1.

Introduction

Laser phototherapy (LPT) has been clinically used to accelerate the wound healing in several diseases including those from the oral cavity.¹ The LPT has proven to be efficient in treating slow healing skin ulcers,² chronic diabetic leg and foot ulcers,^3,4 and several oral diseases, such as chemotherapy- and radiotherapy-induced oral mucositis,^5–8 herpes simplex infections,⁹ and bone osteonecrosis.^10,11 Diverse cell types, like fibroblasts,^12–14 osteoblasts,¹⁵ endothelial,^16,17 and keratinocytes,^18–20 when growing in culture in adverse conditions respond positively to LPT. This treatment improves cell proliferation, migration, and transcription of genes involved in wound healing.²¹

Epithelial cells play an important role in wound healing by restoring the epithelial barrier, followed by re-establishment of tissue homeostasis.²² Rapid migration and proliferation of epithelial cells located in the basal layer of the epithelium adjacent to the injured area are important events of wound healing. Several molecular pathways are activated during wound healing including the AKT/mTOR pathway.^23–26 In fact, augmented epithelial migration and accelerated wound healing have been observed as a result of in vivo overexpression of the AKT/mTOR pathway in genetically defined animal models.²⁷ Additionally, mammalian target of rapamycin (mTOR) signaling pathway was proven to be a key player in the accelerated migration phenotype of epithelial and muscle cells.^23,27

Only three studies analyzed the effects of LPT on epithelial cells, looking for understanding the mechanisms underlying the biostimulation in these cells.^18–20 It is known that the mechanisms involved in the LPT are those related to the improvement in the respiratory metabolism^28,29 and changes in mitochondrial membrane potential,³⁰ leading to an increase in the mitochondrial respiration and ATP synthesis.³¹ However, it remains unclear how LPT influences epithelial cell proliferation and migration. Thus, this study searched the molecular mechanisms underlying the effects of LPT on oral keratinocytes proliferation and migration, and further the molecular circuitry involved in this process was dissected.

2.

Materials and Methods

The study received approval from the Research Committee of School of Dentistry, Universidade Federal do Rio Grande do Sul under the process number 24114.

3.

Results

3.1.

LPT Accelerates Oral Keratinocyte Migration

In vitro scratch-induced wound model was chosen to assess the migratory ability of the cells upon irradiation with diode laser [Fig. 1(a)]. Oral keratinocytes responded to diode laser biostimulation by accelerating the epithelial cell migration. Compared with sham-irradiated control group, LPT-treated cells showed significant acceleration of migration in 12 h after scratch ($4\mathrm{J}/{\mathrm{cm}}^2$ ***$p<0.001$ and $20\mathrm{J}/{\mathrm{cm}}^2$ **$p<0.01$), followed by wound closure within the first 24 h (*$p<0.05$ for 4 and $20\mathrm{J}/{\mathrm{cm}}^2$) [Fig. 1(b)]. Epithelial cells did not show significant differences in migration when irradiated by either 4 or $20\mathrm{J}/{\mathrm{cm}}^2$ energy density (NS $p>0.05$).

Fig. 1

Oral keratinocyte migration upon laser irradiation. (a) Scratch wound assays in NOK-SI oral keratinocytes cell line monolayers from control and LPT groups (4 and $20\mathrm{J}/{\mathrm{cm}}^2$) during whole experimental time (T0 to T48). Wounds were generated after cell confluence (T0). LPT irradiations were applied three times with 6-h interval. In vitro cell migration was assessed every 12 h. Scale bars represent 50 μm. (b) Graph shows epithelial cell migration, represented as the percentage of open wound of all groups in each experimental time (h). Compared with control group, LPT-treated cells show significant accelerated migration by 12 h after scratch (***$p<0.001$—$4\mathrm{J}/{\mathrm{cm}}^2$ and **$p<0.01$—$20\mathrm{J}/{\mathrm{cm}}^2$), followed by accelerated wound closure within the first 24 h (*$p<0.05$). There were no significant differences in migration when comparing both irradiated groups (4 and $20\mathrm{J}/{\mathrm{cm}}^2$) (NS $p>0.05$).

3.2.

LPT Did Not Influence the Proliferation Rate of Oral Keratinocytes

Cell viabilities of control and irradiated groups 24 h after irradiation are represented in Fig. 2. There were no differences in the amount of viable cells among the groups ($p>0.05$).

Fig. 2

Lack of proliferation of oral keratinocyte upon laser irradiation. Graphical representation of the number of viable cells 24 h after irradiation. Cell viability was determined by the reduction of MTS. Cells ($2.5\times {10}^3$) were seeded onto the 96-well plates 24 h prior to irradiation. Culture media from all the samples were replaced with the nutritional-deficient media (2% FBS) after cellular attachment (6 h after seeding). There is no difference in the amount of viable cells among the established groups [control (sham group), $4\mathrm{J}/{\mathrm{cm}}^2$ laser group, and $20\mathrm{J}/{\mathrm{cm}}^2$ laser group]. Irradiation with LPT does not influence the proliferation rate of oral epithelial cells (NS $p>0.05$).

3.3.

Diode Laser Irradiation Activates the AKT/mTOR Signaling Pathway in Oral Keratinocytes

To further explore the mechanistic details of accelerated epithelial cell migration, the involvement of the AKT/mTOR signaling pathway especially the phosphorylation levels of S6 protein, a downstream marker of mTOR signaling, was tested. LPT was able to induce the activation of the mTOR signaling in both irradiated groups (4 and $20\mathrm{J}/{\mathrm{cm}}^2$). Further, treatment of cells with rapamycin, a specific pharmacological inhibitor of mTOR, completely ablated the LPT-induced phosphorylation of S6 protein (Fig. 3). AKT phosphorylation at serine 473 (${\mathrm{AKT}}^{473}$) was not affected by LPT. As expected, an increase in AKT phosphorylation upon EGF treatment was noticed. GAPDH was used as a loading control.

Fig. 3

Upregulation of the AKT-mTOR pathway during laser-induced accelerated wound healing. Western blot analysis of phosho S6 and phospho AKT in NOK-SI oral keratinocytes cell line. Oral keratinocytes were seeded on 6-well culture dishes supplemented with 10% FBS. Six hours before irradiation, the medium was substituted by nutritional-deficit medium in all groups (DMEM with 2% FBS). Three groups were established: control ($0\mathrm{J}/{\mathrm{cm}}^2$), $4\mathrm{J}/{\mathrm{cm}}^2$ laser group, and $20\mathrm{J}/{\mathrm{cm}}^2$ laser group. The laser groups received three irradiations with 6-h intervals. After 18 h of initial irradiation, Western blot analysis was performed for anti-phospho S6, anti-phospho ${\mathrm{AKT}}^{473}$, and GAPDH (loading control). EGF was used as a positive control. LPT induced the activation of the mammalian target of rapamycin (mTOR) signaling in both irradiated groups (4 and $20\mathrm{J}/{\mathrm{cm}}^2$). Treatment of cells with rapamycin, a specific pharmacological inhibitor of mTOR, completely ablated the LPT-induced phosphorylation of S6 protein. AKT phosphorylation at serine 473 (${\mathrm{AKT}}^{473}$) was not affected by LPT. An increase in AKT phosphorylation upon EGF treatment is noticed.

3.4.

Low-Power Diode Laser Promotes F-Actin Polymerization

Cell migration requires directional reorganization of the actin cytoskeleton and can be mediated by a variety of extracellular factors including growth factors and extracellular matrix. To test if LPT interferes with the cytoskeletal reorganization during epithelial cell migration, changes in F-actin organization were observed at two time points: 10 min and 12 h after the first irradiation using the optimal energy density of $4\mathrm{J}/{\mathrm{cm}}^2$ as previously established (Fig. 1). It was observed that LPT induced a robust accumulation of F-actin in NOK-SI cells compared with sham irradiation (Fig. 4). It was also observed that LPT induced the polarization of F-actin as early as 10 min after the initial irradiation, which was intensified after 12 h (Fig. 4, arrows). This was also accompanied by changes in cellular morphology (Fig. 4) to one that is typical of highly motile cells.^34,35

Fig. 4

Low-power diode laser promotes F-actin polymerization. Immunofluorescence images of F-actin staining in NOK-SI oral keratinocytes cell line. Oral keratinocytes cells were seeded on 6-well culture dishes supplemented with 10% FBS. Culture media were substituted by DMEM supplemented with 2% FBS 6 h before the initial irradiation. Two groups were established: control group ($0\mathrm{J}/{\mathrm{cm}}^2$) and $4\mathrm{J}/{\mathrm{cm}}^2$ laser group. The laser groups received three irradiations with 6-h intervals. F-actin staining was performed 24 h after administration of nutritional-deficit medium. Phalloidin (F-actin staining—red), DAPI (DNA staining—blue). LPT induced a robust accumulation of F-actin in treated cells (arrows), associated with the changes in the cellular morphology. Scale bars represent 50 μm.

4.

Discussion

Wound healing is a key process essential for tissue repair and restoration of cellular homeostasis. It plays important roles in several physiological and pathological conditions involving ulcerated lesions that constitute significant clinical challenges such as burn ulcers, mucositis, diabetes with slow or nonhealing ulcers, pressure, and chronic venous ulcers.^36–38 In an epitheliocentric view, wound healing is characterized by a multistep process comprising epithelial proliferation and migration. Notably, epithelial proliferation is often observed at the original wound edge, characterized by an increase in epithelial thickness. However, the initial stages in epithelial cell migration are mainly observed in the epithelial tongue, characterized by the layers of epithelial cells that migrate into the wound bed, thereby promoting healing of the wound. The epithelial tongue is primarily composed of migratory cells and a very few proliferating cells. It is interesting to note that the cells from the epithelial tongue do not undergo a terminal differentiation program compared with the cells localized at proliferating wound edge, suggesting that these two compartments are driven by distinct sets of active molecular processes.³⁹

Clinically, LPT has been shown to be effective in wound healing by accelerating the closure of lesions in addition to reducing pain and discomfort.^1–11,40 These observations suggest that the LPT may influence mucosal wound healing by accelerating epithelial cell migration over the wounded site. However, only three studies analyzed the effects of LPT on epithelial cells and the molecular mechanisms by which LPT can promote biostimulation in these cells.^18–20 In the present study, we sought to explore the specific effects of diode laser irradiation on oral keratinocytes during wound healing using a keratinocyte cell line originally isolated from the retromolar area of the oral cavity.³² Our results suggest that the LPT accelerates the healing of oral mucosa by activation of oral keratinocytes migration and mTOR signaling pathway independent from cellular proliferative mechanisms.

Recent studies including those from our laboratory have identified key molecules responsible for enhancing and impairing epithelial cell migration.^27,32 We had shown that the PI3K/mTOR signaling pathway plays a crucial role in driving the re-epithelialization process. In fact, depletion of phosphatase and tensin homolog (PTEN), a negative modulator of the PI3K/mTOR signaling pathway, resulted in accelerated epithelial cell migration and overall wound healing.²⁷ The identification of new therapeutic strategies to enhance the epithelial wound healing is aimed at the discovery of clinically viable solutions for slow healing ulcers. Recent advances in the field of photobiomodulation^41,42 have shown a great deal of promise in this regard.^1,12,18,21 However, molecular mechanisms have not been completely clear. The use of low-power diode laser or LPT has been shown to modulate several biological processes including wound healing; however, only a few reports have showed the effect of LPT on epithelial cells.^18–20 In addition, the impact of LPT over keratinocytes from the oral mucosa and the associated molecular mechanisms involved in accelerated migration remain unknown. In the present study, we investigated the effect of LPT on wound-healing process of oral keratinocytes and explored the molecular circuitry involved in epithelial cell migration. To get in-depth analysis on cellular response to laser stimulation, we used two different LPT parameters: 660-nm diode $\text{laser}/40\mathrm{mW}$, 4 and $20\mathrm{J}/{\mathrm{cm}}^2$ with same frequency of irradiation and irradiation time intervals. This choice was made based on the literature which showed that the small energy densities of irradiation lead to better effects compared with higher energy densities in wound healing.^43–48 These studies were in accordance to the Arndt–Schultz law⁴⁹ (e.g., a small stimulus can excite physiological activities, whereas a higher stimulus can inhibit them). Then, 4 and $20\mathrm{J}/{\mathrm{cm}}^2$ were used as lower and higher energy densities, respectively. By maintaining the same power in both groups, the cells were irradiated with energy per point of 0.16 J in the $4\mathrm{J}/{\mathrm{cm}}^2$ laser group and an energy per point five times higher (0.8 J) in the $20\mathrm{J}/{\mathrm{cm}}^2$ laser group. With these parameters, it was possible to observe the response of the cells to very different energies, and even though both 0.16 and 0.8 J per point could be considered low energies, they were different enough to induce diverse responses.

Our results revealed that, independent of the energy density and energy per point, the laser irradiation had no effect on cell proliferation rate, but accelerated epithelial cell migration. In fact, the laser irradiation closed the open wound in half of the time compared with that of the control group. In addition to the accelerated cell migration, LPT also enhanced actin polymerization in epithelial cells, a hallmark of the highly motile cells. Using the same energy density in an in vivo animal model, our group observed that LPT was capable of accelerating the oral mucosa wound-healing process. Moreover, faster and more organized re-epithelialization and tissue healing of the oral mucosa were achieved with an energy density of $4\mathrm{J}/{\mathrm{cm}}^2$ in comparison with $20\mathrm{J}/{\mathrm{cm}}^2$.⁴⁰ Nonetheless, an increase in energy density has been shown to impair the wound healing in vivo.⁴⁵ Other studies have predominantly attributed the inhibitory effect to higher power levels rather than energy density per se.⁵⁰ We agree with this point of view, that the tissue response could not be only related to the energy density, but with the energy per point or total energy applied in tissue. In our study, the epithelial cells after LPT irradiation with 0.16 J energy per point ($4\mathrm{J}/{\mathrm{cm}}^2$ laser group) and 0.8 J energy per point ($20\mathrm{J}/{\mathrm{cm}}^2$ laser group) showed similar in vitro proliferative, migratory, and mTOR signaling pathway protein expression.

The current mechanism by which LPT induces cellular response involves the activation of mitochondrial respiratory chain.¹ LPT irradiation activates cellular cytochromes that promote a cascade of events leading to the generation of reactive oxygen species (ROS), changes in ${\mathrm{Ca}}^{2+}$ flux, no binding to cytochrome c oxidases that affect the levels of cyclic nucleotides, and DNA and RNA synthesis, thereby modulating a variety of cellular functions.⁵¹ We also found that LPT activates the mTOR signaling pathway in epithelial cells. Apart from its role in cell growth, shape, polarity, and size,^52–58 mTOR also acts as a metabolic sensor and has been shown to regulate both the resting oxygen consumption and oxidative capacity. Therefore, mTOR activity correlates with the overall mitochondrial function, and pharmacological inhibition using rapamycin significantly reduces mitochondrial membrane potential.⁵⁹ In all our experimental conditions, we noticed that LPT increased the phosphorylation of S6 protein and rapamycin treatment impaired this phosphorylation, indicating that LPT triggers mTOR signaling pathway. AKT phosphorylation was not influenced by LPT, suggesting that LPT activation of mTOR may be through mTORC1 and independent of mTORC2, which is known to phosphorylate AKT at serine 473. The physiological implications of this mechanism are yet to be elucidated in detail.

Our findings have advanced the current understanding on the molecular circuitry involved in the use of a low-power diode laser in oral epithelial cells. Our data strongly indicate that LPT is a promising therapeutic strategy for diseases of the oral cavity such as oral mucositis, resulting from chemotherapy and radiotherapy, herpes simplex infections, and postsurgical management. Nonetheless, new studies are under way to better understand the effect of LPT over oral keratinocyte physiology and to further dissect the molecular mechanisms involved in photobiomodulation.

5.

Conclusion

Based on the limits of this in vitro study, we concluded that the improvement of keratinocyte migration after LPT can occur through activation of mTOR signaling pathway.