In Vitro Cytological Responses against Laser Photobiomodulation for Periodontal Regeneration

Periodontal disease is a chronic inflammatory disease caused by periodontal bacteria. Recently, periodontal phototherapy, treatment using various types of lasers, has attracted attention. Photobiomodulation, the biological effect of low-power laser irradiation, has been widely studied. Although many types of lasers are applied in periodontal phototherapy, molecular biological effects of laser irradiation on cells in periodontal tissues are unclear. Here, we have summarized the molecular biological effects of diode, Nd:YAG, Er:YAG, Er,Cr:YSGG, and CO2 lasers irradiation on cells in periodontal tissues. Photobiomodulation by laser irradiation enhanced cell proliferation and calcification in osteoblasts with altering gene expression. Positive effects were observed in fibroblasts on the proliferation, migration, and secretion of chemokines/cytokines. Laser irradiation suppressed gene expression related to inflammation in osteoblasts, fibroblasts, human periodontal ligament cells (hPDLCs), and endothelial cells. Furthermore, recent studies have revealed that laser irradiation affects cell differentiation in hPDLCs and stem cells. Additionally, some studies have also investigated the effects of laser irradiation on endothelial cells, cementoblasts, epithelial cells, osteoclasts, and osteocytes. The appropriate irradiation power was different for each laser apparatus and targeted cells. Thus, through this review, we tried to shed light on basic research that would ultimately lead to clinical application of periodontal phototherapy in the future.


Introduction
Periodontal tissue consists of "gingiva, periodontal ligament, cementum, and alveolar bone" [1]. Periodontal diseases cause a wide range of inflammatory conditions that affect the periodontal tissue, which could lead to loss of teeth and contribute to systemic inflammation [2]. Basic periodontal therapy eliminates etiological factors for periodontal disease and relieves inflammation in periodontal tissues [3]. Recently, periodontal therapy using a laser, "periodontal phototherapy," has attracted much attention [4]. Many reports have been published on the methods recommended for periodontal therapy using lasers [5,6]. In addition, laser irradiation is also applied to treat pressure ulcers [7] and pain associated with temporomandibular dysfunction [8]. However, basic research related to clinical research is inadequate. We propose that it is necessary to understand the molecular biological effects of periodontal phototherapy for periodontal therapy. Photobiomodulation (PBM) is a treatment method based on research findings suggesting that irradiation with specific wavelengths of red or infrared light produces a wide range of physiological effects in cells, tissues, animals, and humans [9]. A previous study reported that the effect was a nonthermal process involving endogenous chromophores eliciting photophysical and photochemical phenomena at various biological scales, resulting in beneficial therapeutic outcomes [10].
The purpose of this review was to investigate cytological responses against laser irradiation for periodontal regeneration. We tried to provide insights on basic research that would subsequently lead to clinical research on periodontal phototherapy in the future.

Interaction with Tissues
When laser energy reaches a tissue surface, it can be reflected, scattered, absorbed, or transmitted to the surrounding tissues. The performance of a laser is determined by the degree of absorption. In particular, absorption in biological tissues is strongly influenced by the absorption coefficient in water, which is inherent to each wavelength [11,12]. Thus, lasers are clinically classified into two types depending on their wavelength: (1) a deeply penetrating type where the laser light penetrates and scatters into the tissue more deeply, such as the neodymium-doped yttrium-aluminum-garnet (Nd:YAG) (1064 nm) and diode lasers (810-980 nm available for clinical application and (2) a superficially absorbed type (shallowly penetrating type) where the laser light is absorbed in the superficial layer and does not penetrate or scatter deeply, such as the carbon dioxide (CO 2 ) (10,600 nm), erbium-doped yttrium-aluminum-garnet (Er:YAG) (2940 nm), and erbium, chromium: yttrium -scandium-gallium-garnet (Er,Cr:YSGG) (2780 nm) lasers [4,13].

Effects of Laser Irradiation on Osteoblasts
The number of reports about the effects of laser irradiation on osteoblasts is increasing. For periodontal regeneration, osteoblasts play essential roles in bone formation and remodeling [14]. Therefore, the laser irradiation of osteoblasts is an important focus of research.
Most reports on laser-irradiated osteoblasts in vitro used diode lasers, including a blue diode laser (λ = 450 nm), a red diode laser (λ = 635-660 nm), and a Ga-Al-As laser (λ = 780-980 nm). Several studies have reported the effects of the Nd:YAG laser (λ = 1064 nm) on osteoblasts in vitro. A few reports on the effects of CO 2 and Er:YAG lasers (λ = 2940 nm) have been published.

Nd:YAG Laser
Nd:YAG laser is the second-most studied laser for its effect on osteoblasts or osteoblast-like cells after diode lasers. The effects of Nd:YAG laser on Saos-2 human osteoblast-like cells have been investigated in many studies [75][76][77][78][79]. Nd:YAG laser irradiation has been reported to have positive effects on osteoblasts or osteoblast-like cells. Irradiation at 10 Hz and 20 mJ for 10 s had a stimulatory effect on the cell viability and proliferation of Saos-2 cells at 7, 14, and 21 days [75]. Cell proliferation of Saos-2 was also significantly increased at 48 h following irradiation at 50 or 70 Hz and 20 mJ/pulse for 10 s [76]. Another study reported that irradiation 3 times at 0.5-2 W enhanced cell proliferation rates of Saos-2 cells on day 4 compared to one-time irradiation [77]. Irradiation at 10.3 J/cm 2 accelerated cell migration until 24 h after irradiation and significantly enhanced ATP production in Saos-2 cells at 24 h following irradiation [78]. However, a Q-switched Nd:YAG laser irradiation at 1.5, 3, or 5 J/cm 2 significantly decreased proliferation in MC3T3-E1 cells [80].
Nd: YAG laser irradiation showed various effects regarding gene and protein expression related to osteogenic differentiation or bone remodeling. ALP activity was significantly increased in MC3T3-E1 cells at 3, 7, and 14 days after irradiation at 1.5, 3, or 5 J/cm 2 , with or without nonglycosylated human recombinant BMP-2 (100 ng/mL) treatment [80]. ALP gene (Alpl) expression was significantly increased in Saos-2 cells at 24 h and 7 days after irradiation at 17.3 and 1.5 J/cm 2 , respectively [76,79]. An increase in Runx2 and osteopontin mRNA expression was also significantly induced at 7 days after irradiation at 1.5 J/cm 2 in Saos-2 cells [76]. Expression of BMP2 was significantly increased in MC3T3-E1 cells 2 days after irradiation at 3 J/cm 2 [80]. Irradiation at 17.3 J/cm 2 significantly increased mRNA expression of RANKL and OPG in Saos-2 cells at 24 h [79]. In a previous study, highly intensified calcium deposition on day 12 and significantly enhanced mineralization on day 21 were observed in MC3T3-E1 cells by Nd:YAG laser irradiation at 1.5-5 J/cm 2 with or without rhBMP-2 treatment [80]. In addition, intracellular Ca 2+ in Saos-2 cells was increased by irradiation at 50 Hz with a fluence of 1.5 J/cm 2 through the activation of the transient receptor potential 1 (TRPC1) ion channels [76]. Gene expression of insulin-like growth factor-1 (IGF-1; IGF1), an important regulator of bone formation [81], was significantly enhanced in MC3T3-E1 cells on day 2 after irradiation at 3 J/cm 2 with or without rhBMP-2 treatment [80].

Er:YAG Laser
Three previous studies reported the effects of Er:YAG laser irradiation on osteoblasts or osteoblast-like cells in vitro [82][83][84]. Er:YAG laser irradiation at 5.1-12.7 J/cm 2 significantly reduced mitochondrial activity in Saos-2 cells compared to nonirradiated cells. However, mitochondrial activity was significantly increased with decreasing energy settings and/or increasing the distance between the laser application tip and the bottom of the culture plate [82]. Er:YAG laser irradiation at a fluence of 1.0-4.3 J/cm 2 significantly increased MC3T3-E1 cell proliferation by irradiation in the absence of a culture medium. When irradiated at higher fluences (6.7 and 8.6 J/cm 2 ), cell cytotoxicity of MC3T3-E1 was significantly increased. In the presence of a culture medium during irradiation, Er:YAG laser irradiation at much higher fluences (12.9 and 15.1 J/cm 2 ) significantly increased MC3T3-E1 cell proliferation on day 1 and 3 without increasing cell cytotoxicity. The effect of Er:YAG laser on cell proliferation seemed to be induced by the activation of ERK [83], which plays a central role in the control of cell proliferation [85]. Another study reported that Er:YAG laser irradiation did not affect cell proliferation but significantly enhanced calcification of primary osteoblast-like cells from rat calvaria [84]. Irradiation at 3.3 J/cm 2 significantly promoted mineralization of primary osteoblast-like cells on day 7, possibly via enhanced Bglap expression, without major thermal effects. Microarray analysis revealed that irradiation at 3.3 J/cm 2 caused an upregulation of inflammation-related genes and downregulation of Wisp2, which plays an important role in the differentiation and mineralization of osteoblasts [86]. Gene set enrichment analysis showed that Er:YAG laser irradiation enriched Notch signaling, which plays a critical role in various cellular functions, including the promotion of osteogenic differentiation of osteoblasts in synergy with BMP [87].

Er,Cr:YSGG Laser
There are no reports on the direct effects of Er,Cr:YSGG laser irradiation on osteoblasts or osteoblast-like cells. Hence, the molecular biological effects of Er,Cr:YSGG laser irradiation on osteoblasts remains unclear.

CO 2 Laser
The effect of CO 2 laser irradiation on osteoblast-like cells was reported in a previous study [88]. The study investigated the effect of CO 2 laser irradiation on rat osteoblast-like ROS 17/2.8 cells at 0.5-2 W for 20 s, resulting in a power density of 0.4-1.43 J/cm 2 . CO 2 laser irradiation at 1.43 J/cm 2 enhanced the mRNA expression of bone sialoprotein (BSP) at 12 h after irradiation. Transcription of BSP (IBSP) gene was also enhanced via the tyrosine kinase, Src tyrosine kinase, and ERK 1/2 signaling pathways, and fibroblast growth factor 2 response element in the rat IBSP gene promoter by CO 2 laser irradiation.

Summary
The contents of this section are summarized in Table 1. Several reports have revealed the favorable effects of laser irradiation on osteoblasts or osteoblast-like cells. Laser irradiation enhances or increases cell proliferation, viability, migration, calcification, and expression of genes and proteins related to osteogenic differentiation, thereby promoting bone formation. These effects were observed in many studies using various types of lasers with different wavelengths; however, most of the effective energy fluences were low (under 6.0 J/cm 2 ). In some studies, high power as well as low power irradiation was reported to have biological effects on osteoblasts or osteoblast-like cells. However, in vitro evidence related to Nd:YAG, CO 2 , Er:YAG, and Er,Cr:YSGG lasers are still limited. Further research is needed to elucidate the molecular biological effects of laser irradiation on osteoblasts.

Effects of Laser Irradiation on Fibroblasts
Fibroblasts are components of the connective tissue, which migrate to a lesion from the late inflammatory phase until epithelialization is completed [89]. Fibroblasts play an essential role in supporting other cells, are associated with wound healing or regeneration, and function to break down blood clots, thereby secreting various growth factors and cytokines and creating new extracellular matrix (ECM) and collagen structures [90]. Additionally, fibroblasts play a critical role in wound contraction [91]. Therefore, fibroblasts are essential for effective wound healing and tissue regeneration.
Since various types of lasers have been shown to enhance wound healing through tissue repair and anti-inflammatory effects in previous studies [92,93], the biological and molecular mechanism of this event has been pursued over the years. In particular, the effect of lasers on fibroblasts has been focused on this field. In this section, we focus on gingival fibroblasts. Osteoblast proliferation increased significantly after 830 nm laser irradiation (10 J/cm 2 ) but decreased after 780 nm laser irradiation (at 1, 5, and 10 J/cm 2 ). MG-63 cell proliferation increased significantly after 670 nm (at 5 J/cm 2 ) and 780 nm (at 1, 5, and 10 J/cm 2 ) laser irradiation, but not after 830 nm laser irradiation. Alkaline phosphatase (ALP) activity in the osteoblast line was increased after 830 nm laser irradiation at 10 J/cm 2 Laser irradiation significantly accelerated cell proliferation activity on day 3 and ALP activity on day 7 via transient receptor potential vanilloid 1(TRPV1).
Expression of Alpl, Sp7, and Runx2 mRNAs was significantly increased. Calcification was significantly increased 3 weeks later after final irradiation on day 2, 4, 6, 9, and 11.  Cell viability, alkaline phosphatase activity, and the expression of osteopontin and collagen type I mRNA were slightly enhanced in cells irradiated with 1 J/cm 2 . Combined application of rhBMP-2 and laser irradiation was more effective than application of either modality alone. Expression of RANKL, OPG, and M-CSF in hFOB cells were increased following application of rhBMP-2 and laser irradiation. The expression of TGFB1, BMP2, collagen type I, and osteopontin were increased following combined application of rhBMP-2 and laser irradiation.  Greater cell proliferation in the irradiated groups was observed first after 96 h. Osteocalcin synthesis and TGF-b1 production were significantly greater on the samples exposed to 3 J/cm 2 [56] Diode Rat primary calvarial osteoblastic cells Laser irradiation at both wavelengths promoted cell proliferation and wound healing. ALP activity and mineralization were significantly increased. Irradiation stimulated BMP2-induced phosphorylation of Smad1/5/8 and Bmp2 expression, but had no effect on the expression of inhibitory Smads6, Smad7, Bmp4, or insulin-like growth factor 1.
Laser irradiation enhanced Smad-induced Id1 reporter activity and BMP-induced transcription factors such as Id1, Osterix, and Runx2. Laser irradiation also stimulated BMP-induced expressions of type I collagen, osteonectin, and osteocalcin mRNA.
[ The proliferation rates on day 4 increased as the number of applications increased, especially in those cases in which the irradiation was performed 2 or 3 times more.

Diode Laser
Diode lasers are representative lasers used in PBM for wound healing, and their biostimulatory effects, such as anti-inflammatory effects, have been reported in previous studies [4]. To determine the physiological mechanisms related to the biological effects, lipopolysaccharide (LPS)-challenged human gingival fibroblasts (HGFs) were irradiated using an 830 nm diode laser, with a total energy of 1.9-12.6 J corresponding to 3-20 min exposure, and PGE 2 production and cyclooxygenase (COX)-1 (COX1) and COX-2 (COX2) gene expression were analyzed. The results suggested that PGE 2 production and COX-2 mRNA levels were significantly suppressed in a dose-dependent manner upon laser exposure [94]. Additionally, dramatic downregulation of plasminogen activator (PA) activity, implicated in the degradation of extracellular matrix and synthesis of kinin in the process of inflammation, and downregulation of tissue PA mRNA levels were observed in the HGFs irradiated with 830 nm laser at 7.9 J/cm 2 compared to that in the control group [95]. In addition, under similar conditions, interleukin (IL)-1β production was reduced, and further investigation by RT-PCR showed that mRNA expression of IL-1β was inhibited, whereas that of IL-1β-converting enzyme (ICE) was invariable [96].
The effect of diode laser on the proliferation and migration of fibroblasts has been reported previously [97][98][99], and the exposure time is more relevant to cell proliferation and cell survival than to power output [97]. Moreover, the cell proliferation rates in the single-dose and double-dose groups were compared using a 685 nm diode laser. Although cell proliferation was enhanced in both groups, no significant difference was observed between the two laser-irradiated groups. In addition, a single dose of 2.0 J/cm 2 in the irradiated group resulted in a higher proliferation and viability rate than the nonirradiated control group. They also evaluated the secretion of growth factors such as basic fibroblast growth factor (bFGF), IGF-1, and the receptor of IGF-1 (IGFBP3). Single-dose irradiation significantly increased the secretion of bFGF and IGF-1 in irradiated cells, but the secretion of IGFBP3 was not significantly increased compared to that in control cells. All growth factors were significantly increased in the double-dose group compared to the nonirradiated group [100]. Similar to this result, the upregulation of mRNA expression for other growth factors such as IGF, VEGF, and TGF-β has also been confirmed [101][102][103]. In contrast, irradiation with an 810 nm laser caused a dramatic reduction in HGF cell numbers in vitro, with variable parameters, i.e., fluence of 24.6-492.8 J/cm 2 [104].
Diode lasers have also been reported to have positive effects on collagen synthesis [102]. Investigation of the effect of a 904 nm diode laser on cell growth and procollagen synthesis of NIH-3T3 fibroblasts was approximately three-to sixfold after irradiation with 3 and 4 J/cm 2 , although no significant increase in procollagen synthesis was observed. However, neither cell growth nor procollagen synthesis was observed at 5 J/cm 2 [105]. In contrast, in another study, gene expression of collagen type 1 was upregulated in the HGF cell line (HGF3-PI 53) 3 days after irradiation (4 J/cm 2 ) [99].
To examine the laser's effect on fibroblast-myofibroblast differentiation, NIH/3T3 fibroblasts were irradiated with a 635 nm diode laser at 0.3 J/cm 2 , and morphological, biochemical, and electrophysiological assays were conducted. Expression of matrix metalloproteinase (MMP)-2 and MMP-9 (MMPs play a pivotal role in physiological processes such as tissue remodeling) was upregulated, whereas tissue inhibitors of MMPs (TIMP)-1 and TIMP-2 were suppressed. Additionally, TGF-β1/Smad3-mediated fibroblast-myoblast transition was inhibited. These results suggest that the diode laser modulates the TRPC1 ion channel, which in turn contributes to an antifibrotic effect by interfering with TGF-β1 signaling [106].
Bisphosphonate treatment is known to have a negative effect on wound healing [107]. In a study, diode laser irradiation tended to increase the viability of HGFs, although no significant difference was observed compared to that of nonirradiated control HGFs. However, when HGFs were cultured in a bisphosphonate-conditioned medium, laser irradiation significantly increased cell viability. Furthermore, laser irradiation on cell-free bisphosphonate-conditioned medium before culturing HGFs had no significant effect on cell viability, which indicated that laser irradiation directly affected the HGFs rather than suppressing the medicinal effect of bisphosphonate [43]. Thus, diode lasers may have the potential to become a supportive tool for preventing and treating of bisphosphonate-related diseases, such as osteonecrosis of the jaw.
However, adverse effects of lasers have also been reported. Diode laser irradiation (904 nm) at 3 J/cm 2 on fibroblast cell line changed the ultrastructure of the cells' cytoplasmic organelles; concurrently, a significant reduction in protein synthesis was observed [108]. Therefore, the cytotoxicity of the lasers should also be investigated for their safe usage.

Nd:YAG Laser
Previous studies have indicated that Nd:YAG lasers have various biological effects on cells, both in vivo and in vitro [4]. Nd:YAG laser (wavelength: 1060 nm) reduced collagen synthesis in human skin fibroblasts at energy levels as low as 1.1 × 10 3 J/cm 2 , without altering DNA replication or cell viability [109]. Furthermore, DNA replication and collagen synthesis in human skin fibroblasts have been compared between Nd:YAG laser irradiation at 1.2-4.7 × 10 3 J/cm 2 for 3-12 s and under halogen lamp heat. Marked inhibition of DNA replication and collagen production was observed in the laser-irradiated fibroblasts, although no such decrease was noted in the halogen lamp-heated fibroblasts. Therefore, several characteristics other than the thermal effect may be critical in altering the biological functions of fibroblasts [110].
Nevertheless, histological analysis of laser-treated skin areas showed new collagen formation and increased the number of fibroblasts [111]. In addition, many other studies showed an increase in procollagen and collagen type-1 levels after Nd:YAG laser irradiation [76,[112][113][114][115]. Nd:YAG laser downregulated the expression of MMP-1 and MMP-2 enzymes in the injured skin [112]. The reduction in MMP-1 was observed in keratinocyte-fibroblasts after Q-switched Nd:YAG laser irradiation at 8 J/cm 2 [113]. Moreover, the effects of different wavelengths (532 nm and 1064 nm) of a Q-switched Nd:YAG laser on human skin fibroblasts were investigated. Both the lasers significantly increased the expression of type I and III procollagen and tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and decreased MMP-2 and MMP-3 expression. Higher increased/decreased rates were observed in the 1064 nm Nd:YAG laser irradiation. Additionally, the 532 nm Nd:YAG laser increased Hsp70 and IL-6 expression, whereas the 1064 nm Nd:YAG laser upregulated TGF-β expression, suggesting that the molecular biological effects of Nd:YAG laser irradiation may differ according to the wavelengths used [115].

Er:YAG and Er,Cr:YSGG Lasers
The specific absorption characteristics of Er:YAG and Er,Cr:YSGG lasers have been reported to be beneficial for wound healing after soft tissue ablation [116,117]. During ablation, cells underlying the surface layer, including fibroblasts, indirectly receive low energy of the Er:YAG/Er,Cr:YSGG laser irradiation, which has been shown to promote wound healing and tissue regeneration [10]. Therefore, recently, a direct effect of low-level irradiation of Er:YAG/Er,Cr:YSGG laser on fibroblasts has been investigated in vitro. Pourzarandian et al. [118] showed that low-level laser therapy using an Er:YAG laser enhanced the proliferation of cultured HGFs and identified the optimal stimulative energy density of 3.4 J/cm 2 . They also observed a significant increase in PGE 2 production and COX-2 mRNA expression after irradiation, and laser-induced PGE 2 synthesis was completely inhibited by the COX-2 inhibitor, NS398 [119]. Proteomic analysis has been performed to investigate differentially expressed proteins in HGFs induced by low-level Er:YAG laser irradiation. On day 1 after irradiation at 2.1 J/cm 2 , significant cell proliferation without cell damage was observed. In addition, a total of 377 differentially expressed proteins were identified by mass spectrometry, 59 of which were upregulated and 15 were downregulated in laser-irradiated HGFs. Among the upregulated differentially expressed proteins, galectin-7, which is one of the essential proteins in the wound-healing process, was validated by quantitative PCR, Western blotting analysis, and enzyme-linked immunosorbent assay. To confirm the effect of galectin-7, HGFs were treated with recombinant human galectin-7, and cell proliferation was assessed in a dose-dependent manner, which suggested that alteration in protein expression and upregulation of galectin-7 may partly contribute to proliferation in HGFs [120]. Kong et al. [121] observed maximal cell proliferation at 6.3 J/cm 2 on day 3 after irradiation, although it was accompanied by an increase in lactate dehydrogenase (LDH) release. An increase in ATP level, Ki-67 staining, and cyclin-A2 mRNA expression was confirmed, and it was observed that the increase in cell proliferation was due to the effect of Er:YAG laser irradiation on the cell cycle. However, alterations in the mitochondria and ribosomal endoplasmic reticulum (ER) were observed at 3 h postirradiation at 6.3 J/cm 2 ; the changes subsided after 24 h, suggesting the occurrence of transient cellular injury. Furthermore, as the surface temperature of laser-irradiated cells reached 40.9 • C, nonirradiated cells were treated with a medium warmed at 40 • C, which also increased cell proliferation. In addition, laser-induced cell proliferation was suppressed by inhibitors of the thermosensory transient receptor potential channels (TRPV-1), capsazepine, or SKF96365. Finally, 21 genes involved in heat-related biological responses and endoplasmic reticulum-associated degradation were identified by microarray analysis. Therefore, 6.3 J/cm 2 laser irradiation on HGFs may enhance cell proliferation through photothermal effects, despite transient cellular damage.
The effects of Er:YAG and Er,Cr:YSGG irradiation on cultured fibroblast cell lines (NCBI:C-165) were compared at different fluences: 1 W power output (10 Hz and 100 mJ) and 0.5 W power output (10 Hz and 150 mJ), respectively. Cell proliferation was upregulated in both groups compared to that in the control, but Er,Cr:YSGG laser irradiation tended to be more effective in cell proliferation than Er:YAG laser irradiation [122].
The mechanical effects of Er:YAG laser irradiation on fibroblasts have also been studied. As the number of primary human gingival fibroblasts significantly decreased after 3.0 W irradiation, gene expression analysis was conducted for cells irradiated at 0.6, 1.0, and 1.2 W. Cells were divided into four groups: control cells (not undergoing any procedures), cells undergoing only Er:YAG laser irradiation, cells undergoing only centrifugal loading, and a cells undergoing both Er:YAG laser irradiation and centrifugal force loading. Gene expression of COX2, IL1B, TNFA, BMP2, and BMP4 was significantly increased in laser-irradiated cells (in a dose-dependent manner) compared to the control cells at 24 h after irradiation. Additionally, only COX2 gene expression showed a significant increase in the centrifugal-loaded cells compared to control cells. In contrast, gene expression of COX2, IL1B, TNFA, BMP2, and BMP4 was significantly higher in the laser-loaded and centrifugally loaded cells than in the centrifugally loaded cells. These results suggest that bone metabolism genes may be regulated by mechanical stimulation and laser irradiation combined [123].

CO 2 Laser
High-power CO 2 laser irradiation is mainly used in various surgical procedures as an alternative to traditional scalpel procedures [124]. Recently, low-level laser irradiation with a CO 2 laser has gained attention in dentistry due to its promotive effect on wound healing [125][126][127]. The secretion of TGF-β1 was downregulated, whereas that of bFGF was upregulated by high-frequency CO 2 laser irradiation, which occurred maximally at 4.7 J/cm 2 in both normal and keloid dermal fibroblasts in vitro, resulting in enhancement of cell replication [128]. Thus, CO 2 laser irradiation may have the ability to balance collagen organization in fibrosis. Furthermore, PBM with a CO 2 laser on proliferation and migration was examined at the cellular level. Promotion of cell proliferation and migration of cultured human dermal fibroblasts (HDFs) were examined by MTS assay and cell migration assay, respectively, with irradiation of 1.0 J/cm 2 . In addition, with the same power, Western blotting analysis showed activation of Akt, ERK, and JNK signaling pathways. However, suppression of Akt, ERK, or JNK signaling pathways significantly inhibited both the proliferation and migration of laser-irradiated HDFs. The study indicated that low-level laser irradiation with a CO 2 laser might promote proliferation and migration of fibroblasts via activation of Akt, ERK, or JNK signaling pathways [129].
From another perspective, as the clinical use of CO 2 lasers has increased, the safety of laser irradiation has been investigated. Apfelberg et al. [130] exposed cultured fibroblasts to CO 2 laser irradiation before the occurrence of malignancy was examined. The results showed that CO 2 laser-irradiated cells did not exhibit a greater incidence of malignancy compared to controls, indicating that CO 2 laser seems to be noncarcinogenic in laboratory cells.

Summary
The contents of this section are summarized in Table 2. The effects of PBM by laser irradiation on fibroblasts appear to be comparable despite the different wavelengths. Proliferation, migration, and secretion of cytokines/chemokines are the main functions affected by laser irradiation; which may lead to early wound healing; although, the biological/molecular evidence to support this phenomenon is still partial and inadequate. Moreover, excessive power or irradiation time results in cell damage and ineffective treatment. However, concurrently, lasers have the potential to regulate collagen synthesis through fibroblast stimulation depending on the target disease. Thus, further investigation on optimal configuration, which is consistent in vivo and in vitro, and more profound bioinformatic studies are required in the future to clarify the critical mechanisms of the effects of lasers on fibroblasts.    Laser irradiation caused ultrastructural changes. Procollagen synthesis was unaffected, but significant reduction in the amount of protein was observed in the medium conditioned by irradiated cells.   Gene expression of COX2, IL1B, TNFA, BMP2, and BMP4 significantly increased in laser-irradiated group in a dose-dependent manner compared to the control group. COX2 gene expression showed significant increase in the centrifugal load group compared to control group, whereas gene expression of COX2, IL1B, TNFA, BMP2, and BMP4 was significantly higher in the laser and centrifugal loaded group than in the centrifugal load group.

Effects of Laser Irradiation on Periodontal Ligament Cells
The periodontal ligament is the only ligament in the body that connects two distinct hard tissues. It is a fibrous, complex, and soft connective tissue that attaches the tooth root to the inner wall of the alveolar bone. The periodontal ligament thickness decreases with age. It is functionally essential for tooth support and for allowing teeth to withstand the forces generated during mastication [131].

Diode Laser
Diode laser irradiation has been reported to have positive effects on human periodontal ligament cells (hPDLCs). PBM at energy doses of 2 and 4 J/cm 2 upregulated gene expression related to osteogenic differentiation, including BMP2, OC (BGLAP), RUNX2, and ALPL in hPDLCs. PBM enhanced the osteogenic differentiation of hPDLCs via cAMP regulation [132]. Additionally, it significantly increased cellular viability, decreased cellular inflammatory marker expression, and increased OC activity in hPDLCs at two energy densities (5 and 10 J/cm 2 ) [133]. Suppression of inflammation is one of the positive effects of laser irradiation. After 670 nm Ga-Al-As laser irradiation (5 and 10 J/cm 2 ), the mRNA expression of inducible NO synthase (INOS), COX2, and IL1B were decreased compared to that in nonirradiated control cells.
Another study using an 830 nm laser showed that laser irradiation at 3.8 J/cm 2 decreased COX-2 and cytosolic phospholipase A 2 -α mRNA expression after 24 h in mechanically stretched hPDLCs [134]. The increase in PGE 2 production was significantly inhibited by diode laser irradiation at 346-1152 J/cm 2 in a dose-dependent manner. The increase in IL-1β production was also significantly inhibited by diode laser irradiation, although the inhibition was observed only with high-power irradiation [135]. Diode laser irradiation (4.0-7.9 J/cm 2 ) significantly inhibited a marked increase in plasminogen activator activity in hPDLCs in response to stretching [136]. Additionally, Huang et al. [137] reported that the gene expression levels of INOS, TNFA, and IL1B in LPS-exposed periodontal ligament cells were decreased after irradiation, and phospho-ERK expression was significantly increased in the laser-irradiated cells compared to that in nonirradiated cells.
hPDLCs irradiated with an 810 nm diode laser, showed promotion of proliferation and differentiation. Irradiation at 3.9 J/cm 2 increased proliferation of human periodontal ligament fibroblasts (PDLFs) between 24 and 48 h, and ALP activity at 48 and 72 h. The phosphorylated ERK level was also more prominent after irradiation at 3.9 J/cm 2 energy fluency [138]. Additionally, the protein expression of MMP-8 in hPDLFs was decreased by 810 nm diode laser irradiation at 10 J/cm 2 [139]. Moreover, the 809 nm diode laser irradiation at 2.0-7.8 J/cm 2 of PDLFs significantly upregulated their proliferation up to 72 h [140].

Er:YAG Laser
Er:YAG laser irradiation at 4.2 J/cm 2 on hPDLFs promoted cell proliferation, migration, and invasion abilities. The report also revealed that the silencing of galectin-7 abrogated the effects of Er:YAG laser on cell proliferation, migration, and invasion, suggesting that the Er:YAG laser promoted these effects through the induction of galectin-7 [141].

Nd:YAG Laser, Er,Cr:YSGG Laser, and CO 2 Laser
There are no reports on the effects of Nd:YAG laser, Er,Cr:YSGG laser, and CO 2 laser irradiation on periodontal ligament cells.

Summary
The contents of this section are summarized in Table 3. Laser irradiation on PDLs enhanced cell proliferation, migration, calcification, and differentiation. In addition, gene expression was altered by laser irradiation, especially with suppression of inflammatory products. However, the effects of laser irradiation on hPDLCs and hPDLFs were only investigated using diode and Er:YAG lasers.
It is necessary to generate more evidence and reveal the mechanisms by which laser irradiation affects hPDLCs. The proliferation, migration, and invasion abilities were induced through the upregulation of galectin-7 after laser irradiation.

Effects of Laser Irradiation on Endothelial Cells
The mechanisms related to the effects of laser irradiation on wound healing are not completely clear. However, some studies reported that laser treatment could accelerate wound healing, especially in acute, chronic, and impaired wound-healing conditions [142] as well as in periodontal disease [143]. Endothelial cells play important roles in the process of wound healing and regeneration of periodontal tissue [144]. Hence, in this section, we summarized the direct effects of laser irradiation on endothelial cells.

Diode Laser
Some researchers have investigated the effect of diode laser irradiation on endothelial cells. The human vascular endothelial cell line (HECV) irradiated with an 808 nm diode laser (60 J/cm 2 ) demonstrated no significant difference in viability but demonstrated higher proliferation than non-treated cells. Moreover, the study reported that diode laser stimulated mitochondrial oxygen consumption and ATP synthesis in HECV [145]. Another study using the 808 nm diode laser reported that CD54, CD62E, monocyte chemotactic protein-1 (MCP-1) expression, and von Willebrand factor release were altered in human umbilical vein endothelial cells (HUVECs) stimulated with IL-1β followed by laser irradiation. MCP-1 expression in HUVECs was significantly lower 6 h after 4.5 J/cm 2 stimulation than in IL-1β stimulated cells. In addition, both 1.5 and 4.5 J/cm 2 of laser irradiation inhibited IL-1β-induced increase in CD54 and CD62E concentration in the supernatant. Therefore, this study suggested that low-power laser irradiation decreased the pro-inflammatory and procoagulant activity of IL-1β-stimulated endothelial cells [146]. Moreover, a 670 nm diode laser irradiation caused a stimulatory effect on the proliferation of HUVECs [147] and increased their viability [43]. Using a 635 nm diode laser at different doses (2, 4, and 8 J/cm 2 ), all doses of irradiation significantly increased the proliferation of HUVECs and significantly reduced the concentration of soluble vascular endothelial growth factor (sVEGFR-1), an inhibitor of vascular endothelial growth factor (VEGF), compared with nonirradiated cells [148]. A study using rhesus macaque choroid-retinal endothelial cells (RF/6A) reported that an 810 nm diode laser (over 84.0 J/cm 2 ) irradiation caused significant cell death, and irradiation at a fluence of 45.9-76.4 J/cm 2 induced Hsp70 hyperexpression at 12-18 h postirradiation [149].

Nd:YAG Laser
Some studies have reported the effect of Nd:YAG laser irradiation on endothelial cells. A significant induction in vinculin expression, a focal adhesion protein involved in cell adhesion and migration, in human endothelial H-end cells was observed in the Nd:YAG-irradiated (fluence, 1.5 J/cm 2 ) cells. Moreover, this study showed that Nd:YAG laser irradiation did not affect cell viability and stimulated cell growth [76]. In another study, cultured rat aortic endothelial cells at 5 h after Nd:YAG (1.6 J/cm 2 ) laser irradiation were examined using a DNA array chip. This study showed that 20 genes in laser-treated cells were upregulated by more than four-fold compared to those in the control, and Nd:YAG laser irradiation also upregulated gene expression related to cell migration, cell structure neurotransmission, and inflammation [150]. Moreover, Nd:YAG laser irradiation (1.5 J/cm 2 ) of HUVECs cultured on titanium disks coated with Porphyromonas gingivalis LPS caused downregulation of endothelial adhesion molecules, including that of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM) levels compared to nonirradiated HUVECs [151].

Er:YAG, Er,Cr:YSGG, and CO 2 Lasers
There are no reports on the direct effects of irradiation on endothelial cells. Therefore, the molecular biological effects of Er:YAG, Er,Cr:YSGG, and CO 2 laser irradiation on endothelial cells are not apparent to date.

Summary
The contents of this section are summarized in Table 4. In conclusion, studies on the molecular biological effects on endothelial cells using lasers have not been so much reported and are inconclusive. Further research is needed to reveal the effects of diode lasers on endothelial cells.

Effects of Laser Irradiation on Cementoblasts
Cementum is a unique, avascular, and mineralized tissue formed by cementoblasts [152]. Only one study has reported the effects of laser irradiation on cementoblasts. Diode laser irradiation at 940 nm was performed on root plate-or microplate-seeded cementoblasts at a fluence of 18 J/cm 2 . Cell proliferation was not different until 96 h, but laser irradiation significantly retarded the decrease in cell proliferation after 96 h compared to the untreahted control group. Additionally, Ibsp and Bglap, which are transcripts required for cementum formation, were significantly increased in laser-irradiated cells compared to nonirradiated cells. Moreover, the expression levels of Bmp-2,3,6,7 were significantly increased. These results indicate that biostimulation can be used during regenerative periodontal therapies to trigger cells with a periodontal attachment apparatus [153]. The contents of this section are summarized in Table 5. The mRNA expression related to only cementoblast and bone morphogenetic protein were increased in laser-irradiated cells.

Effects of Laser Irradiation on Epithelial Cells
Epithelial cells are found on the surfaces of tissues and organs. Although they share some common characteristics, they vary in size, shape, and general appearance, according to their location [154]. Moreover, they protect deeper tissues against the external environment and possess secretory and supportive functions, thus contributing to homeostasis maintenance. Epithelial cells play an important role in wound healing [155]. There are many studies on the effects of laser irradiation on epithelial cells associated with various tissues [156][157][158], with the exception of oral tissues. Herein, we summarize the effects of laser irradiation on oral epithelial cells. To our knowledge, no research has been published regarding the effects of Nd:YAG, Er:YAG, Er,Cr:YSGG, or CO 2 lasers on oral epithelial cells. Although some studies have reported increased proliferation in Nd:YAG laser-irradiated epithelial cells [77], there are no studies on oral tissues.

Diode Laser
Diode laser irradiation has been previously reported to enhance wound healing [159]. Diode lasers can penetrate superficial tissues to exert their effects in deeper tissues. However, epithelial cells are the first cells that receive laser energy. Thus, epithelial cells absorb the highest amount of energy compared to other underlying cells. Therefore, the effects of diode lasers have been studied in vitro on various epithelial cells, including keratinocytes [160], a keratinocyte cell line (Hacat) [161,162], epithelial adenocarcinoma (HeLa) cells [163,164], pigment epithelial cells [165][166][167], and human breast epithelial cell lines (SVCT and Bre80hTERT) [168]. In this section, we focus on epithelial cells or cell lines related to oral tissues.
To examine the effect of a low level diode laser irradiation on oral epithelial cells, cultured, normal human oral keratinocyte (NOKSI) cells were irradiated using an 810 nm diode laser in continuous wave mode for 5 min, at a distance of 14.5 cm. Upregulated gene and protein expression of human β defensin-2 (HBD-2), a potent antimicrobial and wound-healing factor, were confirmed by qPCR, Western blotting, and immunostaining. Increased expression of HBD-2 was mediated by laser-activation of the TGF-β1 pathway [169].
A pulsed diode laser has also been used for in vitro studies on epithelial cells. In a study by Ejiri et al. [170], the effect of low-level diode laser irradiation was examined on primary human gingival epithelial cells (HGECs). Using a 904-910 nm diode laser applied at a high frequency of 30 kHz for 1-10 min, at 5.7-56.7 J/cm 2 , a significant increase in the proliferation of laser-irradiated cells was detected by WST-8 assay, 24 h after laser irradiation. The maximum proliferative effect was observed after 5 min of laser irradiation. The in vitro wound-healing assay showed a dramatic increase in migration among the laser-irradiated cells. Moreover, phosphorylation of MAPK/ERK was observed at 5, 15, 60, and 120 min after irradiation, whereas stress-activated protein kinases/c-Jun N-terminal kinase and p38 MAPK remained unphosphorylated. These results indicate that proliferation and migration of HGECs may be promoted via activation of MAPK/ERK.
The antimicrobial effects of diode lasers have been reported previously [171]. To investigate the cellular mechanisms of these effects, human oral squamous epithelial carcinoma cell lines (Ca9-22 and SCC-25) were treated with LPS. An 805 nm diode laser was then used to irradiate cells in a repeated pulse mode for 60 s at a 1 cm distance. The expression of DEL1, which encodes a protein with anti-inflammatory effects, was significantly increased following laser irradiation. In contrast, LPS-induced IL-6 and IL-8 expression was significantly suppressed following laser irradiation. A significant increase in migration was also observed in laser-irradiated cells [172]. These results indicate a suppressive effect of laser irradiation on the inflammatory response.

Summary
The contents of this section are summarized in Table 6. Many studies have examined the effect of laser irradiation on the epithelium in vivo [156][157][158]. However, despite the importance of epithelial cells in wound healing, in vitro studies are limited, especially with regard to epithelial cells associated with oral tissues. Epithelial cells have different characteristics based on their location and associated tissues, and further investigation of oral epithelial cells is required to enable the advancement of laser therapies for use in oral wound healing. The mRNA expression of DEL1 was significantly upregulated by laser irradiation (p < 0.01). Lipopolysaccharide (LPS)-induced interleukin (IL)-6 and IL-8 expression was significantly suppressed in the LPS+laser group (p < 0.01). Intercellular adhesion molecule (ICAM)-1 expression was significantly higher in the LPS+laser group than in the LPS only or control groups. Compared with the control, the migration of epithelial cells was significantly increased by diode laser irradiation.

Effects of Laser Irradiation on Osteocytes
Only a few studies have reported the effects of laser irradiation on osteocytes. Suppression of Sost expression was observed in primary osteocyte-like cells isolated from rat calvaria after CO 2 laser irradiation at 0.7-2.8 J/cm 2 without an increase in temperature. The study reported an increase in Dmp1 expression was after CO 2 laser irradiation at 1.4-2.8 J/cm 2 [173]. Ohsugi et al. [174] reported that Sost expression in osteogenic cells (osteoblast-like cells isolated from rat calvaria cultured with osteoinduction medium for 21 days) was decreased at 6 h after Er:YAG laser irradiation (2940 nm) at energy densities of 1.5 and 3.1 J/cm 2 . Following the results obtained by quantitative PCR, sclerostin (coded by Sost) expression in the cultured supernatant was significantly decreased. As sclerostin produced by osteocytes can inhibit osteoblast activity and suppress bone formation, it is thought that Er:YAG laser irradiation may promote bone formation via the suppression of Sost expression. The contents of this section are summarized in Table 7. The mRNA expression of Sost was decreased and that of Mef2c was increased in laser-irradiated cells.

Effects of Laser Irradiation on Osteoclasts
Osteoclasts are multinucleated giant cells that have the capacity to resorb mineralized tissues [175]. The development of osteoclasts proceeds within the local microenvironment of the bone.
Only a single report has been published on the effect of laser irradiation on osteoclasts in vitro. Rat osteoclast precursor cells (osteoclast-like cells) purified from rat bone marrow were subjected to laser irradiation with 810 nm diode and a maximum power output of 50 mW at exposure times of 1, 3, 6, or 10 min/day, which corresponded to 9.3, 28.0, 56.0, or 93.3 J/cm 2 , respectively. Laser irradiation at 9.3-56.0 J/cm 2 increased the number of tartrate-resistant, acid phosphatase-positive multinucleate cells. Furthermore, osteoclasts appeared on day 2 in the laser-irradiated groups but not until day 3 in the nonirradiated control groups. Receptor activator of NF-kappaB (RANK) in the laser-irradiated groups showed significantly greater staining compared to the control group on day 2 and 3 by immunohistochemistry, and the mRNA expression of RANK was upregulated, consistent with the immunohistochemistry results. The study suggested that irradiation with an 810 nm diode laser facilitated the differentiation and activation of osteoclasts via RANK expression [176]. The contents of this section are summarized in Table 8.

Effects of Laser Irradiation on Stem Cells
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more numbers of the same stem cells. They are the earliest type of cells in the cell lineage [177]. Mesenchymal stem cells (MSCs) are multipotent cells found in adult tissues. Adult MSCs were isolated from almost every type of connective tissue, such as adipose [178], bone marrow, periodontal ligament [179], and dental pulp tissues [180]. Stem cell therapy is the use of stem cells to treat or prevent diseases. Stem cell therapy is applied to many types of treatments, including regeneration and wound healing [181]. Recently, the PBM effects of laser irradiation on MSCs have attracted much attention. In the next paragraph, we have reviewed the effect of laser irradiation on MSCs.

Diode Laser
Although studies on PBM's effect on MSCs are limited, several studies using diode lasers with different wavelengths have been reported. PBM (using a combination of 630 and 810 nm lasers) stimulated the viability of human adipose-derived stem cells (hASCs) and human bone marrow mesenchymal stem cells (hBM-MSCs). In addition, PBM (irradiation once or twice at 630 nm and 0.6 and 1.2 J/cm 2 ) increased the viability of hASCs compared to the control and laser-treated hBM-MSCs. Furthermore, PBM (using a combination of 630 and 810 nm lasers, 3 times irradiation at 2.4 J/cm 2 ) increased hASC viability compared to control and laser-treated hBM-MSCs [182]. Some studies have reported the effect of laser irradiation at around 630 or 810 nm on MSCs. Diode laser irradiation at 635 nm in MSCs derived from femurs and tibias in rats caused an increase in the expression levels of v-akt murine thymoma viral oncogene homolog 1 (Akt1), cyclin D1 gene (Ccnd1), phosphatidylinositol 3-kinase, catalytic alpha polypeptide gene (Pik3ca), in addition to a decrease in protein tyrosine phosphatase nonreceptor type 6 (Ptpn6), and serine/threonine kinase 17b (Stk17b) expression. Microarray analysis was also performed in this study, which revealed that 119 genes were differentially expressed, and various genes involved in cell proliferation, apoptosis, and the cell cycle were affected. The study suggested that the increase in MSC proliferation was mediated through the PI3K/Akt/mTOR/eIF4E pathway [183]. In addition, cytotoxicity evaluated by LDH assay did not show a significant difference between nonirradiated and 635 nm diode laser-irradiated (0.5-5.0 J/cm 2 ) MSCs obtained from rat bone marrow. Diode laser irradiation at 0.5 J/cm 2 was found to be an optimal energy density to stimulate the proliferation of bone marrow stromal cells (BMSCs); additionally, irradiation at 5.0 J/cm 2 significantly stimulated the secretion of VEGF and NGF. Furthermore, after 5-aza induction, myogenic differentiation was observed in all the groups, and diode laser irradiation at 5.0 J/cm 2 dramatically facilitated the differentiation [184]. The effect of 635 nm diode laser irradiation on the osteogenic differentiation of MSCs has also been reported. Laser irradiation (0.4 J/cm 2 ) on human mesenchymal stromal cells (hMSCs) increased vinculin-rich clusters, osteogenic expression of markers (e.g., Runx-2, alkaline phosphatase, osteopontin), and mineralized bone-like nodule structure deposition as well as induced stress fiber formation and upregulated the expression of the proliferation marker Ki67. The study suggested that 635 nm diode laser irradiation may be a potentially effective option for promoting/improving bone regeneration [33]. In addition, 635 nm diode laser irradiation (0.3 J/cm 2 ) on MSCs derived from the femora and tibia of male C2F1 mice significantly enhanced MSC proliferation, without a change in cell viability. They also found that the increase in proliferation after 635 nm diode laser irradiation was associated with the upregulation and activation of the Notch-1 pathway and increased membrane conductance through voltage-gated K+, BK, and Kir channels and T-and L-type Ca 2+ channels [185].
Recently, studies on diode laser irradiation at 808 nm related to MSCs have also been reported. At 0.5-4.0 J/cm 2 , irradiation of human gingival mesenchymal stem cells (HGMSCs) promoted their migration but not proliferation. Furthermore, diode laser irradiation could activate mitochondrial ROS, which could elevate the phosphorylation levels of JNK and IKB in HGMSCs, further activating NF-κB concomitantly with the elevation of the nuclear translocation of p65. Taken together, these results indicate that PBM may promote cell migration via the ROS/JNK/NF-κB pathway [186]. High power 808 nm diode laser irradiation (64 J/cm 2 ) enhanced osteogenesis. Laser irradiation of BMSCs from 3-old female BALB/c mice increased the protein expression of Runx2 and Osterix and suppressed PPARγ, a pivotal transcription factor in adipogenic differentiation. Positive areas of ALP and Alizarin Red S histochemical staining were significantly increased after laser irradiation [187].
Regarding the 606 nm diode laser irradiation, irradiation with 1.9 J/cm 2 enhanced the proliferation of BMSCs, although irradiation with 11.7 J/cm 2 suppressed the proliferation. The cytotoxic effect of 50 µg/mL carboplatin was eliminated, and the inhibitory effect of 0.1 µg/mL vincristine was attenuated by laser irradiation at 1.9 J/cm 2 [188]. In addition, 660 nm diode laser irradiation (5 J/cm 2 ) on stem cells from human exfoliated deciduous teeth (SHEDs) increased cell proliferation and expression of mesenchymal stem cell markers, including OCT4, Nestin, and CD90 [189]. However, another study showed that human dental pulp stem cells (hDPSCs) irradiated at 660 nm and 5 J/cm 2 showed signs of apoptosis and necrosis as observed by transmission electron microscopy (TEM). Diode laser irradiation at 3 J/cm 2 increased fibronectin production in hDPSCs [190]. The effects of 660 nm diode laser irradiation (1.6 J/cm 2 ) were also evaluated in hDPSCs. Gene expression of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GNDF), matrix-associated protein 2 (MAP2), nuclear receptor-related 1 protein (NURR1), and dopamine transporter (DAT) were increased, especially in the first 7 days of dopaminergic induction. However, the hDPSCs were not able to differentiate into functional dopaminergic neurons either in nonirradiated control or laser-irradiated groups [191].

Nd:YAG Laser
There are some reports on the effects of Nd:YAG laser irradiation on MSCs. Nd:YAG laser irradiation (2 and 4 J/cm 2 ) on hBMSCs promoted proliferation and osteogenesis, although irradiation at an energy density of 16 J/cm 2 significantly suppressed the proliferation and osteogenesis of hBMSCs [192]. In addition, Nd:YAG laser irradiation at 9.8 J/cm 2 on MSCs obtained from horses did not show a difference in viability between irradiated and control MSCs. However, laser-irradiated MSCs exhibited slightly lower proliferation and significantly increased expression of IL-10 and VEGF compared to nonirradiated control MSCs [193]. Frequency-doubled Nd:YAG laser irradiation (532 nm) of human adipose tissue-derived stem cells (hADSCs) was performed at densities of 5-45 J/cm 2 for 30-300 s. Mitochondrial activity of hADSCs was evaluated by autofluorescence emission at wavelengths associated with nicotinamide adenine dinucleotide (NADH) and flavoproteins. Laser irradiation at 5-9.2 J/cm 2 significantly increased the proliferation of hADSCs, which was attributed to an increase in mitochondrial activity, although hADSCs irradiated at 28 and 45 J/cm 2 showed a significant decrease in proliferation and autofluorescence [194].

CO 2 Laser
We found only one report that mentioned the effect of CO 2 laser irradiation on MSCs in an in vitro study. CO 2 laser irradiation (9 W, exposure time 4 ms/shot and a medium pattern of the spots) on hADSCs increased their proliferation when cultured under nutrient-deprived conditions (0.5% FBS) and reduced cell proliferation in a medium supplemented with 10% FBS. CO 2 laser irradiation caused a transient increase in mitochondrial ROS and the capacity to restore ∆ψm after rotenone-induced depolarization, and increased the secretion of MMP-2 in conditioned media comprising MMP-9, VEGF, and adiponectin, which have the capacity to support the angiogenesis of endothelial progenitor cells. The study concluded that CO 2 laser irradiation on ADSCs might activate the redox pathways that increase cell proliferation and enhance the secretion of angiogenic molecules [195].

Summary
The contents of this section are summarized in Table 9. Reports on the effects of laser irradiation on MSCs are limited. However, some studies have shown that laser irradiation, especially low-power irradiation, causes cell proliferation and favorable gene expression changes in MSCs. MSCs are already clinically applied for periodontal regeneration. MSC sheets transplanted to root surfaces can induce regeneration of periodontal tissue [196]. Although further research is required to clarify the effects of laser irradiation on MSCs, laser irradiation may enhance MSCs regenerative capabilities in periodontal tissues. Laser irradiation at 1.6 J/cm 2 increased mRNA expression of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), matrix-associated protein 2 (MAP2), nuclear receptor-related 1 protein (NURR1), and dopamine transporter (DAT) in hDPSCs. [192] Nd:YAG  Laser irradiation (output power 9 W, exposure time 4 ms/shot) increased proliferation, mitochondrial ROS, the capacity to restore ∆ψm after rotenone-induced depolarization and the secretion of matrix metalloproteinase (MMP)-2.

Conclusions
This review summarizes the effects of laser irradiation on cells related to periodontal tissues ( Figure 1) and clearly shows that laser irradiation can have many positive effects on various cell types in periodontal tissues. Numerous studies have reported that laser irradiation enhances cell proliferation, migration, viability, calcification, gene expression, and protein expression. Additionally, the favorable effects on cells vary depending on the fluences and type of lasers. Irradiation using diodes or Nd:YAG lasers is clinically feasible and can be applied in association with any periodontal procedure, since it reaches deep tissues due to its deeply penetrating wavelength. By contrast, Er:YAG and CO 2 lasers, which are only superficially absorbed, are only effective on epithelial cells and connective tissue surfaces, during nonsurgical periodontal treatments, or exposed bone and connective tissues, during periodontal surgeries. Although the purposes of periodontal treatment include anti-inflammation, tissue repair, and tissue regeneration, a single laser irradiation under a single specific irradiation condition cannot achieve all desired positive effects. Furthermore, a certain irradiation condition might have negative effects on some cells in periodontal tissues, since appropriate irradiation conditions vary with cell type. When applying a laser to regenerate periodontal tissues in the clinic, it is necessary to consider which cells need to be targeted and activated and then select the suitable laser type and energy fluence to match the cell type. However, there is still insufficient basic research on the PBM of lasers from this review. Because the type of lasers, irradiation time, distance, and fluence are quite varied, it is difficult to critically determine the optimal criteria for laser usage. From this review, we realize the promising PBM effects of lasers in periodontal therapy, and we can gain insights regarding the appropriate fluences to be utilized in laser applications to osteoblasts, fibroblasts, and MSCs. We will continue to research lasers for periodontal phototherapy, including regeneration of periodontal tissues in the future.
there is still insufficient basic research on the PBM of lasers from this review. Because the type of lasers, irradiation time, distance, and fluence are quite varied, it is difficult to critically determine the optimal criteria for laser usage. From this review, we realize the promising PBM effects of lasers in periodontal therapy, and we can gain insights regarding the appropriate fluences to be utilized in laser applications to osteoblasts, fibroblasts, and MSCs. We will continue to research lasers for periodontal phototherapy, including regeneration of periodontal tissues in the future.

Conflicts of Interest:
The authors declare no conflict of interest.