Mechanosensitive response of murine C2C12 myoblasts to focused Low-Intensity Pulsed Ultrasound (FLIPUS) stimulation

Quantitative Ultrasound (QUS) is a promising technique for bone tissue evaluation. Highly focused transducers, used for QUS, have also a capability to be applied for tissue-regenerative purposes and can provide geometrically controlled deposition of an accurate acoustic dose. This allows for overcoming of unwanted artifacts, i.e. standing waves, ring interferences, temperature elevations, cross-stimulation of adjacent wells, which often lead to irreproducible biological results, associated with existing Low-Intensity Pulsed Ultrasound (LIPUS) in-vitro set-ups. Focused LIPUS (FLIPUS) is a novel reliable tool, which is minimizes or avoids the aforementioned artifacts. In this study we evaluated mechanical response of murine C2C12 myoblastic cells, possessing osteogenic-lineage differentiation potential, to the well-controlled FLIPUS dose. The optimized acoustic dose (3.6 MHz, pulsed at 100 Hz with 28% duty cycle, corresponding to I_SATA = 29.4 ± 4.8 mW/cm²) induced 1.5 fold increase (p<;0.05) in AP-1 promoter activity already after 5 minutes of FLIPUS exposure. The Sp1 binding was enhanced by about 20 % (p<;0.05) after 5 min FLIPUS stimulation and the trend persisted for 30 min (p<;0.01) and 1 hour (p<;0.01) stimulation times. Activation of TEAD-binding sequences to about 2 fold (p<;0.01) was observed after 5 min FLIPUS stimulation. The proliferation of the C2C12 cells was up-regulated in starving conditions on day 1 and 3 of daily short-term FLIPUS-treatments. These results imply the pro-proliferative nature of the selected FLIPUS-dose in C2C12s, with potential functional role of the described mechanosensitive transcription factors. The use of focused ultrasound for both visualization and therapeutic purposes may be applied in clinical settings to provide monitoring and support healing of damaged tissues.