Introduction
Low-level laser therapy (LLLT) is a treatment that utilizes focused light to stimulate a process known as photobiomodulation (PBM), which is “a non-thermal process involving endogenous chromophores that elicit a photophysical and photochemical reaction at various biological scales” [
1]. This therapy uses nonionizing light sources, including lasers, light-emitting diodes, and broadband light in the visible (400–700 nm) and near-infrared (700–1100 nm) electromagnetic spectrum [
2]. During PBM, photons penetrate cells and are absorbed by the cytochrome-c complex within the mitochondria. This triggers a biological cascade that increases cellular metabolism, which has beneficial outcomes such as pain relief, immunomodulation, wound healing, and tissue regeneration [
3,
4].
Previous studies have demonstrated that PBM attenuates proliferation, survival, and differentiation in various cell types, which makes it a useful tool for achieving the objectives of tissue engineering, such as reconstructing or regenerating deteriorated or damaged tissues [
5,
6]. Tissue reconstruction is achieved by the combination of scaffolds, stem cells, and inducing factors that act together to regenerate injured or missing tissues [
7]. Stem cells are capable of self-renewal, and because of their undifferentiated nature, they can be stimulated to perform specific biological tasks and differentiate into several cell types [
8]. Adult mesenchymal stem cells (MSCs) can be isolated from adipose tissue [
9], bone marrow [
10], periodontal ligaments [
11], and human dental pulp stem cells (hDPSCs) [
12]. Currently, the latter is of significant interest for tissue engineering because of the prior development of standardized extraction protocols, which facilitate collection and the fact that they can be obtained from disposable samples. Another advantage is their capacity for proliferation and differentiation into multiple phenotypes, such as osteoblasts, chondrocytes, and adipocytes [
13‐
15].
The effects of PBM on viability, proliferation, and differentiation into osteogenic lineage of MSCs remain controversial. Although there are no clear conclusions, several studies have shown that PBM enhances MSCs viability and proliferation, particularly when red laser wavelengths are used [
16‐
21]. However, other studies have found that laser treatment does not increase osteoblast proliferation and differentiation compared with control cells [
22‐
24]. These discrepancies may result from differences in the methodologies used in the studies and the fact that it is unclear how the variation in irradiation parameters (e.g., wavelength, fluence, power density, emission mode, and application time) influence the biomodulatory effect of the laser on mesenchymal cells.
Because the underlying mechanism that regulates the increase in cell proliferation, differentiation, and migration is not yet precise, the use of LLLT remains controversial. Among the best-known proposed mechanisms of PBM is the action of cytochrome-c oxidase (CCO), an essential photoreceptor in mitochondria that contributes to the maintenance of the mitochondrial transmembrane potential and, consequently, influences ATP production. In addition, functional changes in the mitochondrial electron transport chain can generate reactive oxygen species (ROS) and changes in the mitochondrial membrane potential (∆Ψm), which affect ATP production [
2‐
25]. ROS plays an important role in cell signaling, proliferation, cycle regulation, and protein synthesis [
21,
26]; however, the specific role of ROS in the differentiation of MSCs toward PBM-treated osteoblasts is unknown.
The clinical effects of laser therapy depend on the correct selection of irradiation parameters, including wavelength, fluence, power density, and application time [
2,
17]. Therefore, the purpose of this study was to determine and compare the effect on the proliferation and osteoblastic differentiation of hDPSCs cells treated with two types of lasers of different wavelengths and different exposure times. In addition, we also evaluated the changes in ∆Ψm and ROS’s participation in the osteoblastic differentiation of mesenchymal cells stimulated by low-level laser irradiation.
Discussion
In the present study was determined the effect of LLLT on proliferation, ROS production, changes in ∆Ψm, and osteoblastic differentiation of hDPSCs treated with two wavelengths (660 nm and 940 nm) during different exposure times (5 s, 50 s, and 180 s). When evaluating postirradiation effects, a decrease in cell proliferation was observed 8 days after irradiation, irrespective of wavelength and irradiation time. This may be related to increased differentiation toward the osteoblastic lineage, as evidenced by the expression of RUNX2 and BMP2, which increased 21 days after treatment in all experimental groups. Similarly, a significant increase in the presence of calcification nodules was observed mainly in the group exposed to the 940-nm laser at 50 s, which also exhibited the highest values of alizarin red S. Additionally, an increase in ROS and ΔΨm levels was evident, which has previously been reported as a possible effect associated with PBM [
2,
22,
39].
It was previously reported that the effect of LLLT, both in vivo and in vitro, depends on the application parameters used [
40]. The treatment will likely be ineffective if the wrong parameters are applied [
41]. Several studies [
3,
42] evaluating different doses of LLLT have reported a biphasic response, and the Arndt–Schulz law is accepted as a suitable model to describe the effects of this therapy. According to this law, when energy is insufficient, there is no response (because the minimum threshold has not been reached). If more energy is applied, the threshold is exceeded, and biostimulation is achieved; however, when energy is excessive, stimulation disappears and is replaced by bio inhibition [
43]. Therefore, it is essential to determine, through scientific studies, the precise and appropriate parameters of low-level laser irradiation to achieve the desired objectives in a reproducible manner.
The criteria for selecting the application parameters in this study were based on a previous systematic review that analyzed dosimetry during in vitro studies [
21] and the range of established irradiation parameters (wavelength, power, power density, energy density, application time) in which PBM was reported to be effective [
44], considering the upper and lower limits of these parameters. Since we intended to evaluate wavelengths belonging to the red and near-infrared electromagnetic spectrum and according to previous literature reports, the two wavelengths selected in the present study were 660 nm and 940 nm (Table
2). They belonged to the red and near-infrared range of the electromagnetic spectrum. This allowed us to observe the behavior of LLLT in hDPSC cultures.
Systematic reviews [
16,
19] indicated that 660 nm red laser had been used in most of the included studies. Another study reported using 810 nm and 980 nm diode lasers, with output power ranging from 20 to 100 mW. This is consistent with a more recent systematic review [
21], which indicated that 13 studies used diode lasers with wavelengths between 635 and 980 nm. Only one study used an Nd: YAG laser (λ1064 nm). Energy densities ranged from 0.378 to 78.75 J/cm
2 and irradiation times were between 1.5 and 300 s. All these reviews concluded that there is a positive effect of LLLT on cell proliferation and differentiation; however, the evidence was weak due to the heterogeneity of the methods used and the moderate risk of bias in the various studies.
In the present study, no significant changes in proliferation were observed in the short term (2nd day); however, by the 6th day, a decrease in proliferation was observed in the group exposed at 660 nm, as well as an increase in proliferation in the group exposed at 940 nm 50 s, which was subsequently reduced by the 8th day, when both wavelengths caused a significant decrease in proliferation compared to the control group, with no apparent reduction in cell viability. Furthermore, this reduction in cell number correlated with morphological changes, increased formation of calcification nodules, and expression of osteoblast differentiation marker genes, suggesting that both 660-nm and 940-nm lasers induced differentiation of hDPSCs toward an osteoblastic lineage.
Previous studies have demonstrated a differentiation-inducing effect in cells exposed to LLLT. Miglario et al. [
39] used a 980-nm diode laser with irradiation times of 1, 5, 10, 25, and 50 s and energy fluences of 1.57, 7.87, 15.74, 39.37, and 78.75 J/cm
2, to evaluate the effect on murine MC3T3-E1 preosteoblasts. Increased proliferation was observed when treated with 7.87 J/cm
2 but decreased with 78.75 J/cm
2 during 24 and 48 h following irradiation, suggesting that higher energy densities may have an inhibitory effect on the proliferation of this cell type. This agrees with the results presented in this study.
A study using a 635 nm diode laser at 30 mW/cm
2 for 0, 34, 67, or 102 s (0, 1, 2, or 3 J/cm
2) applied to MC3T3-E1 cells revealed that LLLT induced osteoblast differentiation into primary MSCs and osteoblast precursor cells, as evidenced by functional assays (calcium deposition) and expression of differentiation markers, including alkaline phosphatase (ALP), osteocalcin and BMP2. Furthermore, the group that showed a significant increase in BMP2 expression was those treated with 635 nm at 3 J/cm
2 [
45]. Another study exposed Saos-2 osteoblasts to three different PBM treatments, including 635 ± 5 nm, 808 ± 10 nm, and 405 nm. Compared to controls, a statistically significant increase in RUNX2 and ALP expression was observed after 635-nm laser PBM. Treatment with 808 nm increased the expression of RUNX2 but not ALP. No variations in the expression of these genes were detected in cells subjected to PBM at 405 nm compared to control cells [
2].
When studying the effect of 808-nm laser on human periodontal ligament stem cells, a significant increase in cell proliferation and osteogenic differentiation (through the expression of RUNX2, Col-1, ALP, and osteonectin) was observed, especially at 1 and 2 J/cm
2 combined with vitamin D [
46]. Similarly, in another study [
47] comparing the effect of a single and double dose 808-nm laser with a control group in DPSC, the double dose irradiation groups were consistent with an increase in calcium and ALP formation compared to the single irradiation group. In addition, osteopontin expression was significantly increased in the double-dose group compared to the single-dose group. Positive staining with alizarin red S and ALP confirmed the presence of calcium deposits in the analyzed samples. The above reports agreed with the results of increased RUNX2 expression 21 days after irradiation, especially in the 660 nm (red laser) at 5 s, 50 s, and 180 s, and 940 nm (near-infrared laser) groups at 50 s and 180 s, with PBM treatment at 940 nm for 50 s inducing the highest expression. In cells treated with 940 nm for 5 s, a lower expression of RUNX2 was observed compared to the other groups, which can be attributed to a low fluence applied (0.64 J/cm
2), which was not sufficient to produce an effect on RUNX2 expression. As for BMP2 expression, higher expression was observed at 21 days in the 660 nm groups with application times of 50 and 180 s and in the 940 nm groups at 50 and 180 s. The group treated with 940 nm for 50 s exhibited the highest gene expression (4.5-fold).
Regarding the appearance of calcification nodules, the 940-nm laser group for 50 s (6.4 J/cm
2) presented the highest alizarin red values, similar to the results reported by Sivakumar et al. [
47], when applying a laser with a double dose of irradiation. However, in contrast, we found a significant increase in calcification nodule formation with a single irradiation dose.
Regarding intracellular redox state, when evaluating the relationship between ROS production and Δψm with low-level laser irradiation, an increase in Δψm was observed, which corresponded to an accumulation of intracellular ROS after irradiation with the two lasers (660 nm and 940 nm). The use of DCFH2-DA as a fluorescent probe to detect the redox state was useful for the identification of oxidative species, such as peroxides, superoxides, and nitric oxide [
48], generated by laser PBM. ROS produced by the application of low-power lasers is presumably derived from endogenous sources, such as mitochondria, which are sensitive to red and near-infrared light. The effect of LLLT on metabolic activity is associated with the ability to stimulate electron transfer during oxidative phosphorylation, which promotes ATP production [
25,
49]. This, combined with the results obtained from stimulation of the mitochondrial membrane potential, suggests an effect of LLLT on the inner mitochondrial membrane or mitochondrial protein complexes. The result is efficient electron transfer and adequate proton flux to catalyze ATP synthesis through activation of the transmembrane enzyme ATP synthase. Specifically, increased ATP synthesis is associated with light absorption by unit IV of the mitochondrial respiratory chain, which contains the chromophore CCO [
50]. Consistent with these results, previous studies have indicated that ROS levels and ΔΨm are directly proportional. The rate of ROS production increases significantly when ΔΨm is above 140 mV and decreases by approximately 70% when the ΔΨm level drops to 10 mV [
51]. This effect resulting from laser PBM is opposite to that obtained by exposure to TBPH, a prooxidant agent that induces excessive ROS production leading to a concomitant decrease in ΔΨm. This phenomenon has been termed “ROS-induced R.O.S. release,” a cycle of mitochondrial ROS formation and release through the mitochondrial permeability transition pore (mPTP). During prolonged oxidative stress, sustained opening of the mPTP leads to increased ROS production that causes cellular injury associated with decreased ΔΨm and ultimately affects cell survival, migration, proliferation, and differentiation.
Consequently, mitochondria are known to play an essential role in the regulation of apoptosis. The results showed that the Δψm of treated cells increased compared with TBPH-treated cells and even with untreated cells. Since Δψm decreases during apoptosis, LLLT may have a positive effect on reducing apoptosis, in part, through the PI3K/Akt signaling pathway. The increase in ROS because of LLLT exposure agrees with results reported by other authors [
49,
52], who have described the beneficial effects of ROS accumulation at safe concentrations, in addition to improving total cellular antioxidant capacity and shifting the oxidative/antioxidative balance toward the antioxidant state.
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