Ultrasound-mediated mechanotransduction and its applications

2019-04-05In recent years, ultrasound has regained attention for use in various therapeutic applications due to its capability to induce specific biological processes. For instance, low-intensity pulsed ultrasound (LIPUS) has been used in clinics for decades for the treatment of bone fracture and other osseous defects. The clinical effects of LIPUS have been well-documented over the years, and numerous in vitro and in vivo studies also suggested that ultrasound influences different processes of bone regeneration. Although the biophysical mechanisms underlying the interaction between ultrasound and cells have not been fully understood, many agree on a pivotal role of Ca²⁺ signaling through mechanotransduction pathways. Because Ca²⁺ regulates a vast range of downstream cellular processes including differentiation, gene transcription, and hormone secretion, a better understanding of how ultrasound influences Ca²⁺ signaling could lead to new therapeutic applications. ❧ Despite its limitations in clinical applications, high-frequency ultrasound (>30 MHz) is a powerful tool for mechanotransduction studies due to its single-cell stimulation capability. In this study, High-frequency (∼50 MHz) focused ultrasonic transducers along with a customized stimulation and monitoring system was utilized to analyze ultrasound-induced intracellular Ca²⁺ dynamics from single cells. Various pharmacological agents were also used in our efforts to reveal the underlying biophysical mechanisms of the Ca²⁺ response. ❧ The characterization of the ultrasound-triggered Ca²⁺ transients in human mesenchymal stem cells (hMSCs) revealed that there is minimum ∼20 seconds of latency from onset of ultrasound; response latency increases as acoustic pressure decreases. The inclusion of the agents U-73312 and 2-APB completely abolished the response indicating that phospholipase C (PLC)-derived inositol 1,4,5-trisphosphate (IP₃) activates IP₃ receptors on the endoplasmic reticulum (ER) to cause the Ca²⁺ response. Further study revealed that ultrasound opens hemichannels, particularly connexin 43, on the membrane to release ATP into the extracellular space which, in turn, activates P₂Y purinergic receptors on the stimulated cells and its adjacent cells. The results of this study also suggest that ultrasound stimulation can elicit Ca²⁺ oscillation in various cells. As specificity and efficiency of intracellular Ca²⁺ signaling rely on the frequency and amplitude mode of the oscillatory signal, verification for the degree of modulation is critical in order to develop an ultrasonic energy based Ca²⁺ modulator. It was found that the US-induced Ca²⁺ signal oscillates in a similar frequency to spontaneous oscillation, and repetitive stimulation can modulate the frequency within the limited range which is time-locked to the stimulus. ❧ Lastly, we explored the medical application of the technique. Since insulin is known to be secreted in a pulsatile manner via Ca²⁺ evoked exocytosis, activation of Ca²⁺ oscillation from silent or sick pancreatic β-cells can increase the rate of secretion. It was found that ultrasound stimulation increased the percentage of HIT-T15 pancreatic β-cells showing active intracellular Ca²⁺ oscillation by up to 5-fold. Furthermore, the two distinct oscillatory patterns, fast irregular, and slow periodic, were evoked by ultrasound via L-type Ca²⁺ channels and store-operated channels which coupled to P₂X and P₂Y purinergic receptors, respectively. These findings suggest that ultrasound-based Ca²⁺ modulation could be used as a therapeutic tool in medicine