Nonuniform temperature rise in in vitro osteoblast ultrasound exposures with associated bioeffect

There is a growing interest to use ultrasound to stimulate cellular material in vitro conditions for the treatment of musculoskeletal disorders. However, the beneficial effect resulting from ultrasound exposure is not accurately specified. Many in vitro ultrasound setups are very vulnerable to temperature elevation due to sound absorption, sound reflections, and inadequate heat transfer. The objective of this study is to show that temperature variations capable of modifying biological results may exist in common in vitro exposure system. Human osteoblastic MG-63 cells plated on a 24-well cell plate were treated with pulsed ultrasound in 37 °C water bath (10 min, frequency = 1.035 MHz, burst length = 200 μs, pulse repetition frequency = 1 kHz, duty cycle = 0.2, temporal-average acoustic power = 2 W, and peak pressure = 670-730 kPa) and the activation of heat-dependent canonical Wnt cell signaling was measured. The ultrasound-induced temperature rise was measured with thermocouples and infrared imaging. Chamber-to-chamber comparison showed substantial temperature variation (41.6 °C versus 49.1 °C) among the different chambers. The chamber walls were the most susceptible to heating. The variations in the chamber temperatures correlated to variations in the cell signaling levels (1.3-fold versus 11.5-fold increase). These observations underline the need for system-specific temperature measurements during in vitro exposures.