Abstract
Introduction: Biomechanical stimulation of cultured human osteoblast-like cells, which is based on controlled mechanical vibration, has been previously indicated, but the exact mechanical parameters that are effective for cells' proliferation enhancement are still elusive due to the lack of direct data recordings from the stimulated cells in culture. Therefore, we developed a low friction tunable system that enables recording of a narrow range of mechanical parameters, above the infrasonic spectrum, that applied uniformly to human osteoblast-like cells in monolayer culture, aiming to identify a range of mechanical parameters that are effective to enhance osteoblast proliferation in vitro. Methods: Human osteoblast-like cells in explant monolayer culture samples were exposed to mechanical vibration in the 10-70Hz range of frequencies for two minutes, in four 24 hours intervals. Cell numbers in culture, cellular alkaline phosphatase activity (a marker of cell maturation), and lactate dehydrogenase activity in culture media (representing cell death) were measured after the mechanical stimulation protocol application and compared statistically to the control cell cultures kept in static conditions. The cell proliferation was deduced from cell number in culture and cell death measurements. Results: We found that 50-70 Hz of vibration frequency protocol (10-30 μm of maximal displacement amplitude, 0.03g of peak-to-peak acceleration) is optimal for enhancing cells' proliferation(p<0.05), with a parallel decrease of their maturation (p<0.01). Discussion: We detected the optimal mechanical parameters of excitation protocol for induction of osteoblast proliferation in vitro by a mechanical platform, which can be used as a standardized method in the research of mechanotransduction in human osteoblast.
Highlights
Biomechanical stimulation of cultured human osteoblast-like cells, which is based on controlled mechanical vibration, has been previously indicated, but the exact mechanical parameters that are effective for cells' proliferation enhancement are still elusive due to the lack of direct data recordings from the stimulated cells in culture
We determined the narrow range of high frequencies spectrum of 50-70 Hz mechanical vibration that is effective in enhancing human osteoblast proliferation in vitro
This phenomenon was not apparent following 20Hz excitation. This conclusion is independently supported by the evidence of the lower maturation state of the cells exposed to the 50-70 Hz mechanical vibration protocol as expressed by lower alkaline phosphatase cellular activity
Summary
Biomechanical stimulation of cultured human osteoblast-like cells, which is based on controlled mechanical vibration, has been previously indicated, but the exact mechanical parameters that are effective for cells' proliferation enhancement are still elusive due to the lack of direct data recordings from the stimulated cells in culture. Large numbers of cells can be studied in the same mechanical conditions, which are determined by the dimensions of the well plate and its weight, its surface biophysical properties, volume of the culture media in the wells and by a profile of the force generated by a vibration actuator This experimental setup can be used for application of vibration in higher frequencies, i.e., above the infrasonic range (20-70 Hz). To determine the role of mechanical excitation of osteoblast by high-frequency vibration, we developed an experimental model that utilizes a low friction system to deliver vibration force to a cell culture well plate This system provides a relatively stable sinusoidal vibration force that can be effectively controlled and measured. We hypothesize that by using the designed method of high frequency vibration application, we will be able to provide reliable evidence that human osteoblast is stimulated for enhanced proliferation and will determine the range of on the effective mechanical parameters that trigger this effect
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