Study of factors affecting the magnitude and nature of ultrasound exposure with in vitro set-ups.

Therapeutic ultrasound is a clinically applied method to improve fracture healing and holds great potential as a manipulator of biologic material relevant to tissue engineering approaches. Unfortunately, the cell stimulating property of ultrasound is not known, which inhibits the optimal use of this technique. Additionally, many in vitro studies in this field use ultrasound configurations that are vulnerable to errors during calibration and use. These errors arise from the structural simplicity and incomplete characterization of these configurations. In this study, pulse-echo ultrasound, laser Doppler vibrometry and Schlieren imaging were applied to noninvasively characterize common in vitro experimental configurations. Fine wire thermocouple measurements were conducted to characterize any possible temperature rise during the ultrasound exposures. The results quantified the frequency dependent sound transmission through culture wells and the standing wave effect within the cell volume. These effects can cause uncertainty of up to 700% in the actual ultrasound exposure experienced by the cell. A temperature rise of 2.7°C was measured from an ultrasound configuration commonly used in vitro ultrasound studies. Furthermore, wave mode conversion in culture wells was observed, emphasizing the complexity of these sonications. Similar type Lamb waves have been observed in bone in vivo. Thus, Lamb waves may be a mechanism for stimulating the cells.