Mechanical Vibration Promotes Osteogenic Differentiation of Human Periodontal Ligament Cells and Its Regulation of Osteoclastogenesis

Objectives: Recently mechanical vibration has been shown to be able to enhance PDL’s maintenance. However, its cellular and molecular mechanisms are unknown. The purpose of this study is to investigate the effects of mechanical vibration on the osteogenic differentiation of human periodontal ligament cells (hPDLCs) and its potential regulation of osteoclast formation.
Methods: Human PDL cells (hPDLCs) were cultured in DMEM with 10% FBS and 1% Pen/Strep at 37°C and 5% CO₂. The cells were seeded at a density of 2000 cells/well in 6-well culture plates, and exposed to a sinusoidal mechanical vibration (0.3g) at various frequencies i.e. 0, 30, 60, 90 Hz. For differentiation study, the cells were subjected to vibration 1 hour a day for consecutive 14 days followed by ALP and Alizarin Red stainings. For signaling pathway study, RNA and protein samples were collected after 1 hour of vibration and 1 hour of vibration followed by 5 hours of post incubation, respectively. To induce osteoclast formation, RAW264.7 cells were treated with 20 ng/ml RANKL with or without the conditioned medium (CM) collected from the vibrated hPDLCs (v:v = 1:1, refreshed every 2 days for 5 days total). After TRAP staining, the TRAP+ multi nuclei (>=3) cells were counted and calculated. One-way ANOVA with Tukey post hoc comparison was used to compare the results among the experimental groups, with p value being set at 0.05.
Results: Mechanical vibration enhanced osteogenic differentiation of hPDLCs, most significantly at 60 Hz (p<0.05, n=3). Wnt/β-catenin signaling was activated by the vibration, which was paralleled with up-regulation of osteogenic-related marker genes enhanced by LiCl (Wnt/β-catenin signaling agonist). In addition, LiCl treatment also enhanced the vibration induced up-regulation of OPG/RANKL ratios at both mRNA and protein levels. The CM collected from the vibrated hPDLCs significantly inhibited osteoclastic formation. Comparing to control group (0 Hz), 30 Hz but not 60 and 90 Hz of vibration showed significant reduction of osteoclast formation by about 21% (p<0.05, n=6).
Conclusions: Mechanical vibration promotes osteogenic differentiation of hPDLCs via potentiation of Wnt/β-catenin signaling. This osteogenic response also inhibits osteoclastogenesis through regulation of downstream OGP/RANKL axis.