Effects of Acceleration Amplitude and Frequency of Mechanical Vibration on Osteoblast-Like Cells

Abstract

Bone formation is subject in vivo to mechanical stimulation. Although many researches for bone cells of osteoblastic lineage sensing and responding to mechanical stimulation have been reported mainly in the biochemical field, effects of mechanical stimulation on bone cells are not well understood. In this study, in order to clarify effects of acceleration amplitude and frequency of mechanical stimulation on MC3T3-E1, which is an osteoblast-like cell line derived from mouse calvaria, in the sense of mechanical vibrations, their cell proliferation, cell morphology, bone matrix generation and gene expression of alkaline phosphatase (ALP) were investigated when sinusoidal inertia force was applied to the cells. 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 reverse transcription polymerase chain reaction (real-time RT-PCR) method. After the vibrating groups for the PCR were excited for 7 days, 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. ALP gene level in each sample was normalized to the measured housekeeping gene level. The results to be obtained are as follows. In the range from 12.5 to 200 Hz, saturation cell density for the cell proliferation shows tendency of increase as frequency decreases and ALP gene expression shows a peak to frequency at 50 Hz. Among 0, 0.25 and 0.5 G, saturation cell density and ALP gene expression show tendency of increase as acceleration amplitude increases.