Photobiomodulation Exerts a Biphasic Dose Response on the 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 whether irradiation with a 940-nm diode laser elicits a biphasic dose-response in proliferation and osteoblastic differentiation assays of human PDLSCs. Methods: Six human PDLSC lines were established and characterized using flow cytometry and multilineage differentiation assays. Cell proliferation was assessed after irradiation with fluences of 1, 2, 4, and 10 J/cm². Osteoblastogenesis was induced 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 lines 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 at 2 J/cm² but was markedly reduced at 10 J/cm². Gene expression analysis confirmed this biphasic response under optimal culture conditions. Irradiation with 2 J/cm² upregulated all the analyzed genes, whereas 10 J/cm² 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 biphasic effect on human PDLSCs that critically depends on PBM fluency. A lower energy density of 2 J/cm² promotes proliferation and osteoblastogenesis, whereas higher energy densities, particularly 10 J/cm², are inhibitory.