrice for repeatability. LDH, lactate dehydrogenase; LLL, low-level laser.

Scratch wound healing assay

The images of the scratch assay of 405 nm and 640 nm (Fig. 3) were acquired at 0, 24, and 48 hr after irradiation. At 24 and 48 hr, the irradiated fibroblasts at wavelengths of 405 and 640 nm showed faster wound closure than non-irradiated cells (Fig. 4). At 24 and 48 hr, the 360-sec group (0.51 J/cm2) irradiated at a wavelength of 405 nm presented a rapid healing rate than other groups. Significant differences between irradiated (360 sec) and non-irradiated fibroblasts were observed at 48 hr. Meanwhile, at 24 and 48 hr, a rapid healing rate was found in the 120-sec group (0.26 J/cm2) irradiated at a wavelength of 640 nm than in other groups. Statistical differences were observed between the non-irradiated and irradiated groups (120 and 360 sec) at 24 hr. Meanwhile, at 48 hr, significant differences were observed between the irradiated (120 sec) and non-irradiated groups.

Fig. 3.

Fig. 3.

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Evaluation of wound healing of canine oral mucosa fibroblasts after being irradiated with different doses at a wavelength of 405 nm and 640 nm using scratch assay. Dotted lines indicate the scratch area at 0, 24, and 48 hr after laser irradiation. At a wavelength of 405 nm, fibroblasts that were irradiated for 360 sec showed the narrowest scratch at 48 hr. Meanwhile, at a wavelength of 640 nm, fibroblasts that were irradiated for 120 sec showed the narrowest scratch at 48 hr. Scale bars=20 µm.

Fig. 4.

Fig. 4.

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Effects of LLL therapy at wavelengths of 405 and 640 nm on cell migration. The scratch wound area was measured at 0, 24, and 48 hr after laser irradiation. *P<0.05 represents statistically significant differences between irradiated and non-irradiated groups (control). LLL, low-level laser.

DISCUSSION

In the present study, we evaluated the effects of irradiation using two laser wavelengths on the cell viability and migration of canine oral mucosa fibroblasts. In both laser wavelengths (405 and 640 nm) at different irradiation times (120, 360, and 1,800 sec), the fibroblast viability and migration tended to be higher compared with the control group (0 sec). All the treatment groups did not induce a higher level of cytotoxicity compared with the control group. This finding indicates that laser therapy under these conditions stimulates the proliferation and migration of oral mucosa fibroblasts, is not harmful, and might be safely employed for irradiation of canine oral mucosa fibroblasts.

LDH assay was performed to assess cell damage by releasing the cytosolic glycolytic enzyme LDH into the medium due to plasma membrane permeabilization after irradiation [26]. No statistically significant difference in LDH concentration was observed between the non-irradiated and irradiated groups, indicating that both laser wavelengths and their fluences were not harmful to living fibroblasts. This result is in line with a previous study [23], where LLL irradiation did not show a significant difference in LDH levels between the non-irradiated and irradiated groups.

Fibroblast viability and migration were investigated in vitro to evaluate the effects of two laser wavelengths on canine oral mucosa fibroblasts. Fibroblast viability was evaluated by assessing dehydrogenase activity in living cells. CCK-8 (also known as WST-8 assay) has been widely used to assess cell viability and cytotoxicity by detecting high NAD (P) H levels using tetrazolium salt, which directly corresponds to dehydrogenase activity and provides a measure of cellular metabolic activity [5, 12]. A scratch assay was performed to evaluate and compare cell migration. Scratch assay (wound healing assay) is a technique to evaluate cell migration in vitro by scratching a confluent cell monolayer to create a new artificial gap and observing the movement of cells in the scratch area [18, 31, 44]. Cell migration is fundamental in various biological phenomena, including tissue homeostasis, wound healing, and immune response [7, 53]. Fibroblasts migrate and proliferate into the wound site and are required to produce and secrete proteases from the ECM to heal the wound site [29, 53].

In the present study, the proliferation, viability, and migration of fibroblasts tended to be higher in all irradiated groups than in the non-irradiated groups, which was expected. This finding indicates that the laser with a wavelength of 405 and 640 nm and a specific fluence rate could promote fibroblast proliferation and migration, both essential in normal wound healing processes. Fibroblasts that were irradiated for 360 sec at a wavelength of 405 nm showed higher proliferation and viability; however, no significant differences were observed compared with other groups. In contrast, fibroblasts that were irradiated for 120 sec at a wavelength of 640 nm showed higher proliferation and viability; however, no significant differences were observed compared with other groups. Wound closure was faster in the irradiated groups than in the non-irradiated groups. The groups that were irradiated for 360 sec at a wavelength of 405 nm and for 120 sec at a wavelength of 640 nm showed the rapid wound closure than other irradiated groups. This finding indicates that using the Erchonia® EVL dual-diode laser in one-time irradiation for 120–360 sec at a fluence of 0.2–0.5 J/cm2 stimulates canine oral mucosa fibroblast proliferation and wound closure. This result is in line with previous in vitro studies using human fibroblasts at a wavelength of 640 nm [21, 48] and animal models [17, 50], which reported the advantages of LLL therapy in wound healing, including cell proliferation and migration. Prado, et al. [43] performed a scoping review of the effects of photobiomodulation with blue light (400–500 nm) on wound healing. They summarized that blue light at a low energy density (<20 J/cm2) stimulated different cell types and proteins, but blue light at a high energy density (20.6–50 J/cm2) reduced cell proliferation, migration, and metabolism. Studies on human dermal fibroblasts reported that using blue light at a wavelength of 470 nm with an energy density of 5 J/cm2 [35] and 420 nm with an energy density of 3.43 J/cm2 [46] stimulated cell proliferation. Etemadi, et al. [10] also reported that LLL therapy with blue diode laser at power densities of 400 mW/cm2 with irradiation times of 10 and 15 sec corresponding to energy densities of 4 and 6 J/cm2 exerted statistically significant positive effects on the proliferation and migration of gingival fibroblasts. These findings suggest that the combination of blue light and red light stimulates fibroblast proliferation and migration.

Overall, among the irradiated groups, the group that was irradiated for 1,800 sec (2.5–3.9 J/cm2) at wavelengths of 405 and 640 nm showed the slowest proliferation and wound closure rate, suggesting that the time and dose of irradiation are essential to the cell response. Huang, et al. [24, 25] summarized the biphasic dose responses in LLL therapy on culture cells in vitro, animal models in vivo, and clinical studies. Based on the three-dimensional model of the Arndt–Schulz curve, they suggest that too much power density and/or time may lead to the inhibition effect of LLL therapy. Another study reported that using a wavelength of 632.8 nm at high doses (10 and 16 J/cm2) increased cellular damage and decreased cellular viability and proliferation in human skin fibroblasts [21]. Similarly, Flores Luna, et al. [14] proved the principle of the Arndt–Schulz law, wherein small doses provide positive stimuli to the cellular environment and higher doses cause deleterious effects, inhibiting cellular activity. They found a significant reduction in the number of cells using 3.61 and 3.16 J/cm2 of energy compared with 0.45 and 0.75 J/cm2 of energy at 660 nm. The concept of biphasic dose–response is essential in LLL therapy because of the large number of illumination parameters for each treatment and laser characteristic [2, 3, 24, 25].

To date, the exact mechanism of LLL therapy in influencing cellular processes is not yet well established and is likely multifactorial. Studies reported that LLL therapy interacts with mitochondrial chromophores [19, 20, 27], modulates reactive oxygen species that increase energy production (adenosine triphosphate), and stimulates enzymes and transcription factors [3, 6, 20, 38, 45] that improve wound healing. Therefore, further studies should be conducted to investigate these factors in canine oral mucosa fibroblasts.

To the best of our knowledge, this is the first study that uses canine oral mucosa fibroblasts to investigate the effects of LLL irradiation in vitro on the wound healing process, especially cellular proliferation, and migration. We concluded that the Erchonia® EVL dual-diode laser with two wavelengths (405 and 640 nm) has a stimulating effect on the proliferation and migration of canine fibroblasts with an irradiation time of 120–360 sec without causing cytotoxicity. This finding may contribute to a better understanding of the beneficial role of LLL stimulation in wound healing in canines.

CONFLICT OF INTEREST

This study was partially funded by Takabayashi Sangyo Co., Ltd. The Erchonia® EVL laser was provided and lent by Takabayashi Sangyo Co., Ltd.

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