Optimal Ultrasound Exposure Conditions for Maximizing C2C12 Muscle Cell Proliferation and Differentiation

Abstract

Described here is an in vitro systematic investigation of the effects on C2C12 myoblasts of exposure to finely controlled and repeatable low-intensity pulsed ultrasound of different frequencies (500 kHz, 1 MHz, 3 MHz and 5 MHz) and different intensities (250, 500 and 1000 mW/cm²). An in-house stimulation system and an ultrasound-transparent cell culture well minimized reflections and attenuations, allowing precise control of ultrasound delivery. Results indicated that a 3 MHz stimulation at 1 W/cm² intensity maximized cell proliferation in comparison with the other exposure conditions and untreated controls. In contrast, cell differentiation and the consequent formation of multinucleated myotubes were maximized by 1 MHz stimulation at 500 mW/cm² intensity. The highly controlled exposure conditions employed allowed precise correlation of the ultrasound delivery to the bio-effects produced, thus overcoming the inconsistency of some results available in the literature and contributing to the potential of ultrasound treatment for muscle therapy and regeneration.

Keywords: Bio-effects; C2C12; Differentiation; Frequency; Intensity; Low-intensity pulsed ultrasound; Muscle regeneration; Myoblasts; Proliferation; Ultrasound stimulation.