| The rapid growth and potential rewards of the fields of tissue engineering and cellular mechanotransduction have necessitated the investigation of all types of mechanical stimulation and their affect on cells and tissues. The purpose was to test the hypothesis that mechanical vibrations and impacts modulate proliferation and differentiation of skeletal myoblasts. The instrumentation to test this hypothesis on vibrations from 1Hz to 1000 Hz (∼0.4g) and impacts from 1 Hz to 0.0001 Hz (∼50g) was designed and built. C2C12 myoblasts stimulated with vibrations in the ranges of 20--60 Hz, 60--200 Hz, 200--500 Hz, and 500--1000 Hz (∼0.4g), experienced the following approximate changes in proliferation rate; 16% increase, 20% increase, 30% decrease, and no change, respectively. Explanations for these results include direct sensing of the vibration stimulus, generation of indirect fluid shear stimuli, and changes in bulk transport and metabolism. Subjecting C2C12 myoblasts to impact loads (1Hz, 50g) caused a drastic decrease in the number of cells present in culture that was not seen when either frequency or amplitude were lowered to 0.017Hz and 0.3g, respectively, while the other parameter remained unchanged. Whether it was apoptosis or necrosis that was responsible for the decrease in viable cells over time is unclear. Phosphorylation of a group of proteins thought to be involved in the cellular transduction of mechanical stimuli (FAK, AKT, p-38 MAPK, p70-S6 Kinase, and ERK 1/2) was unaffected by the 60--200 Hz vibration stimulus at exposure times of fifteen and ninety minutes. Myogenin expression nearly doubled in cultures under chronic 60--200 Hz vibration at 48 hours after switching to a low horse serum differentiation media. Also, myosin heavy chain expression was increased with vibration stimulation by approximately 60% at both 24 hours and 48 hours of exposure. These findings suggest that this protocol accelerates terminal differentiation of C2C12 myoblasts into myotubes. Myotube alignment was observed to approximate the axis of vibration. Research on the effects of mechanical impacts and vibrations on myoblasts may lead to a better understanding of skeletal muscle formation and regeneration as well as advances in the engineering of skeletal muscle and other tissues. |
