Cyclic Hydrostatic Pressure Suppresses Osteogenic Differentiation via Actomyosin Contractility.

PURPOSE: The periodontal ligament (PDL), which contains osteoblasts, is a unique connective tissue that resists mineralization despite being located between mineralized cementum and alveolar bone. The role of physiological mechanical stress, particularly the cyclic hydrostatic pressure (HP) generated during mastication, in regulating osteogenic potential remains poorly understood. This study aimed to compare the effects of static and cyclic HP on osteogenic differentiation and to elucidate the underlying mechanisms. METHODS: A custom-built pressure-loading device was developed to apply either static or cyclic HP to MC3T3-E1 pre-osteoblastic cells. These cells, which are widely used as models for osteogenic differentiation, were subjected to conditions mimicking PDL-relevant mechanical environments. Osteogenic differentiation was evaluated using alkaline phosphatase (ALP) activity assays. Actomyosin contractility was assessed through immunofluorescence staining of focal adhesions using vinculin, along with phosphorylated myosin light chain (p-MLC) to assess myosin activity. To examine the role of actomyosin contractility, cells were treated with blebbistatin, a myosin II inhibitor. RESULTS: Compared to static HP (5 MPa), cyclic HP (5 MPa, 0.2 Hz) more strongly inhibited ALP activity. HP stimulation enhanced both focal adhesion formation and p-MLC levels, with the latter showing a markedly greater increase under cyclic HP than static HP. Notably, inhibition of p-MLC phosphorylation with 10 μM blebbistatin attenuated these HP-induced effects, indicating that actomyosin contractility plays a critical role in mediating the suppression of osteogenic differentiation. CONCLUSIONS: Cyclic HP more effectively inhibited osteogenic differentiation than static HP, likely through enhanced actomyosin contractility associated with increased p-MLC expression in MC3T3-E1 pre-osteoblastic cells. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-025-00875-w.