A Focused Low-Intensity Pulsed Ultrasound (FLIPUS) System for Cell Stimulation: Physical and Biological Proof of Principle

Quantitative ultrasound (QUS) is a promising technique for bone tissue evaluation. Highly focused transducers used for QUS also have the capability to be applied for tissue-regenerative purposes and can provide spatially limited deposition of acoustic energy. We describe a focused low-intensity pulsed ultrasound (FLIPUS) system, which has been developed for the stimulation of cell monolayers in the defocused far field of the transducer through the bottom of the well plate. Tissue culture well plates, carrying the cells, were incubated in a special chamber, immersed in a temperature-controlled water tank. A stimulation frequency of 3.6 MHz provided an optimal sound transmission through the polystyrene well plate. The ultrasound was pulsed for 20 min daily at 100-Hz repetition frequency with 27.8% duty cycle. The calibrated output intensity corresponded to ISATA = 44.5 ± 7.1 mW/cm
²
, which is comparable to the most frequently reported nominal output levels in LIPUS studies. No temperature change by the ultrasound exposure was observed in the well plate. The system was used to stimulate rat mesenchymal stem cells (rMSCs). The applied intensity had no apoptotic effect and enhanced the expression of osteogenic markers, i.e., osteopontin (OPN), collagen 1 (Col-1), the osteoblast-specific transcription factor-Runx-2 and E11 protein, an early osteocyte marker, in stimulated cells on day 5. The proposed FLIPUS setup opens new perspectives for the evaluation of the mechanistic effects of LIPUS.