Low-Frequency Ultrasound Enhances Osteogenic Differentiation Potential of Mesenchymal Stem Cells

Effects of ultrasound on mesenchymal stem cells (MSCs) have been investigated widely at MHz frequencies. However, the impact of kHz ultrasound frequencies on MSCs is unknown. This study investigated the effects of 25 kHz ultrasound on bone marrow-derived human MSCs. A 25 kHz transducer manufactured in-house was characterised and used to deliver ultrasound to MSCs in an in vitro experimental apparatus comprising of a degassed water-filled tank lined with ultrasound-absorbing tiles and an acoustically transparent non-traditional cell culture vessel called CLINIcell® cassette. Ultrasound field scans inside the tank confirmed absence of standing waves and peak negative pressure (PNP) ranges of 0, 0-45, 0-108 and 0-185 kPa with highest PNP at the centre of the cassette. Cells seeded in the cassette were exposed to continuous 25 kHz ultrasound for 5 min and immediately after treatment, cell death increased and metabolic activity decreased with increasing PNP, with most cell detachment at the centre of the cassette. Necrosis or apoptosis, accompanied by altered mitochondrial homeostasis (visualised via PINK1 and IP₃R immunocytochemistry), was found to be the mechanism of cell death. Two and five days post-treatment, a PNP of less than 108 kPa upregulated early osteogenic genes (OSX, RUNX2 and bGLAP). Fourteen days post-treatment, a PNP of less than 45 kPa enhanced collagen production and mineralisation by 10% (measured via Sirius Red and Alizarin S Red staining). These findings suggest that a low ultrasound PNP (<45 kPa) at 25 kHz may offer potential therapeutic benefits applicable for bone regeneration.