Fluence-Dependent Effects of 940-nm Photobiomodulation on the Proliferation and Osteoblastic Differentiation of Human Periodontal Ligament Stem Cells

Background/Objectives: Photobiomodulation (PBM) has been proposed to regulate periodontal tissue remodeling, but its effects on osteoblastogenesis in periodontal ligament stem cells (PDLSCs) remain highly dependent on laser parameters. This study investigated the fluence-dependent effects of 940 nm diode laser irradiation on the proliferation and osteoblastic differentiation of human PDLSCs. Methods: Six primary human PDLSC cultures were established and characterized using flow cytometry and multilineage differentiation assays. Cell proliferation was assessed after a single irradiation at fluences of 1, 2, 4, and 10 J/cm2. Osteoblastogenesis was induced using repeated irradiation regimens initiated at 2, 4, or 10 J/cm2 under suboptimal or complete osteogenic conditions and evaluated by Alizarin Red staining on day 21. The expression of osteogenic genes (RUNX2, BMP2, SPP1, TGFB1, COL1A1, ALPL, and BGLAP) was evaluated by qRT-PCR at different time points. Culture supernatants were analyzed for soluble osteogenic mediators. Results: The established PDLSC cultures had an MSC-like phenotype, robust osteogenic potential, moderate chondrogenic potential, and weak or no adipogenic potential. Proliferation of PDLSCs was considerably increased by PBM at 2 J/cm2 on day 7, whereas fluences of 4 and 10 J/cm2 inhibited proliferation. Osteoblastic mineralization was increased with the 2 J/cm2 irradiation regimen but was markedly reduced with the 10 J/cm2 regimen. Gene expression analysis confirmed this fluence-dependent response under optimal culture conditions. The 2 J/cm2 irradiation regimen significantly upregulated each analyzed osteogenic gene at one or more time points, whereas the 10 J/cm2 regimen downregulated RUNX2, BMP2, SPP1, COL1A1, and BGLAP. PBM at 2 J/cm2 also increased production of soluble BMP-2, OPG, PDGF-BB, ALP, and Leptin in a time-dependent manner. Conclusions: PBM exerts a fluence-dependent effect on human PDLSCs. A single irradiation at 2 J/cm2 increased proliferation, whereas the irradiation regimen initiated at 2 J/cm2 promoted osteoblastogenesis. In contrast, higher-fluence conditions, particularly the regimen initiated at 10 J/cm2, were inhibitory.