The photobiomodulation effects of continuous and pulsed blue diode laser on proliferation and osteogenic differentiation of periodontal ligament stem cells

Abstract This study investigated the photobiomodulation effect of pulsed and continuous blue diode laser on osteogenic differentiation and proliferation of periodontal ligament mesenchymal stem cells. Periodontal Ligament Stem cells were seeded in 96‐well plates, and 450 nm blue laser irradiation procedure was performed a day after cell seeding. Each experimental group was divided into two subgroups according to their energy density and irradiation duration: Continuous wave (100 mW, 10s, 2 J/cm 2 and 100 mW, 20 s, 4 J/cm 2 ) and pulse wave (200 mW, 10 s, 2 J/cm 2 and 200 mW, 20 s, 4 J/cm 2 and duty cycle 50% for both). Then, all groups were evaluated with a cell viability test (MTT), cell apoptosis (Annexin V) on the second and fourth days after irradiation, Alizarin Red staining on the 14th day after irradiation based on genes. Real‐time PCR was conducted 7 and 14 days after irradiation. GAPD gene primers were used as internal control, and OPN, OCN, ALP, and RUNX2 gene primers were used as tests. The one‐way ANOVA statistical analysis revealed that cell proliferation in the continuous‐irradiated groups was significantly higher than in pulsed groups. However, there is no significant difference in comparison with the control group. Also, pulsed‐irradiated groups demonstrated a higher rate of necrosis. The osteogenic differentiation in the continuous groups was more substantial than in the pulsed and the control groups. In comparison to all other study groups, the group that received continuous mode irradiation at an energy density of 2 J/cm 2 , power of 100 mW, and a radiation time of 10 s exhibited significantly higher numbers of calcified nodules and increased expression of OPN, OCN, and ALP genes ( p < 0.05). Overall, treating periodontal ligament stem cells with a continuous blue diode laser and appropriate parameters can enhance their osteogenic differentiation and proliferation, accelerating the regeneration of periodontal tissues.