Tetrazolium Red

Irradiation with red light-emitting diode enhances proliferation and osteogenic differentiation of periodontal ligament stem cells

Yan Wu1,2 • Tingting Zhu 3 • Yaoyao Yang4 • Hong Gao 3 • Chunxia Shu 1 • Qiang Chen1 • Juan Yang1 • Xiang Luo1 • Yao Wang5

Abstract

This study aimed to evaluate the effects of low-energy red light-emitting diode (LED) irradiation on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). PDLSCs were derived from human periodontal ligament tissues of premolars and were irradiated with 0 (control group), 1, 3, or 5 J/cm2 red LED in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reaction (RT-PCR) results. Osteoblast-associated proteins (Runx2, OCN, OPN, and BSP) were detected using western blotting. The results of the MTT assay indicated that PDLSCs in the irradiation groups exhibited a higher proliferation rate than those in the control group (P < 0.05). ALP results showed that after 7 days of illumination, only 5 J/cm2 promoted the expression of ALP of PDLSCs. However, after 14 days of illumination, the irradiation treatments did not increase ALP activity. The results of alizarin red staining showed that red LED promoted osteogenic differentiation of the PDLSCs. The real-time polymerase chain reaction (RT-PCR) results demonstrated that red LED upregulated the expression levels of osteogenic genes. Expression of the proteins BSP, OPN, OCN, and Runx2 in the irradiation groups was higher than that in the control group. Our results confirmed that low-energy red LED at 1, 3, and 5 J/cm2 promotes proliferation and osteogenic differentiation of PDLSCs. Keywords Mesenchymal stem cells . LED . Proliferation . Osteogenic differentiation . PDLSCs Introduction According to data from the Fourth National Oral Health Epidemiological Survey, the prevalence of periodontal dis- ease among adults in China is as high as 87.4% [1]. In most patients, periodontal disease is accompanied by some degree of alveolar bone resorption. At present, the main treatment methods are periodontal nonsurgical treatment, surgical treat- ment, and bone replacement material repair. Conventional periodontal nonsurgical treatment can con- trol periodontal inflammation and prevent the development of periodontal disease but cannot restore the damaged alveolar bone. Although surgical treatment and related bone replace- ment materials can increase alveolar bone to a certain extent, this approach is limited by many factors, and its long-term effects are still unclear [2]. Periodontal ligament stem cells (PDLSCs) derived from periodontal ligaments can be obtained from teeth that need to be removed for treatment and have the basic characteristics of stem cells. Under suitable conditions, they can differentiate into osteoblasts and cementum cells and are expected to seed cells for periodontal tissue regeneration. Photobiology therapy is a new type of treatment [3]. A newly used energy source, light can have a resonance effect on cells and cause chemical reactions. Light contains lasers and light-emitting diodes (LEDs), and LEDs have gradually replaced lasers in many research fields. Red LED produce light with a wavelength of 600–700 nm, which is advanta- geous for its easy acquisition, low price, and relative safety. In addition, red LED illuminate tissues and cells uniformly, reach deep tissues, are easy to operate, and can accelerate wound healing [4]. Previous studies have found that red LED can promote osteogenic differentiation of bone marrow stem cells [5]. In addition, red LED reduces osteoclasts by controlling reactive oxygen species and HSP27 and promotes mucosal bone flaps and periodontal repair [6–8]. Whether low-energy red LED can affect the proliferation and osteogenic differentiation of PDLSCs remains to be stud- ied. In this study, we irradiated PDLSCs with a low-energy red LED at various energy densities to elucidate the effects of light energy on the proliferation and osteogenic differentiation of PDLSCs. Materials and methods The isolation of PDLSCs for this study was performed accord- ing to the Ethics Committee of the Affiliated Hospital of Stomatology Southwest Medical University Certificate (con- tract grant 20180314001). PDLSCs were derived from the middle third of the root surface of premolars extracted for orthodontic treatment from 3 healthy individuals (ages 15– 20). Each analysis in this study was repeated on three inde- pendent samples. Cultivation of PDLSCs We isolated, cultured, and identified periodontal ligament stem cells based on methods described in previous studies [9]. The usage of periodontal ligament tissue was allowed with the informed consent of the patients’ parents and ap- proved by the biomedical science research ethics committee of the Affiliated Hospital of Stomatology Southwest Medical University. All cells in this study were used at passage number 3. Irradiation procedure The light source device used in this study was a red LED (Resen, Beijing, China) with continuous output and a wave- length of 600–700 nm. The distance from the light source to the cell layer was approximately 2 cm. Under these conditions, the power density measured at the cell level was approximately 66.7 mW/cm2. The intensity of red LED is 240–350 Lux, and the maximum output power is about 10 W. Based on past stud- ies and the formula (Energy density = Power density × Time), the study was divided into four groups: 1 J/cm2 (irradiated for 15 s), 3 J/cm2 (irradiated for 45 s), 5 J/cm2 (irradiated for 75 s), and a nonirradiated (control group). Therefore, the LED exposure times for each group of cells were 15, 45, 75, and 0 s, respec- tively, every other day. The first day of irradiation was denoted day 1. Corresponding irradiation and tests were conducted ac- cording to the experimental design described. Nonirradiated cells were cultured under the same conditions as the irradiated cells. All irradiations were performed by the same operator. Cell proliferation assays 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bro- mide (MTT) assays were performed to detect cell proliferation as previously described [10]. ALP staining and ALP activity assay The detection of ALP activity was carried out using methods described in previous research [10]. Both ALP staining and ALP activity assay were assessed on the 7th and 14th days after irradiation. Alizarin red staining and calcium quantitation analysis Alizarin red staining and calcium quantitation analysis were performed and analyzed as previously described [10]. The alizarin red activity test was completed on the 21st day after irradiation. Real-time polymerase chain reaction analysis RT-PCR results were analyzed as previously described [10]. Briefly, RT-PCR was performed to analyze the expression levels of ALP, osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2) in each group of cells on day 7. The primers for the specific genes are shown in Table 1. Real-time polymerase chain reaction was performed strictly in accordance with prod- uct specifications. Western blotting Western blotting was used to detect the osteogenic differentiation- related proteins OCN, OPN, Runx2, and BSP [11]. Proteins were extracted, and the quality of proteins was quantified. The proteins were resolved and separated by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. After blocking for 2 h at room temperature, the membranes were washed and incubated at 4 °C overnight with primary antibodies specific for the follow- ing proteins: BSP, ALP, Runx2, and OPN (1:1000, Bioswamp, Wuhan, China). After washing, the membranes were incubated with a secondary antibody, goat anti-rabbit IgG conjugated to peroxidase (1:20,000, Bioswamp, Wuhan, China), at 37 °C for 1 h. Enhanced chemiluminescence (ECL, Millipore, USA) was used to observe the bands, and ImageJ software (version 1.48u, Bethesda, USA) was used to quantify the protein levels; β-actin (1:10,000, Bioswamp, Wuhan, China) acted as the internal control. Statistical analysis The results were statistically analyzed using SPSS 17.0 soft- ware. All test results were analyzed using independent sample t tests. Significance was defined as P < 0.05. Results Promotion of cell growth following red LED irradiation A growth curve was plotted for each group of PDLSCs using the results of the MTT assay (Fig. 1a). The results of the MTT assay showed that the proliferation rate of PDLSCs on days 1, 3, 5, 7, and 9 in the irradiation-treated groups was higher than that in the control group (P < 0.05) (Fig. 1b). Cell proliferation ability of the irradiation-treated groups on day 1 was stronger than that in the control group only at 5 J/cm2 (P < 0.05). On day 3, the cell proliferation ability of the 1, 3, and 5 J/cm2 groups was stronger than that in the control group (P < 0.05). Additionally, the cell proliferation ability of the 3 J/cm2-treat- ed group was stronger than that of the control group on day 5 (P < 0.05). On the seventh day, the cell proliferation ability of the 1 J/cm2-treated group was stronger than that of the control group (P < 0.05). Finally, the test results on the ninth day showed no significant difference in cell proliferation activity between the irradiation-treated groups and the control group (P ˃ 0.05). Effect of red LED irradiation on osteogenesis The results of the ALP staining are shown in Fig. 2a (days 7 and 14), and the results of the ALP activity assay are shown in Fig. 2b (days 7 and 14). On the 7th day of illumination, the results of the ALP staining indicated that the levels of ALP were higher in irradiation groups than in the control group, but the results of the ALP activity assay showed that only 5 J/cm2 irradiation promoted the ALP activity of PDLSCs (P < 0.05). The ALP activity of PDLSCs treated with 1 or 3 J/cm2 was slightly higher than that of cells in the control group, but there was no significant difference (P > 0.05). On the 14th day of illumination, the staining results showed no significant differ- ence in the coloration of the irradiation groups, and the activity test results showed that there were no significant differences between the irradiation groups and the control group (P > 0.05). As shown in Fig. 2a, the levels of ALP were higher on day 14 than on day 7.

Alizarin red staining and calcium quantitation analysis

The results of alizarin red staining are shown in Fig. 3a, and the quantitation results of total matrix mineraliza- tion are shown in Fig. 3b (P < 0.05). Irradiation pro- moted the mineralization of PDLSCs. Irradiation at 5 J/cm2 had the strongest effect in promoting osteogenic differentiation, and there were significant differences in total matrix mineralization between the 5, 1, and 3 J/cm2 groups. Changes in gene expression following irradiation The relative expression levels of the osteogenic genes ALP, BSP, Runx2, OCN, and OPN are shown in Fig. 4. There were differences in the expression of osteogenic genes among the irradiation groups. Irradiation promoted the expression of ALP, but there were no significant differences between the irradiation groups and the control group (P > 0.05). Irradiation also pro- moted the expression of the osteogenic gene BSP, which was significantly different compared with that in the control group (P < 0.05). The promotion effect of 3 and 5 J/cm2 was the most pronounced, and the two groups were significantly different from the 1 J/cm2 group (P < 0.05). Irradiation at 1, 3, and 5 J/cm2 promoted the expression of the osteogenic gene Runx2, but that in the 3 J/cm2 group was significantly different from that in the control group (P < 0.05). The irradiation groups demonstrated increased expression of the osteogenic gene OCN, but the 1 J/cm2 group was not significantly different from the control group (P > 0.05). Irradiation also promoted the expression of the osteogenic gene OPN to a level that was significantly different from that in the control group (P < 0.05), but the effect of 5 J/cm2 was more robust and was significantly different from that of 1 J/cm2 (P < 0.05). Results of western blotting Figure 5a shows the electrophoretic bands of osteogenic pro- teins from red LED-illuminated PDLSCs. The relative expres- sion levels of the osteogenic proteins BSP, Runx2, OCN, and OPN are shown in Fig. 5b. Among the irradiation groups, there were differences in the expression of osteogenic proteins. Irradiation promoted the expression of the osteogenic protein BSP at a level that was significantly different than that in the control group (P < 0.05). This promotion effect was more ro- bust at energy density of 3 and 5 J/cm2, and these two groups were significantly different from the 1 J/cm2 group (P < 0.05). Irradiation energy density of 1, 3, and 5 J/cm2 promoted the expression of the osteogenic genes Runx2 and OCN, but the effect at 3 J/cm2 was significantly different from that in the control group (P < 0.05). In the irradiation groups, the expres- sion of the osteogenic genes BSP and Runx2 was consistent with the expression of the osteogenic proteins BSP and Runx2. Irradiation promoted the expression of the osteogenic protein OPN at a level significantly different from that in the control group (P < 0.05). In the irradiation-treated groups, the expres- sion of the osteogenic genes OPN and OCN was inconsistent with the expression of the osteogenic proteins OPN and OCN. Discussion PDLSCs are cells that have not yet differentiated in the peri- odontal ligament. PDLSCs have the basic characteristics of stem cells and form dentin or cementum cells in a suitable induction environment [12], which is beneficial to the regen- eration and repair of teeth and periodontal tissues. PDLSCs have strong proliferation and self-renewal abilities and can generate a large number of cells in a short time. Moreover, PDLSCs have stronger osteogenic differentiation ability than other dental-derived stem cells [13] and are ideal periodontal regenerative cells. Tooth movement, alveolar bone recon- struction, and periodontal repair all depend on the prolifera- tion and differentiation of PDLSCs, making PDLSCs ideal for repairing periodontal and alveolar bone. Red LED is emitted by diodes and has various biological regulatory effects, such as reducing inflammation, promoting wound healing, and affecting cell proliferation and differenti- ation in numerous ways [14–18], including promoting the proliferation of mesenchymal stem cells [15], accelerating the growth of hepatocytes [16], promoting the proliferation and osteogenic differentiation of osteoblast-like cells MC3T3-E1 [17], and promoting umbilical cord mesenchymal osteogenic differentiation [18]. In addition, red LED reduces osteoclasts by controlling reactive oxygen species and HSP27 and promotes mucosal bone flaps and periodontal repair [6–8]. However, research on the effects of red LED on dental stem cells has not been extensively conducted. Whether red LED can affect the proliferation and osteogenic differentiation of PDLSCs and the specific mechanism by which this occurs remain to be studied. Red LED can promote the osteogenesis of stem cells only in an osteogenic induction environment [5], and cells need 48 h to exhibit to the impact of light energy [19]. Therefore, the culture medium selected in this study was osteoinduction liquid, and the cells were illuminated the day after seeding. According to previous research, light energy of 1–10 J/cm2 can have biological effects on cells and tissues [20], and 1–4 J/cm2 has obvious biological effects on mesenchymal stem cells [21]. Pagin [22] found that 5 J/cm2 also had a significant biological effect on preosteoblast MC3T3 cultures in vitro. Therefore, in this study, low light energy density (1–5 J/cm2) was chosen to explore the effect of low-energy red LED of different energies on the proliferation and osteogenic differentiation of PDLSCs. Mamalis [23] found that the effect of light on cell prolifer- ation may be related to the promotion of reactive oxygen species, and Mandrillo [24] found that LED promotes in- creased secretion of proliferation-related proteins. According to these research, low-energy red LED can promote cell pro- liferation, but the specific mechanism is not yet clear and warrants further study. Our study found that low-energy red LED can also promote the proliferation of PDLSCs in a time- dependent and energy density-dependent manner. On the first day after irradiation, we found that only a 5 J/cm2 of red LED had a proliferation effect on PDLSCs, which was consistent with the results of Yamauchi [25]. It can be reminded that the higher energy red LED can promote cell proliferation in the early stage of proliferation. Irradiations 1 3, and 5 J/cm2 were found to promote cell proliferation on days 3–7, which is consistent with the results of the study by Peng Fei [5] on the effects of red LED on the proliferation of bone marrow stem cells. Low-energy red LED has been suggested to robust- ly promote cell proliferation in the middle period. In the late period of illumination, it was found that irradiation did not significantly promote the proliferation of PDLSCs. This study suggests that red LED can obviously promote the proliferation of PDLSCs in the early and middle stages but has no obvious effect during the later stage of proliferation. Furthermore, our findings show that under the same light treatment conditions, the time-dependent and energy density-dependent effect of red LED on stem cell proliferation can be selected with spe- cific light energy and time to quickly and effectively promote stem cell proliferation. These data provide support for further research on the effects of red LED on cell proliferation. Through previous researchers’ exploration of the mecha- nism by which red LED can promote osteogenic differentia- tion, it is known that red LED can regulate the differentiation of cells into osteogenic cell types by regulating the ERK sig- naling pathway [25]. Holder MJ found that increased osteo- genic differentiation of cells induced by red LED may also be related to increased mitochondrial activity [26], but the spe- cific mechanism remains unclear. To explore the effects of red LED on the osteogenic differentiation of PDLSCs, this study examined the effects of low-energy red LED on the osteogenic marker ALP activity, the formation of mineralized nodules, and changes in the expression levels of bone-related genes and proteins (ALP, Runx2, OPN, OCN, and BSP). This study found that low-energy red LED can promote the ALP activity of PDLSCs and increase the secretion of calci- fied nodules. ALP is an extracellular enzyme that provides the necessary phosphoric acid for osteogenic differentiation of cells, and is beneficial for the osteogenic effect of cells. ALP activity is an important indicator of early osteogenic differen- tiation of cells. Green DE found that ALP activity peaks oc- curred within 7-14 days after cell inoculation and culture [27]. It has been reported that LED has a significant effect on ALP activity on day 7 [7], and Yang D [18] found that LED still affects cell ALP activity after 14 days. Yamauchi N [25] found that red LED can increase the ALP activity of cells on days 7 and 14, so our experiment was designed to detect changes in ALP activity in PDLSCs after 7 days and 14 days of red LED irradiation. By exploring changes in ALP activity and early osteogenic markers, it was found that low-energy red LED promotes increased ALP enzyme activity of PDLSCs on day 7, but it cannot affect ALP enzyme activity on day 14. Additionally, we found that low-energy red LED does not increase the ALP mRNA level. These findings are inconsistent with the research results of Yamauchi N [25], who found that red LED pro- motes cellular ALP enzyme activity on days 7 and 14. The reason for this difference may be related to the illumination mode and the higher energy red LED chosen for our experi- ment. However, these findings are consistent with the exper- imental results of Peng Fei [5]. The reason for this result may be that ALP is inactive during synthesis and is called the ALP precursor. ALP precursors are hydrolyzed to form active pro- teases [28]. Low-energy red LED can increase the ALP en- zyme activity of PDLSCs, but it cannot upregulate ALP mRNA levels. Moreover, brief red LED illumination can pro- mote only the hydrolysis of ALP precursors but cannot affect the level of ALP mRNA. As the mineralized nodules in late- phase osteogenesis gradually begin to form, the effect of red LED on the ALP activity of PDLSCs is also gradually weakened. Mineralized nodules are the basis of osteogenic differenti- ation of cells. They appear in the late stage of osteogenic differentiation and are the markers of mature (mineralized) osteogenic differentiation. It has been reported that after 21 days of osteogenic induction of mesenchymal stem cells, cal- cium nodules appear in cells [29]. This study was designed to detect the calcium nodules of PDLSCs after 21 days of red LED. We found that low-energy red LED can promote the formation of calcium nodules, indicating that red LED still has a significant promoting effect on the mineralization stage. These experimental results suggest that the low-energy red LED can obviously promote the differentiation and minerali- zation of PDLSCs in the osteogenic differentiation stage. Additionally, this study found that low-energy red LED can upregulate the bone formation-related genes Runx2, OPN, OCN, and BSP of PDLSCs and can promote the corre- sponding increase in protein secretion. Runx2 is a specific transcription factor in osteogenic differentiation and is an im- portant component in regulating the osteogenic differentiation of cells. The expression level of Runx2 increases in the early and middle stages of osteogenic differentiation. We found that low-energy red LED can significantly increase the level of Runx2 gene expression and promote the increased expression of Runx2 protein. These experimental results are consistent with the findings of Kim [30] and suggest that low-energy red LED may promote the osteogenic differentiation of PDLSCs by affecting Runx2. OPN is a secreted phosphorylated protein and encoded by an important gene that regulates cell mineralization and tissue remodeling, which is expressed early in osteogenic differenti- ation [31]. OCN is a noncollagen component of the extracel- lular matrix, an important gene that regulates cell mineraliza- tion, and a mid-to-late marker of osteogenic differentiation. BSP is a noncollagen protein in the extracellular matrix. It is located at the initiation of hydroxyapatite formation and pro- motes the differentiation and mineralization of cells into oste- oblasts. It is expressed at the late stage of osteogenic differen- tiation and early mineralization [32]. This study found that low-energy red LED can significantly upregulate the mineralization-related genes OPN, OCN, and BSP, and their corresponding proteins. Moreover, we found that the higher light energy produced a more robust promoting effect. The expression levels of the OPN and BSP genes in the irradiated groups increased exponentially, and the maximum increase was 8-fold. The results of this study are consistent with the detection results of mineralized nodule formation. Red LED may promote the bone mineralization of PDLSCs by signifi- cantly increasing the expression of OPN, OCN, and BSP. The results of this study are also consistent with those reported by Stein A [33]. This study found that the increased expression of the corresponding protein by red LED irradiation is largely consistent with the observed increase in gene expression. From the protein level, it was further demonstrated that low- energy red LED promotes the osteogenesis of PDLSCs. Our research results are consistent with the findings of Yang D [18]. It has been suggested that low-energy red LED can act on proteins in the extracellular matrix of PDLSCs, regulating cell mineralization and promoting tissue osteoblastic differentia- tion. This effect persists through the entire process of osteogen- ic differentiation, including high expression of OPN in early osteogenic differentiation and OCN and BSP expression in the middle and late stages. This study initially explored the effect of low-energy red LED on the proliferation and osteogenic differentiation of PDLSCs in vitro. Our data provide support for the continued study of the effects of low-energy red LED on the proliferation and differentiation of dental stem cells. LED is expected to become a new treatment approach to promote bone formation and slow local bone tissue absorption. However, the mecha- nism by which LED affects osteogenic differentiation is not yet clear, and further experiments are needed to explore it in depth. Conclusion Low-energy red LED plays a significant role in promoting proliferation and osteogenic differentiation, and this effect is energy density dependent, with higher energy levels promot- ing osteogenesis to a greater degree. The mechanism underly- ing the promoted osteogenic differentiation of PDLSCs may occur via upregulated expression of the BSP, OPN, OCN, and Runx2 genes. References 1. Sun HY, Jiang H, Du MQ, Wang X, Feng XP, Hu Y, Lin HC, Wang B, Si Y, Wang CX, Zheng SG, Liu XN, Rong WS, Wan WJ, Tai BJ (2018) The prevalence and associated factors of peri- odontal disease among 35 to 44-year-old Chinese adults in the 4th National Oral Health Survey. Chin J Dent Res 21(4):241–247. https://doi.org/10.3290/j.cjdr.a41082 2. Sculean A, Nikolidakis D, Nikou G, Ivanovic A, Chapple IL, Stavropoulos A (2015) Biomaterials for promoting periodontal re- generation in human intrabony defects: a systematic review. Periodontol 68(1):182–216. https://doi.org/10.1111/prd.12086 3. de Freitas LF, Hamblin MR (2016) Proposed mechanisms of photobiomodulation or low-level light therapy. IEEE J Select Top Quantum Electron 22(3):7000417. https://doi.org/10.1109/JSTQE. 2016.2561201 4. Li Y, Zhang J, Xu Y, Han Y, Jiang B, Huang L, Zhu H, Xu Y, Yang W, Qin C (2016) The histopathological investigation of red and blue light emitting diode on treating skin wounds in Japanese big- ear white Tetrazolium Red rabbit. PLoS One 11(6):e0157898. https://doi.org/10. 1371/journal.pone.0157898
5. Peng F, Wu H, Zheng Y, Xu X, Yu J (2012) The effect of nonco- herent red light irradiation on proliferation and osteogenic differen- tiation of bone marrow mesenchymal stem cells. Lasers Med Sci 27(3):645–653. https://doi.org/10.1007/s10103-011-1005-z
6. Tao CY, Lee N, Chang HC, Yang C, Yu XH, Chang PC (2016) Evaluation of 660 nm LED light irradiation on the strategies for treating experimental periodontal intrabony defects. Lasers Med Sci 31(6):1113–1121. https://doi.org/10.1007/s10103-016-1958-z
7. Sohn HM, Ko Y, Park M, Kim B, Park JE, Kim D, Moon YL, Lim W (2017) Comparison of the alendronate and irradiation with a light-emitting diode (LED) on murine osteoclastogenesis. Lasers Med Sci 32(1):189–200. https://doi.org/10.1007/s10103-016- 2101-x
8. Lee G, Kim B, Ko Y, Park M, Kim D, Ryu KH, Jun YC, Sohn HM, Lim W (2017) Regulation of RANKL-induced osteoclastogenesis by 635-nm light-emitting diode irradiation via HSP27 in bone marrow-derived macrophages. Photomed Laser Surg 35(2):78–86. https://doi.org/10.1089/pho.2016.4134
9. Gu X, Li M, Jin Y, Liu D, Wei F (2017) Identification and inte- grated analysis of differentially expressed lncRNAs and circRNAs reveal the potential ceRNA networks during PDLSC osteogenic differentiation. BMC Genet 18(1):100. https://doi.org/10.1186/ s12863-017-0569-4
10. Zhu T, Wu Y, Zhou X, Yang Y, Wang Y (2019) Irradiation by blue light-emitting diode enhances osteogenic differentiation in gingival mesenchymal stem cells in vitro. Lasers Med Sci 34(7):1473–1481. https://doi.org/10.1007/s10103-019-02750-3
11. Sun X, Lou L, Zhong K, Wan L (2017) MicroRNA-451 regulates chemoresistance in renal cell carcinoma by targeting ATF-2 gene. Exp Biol Med (Maywood NJ) 242(12):1299–1305. https://doi.org/ 10.1177/1535370217701625
12. Yang ZH, Zhang XJ, Dang NN, Ma ZF, Xu L, Wu JJ, Sun YJ, Duan YZ, Lin Z, Jin Y (2009) Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues. J Periodontal Res 44(2):199–210. https://doi.org/10.1111/j.1600-0765.2008. 01106.x
13. Gao Y, Zhao G, Li D, Chen X, Pang J, Ke J (2014) Isolation and multiple differentiation potential assessment of human gingival mesenchymal stem cells. Int J Mol Sci 15(11):20982–20996. https://doi.org/10.3390/ijms151120982
14. Mamalis A, Jagdeo J (2015) Light-emitting diode-generated red light inhibits keloid fibroblast proliferation. Dermatol Surg 41(1): 35–39. https://doi.org/10.1097/01.DSS.0000452650.06765.51
15. Babaee A, Nematollahi-Mahani SN, Shojaei M, Dehghani-Soltani S, Ezzatabadipour M (2018) Effects of polarized and non-polarized red-light irradiation on proliferation of human Wharton’s jelly- derived mesenchymal cells. Biochem Biophys Res Commun 504(4):871–877. https://doi.org/10.1016/j.bbrc.2018.09.010
16. Feng R, Morine Y, Ikemoto T, Imura S, Iwahashi S, Saito Y, Shimada M (2018) Photobiomodulation with red light-emitting di- odes accelerates hepatocyte proliferation through reactive oxygen species/extracellular signal-regulated kinase pathway. Hepatol Res 48(11):926–936. https://doi.org/10.1111/hepr.13182
17. Asai T, Suzuki H, Kitayama M, Matsumoto K, Kimoto A, Shigeoka M, Komori T (2014) The long-term effects of red light- emitting diode irradiation on the proliferation and differentiation of osteoblast-like MC3T3-E1 cells. Kobe J Med Sci 60(1):E12–E18
18. Yang D, Yi W, Wang E, Wang M (2016) Effects of light-emitting diode irradiation on the osteogenesis of human umbilical cord mes- enchymal stem cells in vitro. Sci Rep 6:37370. https://doi.org/10. 1038/srep37370
19. Horvát-Karajz K, Balogh Z, Kovács V, Drrernat AH, Sréter L, Uher F (2009) In vitro effect of carboplatin, cytarabine, paclitaxel, vin- cristine, and low-power laser irradiation on murine mesenchymal stem cells. Lasers Surg Med 41(6):463–469. https://doi.org/10. 1002/lsm.20791
20. Parker S (2007) Low-level laser use in dentistry. Br Dent J 202(3): 131–138. https://doi.org/10.1038/bdj.2007.75
21. Fekrazad R, Asefi S, Allahdadi M, Kalhori KA (2016) Effect of photobiomodulation on mesenchymal stem cells. Photomed Laser Surg 34(11):533–542. https://doi.org/10.1089/pho.2015.4029
22. Pagin MT, de Oliveira FA, Oliveira RC, Sant’Ana AC, de Rezende ML, Greghi SL, Damante CA (2014) Laser and light-emitting diode effects on pre-osteoblast growth and differentiation. Lasers Med Sci 29(1):55–59. https://doi.org/10.1007/s10103-012-1238-5
23. Mamalis A, Garcha M, Jagdeo J (2015) Light emitting diode- generated blue light modulates fibrosis characteristics: fibroblast proliferation, migration speed, and reactive oxygen species gener- ation. Lasers Surg Med 47(2):210–215. https://doi.org/10.1002/ lsm.22293
24. Mandrillo PM, Fischetto G, Odorisio P, Cura F, Avantaggiato A, Carinci F (2017) Effects of light-emitting diode (led 640 nm) on human gingival fibroblasts: a comparative in vitro study. ORAL Implantol 10(2):151–161. https://doi.org/10.11138/orl/2017.10.2. 151
25. Yamauchi N, Taguchi Y, Kato H, Umeda M (2018) High-power, red-light-emitting diode irradiation enhances proliferation, osteo- genic differentiation, and mineralization of human periodontal lig- ament stem cells via ERK signaling pathway. J Periodontol 89(3): 351–360. https://doi.org/10.1002/JPER.17-0365
26. Holder MJ, Milward MR, Palin WM, Hadis MA, Cooper PR (2012) Effects of red light-emitting diode irradiation on dental pulp cells. J Dent Res 91(10):961–966. https://doi.org/10.1177/ 0022034512456040
27. Green DE, Longtin JP, Sitharaman B (2009) The effect of nanoparticle-enhanced photoacoustic stimulation on multipotent marrow stromal cells. ACS Nano 3(8):2065–2072. https://doi.org/ 10.1021/nn900434p
28. Klionsky DJ, Emr SD (1989) Membrane protein sorting: biosyn- thesis, transport and processing of yeast vacuolar alkaline phospha- tase. EMBO J 8(8):2241–2250
29. Saberi E, Farhad-Mollashahi N, Sargolzaei Aval F, Saberi M (2019) Proliferation, odontogenic/osteogenic differentiation, and cytokine production by human stem cells of the apical papilla in- duced by biomaterials: a comparative study. Clin Cosmet Investig Dent 11:181–193. https://doi.org/10.2147/CCIDE.S211893
30. Kim HK, Kim JH, Abbas AA, Kim DO, Park SJ, Chung JY, Song EK, Yoon TR (2009) Red light of 647 nm enhances osteogenic differentiation in mesenchymal stem cells. Lasers Med Sci 24(2): 214–222. https://doi.org/10.1007/s10103-008-0550-6
31. Liu YK, Uemura T, Nemoto A, Yabe T, Fujii N, Ushida T, Tateishi T (1997) Osteopontin involvement in integrin-mediated cell signal- ing and regulation of expression of alkaline phosphatase during early differentiation of UMR cells. FEBS Lett 420(1):112–116. https://doi.org/10.1016/s0014-5793(97)01498-1
32. Vahabi S, Torshabi M, Esmaeil Nejad A (2016) In vitro comparison of the efficacy of TGF-β1 and PDGF-BB in combination with freeze-dried bone allografts for induction of osteogenic differentia- tion in MG-63 osteoblast-like cells. J Mater Sci Mater Med 27(12): 182. https://doi.org/10.1007/s10856-016-5802-6
33. Stein A, Benayahu D, Maltz L, Oron U (2005) Low-level laser irradiation promotes proliferation and differentiation of human os- teoblasts in vitro. Photomed Laser Surg 23(2):161–166. https://doi. org/10.1089/pho.2005.23.161

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