
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39232133
71386
10.1038/s41598-024-71386-3
Article
Effect of photobiomodulation therapy with 660 and 980 nm diode lasers on differentiation of periodontal ligament mesenchymal stem cells
Etemadi Ardavan 1
Aghaie Milad 1
Sayar Ferena sayar_f@yahoo.com

1
Chiniforush Nasim nasimch2002@yahoo.com

23
1 grid.411463.5 0000 0001 0706 2472 Department of Periodontics, Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2 https://ror.org/01c4pz451 grid.411705.6 0000 0001 0166 0922 Dentistry Research Institute, Tehran University of Medical Sciences, Tehran, Iran
3 https://ror.org/0107c5v14 grid.5606.5 0000 0001 2151 3065 Department of Surgical Sciences and Integrated Diagnostics, University of Genoa, Genoa, Italy
4 9 2024
4 9 2024
2024
14 2058731 10 2023
27 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
This study aimed to compare the effects of photobiomodulation therapy (PBMT) with 660 and 980 nm diode lasers on differentiation of periodontal ligament mesenchymal stem cells (PDLMSCs). In this in vitro, experimental study, PDLMSCs were obtained from the Iranian Genetic Bank and cultured in osteogenic medium. They were then subjected to irradiation of 660 and 980 nm diode lasers, and their viability was assessed after one, two, and three irradiation cycles using the methyl thiazolyl tetrazolium (MTT) assay. The cells also underwent DAPI staining, cell apoptosis assay by using the Annexin V/PI, Alizarin Red staining, and real-time polymerase chain reaction (PCR) for assessment of the expression of osteogenic genes. Data were analyzed by two-way ANOVA. The two laser groups had no significant difference in cell apoptosis according to the results of DAPI staining. Both laser groups showed higher cell viability in the MTT assay at 4 and 6 days compared with the control group. Annexin V/PI results showed higher cell viability in both laser groups at 4 days compared with the control group. Rate of early and late apoptosis was lower in both laser groups than the control group at 4 days. Necrosis had a lower frequency in 980 nm laser group than the control group on day 6. Alizarin Red staining showed higher cell differentiation in both laser groups after 3 irradiation cycles than the control group. The highest expression of osteopontin (OPN), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2) was noted in 660 nm laser group with 3 irradiation cycles at 14 days, compared with the control group. PBMT with 660 and 980 nm diode lasers decreased apoptosis and significantly increased PDLMSC differentiation after 3 irradiation cycles.

Keywords

Photobiomodulation therapy
Cell Survival
Cell differentiation
Mesenchymal stem cells
Subject terms

Dentistry
Periodontics
issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Periodontal disease is an inflammatory condition with a high prevalence rate and has a chronic nature and results in periodontal tissue destruction, bone resorption, periodontal ligament (PDL) destruction, cementum loss, and eventual tooth loss1,2.

Mesenchymal stem cells (MSCs) are multipotent undifferentiated cells that can differentiate to different tissues depending on the received signals3,4. MSCs can be isolated from different tissues such as the bone marrow, orofacial region, skin, and adipose tissue5,6. The first MSCs obtained from the orofacial region were isolated from the third molar pulp tissue followed by deciduous teeth, apical papilla, and PDL7,8. MSCs can be used for allogenic grafts since they are well tolerated by the immune system and can enhance healing and induce osteogenesis9,10. PDL is a fibrous connective tissue that supports the teeth and has a small population of PDLMSCs, which are responsible for preservation and regeneration of periodontal tissue in terms of structure and function. They can differentiate to osteoblasts, fibroblasts, and cementoblasts, and form cementum and tissues similar to PDL7. A previous study on rats with bone defects showed that application of PDLMSCs enhanced the healing of periodontal defects through osteogenesis, and regeneration of PDL and cementum-like tissue11. In another study, Yu et al.12 observed that application of PDLMSCs enhanced the regeneration of periodontal hard and soft tissues.

The ultimate goal of surgical and non-surgical periodontal treatments is to achieve a predictable level of regeneration of periodontium at the diseased site13. Over the years, several methods have been proposed to enhance tissue regeneration in periodontal therapy and accelerate the treatment course. Low level laser therapy (LLLT) is a relatively novel modality suggested for this purpose13.

LLLT was first used by Mester et al.14 for hair growth and enhancement of wound healing in rats. In 1993, LLLT was used by Fernando et al. aiming to assess its efficacy for reduction of pain and swelling following bilateral extraction of impacted mandibular third molars. They found no significant difference between the laser and placebo groups15. Subsequent investigations confirmed the optimal efficacy of LLLT for enhancement of cell proliferation16, osteoblastic differentiation of MSCs17, improvement of functional attachment of titanium implants to bone, and enhancement of bone healing and mineralization18. Also, LLLT has been suggested as an effective adjunct to periodontal therapy following non-surgical periodontal treatments19,20, gingivectomy21,22, and regenerative treatments23.

Photobiomodulation therapy (PBMT) refers to irradiation of tissues with laser light or light emitting diodes (LEDs). Depending on the light parameters, PBMT can either induce or prevent tissue responses24. Acceleration of healing by laser is probably attributed to enhancement of cell proliferation. Laser energy is absorbed by the intracellular chromophores and converted to metabolic energy, which is subsequently used in the mitochondrial respiratory chain for ATP production, increasing the DNA activity and RNA and protein synthesis25. PBMT can improve blood circulation, trigger angiogenesis, exert anti-inflammatory and analgesic effects, and induce cell proliferation and tissue regeneration26. Etemadi et al.27 compared the effects of different laser wavelengths (660 and 980 nm) and doses on proliferation of PDLMSCs in vitro and showed that LLLT effectively increased the proliferation of PDLMSCs.

Considering all the above and limited studies available on the effects of different wavelengths and energy densities of low-level lasers on differentiation of PDLMSCs28–30, this study aimed to assess the effect of PBMT with 660 and 980 nm diode lasers on differentiation of PDLMSCs. This study tests the hypothesis that increased frequency of irradiation with 660 and 980 nm diode laser enhance the differentiation of PDLMSCs.

Results

Cell viability according to the results of DAPI staining

Table 1 presents the rate of apoptosis in the groups at different time points. As shown, rate of apoptosis increased on days 4 and 6 compared with day 2. On days 2 and 4, rate of apoptosis in 980 nm laser group was insignificantly lower than that in 660 nm laser and control groups (P > 0.05). On day 6, rate of apoptosis in 660 nm laser group was insignificantly lower than that in 980 nm laser group, and rate of apoptosis in both laser groups was lower than that in the control group but not significantly (P > 0.05). In total, rate of apoptosis was very low. Figure 1 shows apoptosis in all three groups.Table 1 Rate of apoptosis in the groups (n = 3) at different time points.

Time	Group	Mean	Std. deviation	Std. error	Lower bound	Upper bound	P value	
Day 2	Control	321.0000	3.00000	1.73205	313.5476	328.4524	.111	
660 nm	380.6667	67.26316	38.83441	213.5757	547.7576	.111	
980 nm	404.0000	23.81176	13.74773	344.8483	463.1517	.111	
Day 4	Control	507.0000	75.94077	43.84442	318.3527	695.6473	.260	
660 nm	445.2500	39.22903	19.61451	382.8279	507.6721	.260	
980 nm	642.6667	253.69536	146.47108	12.4525	1272.8809	.260	
Day 6	Control	444.6667	58.18362	33.59233	300.1305	589.2028	.179	
660 nm	367.2500	42.78142	21.39071	299.1752	435.3248	.179	
980 nm	410.0000	45.82576	26.45751	296.1625	523.8375	.179	

Fig. 1 Cell apoptosis in the three groups as revealed by DAPI staining and observation under a fluorescent microscope (magnification 20X). Red arrows indicate apoptotic cells.

Cell viability according to the results of the MTT assay

Table 2 presents cell viability (optical density) of the groups subjected to one, two and three irradiation cycles according to the MTT assay. On day 2, no significant difference was noted in cell viability among the groups (P > 0.05). On day 4, the two laser groups had a significant difference with each other (P = 0.022) and also with the control group in cell viability (P = 0.000 for both), such that cell viability in 660 nm laser group was significantly higher than that in 980 nm laser and control groups (P < 0.05). Cell viability in 980 nm laser group was also significantly higher than that in the control group (P = 0.000). On day 6, both 660 nm (P = 0.006) and 980 nm (P = 0.000) laser groups showed significantly higher cell viability than the control group (P < 0.05) but the cell viability was not significantly different between the two laser groups (P = 0.095) (Fig. 2A).Table 2 Cell viability (optical density) of the groups subjected to one, two and three irradiation cycles according to the MTT assay (n = 4).

Irradiation cycle	Group	Minimum	Maximum	Mean	Std. deviation	
1	Control	95.76	106.67	100.0000	4.77213	
660.00	92.73	103.03	99.5455	4.62556	
980.00	96.36	114.55	103.1818	8.18182	
2	Control	95.50	103.00	100.0000	3.67423	
660.00	115.00	119.00	117.3750	1.79699	
980.00	111.00	112.50	111.7500	.64550	
3	Control	99.01	101.38	100.0000	1.04369	
660.00	106.90	111.64	109.2702	1.95919	
980.0D	107.30	117.95	114.4970	4.89931	

Fig. 2 (A): MTT assay for cell viability (optical density) of the groups after one, two and three irradiation cycles. (B): Early apoptosis, (C): Late apoptosis, (D): Necrosis in the groups at different time points based on the results of Annexin V/PI.

Rate of apoptosis according to the results of Annexin V/PI

Table 3 presents the rate of viable cells in the three groups at different time points based on the results of Annexin V/PI. On day 2, no significant difference was found in this regard among the three groups (P > 0.05). On day 4, the difference in this respect was significant among the groups (P = 0.000) such that the cell viability in both 660 nm (P = 0.008) and 980 nm (P = 0.006) laser groups was significantly higher than that in the control group, but the difference between the two laser groups was not significant (P = 0.683). On day 6, no significant difference existed among the three groups in this regard (P = 0.531).Table 3 Rate of viable cells in the three groups (n = 3) at different time points based on the results of Annexin V/PI.

Time	Group	Mean	Std. deviation	Std. error	Lower bound	Upper bound	P value	
Day 2	Control	82.433	1.1015	.6360	79.697	85.170	.068	
660 nm	81.333	.8021	.4631	79.341	83.326	.068	
980 nm	83.267	.2887	.1667	82.550	83.984	.068	
Day 4	Control	81.600	1.3077	.7550	78.352	84.848	.000	
660 nm	91.000	1.8028	1.0408	86.522	95.478	.000	
980 nm	92.367	.4726	.2728	91.193	93.541	.000	
Day 6	Control	82.300	.7000	.4041	80.561	84.039	.531	
660 nm	82.267	1.6258	.9387	78.228	86.305	.531	
980 nm	80.933	2.1502	1.2414	75.592	86.275	.531	

Early apoptosis

Table 4 presents the rate of early apoptosis in the groups at different time points. On day 2, no significant difference existed among the groups in early apoptosis (P > 0.05). On day 4, rate of apoptosis in both 660 nm (P = 0.008) and 980 nm (P = 0.024) laser groups was significantly lower than that in the control group. On day 6, no significant difference existed among the groups in early apoptosis (P > 0.05) (Fig. 2B).Table 4 Rate of early apoptosis in the groups at different time points (n = 3).

Time	Group	Mean	Std. deviation	Std. error	95% confidence interval for mean	Minimum	
Lower bound	Upper bound	
2 days	0	13.0333	1.82300	1.05251	8.5047	17.5619	11.40	
660	13.0667	.60277	.34801	11.5693	14.5640	12.50	
980	12.4000	.30000	.17321	11.6548	13.1452	12.10	
4 days	0	12.9667	1.45717	.84130	9.3469	16.5865	11.30	
660	5.1667	1.41553	.81726	1.6503	8.6830	3.58	
980	5.4300	.42154	.24338	4.3828	6.4772	4.95	
6 days	0	12.3333	.83267	.48074	10.2649	14.4018	14.4018	
660	13.9667	.55076	.31798	12.5985	15.3348	15.3348	
980	13.2333	2.25462	1.30171	7.6325	18.8341	18.8341	

Late apoptosis

Table 5 presents the rate of late apoptosis in the groups at different time points. On day 2, no significant difference existed among the groups in late apoptosis (P > 0.05). On day 4, rate of apoptosis in both 660 nm (P = 0.002) and 980 nm (P = 0.000) groups was significantly lower than that in the control group. Also, rate of apoptosis in 980 nm laser group was significantly lower than that in 660 nm group (P = 0.011). On day 6, no significant difference existed among the groups in early apoptosis (P > 0.05) (Fig. 2C).Table 5 Rate of late apoptosis in the groups at different time points.

Time	Group	Mean	Std. deviation	Std. error	95% confidence interval for mean	Minimum	
Lower bound	Upper bound	
2 days	0	4.7933	.32083	.18523	3.9963	5.5903	4.46	
660	5.2100	1.07373	.61992	2.5427	7.8773	4.34	
980	4.1600	.31480	.18175	3.3780	4.9420	3.90	
4 days	0	5.0633	.14224	.08212	4.7100	5.4167	4.90	
660	3.0900	.23516	.13577	2.5058	3.6742	2.86	
980	1.7633	.11240	.06469	1.4841	2.0425	1.64	
6 days	0	4.9967	.15503	.08950	4.6116	5.3818	4.82	
660	4.2900	1.92291	1.11020	− .4868	9.0668	2.09	
980	6.4633	1.19571	.69035	3.4930	9.4337	5.61	

Necrosis

Table 6 presents the rate of necrosis in the groups at different time points. As shown, no significant difference was found among the groups in this regard on day 2 and day 4 (P > 0.05). On day 6, however, a significant difference existed among the groups in rate of necrosis (P < 0.05), such that the rate of necrosis in 980 nm laser group was significantly lower than that in the control group (P = 0.036) (Fig. 2D).Table 6 Rate of necrosis in the groups at different time points.

Time	Group	Mean	Std. deviation	Std. error	95% confidence interval for mean	Minimum	
Lower bound	Upper bound	
2 days	0	.4233	.10786	.06227	1554	.6913	.30	
660	.0767	.01528	.00882	.0387	.1146	.06	
980	.1600	.05568	.03215	.0217	.2983	.11	
4 days	0	.3867	.10408	.06009	.1281	.6452	.27	
660	.7467	.18610	.10745	.2844	1.2090	.55	
980	.4400	.05568	.03215	.3017	.5783	.38	
6 days	0	.3867	.09074	.05239	.1613	.6121	.29	
660	.1733	.08737	.05044	− .0437	.3904	.10	
980	.0667	.04726	.02728	− .0507	.1841	.03	

Cell differentiation according to the results of Alizarin Red staining

Table 7 presents cell differentiation in the groups according to the frequency of irradiation cycles. Cell differentiation in 660 nm and 980 nm laser groups with 3 irradiation cycles was significantly higher than that in the control group (P < 0.05). Also, cell differentiation in 660 nm laser group with one irradiation cycle was significantly higher than that in 980 nm laser group with one irradiation cycle, and significantly lower than that in 660 nm and 980 nm laser groups with 3 irradiation cycles (P < 0.05). Moreover, cell differentiation in 660 nm laser group with three irradiation cycles was significantly higher than that in the control group, 660 nm and 980 nm laser group with one irradiation cycle, and also 980 nm laser group with two irradiation cycles (P < 0.05). Cell differentiation in 980 nm laser group with three irradiation cycles was significantly higher than that in the control group, 660 nm and 980 nm laser groups with one irradiation cycle, and 980 nm laser group with two irradiation cycles (P < 0.05). Figure 3 present the results of Alizarin Red staining of the cells. Figure 4 shown a microscopical images for the mineralization experiment.Table 7 Cell differentiation in the groups according to the frequency of irradiation cycles.

Group/Cycle	N	Mean	Std, deviation	Std. error	95% Confidence interval for mean	Minimum	
Lower bound	Upper bound	
Control	10	.6203	.12820	.04054	.5286	.7120	.43	
660/1	14	.6571	.09146	.02444	.6043	.7099	.56	
660/2	17	.7995	.18053	.04380	.7067	.8924	.63	
660/3	24	.9128	.16420	.03352	.8435	.9821	.70	
980/1	17	.5423	.08447	.02049	.4989	.5857	.42	
980/2	14	.6665	.03810	.01018	.6445	.6885	.61	
980/3	16	.8083	.10529	.02632	.7522	.8644	.64	

Fig. 3 Photographs of Alizarin Red staining of cells taken under a microscope.

Fig. 4 Microscopical images for the mineralization experiment.

Differentiation of cells according to the expression of osteogenic genes assessed by real-time PCR

Assessments were made at 7 and 14 days after three laser irradiation cycles and the following results were obtained:

OPN (Fig. 5A,B): The highest expression of OPN compared with the control group at 7 days occurred in 660 nm laser group with three irradiation cycles on day 14 followed by 660 nm laser group with two irradiation cycles on day 14, and 980 nm laser group with three irradiation cycles on day 14. OPN gene expression in the control group significantly increased at 14 days compared with 7 days (P < 0.05). No other significant differences were noted (P > 0.05).Fig. 5 (A): Expression of genes in the groups based on the frequency of laser irradiation cycle, (A): OPN (7 days), (B): OPN (14 days), (C): OCN (7 days), (D): OCN (14 days), (E): RUNX2 (7 days), (F): RUNX2 (14 days).

OCN (Fig. 5C,D): The highest expression of OCN compared with the control group at 7 days occurred in 660 nm laser group with three irradiation cycles on day 14 followed by 660 nm laser group with two irradiation cycles on day 14, and 980 nm laser group with three irradiation cycles on day 14. OCN gene expression in the control group significantly increased at 14 days compared with 7 days (P < 0.05). Also, expression of OCN in the control group at 7 days was significantly different from that in 660 nm laser group with three irradiation cycles at 7 days (P < 0.05). No other significant differences were noted (P > 0.05).

RUNX2 (Fig. 5E,F): The highest expression of RUNX2 compared with the control group at 7 days occurred in 660 nm laser group with three irradiation cycles on day 14 followed by 660 nm laser group with three irradiation cycles on day 7, 660 nm laser group with two irradiation cycles on day 14, 980 nm laser group with three irradiation cycles on day 7, 980 nm laser group with two irradiation cycles on day 7, 980 nm laser group with three irradiation cycles on day 14, and 980 nm laser group with two irradiation cycles on day 14. RUNX2 gene expression in the control group significantly increased at 14 days compared with 7 days (P < 0.05). No other significant differences were noted (P > 0.05).

Discussion

This study assessed the effect of PBMT with 660 and 980 nm diode lasers on differentiation of PDLMSCs. The results showed no significant difference among the groups in cell apoptosis according to the results of DAPI staining. Assessment of cell viability by the MTT assay showed higher cell viability in both laser groups at 4 and 6 days, compared with the control group. Assessment of cell viability by Annexin V/PI showed higher number of viable cells in both laser groups than the control group on day 4. Moreover, on day 4, early and late apoptosis in both laser groups was lower than that in the control group. On day 6, necrosis in 980 nm laser group was lower than that in the control group. Evaluation of cell differentiation by Alizarin Red staining showed higher cell differentiation in 660 nm laser group with three irradiation cycles and 980 nm laser group with three irradiation cycles compared with the control group. Assessment of the expression of osteogenic genes by real-time PCR revealed the highest expression of OPN, OCN, and RUNX2 genes in 660 nm laser group with three irradiation cycles at 14 days compared with the control group at 7 days.

PBMT has beneficial effects such as enhancement of blood circulation and angiogenesis, anti-inflammatory and analgesic effects, and stimulation of cell proliferation and tissue regeneration31. The mechanism of action is based on absorption of photons with these wavelengths by the chromophores present in cytochrome C oxidase, and stimulation of mitochondrial metabolism through increasing the matrix metalloproteinases and oxygen uptake and subsequent enhancement of ATP synthesis31. The present study showed that PBMT increased the number of PDLMSCs and enhanced their differentiation to osteogenic cells. Thus, it appears that PBMT may be able to improve tissue regeneration.

Mylona et al.32 in their systematic review evaluated the effect of PBMT on PDLMSCs and found that different laser types and LED increased the proliferation rate, osteogenic differentiation, and expression of different osteogenic genes, and inhibited inflammation. According to their results, although the near infrared 940 nm wavelength may not bring about favorable results, 630 to 830 nm wavelengths show optimal results. They concluded that PBM can increase the differentiation potential of PDLMSCs, which was in agreement with the present findings. However, they did not assess the effect of number of irradiation cycles, while the present study showed that increasing the irradiation cycles significantly increased cell differentiation and expression of osteogenic genes.

Chaweewannakorn et al.33 investigated the in vitro effect of PBMT with LED on proliferation and osteoblastic differentiation of PDLMSCs. They used 630, 680, and 830 nm wavelengths with a fixed energy density of 3.5 J/cm2. The results indicated that 830 nm laser significantly increased the proliferation of cells on days 6 and 8 compared with the control group, while 630 and 680 nm laser groups showed lower rate of proliferation at 8 days. However, the results of the MTT assay in the present study revealed higher rate of cell proliferation on day 4 in 660 nm laser group. Difference between the results of the two studies can be due to different wavelengths and higher energy density in the present study. Their results regarding higher efficacy of 630 and 680 nm laser wavelengths for osteoblastic differentiation were in agreement with the present findings.

Rigi-Ladez et al.34 assessed the effect of PBMT with near infrared laser on PDLMSCs. They compared 810 and 940 nm laser wavelengths with 0.5, 1.5, and 2.5 J/cm2 energy densities, and 100 mW power. They assessed cell viability after 24, 48, and 72 h by the MTT assay. They found significantly higher cell proliferation in 940 nm laser group with 2.5 J/cm2 energy density at all time points compared with other groups. PI staining showed no significant change in cell nucleus in any group. They concluded that irradiation of PDLMSCs with 940 nm laser and 2.5 2.5 J/cm2 energy density can increase cell proliferation. No significant increase in cell proliferation by irradiation of 810 nm laser may be due to low energy density and frequency of irradiations. They did not assess cell differentiation or the effect of laser irradiation cycles in their study. Gholami et al.35 evaluated the effect of PBMT on proliferation, viability, and osteogenic differentiation of human PDLMSCs. They used 940 nm diode laser with 4 J/cm2 energy density and 100 mW power for 3 sessions every 48 h. They assessed cell viability at 24, 48 and 72 h by the MTT assay and found no significant difference between the test and control groups, which may be due to the use of different wavelengths compared with those used in the present study. At 14 and 21 days, both groups with osteogenic medium with and without laser showed higher mineralization compared with the group with no osteogenic medium, which was in line with the present findings. Etemadi et al.27 indicated that PBMT with 635, 660, 808, and 980 nm laser wavelengths increased the proliferation of PDLMSCs but the highest cell viability was recorded at 3 days after irradiation of 980 nm laser with 4 J/cm2 energy density, with significant differences with the rate in other groups. They evaluated only one irradiation cycle in their study and did not assess cell differentiation. Ateş et al.36 evaluated the effect of PBMT with 635 and 809 nm wavelengths and 0.5, 1, and 2 J/cm2 energy densities on osteogenic differentiation of adipose-derived MSCs by alkaline phosphatase activity test, Alizarin Red staining, and PCR (for expression of collagen type I, ALP, and OCN genes) as well as cell viability by the MTT assay. They found that irradiation of 635 nm and 809 nm laser had no significant effect on cell viability at 7 and 14 days except for 635 nm laser with 0.5 J/cm2 energy density on day 14; their results were different from the present findings which may be due to the use of different laser wavelengths and energy densities. Mineralization significantly increased in 809 nm laser group but no increase occurred in cell differentiation according to the alkaline phosphatase activity test or gene expression results. This finding was in contrast to the present results probably due to the use of different laser wavelengths and energy densities. Moreover, Fekrazad et al.37 evaluated the effect of LLLT with 810 nm laser and 4 J/cm2 energy density in combination with MSCs on regeneration of artificially induced 6-mm bone defects in rabbit calvaria. They reported significantly higher bone regeneration and lower inflammation in LLLT group alone (without MSCs). Their results were different from the present findings probably due to different sources of MSCs, and different laser wavelengths.

Only two laser wavelengths were evaluated in the present study. Future studies are required on the effects of other laser wavelengths with different energy densities and powers on differentiation of PDLMSCs.

Despite the increase in cell apoptosis over time, PBMT with 660 and 980 nm diode lasers decreased apoptosis and increased cell viability. Also, PBMT with 660 nm and 980 nm lasers with three irradiation cycles significantly increased PDLMSC differentiation compared with the control group. Moreover, irradiation of 660 nm laser with three irradiation cycles had the greatest effect on expression of OPN, OCN, and RUNX2 genes on day 14.

Additional research is required to gain a deeper understanding of the impact of PBMT on the growth and specialization of PDLMSC, which play a crucial role in the periodontal regeneration process.

Materials and methods

This in vitro, experimental study was conducted on human PDLMSCs obtained from Dentistry Research Institute, Tehran University of Medical Sciences.

Sample size

The minimum sample size was calculated to be 4 in each group for the methyl thiazolyl tetrazolium (MTT) cell viability assay according to a study by Hanna et al.38 using one-way ANOVA feature of PASS 11 assuming α = 0.05, β = 0.2, mean standard deviation of cell viability to be 0.64, and effect size of 0.98.

The minimum sample size was calculated to be 4 in each group for 4′,6-diamidino-2-phenylindole (DAPI) staining according to a study by Hanna et al.38 using one-way ANOVA feature of PASS 11 assuming α = 0.05, β = 0.2, mean standard deviation of cell viability to be 0.38, and effect size of 0.85.

The minimum sample size for other tests was determined based on their standards, which was 10 in each group for Alizarin Red staining, 4 in each group for Annexin V/PI and 4 in each group for the polymerase chain reaction (PCR).

Methodology

PDLMSCs were cultured in standard Dulbecco’s modified Eagle’s medium (DMEM-LG; Biosera, France) supplemented with 15% fetal bovine serum (Capricorn, Germany) and 1% pen-strep (Biosera, France) and incubated at 37 °C and 5% CO2.

To assess cell differentiation, PDLMSCs were cultured in osteogenic medium containing DMEM, 5 mM β-glycerol phosphate (Sigma, Germany), 50 µg/mL ascorbic acid 2- phosphate (Sigma, Germany), 0.01 µM dexamethasone, and 10% fetal bovine serum along with 1% pen-strep39.

The control group did not undergo laser irradiation. The cells in the first experimental group were subjected to irradiation of 980 nm diode (GaAlAs) laser irradiation (Doctorsmie, Italy) with 100 mW power, 0.25 W/cm2 power density, 16 s of irradiation time, and 4 J/cm2 energy density with a probe tip surface area of 0.384 cm2. The cells in the second experimental group underwent 660 nm diode (InGaAlP) laser irradiation (Konftec, Taiwan), with 150 mW power, 0.25 W/cm2 power density, 16 s of time, 4 J/cm2 energy density, and probe tip surface area of 0.5 cm2. Each experimental group included three subgroups of (I) one irradiation cycle on day 1, (II) two irradiation cycles on days 1 and 3, and (III) three irradiation cycles on days 1, 3, and 5.

Laser was irradiated in continuous-wave mode for 16 s at 1 cm distance from the culture medium. To protect the adjacent wells during irradiation, three empty wells were considered between the experimental wells.

Assessment of cell viability by the MTT assay37

The MTT assay was performed at 2, 4 and 6 days as follows: 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide was added to phosphate buffered saline (PBS) in 5 mg/mL concentration, sterilized by using a 0.2 μm filter paper, and stored at − 20 °C until the experiment. For testing, it was diluted with DMEM in 1:10 ratio, and 10 μL of diluted solution was added to each well. The cells were also seeded in a 48-well plate with a density of 103 cells/well. Upon formation of a purple deposit as ensured under a microscope, the wells were emptied and 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan crystals. Finally, the light absorbance (optical density) of the wells was read by a spectrophotometer (Biotek, USA)at 570 nm wavelength. To calculate the cell viability percentage, the optical density values of each group were divided by the optical density of the negative control group and multiplied by 100.

Assessment of cell viability by DAPI staining38,40

Culture and grouping of the cells were similar to those described for the MTT assay. DAPI staining (Santa Cruz, USA) was conducted at 2, 4, and 6 days. For this purpose, the cells were rinsed with PBS 3 times. Next, DAPI stain solution was added to the cells and they were incubated with the stain at 37 °C for 5 min. The number of viable and apoptotic cells was counted under a fluorescent microscope (Olympus, Japan) at × 40 magnification.

Assessment of cell apoptosis by the Annexin V/PI protocol

Culture and grouping of the cells were similar to those described for the MTT assay. The Annexin V test was conducted at 2, 4, and 6 days using the Annexin V apoptosis detection kit (X-Bio, China). Cell suspension was initially prepared by using 500 μL of X1 bonding buffer, 5 μL of Annexin V-FITC and 5 μL of propidium iodide. After 5 min of incubation, staining was performed at room temperature in the dark, and cell apoptosis was evaluated using a flow cytometer (Invitrogen, Thermo Fisher Scientific, US).

Assessment of cell differentiation by Alizarin Red staining37

Culture and grouping of the cells were similar to those described for the MTT assay. The cells were cultured with a density of 104 cells/well in 24-well plates. After 48 h, osteogenic medium was added and 7 and 14 days after the first cycle of laser irradiation, the cells were rinsed with PBS 3 times, and fixed with 10% formaldehyde for 15 min. The fixing solution was then removed, and the cells were rinsed with PBS 3 times, each time for 5 min. Next, Alizarin Red solution (Sigma, Germany) was prepared with 2 g/100 mL concentration, and added to the plates. After 5 min, the staining solution was removed and the cells were rinsed with distilled water 3 times. Finally, the morphology of the cells and formation of calcified nodules were assessed under a light microscope (Olympus, Japan) at × 10 and × 20 magnifications.

Assessment of cell differentiation based on expression of osteogenic genes using real-time PCR37

Assessment of cell differentiation was performed at 7 and 14 days after cell seeding. For this purpose, RNA was extracted by the phenol chloroform technique using RNA XPlus solution (Cinagen). The concentration and quality of the extracted RNA were assessed by NanoDrop® (UV–Vis 2000c; Thermo Scientific, USA). DNase-I and RNase kits were used to eliminate genomic DNA, and then 1000 ng of the extracted RNA was converted to cDNA using cDNA synthesis kit (Biofact, Korea). GAPDH gene primers were used as internal control and osteopontin (OPN), osteocalcin (OCN), and Runt-related transcription factor 2 (RUNX2) gene primers were used as test primers. Finally, the data obtained from real-time PCR (Roche light cycler 480) were analyzed by the ΔΔCT technique, and relative gene expressions were calculated for each group.

Statistical analysis

Two-way ANOVA was applied to compare the groups at 0.05 level of significance.

Acknowledgements

The authors thankfully acknowledge Azad university of Medical Sciences.

Author contributions

Conceptualization, N.C. and A.E.; methodology, N.C.; software, F.S.; validation, M.A.; formal analysis, M.A.; investigation, N.C.; resources, ; writing—original draft preparation, M.A.; writing—review and editing, A.E and N.C.; All authors have read and agreed to the published version of the manuscript.

Funding

This research was conducted by private financial source and no funding was received.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Ardavan Etemadi and Milad Aghaie.
==== Refs
References

1. Armitage GC Clinical evaluation of periodontal diseases Periodontol 1995 2000 7 39 53 10.1111/j.1600-0757.1995.tb00035.x
Armitage, G. C. Clinical evaluation of periodontal diseases. Periodontol 2000(7), 39–53 (1995).10.1111/j.1600-0757.1995.tb00035.x
2. Armitage GC Periodontal diagnoses and classification of periodontal diseases Periodontol 2004 2000 34 9 21 10.1046/j.0906-6713.2002.003421.x
Armitage, G. C. Periodontal diagnoses and classification of periodontal diseases. Periodontol 2004(34), 9–21 (2000).10.1046/j.0906-6713.2002.003421.x
3. Egusa H Sonoyama W Nishimura M Atsuta I Akiyama K Stem cells in dentistry–Part II: Clinical applications J. Prosthodont. Res. 2012 56 4 229 248 10.1016/j.jpor.2012.10.001 23137671
Egusa, H., Sonoyama, W., Nishimura, M., Atsuta, I. & Akiyama, K. Stem cells in dentistry–Part II: Clinical applications. J. Prosthodont. Res. 56(4), 229–248 (2012).23137671 10.1016/j.jpor.2012.10.001
4. Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R Mosca JD Multilineage potential of adult human mesenchymal stem cells Science 1999 284 5411 143 147 10.1126/science.284.5411.143 10102814
Pittenger, M. F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411), 143–147 (1999).10102814 10.1126/science.284.5411.143
5. Tamaki Y Nakahara T Ishikawa H Sato S In vitro analysis of mesenchymal stem cells derived from human teeth and bone marrow Odontology 2013 101 2 121 132 10.1007/s10266-012-0075-0 22772774
Tamaki, Y., Nakahara, T., Ishikawa, H. & Sato, S. In vitro analysis of mesenchymal stem cells derived from human teeth and bone marrow. Odontology 101(2), 121–132 (2013).22772774 10.1007/s10266-012-0075-0
6. Ivanovski S Gronthos S Shi S Bartold PM Stem cells in the periodontal ligament Oral Dis. 2006 12 4 358 363 10.1111/j.1601-0825.2006.01253.x 16792719
Ivanovski, S., Gronthos, S., Shi, S. & Bartold, P. M. Stem cells in the periodontal ligament. Oral Dis. 12(4), 358–363 (2006).16792719 10.1111/j.1601-0825.2006.01253.x
7. Seo BM Miura M Gronthos S Bartold PM Batouli S Brahim J Investigation of multipotent postnatal stem cells from human periodontal ligament Lancet 2004 364 9429 149 155 10.1016/S0140-6736(04)16627-0 15246727
Seo, B. M. et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364(9429), 149–155 (2004).15246727 10.1016/S0140-6736(04)16627-0
8. Miura M Gronthos S Zhao M Lu B Fisher LW Robey PG SHED: stem cells from human exfoliated deciduous teeth Proc. Natl. Acad. Sci. USA 2003 100 10 5807 5812 10.1073/pnas.0937635100 12716973
Miura, M. et al. SHED: stem cells from human exfoliated deciduous teeth. Proc. Natl. Acad. Sci. USA 100(10), 5807–5812 (2003).12716973 10.1073/pnas.0937635100
9. Fu X Jin L Ma P Fan Z Wang S Allogeneic stem cells from deciduous teeth in treatment for periodontitis in miniature swine J. Periodontol. 2014 85 6 845 851 10.1902/jop.2013.130254 24001042
Fu, X., Jin, L., Ma, P., Fan, Z. & Wang, S. Allogeneic stem cells from deciduous teeth in treatment for periodontitis in miniature swine. J. Periodontol. 85(6), 845–851 (2014).24001042 10.1902/jop.2013.130254
10. Ohkoshi S Hara H Hirono H Watanabe K Hasegawa K Regenerative medicine using dental pulp stem cells for liver diseases World J. Gastrointest. Pharmacol. Ther. 2017 8 1 1 6 10.4292/wjgpt.v8.i1.1 28217369
Ohkoshi, S., Hara, H., Hirono, H., Watanabe, K. & Hasegawa, K. Regenerative medicine using dental pulp stem cells for liver diseases. World J. Gastrointest. Pharmacol. Ther. 8(1), 1–6 (2017).28217369 10.4292/wjgpt.v8.i1.1
11. Han J Menicanin D Marino V Ge S Mrozik K Gronthos S Assessment of the regenerative potential of allogeneic periodontal ligament stem cells in a rodent periodontal defect model J. Periodontal. Res. 2014 49 3 333 345 10.1111/jre.12111 23841948
Han, J. et al. Assessment of the regenerative potential of allogeneic periodontal ligament stem cells in a rodent periodontal defect model. J. Periodontal. Res. 49(3), 333–345 (2014).23841948 10.1111/jre.12111
12. Yu N Oortgiesen DA Bronckers AL Yang F Walboomers XF Jansen JA Enhanced periodontal tissue regeneration by periodontal cell implantation J. Clin. Periodontol. 2013 40 7 698 706 10.1111/jcpe.12113 23675871
Yu, N. et al. Enhanced periodontal tissue regeneration by periodontal cell implantation. J. Clin. Periodontol. 40(7), 698–706 (2013).23675871 10.1111/jcpe.12113
13. Negi S Krishnamurthy M Ganji KK Pendor S Modulatory effects by neodymium-doped yttrium aluminum garnet laser on fibroblast attachment to single rooted tooth surfaces following ultrasonic scaling and root planning: An in vitro study J. Indian Soc. Periodontol. 2015 19 1 25 31 10.4103/0972-124X.145819 25810589
Negi, S., Krishnamurthy, M., Ganji, K. K. & Pendor, S. Modulatory effects by neodymium-doped yttrium aluminum garnet laser on fibroblast attachment to single rooted tooth surfaces following ultrasonic scaling and root planning: An in vitro study. J. Indian Soc. Periodontol. 19(1), 25–31 (2015).25810589 10.4103/0972-124X.145819
14. Mester E Gyenes G Tota JG Experimental studies on the effect of laser beams on wound healing Z. Exper. Surgery. 1969 2 94 101
Mester, E., Gyenes, G. & Tota, J. G. Experimental studies on the effect of laser beams on wound healing. Z. Exper. Surgery. 2, 94–101 (1969).
15. Fernando S Hill CM Walker R A randomised double blind comparative study of low level laser therapy following surgical extraction of lower third molar teeth Br. J. Oral Maxillofac. Surg. 1993 31 3 170 172 10.1016/0266-4356(93)90118-G 8512911
Fernando, S., Hill, C. M. & Walker, R. A randomised double blind comparative study of low level laser therapy following surgical extraction of lower third molar teeth. Br. J. Oral Maxillofac. Surg. 31(3), 170–172 (1993).8512911 10.1016/0266-4356(93)90118-G
16. Ozawa Y Shimizu N Kariya G Abiko Y Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells Bone 1998 22 4 347 354 10.1016/S8756-3282(97)00294-9 9556134
Ozawa, Y., Shimizu, N., Kariya, G. & Abiko, Y. Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells. Bone 22(4), 347–354 (1998).9556134 10.1016/S8756-3282(97)00294-9
17. Abramovitch-Gottlib L Gross T Naveh D Geresh S Rosenwaks S Bar I Low level laser irradiation stimulates osteogenic phenotype of mesenchymal stem cells seeded on a three-dimensional biomatrix Lasers Med. Sci. 2005 20 3–4 138 146 10.1007/s10103-005-0355-9 16292614
Abramovitch-Gottlib, L. et al. Low level laser irradiation stimulates osteogenic phenotype of mesenchymal stem cells seeded on a three-dimensional biomatrix. Lasers Med. Sci. 20(3–4), 138–146 (2005).16292614 10.1007/s10103-005-0355-9
18. Khadra M Rønold HJ Lyngstadaas SP Ellingsen JE Haanæs HR Low-level laser therapy stimulates bone–implant interaction: An experimental study in rabbits Clin. Oral Implants Res. 2004 15 3 325 332 10.1111/j.1600-0501.2004.00994.x 15142095
Khadra, M., Rønold, H. J., Lyngstadaas, S. P., Ellingsen, J. E. & Haanæs, H. R. Low-level laser therapy stimulates bone–implant interaction: An experimental study in rabbits. Clin. Oral Implants Res. 15(3), 325–332 (2004).15142095 10.1111/j.1600-0501.2004.00994.x
19. Qadri T Miranda L Tuner J Gustafsson A The short-term effects of low-level lasers as adjunct therapy in the treatment of periodontal inflammation J. Clin. Periodontol. 2005 32 7 714 719 10.1111/j.1600-051X.2005.00749.x 15966876
Qadri, T., Miranda, L., Tuner, J. & Gustafsson, A. The short-term effects of low-level lasers as adjunct therapy in the treatment of periodontal inflammation. J. Clin. Periodontol. 32(7), 714–719 (2005).15966876 10.1111/j.1600-051X.2005.00749.x
20. Makhlouf M Dahaba MM Tuner J Eissa SA Harhash TA Effect of adjunctive low level laser therapy (LLLT) on nonsurgical treatment of chronic periodontitis Photomed. Laser. Surg. 2012 30 3 160 166 10.1089/pho.2011.3069 22233558
Makhlouf, M., Dahaba, M. M., Tuner, J., Eissa, S. A. & Harhash, T. A. Effect of adjunctive low level laser therapy (LLLT) on nonsurgical treatment of chronic periodontitis. Photomed. Laser. Surg. 30(3), 160–166 (2012).22233558 10.1089/pho.2011.3069
21. Damante CA Greghi SL Sant'ana AC Passanezi E Clinical evaluation of the effects of low-intensity laser (GaAlAs) on wound healing after gingivoplasty in humans J. Appl. Oral Sci. 2004 12 2 133 136 10.1590/S1678-77572004000200010 21365136
Damante, C. A., Greghi, S. L., Sant’ana, A. C. & Passanezi, E. Clinical evaluation of the effects of low-intensity laser (GaAlAs) on wound healing after gingivoplasty in humans. J. Appl. Oral Sci. 12(2), 133–136 (2004).21365136 10.1590/S1678-77572004000200010
22. Ozcelik O Cenk Haytac M Kunin A Seydaoglu G Improved wound healing by low-level laser irradiation after gingivectomy operations: A controlled clinical pilot study J. Clin. Periodontol. 2008 35 3 250 254 10.1111/j.1600-051X.2007.01194.x 18269665
Ozcelik, O., Cenk Haytac, M., Kunin, A. & Seydaoglu, G. Improved wound healing by low-level laser irradiation after gingivectomy operations: A controlled clinical pilot study. J. Clin. Periodontol. 35(3), 250–254 (2008).18269665 10.1111/j.1600-051X.2007.01194.x
23. Ozcelik O Cenk Haytac M Seydaoglu G Enamel matrix derivative and low-level laser therapy in the treatment of intra-bony defects: A randomized placebo-controlled clinical trial J. Clin. Periodontol. 2008 35 2 147 156 10.1111/j.1600-051X.2007.01176.x 18081859
Ozcelik, O., Cenk Haytac, M. & Seydaoglu, G. Enamel matrix derivative and low-level laser therapy in the treatment of intra-bony defects: A randomized placebo-controlled clinical trial. J. Clin. Periodontol. 35(2), 147–156 (2008).18081859 10.1111/j.1600-051X.2007.01176.x
24. Anders JJ Lanzafame RJ Arany PR Low-level light/laser therapy versus photobiomodulation therapy Photomed. Laser Surg. 2015 33 4 183 184 10.1089/pho.2015.9848 25844681
Anders, J. J., Lanzafame, R. J. & Arany, P. R. Low-level light/laser therapy versus photobiomodulation therapy. Photomed. Laser Surg. 33(4), 183–184 (2015).25844681 10.1089/pho.2015.9848
25. Kreisler M Christoffers AB Al-Haj H Willershausen B d'Hoedt B Low level 809-nm diode laser-induced in vitro stimulation of the proliferation of human gingival fibroblasts Lasers Surg. Med. 2002 30 5 365 369 10.1002/lsm.10060 12116329
Kreisler, M., Christoffers, A. B., Al-Haj, H., Willershausen, B. & d’Hoedt, B. Low level 809-nm diode laser-induced in vitro stimulation of the proliferation of human gingival fibroblasts. Lasers Surg. Med. 30(5), 365–369 (2002).12116329 10.1002/lsm.10060
26. Rahman S Mosca R Govindool Reddy S Nunez S Andreana S Mang T Learning from clinical phenotypes: Low-dose biophotonics therapies in oral diseases Oral Dis. 2018 24 1–2 261 276 10.1111/odi.12796 29480614
Rahman, S. et al. Learning from clinical phenotypes: Low-dose biophotonics therapies in oral diseases. Oral Dis. 24(1–2), 261–276 (2018).29480614 10.1111/odi.12796
27. Etemadi A Faghih A Chiniforush N Effects of photobiomodulation therapy with various laser wavelengths on proliferation of human periodontal ligament mesenchymal stem cells Photochem. Photobiol. 2022 98 5 1182 1189 10.1111/php.13588 34970994
Etemadi, A., Faghih, A. & Chiniforush, N. Effects of photobiomodulation therapy with various laser wavelengths on proliferation of human periodontal ligament mesenchymal stem cells. Photochem. Photobiol. 98(5), 1182–1189 (2022).34970994 10.1111/php.13588
28. Ginani F Soares DM Barreto MP Barboza CA Effect of low-level laser therapy on mesenchymal stem cell proliferation: A systematic review Lasers Med. Sci. 2015 30 8 2189 2194 10.1007/s10103-015-1730-9 25764448
Ginani, F., Soares, D. M., Barreto, M. P. & Barboza, C. A. Effect of low-level laser therapy on mesenchymal stem cell proliferation: A systematic review. Lasers Med. Sci. 30(8), 2189–2194 (2015).25764448 10.1007/s10103-015-1730-9
29. Fekrazad R Asefi S Baghaban Eslaminejad M Taghiyar L Bordbar S Hamblin MR Photobiomodulation with single and combination laser wavelengths on bone marrow mesenchymal stem cells: Proliferation and differentiation to bone or cartilage Lasers Med. Sci. 2019 34 1 115 126 10.1007/s10103-018-2620-8 30264177
Fekrazad, R. et al. Photobiomodulation with single and combination laser wavelengths on bone marrow mesenchymal stem cells: Proliferation and differentiation to bone or cartilage. Lasers Med. Sci. 34(1), 115–126 (2019).30264177 10.1007/s10103-018-2620-8
30. Marques NP Lopes CS Marques NCT Cosme-Silva L Oliveira TM Duque C A preliminary comparison between the effects of red and infrared laser irradiation on viability and proliferation of SHED Lasers Med. Sci. 2019 34 3 465 471 10.1007/s10103-018-2615-5 30121722
Marques, N. P. et al. A preliminary comparison between the effects of red and infrared laser irradiation on viability and proliferation of SHED. Lasers Med. Sci. 34(3), 465–471 (2019).30121722 10.1007/s10103-018-2615-5
31. Romanovsky AA Almeida MC Garami A Steiner AA Norman MH Morrison SF The transient receptor potential vanilloid-1 channel in thermoregulation: A thermosensor it is not Pharmacol. Rev. 2009 61 3 228 261 10.1124/pr.109.001263 19749171
Romanovsky, A. A. et al. The transient receptor potential vanilloid-1 channel in thermoregulation: A thermosensor it is not. Pharmacol. Rev. 61(3), 228–261 (2009).19749171 10.1124/pr.109.001263
32. Mylona V Anagnostaki E Chiniforush N Barikani H Lynch E Grootveld M Photobiomodulation effects on periodontal ligament stem cells: A systematic review of in-vitro studies Curr. Stem. Cell Res. Ther. 2022 19 544 10.2174/1574888X17666220527090321
Mylona, V. et al. Photobiomodulation effects on periodontal ligament stem cells: A systematic review of in-vitro studies. Curr. Stem. Cell Res. Ther. 19, 544 (2022).10.2174/1574888X17666220527090321
33. Chaweewannakorn C Santiwong P Surarit R Sritanaudomchai H Chintavalakorn R The effect of LED photobiomodulation on the proliferation and osteoblastic differentiation of periodontal ligament stem cells: In vitro J. World Fed. Orthod. 2021 10 2 79 85 33888447
Chaweewannakorn, C., Santiwong, P., Surarit, R., Sritanaudomchai, H. & Chintavalakorn, R. The effect of LED photobiomodulation on the proliferation and osteoblastic differentiation of periodontal ligament stem cells: In vitro. J. World Fed. Orthod. 10(2), 79–85 (2021).33888447
34. Rigi-Ladez MA Hendi SS Mirzaei A Gholami L Fekrazad R Near infrared laser photobiomodulation of periodontal ligament stem cells Chin. J. Dent. Res. 2022 25 1 57 65 35293711
Rigi-Ladez, M. A., Hendi, S. S., Mirzaei, A., Gholami, L. & Fekrazad, R. Near infrared laser photobiomodulation of periodontal ligament stem cells. Chin. J. Dent. Res. 25(1), 57–65 (2022).35293711
35. Gholami L Parsamanesh G Shahabi S Jazaeri M Baghaei K Fekrazad R The effect of laser photobiomodulation on periodontal ligament stem cells Photochem. Photobiol. 2021 97 4 851 859 10.1111/php.13367 33305457
Gholami, L. et al. The effect of laser photobiomodulation on periodontal ligament stem cells. Photochem. Photobiol. 97(4), 851–859 (2021).33305457 10.1111/php.13367
36. Ateş GB Ak A Garipcan B Gülsoy M Photobiomodulation effects on osteogenic differentiation of adipose-derived stem cells Cytotechnology 2020 72 2 247 258 10.1007/s10616-020-00374-y 32016710
Ateş, G. B., Ak, A., Garipcan, B. & Gülsoy, M. Photobiomodulation effects on osteogenic differentiation of adipose-derived stem cells. Cytotechnology 72(2), 247–258 (2020).32016710 10.1007/s10616-020-00374-y
37. Fekrazad R Sadeghi Ghuchani M Eslaminejad MB Taghiyar L Kalhori KA Pedram MS The effects of combined low level laser therapy and mesenchymal stem cells on bone regeneration in rabbit calvarial defects J. Photochem. Photobiol. B Biol. 2015 151 180 185 10.1016/j.jphotobiol.2015.08.002
Fekrazad, R. et al. The effects of combined low level laser therapy and mesenchymal stem cells on bone regeneration in rabbit calvarial defects. J. Photochem. Photobiol. B Biol. 151, 180–185 (2015).10.1016/j.jphotobiol.2015.08.002
38. Hanna R Agas D Benedicenti S Ferrando S Laus F Cuteri V Lacava G Sabbieti MG Amaroli A A comparative study between the effectiveness of 980 nm photobiomodulation delivered by hand-piece with gaussian vs. flat-top profiles on osteoblasts maturation Front. Endocrinol. (Lausanne) 2019 10 92 10.3389/fendo.2019.00092 30842754
Hanna, R. et al. A comparative study between the effectiveness of 980 nm photobiomodulation delivered by hand-piece with gaussian vs. flat-top profiles on osteoblasts maturation. Front. Endocrinol. (Lausanne) 10, 92 (2019).30842754 10.3389/fendo.2019.00092
39. Etemadi A Taghavi S Hodjat M Kosarieh E Hakimiha N Assessment of the photobiomodulation of a blue diode laser on the proliferation and migration of cultured human gingival fibroblast cells: A preliminary in vitro study J. Laser Med. Sci. 2020 11 4 491 496 10.34172/jlms.2020.77
Etemadi, A., Taghavi, S., Hodjat, M., Kosarieh, E. & Hakimiha, N. Assessment of the photobiomodulation of a blue diode laser on the proliferation and migration of cultured human gingival fibroblast cells: A preliminary in vitro study. J. Laser Med. Sci. 11(4), 491–496 (2020).10.34172/jlms.2020.77
40. Barboza CA Ginani F Soares DM Henriques AC Freitas RA Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells Einstein (Sao Paulo) 2014 12 1 75 81 10.1590/S1679-45082014AO2824 24728250
Barboza, C. A., Ginani, F., Soares, D. M., Henriques, A. C. & Freitas, R. A. Low-level laser irradiation induces in vitro proliferation of mesenchymal stem cells. Einstein (Sao Paulo) 12(1), 75–81 (2014).24728250 10.1590/S1679-45082014AO2824
