Low-Level Laser Facilitating Proliferation,Migration,and Reactive Oxygen Species Production in L929 Mouse Fibroblasts

Abstract

Background:

Photobiomodulation therapy has been widely used in various branches of regenerative medicine because it has the advantages of noninvasive, drug-free, and safe form of light therapy. However, the complexity of the parameters of low-level laser (LLL) hinders its wider clinical application.

Materials and Methods:

Mouse fibroblasts (L929) were cultured in vitro using an 810-nm diode laser with low-level mode (E1C 0.1 W, continuous; E1R 0.1 W, intermittent; E2C 0.5 W, continuous; E2R 0.5 W, intermittent). Cell migration ability was detected by in vitro scratch test. Cell proliferation ability was detected by cell counting kit-8 (CCK-8) assay. Reactive oxygen species (ROS) fluorescence intensity was detected by an inverted fluorescence microscope. Production of ROS was detected by flow cytometry.

Results:

Scratch results show that E1C and E2R significantly increased the cell migration rate compared with the E0 group (p < 0.001). E1R also enhanced cell migration (p < 0.01). CCK-8 results show that the increase in the optical density of E1R, E1C, and E2R was higher than that of E0 (p < 0.001). The E2C group did not facilitate cell proliferation. The results of ROS fluorescence analysis and flow cytometry analysis showed that the ROS produced by L929 cells irradiated with different modes of LLL were different. The ROS production of E2C cells was the highest, followed by E2R, E1C, and E1R, and the E0 group was the lowest, with a significant difference (p < 0.01).

Conclusions:

An 810-nm diode laser of 0.1 W with continuous pulse mode and 2 cm away from the wound can maintain the appropriate level of ROS, as well as effectively promote cell proliferation and migration.

Keywords

wound healing L929 cells reactive oxygen species photobiomodulation

Introduction

The wound healing process can be divided into the following phases: the inflammatory phase, proliferative phase, and remodeling phase.¹ Tissue healing process can facilitate cellular behaviors, including proliferation, migration, and differentiation of fibroblasts, macrophages, and keratinocytes.^2–5 Fibroblasts are the main cellular components of loose connective tissue, and they proliferate massively through mitosis and begin to synthesize and secrete a large amount of collagen fibers and matrix components from 5 to 6 days.³ Accordingly, facilitating the migration and proliferation of fibroblasts could be one of the means of improving wound healing.

Photobiomodulation therapy (PBMT) has been used in regenerative medicine in recent years.^6,7 PBMT is featured with the reach potential between 1 and 1000 mW, a wavelength between 300 and 10,600 nm, and a dose from 0.01 to 100 J/cm².⁸ Franek et al.⁹ demonstrated that tissue illumination with 810 nm photobiomodulation, to a greater extent than other wavelengths, results in a significant increase in the rate of reepithelialization and cellular proliferation. Being a noninvasive, drug-free, and safe form of light therapy, PBMT can exert beneficial effects on a variety of pathological conditions, including promoting healing, alleviating pain and anti-inflammation, preventing tissue death, and restoring function.^10,11 PBMT can perform photophysical and photochemical biological functions without damaging cells or tissue by elevated temperatures.^12–14 It has been approved by the Food and Drug Administration as an important method for promoting healing processes.¹⁵ The Multinational Association for Supportive Care in Cancer/International Society for Oral Oncology guidelines point out the potential use of PBMT as a mucositis intervention.³

Underlying molecular mechanisms of PBMT have been explored. Electron transfer chain systems and metal complexes in mitochondria, in particular, the cytochrome C oxidase (CCO) molecule, are a principal intracellular target of red and near-infrared light.^16,17 Photostimulation of CCO leads to an increase in cellular adenosine triphosphate (ATP) synthesis, the release of nitric oxide, signal transduction, and a transient increase in reactive oxygen species (ROS).¹² Low-level of ROS is generated after PBMT,¹⁸ and it would lead to activation of stress/rescue response in cells and tissues, mostly mediated by nuclear factor kappa-B (NF-κB), p53, nuclear factor erythroid-2-related factor 2 (Nrf-2), redox factor-1, activator protein-1, activating transcription factor/Cyclic adenosine monophosphate-response element-binding protein, and hypoxia-inducible factor (HIF)-1 and HIF-like factors.^19,20 This early burst in ROS production has been associated both to a direct activation of NF-κB and to a transient activation of mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases pathways, which in turn can activate Nrf-2, all events finally resulting in cell proliferation stimulation.^12,21 The probable mechanism of PBMT-induced cell proliferation is summarized in Fig. 1.

FIG. 1.

Schematic representation of the hypothesized molecular mechanism underlying Low-Level Laser Therapy induced cell proliferation stimulation through the activation of redox sensitive transduction pathways.

Although PBMT is now used to treat a wide variety of diseases, it remains controversial for clinical application for the following reason.^22,23 There are significant variations in terms of dosimetry parameters: wavelength, energy, pulse structure, irradiance or power density, irradiation time, contact versus noncontact application, and repetition regimen. Lower dosimetric parameters can result in reduced effectiveness of the treatment, and higher ones can lead to tissue damage.¹⁴ Many studies of PBMT have shown negative results, which is possibly because of an inappropriate choice of laser parameters and dosage.^14,24 Therefore, choosing appropriate laser parameters, controlling appropriate ROS release amount, and maintaining effective ROS concentration during the healing process are the keys to promoting wound healing.

In our study, we conducted an investigation into the effects of low-level laser (LLL) on the proliferation, migration, and ROS generation of mouse fibroblasts (L929) in vitro. The low-level mode of the 810-nm diode laser was used to preliminarily explore the effects of changing power and pulse type on mouse fibroblasts so as to provide an experimental basis and theoretical basis for the application of LLL in skin mucosal wound healing and clinical transformation.

Materials and Methods

Cell culture

The experiment was conducted using fibroblasts derived from the L929 cell line (ATCC CCL-1 NCTC clone 929), supplied by the Central Laboratory of China Medical University. L929 cells were cultured in minimum essential medium (MEM; Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Clark, USA) and 1% penicillin/streptomycin (Gibco, USA) in a 5% CO₂ at 37°C humidified atmosphere. Cell morphology was observed by inverted microscope, and cells between passages six and nine were employed.

Experimental group and LLL irradiation

The experiment was conducted with a diode laser, which was delivered by an optical fiber of 400 nm in diameter that was expanded at the tip of the fiber and irradiated a circular area (spot size 1 cm²) at the cell layer level (Table 1). The laser was positioned vertically above each culture dish at a distance of 2 cm (Fig. 2),²⁵ and the irradiation time was 5 min.¹⁸ A power meter Pronto-250 (Gentec Electro-optics, Inc. Canada) was employed to monitor the power at the target and 2 cm away from the culture plate to ensure the repeatability of the experiment. L929 cells were cultivated and separated into five groups (Table 2). Experimental groups were irradiated twice a day for 3 days,²⁶ whereas the control group was not subjected to LLL but was removed from the incubator for the same time. All groups were performed in the absence of the FBS basal medium.

FIG. 2.

LLL irradiation. LLL, low-level laser.

Table 1.

Detailed Parameters of Laser

Laser brand and model	Diode laser (CAO group, USA)
Wavelength	810 ± 10 nm
Output power	0.1–9 W, adjust with 0.1 W amplitude
Intermittent pulse frequency	10 Hz
Pulse width	0.05 ± 0.005 s
Divergence of the laser beam	13° ± 1°
Working mode	Continuous mode and pulse mode

Table 2.

Five Groups of L929 Cells Cultured Under Different Conditions

	Power output (W)	Energy densities (J/cm²)	Pulse structure
E0 (control group)	0	0	—
E1R	0.1	0.5	Intermittent pulse
E1C	0.1	1	Continuous pulse
E2R	0.5	2.5	Intermittent pulse
E2C	0.5	5	Continuous pulse

Cell migration assay

The migration of L929 cells was measured using a scratch assay. L929 cells were seeded and allowed to form a homogeneous monolayer. After cultivation for 24 h, the complete medium was removed, and basal medium without FBS was added. The monolayer of cells was scratched to induce a gap without cell attachment after 12 h. The other side of the wound perpendicular to the scratch was marked with a blue ink string for reference. Laser irradiation mode in part 2.2 was applied in each irradiated group for 3 days, respectively. Images were taken at the same positions of the plate at 24, 48, and 72 h points post-scratching. Images were quantified using Image J software to measure the mobility.

Mobility = (area of scratch area in the experimental group - area of scratch area in the control group)/area of scratch area in the control group ×100%

Cell proliferation assay

The proliferation of L929 cells was measured using a cell counting kit-8 (CCK-8; Beyotime, China) according to the manufacturer’s instructions. Briefly, L929 cells were seeded, and irradiated groups were performed with laser following 2.2 laser irradiation mode for 3 days. After 1, 2, and 3 days, the medium was replaced with 350 μL of mixed solution, consisting of basal medium and CCK-8 solution at a ratio of 10:1. After incubation for 1 h, the optical density (OD) was measured at 450 nm by a microplate reader (Tecan, Salzburg, Austria).

ROS intensity detection by fluorescence microscopy

Intracellular ROS amount was measured using a ROS assay kit (Beyotime Biotechnology, China), which is a 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Cells were divided into control (E0 and positive control [PC]) or irradiated groups (E1R, E1C, E2R, and E2C) as mentioned above. As PC of ROS generation, cells treated with 1.5 mL MEM (with 2 μL Rosup) were used. After irradiation, the medium was replaced by 1.5 mL of mixed solution, consisting of MEM and DCFH-DA solution at a ratio of 2000:1. All groups were incubated for 0.5 h and then washed with MEM. Thus, ROS production was measured by fluorescence microscopy (Nikon, Japan) with emission of 488 nm. The intensity of fluorescence was measured by Image J and Origin programs.

ROS generation by flow cytometry

After L929 incubation under the same conditions, intracellular ROS levels were detected by flow cytometry. Cells were grouped and loaded with the DCFH-DA probe as mentioned above. After washing the cells with MEM, the cells were digested with trypsin and collected into an endogenous pyrogen tube. After centrifugation at centrifugal radius of 10 cm, 111.8 g for 5 min, 1 mL MEM was suspended in a flow tube. The cells were assayed in a flow cytometer (BD LSRFortessa, USA), and the data were analyzed with Flowjo software.

Statistical and data analysis

Each experiment was conducted in triplicate. All data were expressed as the mean ± standard deviation. The statistical analysis was performed with SPSS 21.0 software via a t-test and one-way analysis of variance, followed by the Tukey’s post-test, where p < 0.05 was considered to be significant.

Results

Effects of the LLL on L929 migration

Cells were fusiform and spread out on the surface of the petri dish (Fig. 3). Cell nuclei were round, and cell division was visible, indicating that the cells were in good condition. The results of the cell migration assay showed that compared with the E0 group, the cell mobility of the E1R group, the E1C group, and the E2R group increased significantly after 72 h. L929 cells exposed to E1C showed the highest migration rate (63.18 ± 1.32%), followed by E2R (26.40 ± 0.79%) and E1R (23.83 ± 0.92%). Cell migration was significantly inhibited in the E2C group (10.97 ± 0.49%) (Fig. 4a). As presented in Figure 4b, the mobility of E1C was significantly higher than that of the E0 group after 24 h (p < 0.01), followed by E2R (p < 0.05). E1R and E2C groups did not show any significant differences compared with E0 (p > 0.05). At 72 h, the number and mobility of cells in the E0 group decreased significantly. In contrast, compared with the E0 group, the mobility of E1C and E2R groups was significantly increased (p < 0.001). The mobility of the E1R group was also increased (p < 0.01). However, there was no significant difference between the E2C and the E0 group (p > 0.05).

FIG. 3.

L929 growing in vitro (×100).

FIG. 4.

(A) L929 cells in each group were observed at 0, 24, 48, and 72 h after scratching in vitro (scale bar = 100 μm). (B) Quantitative analysis of L929 fibroblast cell scratch closure 24, 48, and 72 h after the scratch assay. The data are presented as the mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 by t-test). (C) The proliferation ability of L929 cells in each group was analyzed by CCK-8, and the OD of each group was shown within 24–72 h. Results are expressed as the mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA). ANOVA, analysis of variance; CCK-8, cell counting kit-8; OD, optical density; SD, standard deviation.

Effects of the LLL on L929 proliferation

CCK-8 results showed that after cultivation for 24 h, the OD value of the cells irradiated at E1C was significantly higher than those of the control cells (p < 0.001), followed by E2R (p < 0.01), and the corresponding result is shown in Figure 4c. However, there was no significant difference between the E1R, E2C, and E0 groups (p > 0.05). After 48 h, the number of cells in the laser treatment group increased in different degrees. After 72 h, the number of cells in the E0 group decreased, and proliferation was inhibited. In contrast, the increase in the OD values of the E1R, E1C, and E2R was higher than that of E0 group (p < 0.001); however, there was no statistical difference between the E2C and the E0 group (p > 0.05).

Effects of the LLL on L929 ROS

ROS fluorescence intensity analysis

The results of ROS fluorescence intensity analysis show that ROS levels in the E0, E1R, E1C, E2R, E2C, and PC groups were observed under 10- and 20-fold fluorescence microscopes, as shown in Figure 5A. The green fluorescence intensity indicates the ROS production level. This study observed that the fluorescence intensity of the PC group was the highest, followed by the E2C group. However, the ROS levels were lower in the E1R, E1C, and E2R groups. The ROS levels were lowest in the E0 group. ROS production was also semiquantified using image J software. We found that from group E0 to group E2C, the wave peaks gradually increased, and the area under the curve also gradually increased (Fig. 5B). It was found that the average fluorescence intensity gradually increased from the E0 group to the E2C group (Fig. 5C), and there were significant differences compared with the E0 group (p < 0.01). There was also a significant difference among the E1R, E1C, E2R, and E2C groups (p < 0.05).

FIG. 5.

(A) The production of ROS in L929 cells induced by laser irradiation with different parameters was observed under 10- and 20-fold fluorescence microscopy (scale bar = 100 μm). (B) Quantitative analysis of the different groups effect on L929 cell-induced ROS generation. (C) Fluorescence density analysis of individual cells in each group. The data are presented as the mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 by t-test). ROS, reactive oxygen species; SD, standard deviation.

Flow cytometry analysis

Flow cytometry results showed that ROS generation increased after laser irradiation (Fig. 6A). The increased expression of ROS was elevated upon laser irradiation. As illustrated in Figure 6B, ROS production from left to right was E0, E1R, E1C, E2R, E2C, and PC and increased in turn. Quantitative validation of this result was performed with the mean fluorescence intensity assay. The results of Figure 6C demonstrated that the fluorescence intensity of ROS in the laser irradiation group is significantly higher than that in the E0 group (p < 0.01). There were also significant differences between the laser irradiation groups (p < 0.05).

FIG. 6.

(A) Flow cytometry was used to detect the influence of LLL on ROS under different parameters. (B) Peak fluorescence intensity of each group. (C) Quantitative analysis of the different groups effect on L929 cell-induced ROS generation. The data are presented as the mean ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 by t-test). LLL, low-level laser; ROS, reactive oxygen species; SD, standard deviation.

Discussion

In recent years, the LLL has become recognized in the field of regenerative medicine. The application of PBMT is complicated due to the diversity of the parameters. Therefore, the exploration for parameters related to the process of wound healing and conducive to the proliferation and migration of fibroblasts is promising for an increase in PBMT efficiency in clinical treatments.²⁷ Further study is urgent in order to clarify the effects of the different LLL parameters on the growth of fibroblast cells. The main aim of our experiment was to determine irradiation parameters, which could enhance proliferation and migration of L929 cells by 810 nm low-level diode laser irradiation, and to examine the level of ROS production.

Proliferation and migration of fibroblasts in the wound area are particularly important because fibroblasts are directly liable for depositing extracellular matrix, forming granulation tissues, and contracting wound lesions.^28–31 In our study, the mobility of L929 cells with different LLL parameters differed. The results indicate that the migration of L929 cells is dose-dependent with LLL. Thus, this dose response is expressed by a special graphical depiction, namely “Arndt–Schulz law.”³² In detail, the Arndt–Schulz curve shows the effect of energy density on the biological response of cells. It is asserted that when laser exposure time is too short or irradiance is too weak, it is probable that cells might not be responsive to laser treatment. While laser exposure time is long enough or irradiance is high enough, an inhibitory effect could be observable. In this study, our results were consistent with Arndt–Schulz law. Similarly, other researchers have also suggested an increase in cell migration at low fluences of laser irradiation and a toxic effect at higher fluences.^33,34 Therefore, the different effects on cell migration may be due to the differences in the irradiation parameters used, which is consistent with our results.

On one hand, faster cell migration results in more cell colonization on the wound area, probably leading to more extracellular matrix deposition. On the other hand, higher cell proliferation may result in faster tissue repair in order to promote rapid wound healing. It has been reported that most of the cells in the wound area were in a stress state,^35,36 and irradiation exerts an effect on stressed cells or on cells with altered metabolism, making the stress cells become normal cells. Some studies have shown that one way to stress cells in vitro is to reduce FBS concentration in cell culture medium.^37,38 Therefore, we chose serum-free medium to verify the effect of LLL on the proliferation of L929 cells. CCK-8 results showed that cell proliferation of the E0 and E2C groups was significantly inhibited after 72 h. On the contrary, E1C showed strong proliferation ability at 24 h, followed by the E2R and E1R groups. We analyzed that the proliferation of E0 group was inhibited due to the lack of serum in the medium, whereas the proliferation of E2C group was inhibited due to the excessive laser energy. This result is consistent with the scratch result. Fortuna et al.³⁹ demonstrated that there was a greater fibroblast number and collagen expression after the suspension of the laser therapy. Yin et al.²⁷ demonstrated from the cell mitosis that LLL can promote cell proliferation via acceleration of G1 to S phase progression. Taken together, our experiments show that E1C is an ideal LLL parameter to promote cell proliferation and migration.

The absorption of laser light leads to the production of ROS, being a natural by-product of oxygen metabolism. This plays a key role in cellular signaling pathways from mitochondria to nuclei, regulating nucleic acid synthesis, protein synthesis, enzyme activation, and cell cycle progression.⁴⁰ Absorption of the light increases ATP production and the induction of transcription factors through action on the mitochondria. This leads to increased cell proliferation and migration of cells, including fibroblasts, which play a crucial role in wound healing.⁴¹ Therefore, we conclude that the effect of ROS on cell functions is dose-dependent, and ROS levels determine whether cell proliferation and migration are promoted or inhibited. It has been demonstrated that low-level ROS can activate the MAPK pathway, as well as the Nrf-2 and NF-κB pathways, which can promote cell proliferation, migration, and differentiation.^42,43 We used the DCFH-DA probe to label L929 cells and found that the E2C group had the strongest fluorescence intensity, followed by the E2R group, the E1C group, and the E1R group. Although the E0 group was not stimulated by laser, it was also under oxidative stress in serum-free medium, and weak fluorescence was observed. Quantitative validation of this result was performed with the flow cytometry assay. The E2C group had the strongest fluorescence intensity and was inhibited in the process of cell proliferation and migration. On the contrary, the fluorescence intensity of the E0 group was the weakest. Although ROS was also produced, it also showed significant inhibition after 72 h of cell proliferation and migration. ROS disappeared before inducing the production of the cell pathway. The E1C group showed remarkable proliferation and migration ability after 24 h, and the production of ROS was at the medium level. Although the fluorescence intensity of E2R was higher than that of E1C, the cell proliferation and migration ability of E2R were weaker than that of E1C. We conclude that the effect of ROS on cell functions is dose-dependent, and ROS levels determine whether cell proliferation and migration are promoted or inhibited. At high levels, oxidative damage leads to cell proliferation inhibition and apoptosis, mainly through protein kinase C inactivation and Caspase 3 activation.^44,45 Chen et al.³² reported that both excessive and insufficient amounts of ROS were detrimental to cell viability and function. This is consistent with our results. Therefore, keeping ROS at a proper level and reducing oxidative stress are crucial for the functioning of fibroblasts during wound healing.

At present, the influence of different energy densities or power densities differs on cells for LLL parameters. In this experiment, for the 810-nm diode laser, we found that 0.1 W, continuous pulse mode, and 2 cm from the wound area can promote cell proliferation and migration. Therefore, our experiment provides an experimental and theoretical basis for the application of LLL in wound healing and clinical transformation.

Conclusions

In conclusion, an 810-nm diode laser at 0.1 W, continuous pulse mode, and 2 cm away from the wound can maintain the appropriate level of ROS and can effectively promote cell proliferation and migration. Our study provides a theoretical basis for the application of laser therapy for oral mucosal diseases.

Ethical Statement

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Authors’ Contributions

A.S.: Development or design of methodology, writing original draft, and writing review and editing (lead). M.X.: Management activities to annotate, scrub data, and maintain research data for use. X.S. and Y.Y.: Investigation and data curation (equal). T.G.: Oversight and leadership responsibility for the research activity planning and execution, including mentorship external to the core team. Y.Z.: Ideas, formulation or evolution of overarching research goals and aims, and acquisition of the financial support for the project leading to this publication. All authors have read and approved the final version of the article.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the National Natural Science Foundation of China (82002886).

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