Effects of Amplitude and Frequency of Vibration Stimulation on Cultured Osteoblasts

Mechanical stimulation to bones affects osteogenesis such as decrease of bone mass of astronauts under zero gravity, walking rehabilitation to bone fracture and fracture repair with ultrasound devices. Bone cells have been reported to sense and response to mechanical stimulation at cellular level morphologically and metabolically. In the view of mechanical vibrations, bone cells are deformed according to mechanical stimulation and their mechanical characteristics. Recently, it was reported that viscoelasticity of cells was measured using tensile and creep tests and that there was likely natural frequency and nonlinearity of cells in the sense of structural dynamics. It suggests that there is effective frequency and amplitude of mechanical stimulation on osteogenesis by bone cells. In this study, sinusoidal inertia force was applied to cultured osteoblasts, MC3T3-E1, and effects of frequency and acceleration amplitude of mechanical vibration on the cells were investigated in respect of cell proliferation, cell morphology, bone matrix generation and alkaline phosphatase (ALP) gene expression. After the cells were cultured in culture plates in a CO₂ incubator for one day and adhered on the cultured plane, vibrating groups of the culture plates were set on an aluminum plate attached to a exciter and cultured under sinusoidal excitation in another incubator separated from non-vibrating groups of the culture plates. Acceleration amplitude and frequency were set to several kinds of conditions. The time evolution of cell density was obtained by counting the number of cells with a hemocytometer. The cell morphology was observed with a phase contrast microscope. Calcium salts generated by the cells were observed by being stained with alizarin red S solution and their images were captured with a CCD camera. The vibrating groups for the cell proliferation and the calcium salts staining were sinusoidally excited for 24 hours a day during 28-day cultivation. Gene expression of ALP was measured by a real-time RT-PCR method. After the vibrating groups for the PCR were excited for 6 hours, the total RNAs were extracted. After reverse transcription, real-time RT-PCR was performed. Gene expression for ALP and a housekeeping gene were determined simultaneously for each sample. Gene levels in each sample were normalized to the measured housekeeping gene levels. As a result, it is shown that saturate cell density becomes high and bone matrix generation is promoted by applying mechanical vibration and that there may be some peaks to frequency and a certain threshold value to acceleration amplitude of mechanical vibration for saturation cell density and bone matrix generation.