Hydrostatic Compressive Stress Promotes MC3T3-E1 Cell Proliferation Through ROCK-Dependent Regulation of Cyclin D1 and CDK4

A CellScale MechanoCulture TR hydrostatic stimulation system was used to apply controlled cyclic hydrostatic compressive stress to MC3T3-E1 preosteoblasts adhered to sterile coverslips. The MCTR consisted of a cell-culture assembly connected to a pressure-regulator control box. Compressed air deformed an elastic membrane, transmitting cyclic hydrostatic pressure through the culture medium to the cells without direct platen contact. Before loading, the system was sterilized with ultraviolet light for one hour. Cell-covered coverslips were transferred into the culture assembly and approximately 6 mL of culture medium was added to ensure complete coverage of each well before the chamber was sealed. All experiments used a fixed cyclic frequency of 0.05 Hz consisting of 5 seconds of compression, no hold period, 5 seconds of recovery, and 10 seconds of rest per cycle. Initial experiments exposed cells to 5 kPa for 15, 30, 45, 60, or 90 minutes. Pressure magnitude was then screened at 0, 1, 5, 10, and 20 kPa for 45 minutes, after which duration was further evaluated at the selected 5 kPa pressure. The strongest proliferative response occurred at 5 kPa for 45 minutes, and this protocol was used for subsequent mechanistic experiments. MCTR stimulation increased F-actin organization and ROCK, Cyclin D1, and CDK4 expression and increased cell proliferation. Pretreatment with the ROCK inhibitor Y-27632 attenuated these responses, demonstrating that the mechanically induced proliferative effect depended substantially on ROCK signaling. The MechanoCulture TR therefore enabled the authors to independently control the hydrostatic-pressure component of the osteoblast mechanical environment and investigate pressure-dependent mechanotransduction without introducing direct substrate deformation or platen compression.