Effects of Acceleration Amplitude and Frequency of Mechanical Vibration on Cultured Osteoblasts

This paper describes the effects of the frequency and acceleration amplitude of mechanical vibration on osteoblasts, the bone cells that generate the bone matrix. Their cell proliferation and bone matrix generation were investigated when sinusoidal inertia force was applied to the cells. 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. 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. 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 days of culture. Gene expression of alkaline phosphatase (ALP) and runt-related gene 2 (Runx2) 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 4 days, the total RNAs were extracted. After reverse transcription, real-time RT-PCR was performed. Gene expression for ALP, Runx2, and a housekeeping gene were determined simultaneously for each sample. ALP and Runx2 gene level in each sample was normalized to the measured housekeeping gene level. The following experimental results of sinusoidal excitation of osteoblasts have been shown: (a) Cell density decreased at 0.5 G with increasing frequency in the range from 12.5 to 1000 Hz and increased at 25 Hz with increasing acceleration amplitude from 0 to 0.5 G at 14 days of culture. (b) No calcium salts were observed in the non-vibrating group and the areas of calcium salts observed in the 0.5 G vibration group were larger than those in the 0.25 G group at 25 Hz at 21 days of culture. (c) The mRNA level of ALP at 0.5 G showed the peak at 50 Hz in the range from 12.5 to 1000 Hz and that at 50 Hz showed the peak at 0.5 G in the range from 0.25 to 1 G at 4 days of culture. In the case of Runx2, the same tendency was found. It has been shown that it is important to consider mechanical vibration as well as biochemical aspects in studies of the functional adaptation of cells to mechanical stimulation.