Introduction

Photobiomodulation therapy (PBMT) is a treatment modality that has proven to be effective in accelerating wound healing, pain relief, reducing dentin sensitivity and the severity of xerostomia, and herpes labialis frequency [1,2,3]. The application of PBMT in controlling adverse reactions of cancer therapies has shown promising results and reached significant attention. The Multinational Association for Supportive care in Cancer/International Society for Oral Oncology (MASCC/ISOO) guidelines recommend PBMT to patients undergoing head and neck radiotherapy, associated or not with chemotherapy, due to numerous data in which the incidence and severity of oral mucositis have been positively impacted by PBMT [1, 4, 5].

The therapy operates with photon emission from a low-level laser light that transfers low energy to tissues and does not generate heat [2]. The exposure of biological tissues to low-level laser light induces the modulation of cellular functions by activating several pathways involved in cell growth and survival, proliferation, migration, and transcription [2, 6]. At a cellular level, this therapy promotes local, regional, and systemic action, enhancing mitochondrial activity and increasing the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) [7, 8].

The mechanism of action of PBMT has been discussed and reported in multiple in vitro studies, showing that it can promote stimulation or inhibition, depending on light parameters [9]. A review has analyzed 32 in vitro studies and concluded that an energy density varying from 0.5 to 4.0 J/cm2 and a light wavelength from 600 to 700 nm for PBMT could enhance proliferation of different cell types [10]. These findings are in conformity with previous in vitro studies and literature reviews that show positive biostimulation effects on fibroblasts, keratinocytes, and osteoblasts [11,12,13,14].

Moreover, the primary challenge is applying the optimal laser parameters to deliver an ideal amount of energy to enhance the metabolism and improve clinical outcomes [2, 8, 15]. To test the hypothesis that the same density of energy with lower power could lead to an improvement on cell viability, stimulated gingival fibroblasts were exposed to different densities of energy and two different output powers. Thus, this pilot study aimed to analyze the effects of different parameters of PBMT on cell viability, using human gingival fibroblast cells stimulated with bacterial and ionizing radiation–induced stress.

Materials and methods

Cell isolation and primary culture

The explant technique was used to obtain a cell line of human gingival fibroblasts. Before the fragment’s collections, ethics registration and approval had been obtained from the Human Research Ethics Committee of the Health Sciences College of the University of Brasília (CAAE Nº 78,679,717.6.0000.0030), and all the donors signed the understanding and written consent. Then, the gingival fragments of young donors who underwent third molar extraction surgery were isolated and transported to the Laboratory of Oral Histopathology immersed in cold Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich, St. Louis, MO) supplemented with 20% fetal bovine serum (FBS) (Gibco®, Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO). The fragments were washed twice with phosphate-buffered saline (PBS), explanted into small fragments, placed on 6-well plates, stabilized with a glass coverslip, covered by 2 mL of DMEM supplemented with 20% FBS and antibiotics, and maintained in a humidified incubator with ideal conditions (37 ºC and 5% CO2). The culture medium was replaced every 3 days and when 80–90% confluency was reached, cells were detached with trypsin (0.25%)/EDTA (1 mM) solution (Sigma-Aldrich, St. Louis, MO) and replaced in 100-mm dishes with DMEM plus 10% FBS and antibiotics to expand the culture or stored at − 80 ºC in a freezing solution containing FBS and 8% dimethyl sulfoxide (DMSO).

Bacterial and ionizing radiation–induced stress

For the stressful condition induction, cells were treated with three stimuli, as established in previous experiments of the Laboratory of Oral Histopathology of the University of Brasília (data not shown): lipopolysaccharide (LPS) of Escherichia coli 0111:B4 purchased from Sigma-Aldrich (St. Louis, Missouri, USA), Porphyromonas gingivalis protein extract (Pg), and ionizing radiation (IR). The protein extract of Pg was prepared in the University of Campinas, São Paulo, Brazil, as described by Albiero et al. [16] and donated to the Laboratory of Oral Histopathology of the University of Brasília. In order to achieve stress induction, before beginning the experiments, the cells were treated with LPS (1 µg/mL) and Pg (5 µg/mL), incubated for 1 h, and then irradiated with 8 Grays (Gy).

Photobiomodulation therapy parameters

The laser irradiation sessions were performed using a continuous-wave InGaAlP laser (Photon Lase III DMC, São Paulo, Brazil) in punctual and contact mode. The wavelength 660 nm laser was applied with output powers of 40 and 30 mW. The energy densities were 2, 3, 4, and 5 J/cm2 for each power. The complete treatment was performed in four sessions with 6-h intervals from each session according to Meneguzzo et al. [17] and Moreira et al. [18]. The overall parameters are presented in Table 1.

Table 1 Laser parameters used for photobiomodulation therapy
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Experimental groups

The laser irradiation protocol presented in Table 1 was carried out in nine groups, considering a negative control group/vehicle (stimulated model without PBMT) and four energy densities (2, 3, 4, and 5 J/cm2) irradiated using two power doses (30 mW and 40 mW).

Cell viability

Gingival fibroblasts were seeded into 96-well plates at a density of 5 × 103 and incubated for 24 h. Then, the stressful stimuli protocol was applied in nine biological replicates for each group. The first photobiomodulation session was conducted right after ionizing radiation, according to experimental groups, and repeated three more times, every 6 h. After 24 h from the last session, the cells viability was assessed by the tetrazolium dye MTT (3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. For this, 10 µL of MTT solution (Sigma-Aldrich, St. Louis, Missouri, USA) was added, and the cells were incubated and protected from light for 4 h. Then, the solution was removed, and 100 µL of acidified isopropanol (25 mL of isopropanol + 104 µL of HCl 100%) was added to each well. Cellular viability was analyzed after absorbance measurement using the spectrophotometer Thermo Plate TP Reader at 570 nm (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

Statistical analysis

The Shapiro–Wilk test was applied to assess data normality. As data resulted in parametric distribution, the one-way ANOVA followed by Dunnett’s and Turkey’s post-tests were applied to compare groups. The level of statistical significance was 95% (p < 0.05). The tests were performed using GraphPad Prism 9.3.0 (GraphPad Software, CA, EUA).

Results

Combination of energy and power can differently modulate cell response

The PBMT effect was analyzed in cells stimulated with bacterial and ionizing radiation–induced stress. For cell viability analysis, all treated groups irradiated with different doses of low-level laser were compared to a control group (non-irradiated with low-level laser). After 24 h from the last laser irradiation session, the groups outputted in 30 mW of power presented cell viability when operated with 2, 4, and 5 J/cm2; however, 3 J/cm2 dose significantly decreased mitochondrial activity (p < 0.05). In contrast, when the laser irradiation session was set up in a higher power (40 mW), the cell viability was reduced using 2, 3, and 5 J/cm2 doses, with statistical significance for 5 J/cm2 (p < 0.001). Hence, the results indicated that the combination of energy and power can differently modulate cell response (Fig. 1).

Fig. 1
figure 1

Cell viability after application of different photobiomodulation doses powered at 30 and 40 mW compared to a model of ionizing radiation and bacterial-induced stress. LPS lipopolysaccharide of Escherichia coli; Pg protein extract of Porphyromonas gingivalis; IR ionizing radiation. Analytical statistics: One-way ANOVA for parametric data followed by Dunnett’s post-test (*p < 0.05; *** < 0.001)

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Inhibitory response could be dependent on laser parameters

The same density of energy can be delivered setting different parameters, while performing a PBMT protocol [9]. In this study, two power outputs (30 and 40 mW) were set, using different times of exposition to deliver and compare four energy densities (2, 3, 4, and 5 J/cm2). The results demonstrated that operating the same energy, using lower power, seems to be superior to a higher power, being statically significant for 5 J/cm2 dose (p < 0.001). This pattern followed with all different groups, except by 3 J/cm2. Thus, bacterial and ionizing radiation–induced cells exposed to four PBMT sessions of 2, 4, and 5 J/cm2 were more capable to keep mitochondrial activity, operating at 30 mW than 40 mW (Fig. 2). These results suggested that inhibitory response could be power-dependent.

Fig. 2
figure 2

Comparison of same energies density powered at 30 and 40 mW. Analytical statistics: One-way ANOVA for parametric data followed by Tukey’s post-test (*** < 0.001)

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Discussion

The effects of PBMT depend on laser parameters, including wavelength, power, energy, spot area, and time of exposure. Also, these combined aspects could differently influence cell activity, such as proliferation [15, 19,20,21]. Thus, the effects of different parameters in wound healing are in current interest, since it can enhance cell growth depending on the set output.

This pilot study investigated the effect of low-power InGaAlP laser irradiation comparing the output power of 30 and 40 mW and the corresponding energy densities of 2, 3, 4, and 5 J/cm2, while laser irradiation was performed in four sessions with an interval of 6 h between them. In accordance with published findings, lower power delivering the same density of energy was more capable to maintain cell viability than higher output power which suggests the influence of PBMT parameters in cell response [22,23,24,25,26,27].

Brueghel and Dop Bärr [9] suggested that power density and time of exposure seem to be more important than the total energy dose of PBMT on human fibroblasts. Azevedo et al. [20] found an inverse influence between power density and cell growth [9, 22, 28, 29]. Previous studies showed the same results, indicating that higher output power had inhibitory characteristics [6, 15, 24, 30].

Low doses of PBMT activate a proton gradient, releasing calcium from mitochondria into the cell’s cytoplasm. This process stimulates a cascade of cellular functions and protein secretion enhancing cell proliferation. In contrast, higher doses can release an excessive amount of calcium promoting hyperactivity of calcium-adenosine triphosphatase, inhibiting cell metabolism [30,31,32]. Our results indicated that the same energy density, outputted in the power of 30 mW, showed higher viable cells than 40 mW, suggesting that the power can determine the stimulatory or inhibitory effect of the laser irradiation on cellular responses.

Hawkins and Abrahams [30] demonstrated that the cumulative effect from the accumulated doses determines the biomodulation effect, multiple exposures at higher doses cause additional stress and significantly reduced cell viability, and lower doses and fewer exposures maintained cell viability.

The response of the tissue exposed to PBMT protocols is the combination of time of exposure, total energy delivered, and cell response. The study design was defined with a primary culture of gingival fibroblasts collected from patients. Even though the followed inclusion criteria considered healthy patients, the PBMT results may vary according to patients’ response, cell type, tissue condition, and explant methods used for the culture [24]. Also, it is possible that, in this preliminary study, the total energy delivered in 3 J/cm2 and 30 mW of output power associated with the time that the energy was delivered was unable to produce effect comparable with other parameters; this could be associated with different degrees of the effect produced by PBMT [6].

Considering that wound healing depends on cell proliferation, it is crucial to study and deeper understand the effect of power densities of PBMT in vitro, since this is the first step to understand the cascade process in a complex body [31]. There is not a well-defined standard of output power setting, although various studies have been performed to observe the effects of low-level laser in cellular response [2, 8, 10,11,12]. Comparing different protocols of application can contribute to determine the optimal combination of parameters according to different cell types and expected results. In addition, it is possible to obtain lower or higher doses to reach an energy density, controlling the output of power and the laser irradiation time which can facilitate the replication of protocols even when different equipment is available [6, 9].

There are some limitations in this study that should be addressed. First, this is a pilot study that only focused on cell viability. Considering the importance of laser parameters for different culture conditions, a single trial experiment assessing different PBMT outputs was conducted to allow future analysis since cell response depends on that. Second, the definition of output parameters was based on literature research, which contemplates different cell types and objectives. Thus, the future proposal is to continuously study cellular response after bacterial and ionizing radiation–induced stress, comparing output powers of 30 mW and lower with further experiments to analyze the effects of PBMT on cell morphology, proliferation, migration, gene and protein expression, and specific pathway signalization.

Conclusion

The present pilot study showed that PBMT effects can be influenced by the power outputted parameter. After analyzing PBMT set in 660 nm wavelength, output power of 30 and 40 mW, and energy densities of 2, 3, 4 and 5 J/cm2, it was possible to conclude the PBMT effect. The results using a protocol of PBMT, set with 660 nm of wavelength in four sessions of laser application with 6-h interval suggest that delivering 2, 4, and 5 J/cm2 of density of energy with output power of 30 mW, leading to more time of exposure, presented better results on cell viability compared to the same energy density with 40 mW. However, further studies comparing density energy should be conducted.