IADR Abstract Archives
45kHz Ultrasound Influences Endothelial Cell Growth and Gene Expression
Objectives: Angiogenesis is required in hard and soft tissue repair and involves the differentiation, migration and proliferation of endothelial cells to develop new blood vessels. The majority of the studies investigating the effect of ultrasound on angiogenesis involve the use of ultrasound (US) in the megahertz (MHz) frequency range. Little is known about the effects of kHz frequencies, which are used in ultrasonic surgical devices. The present study aimed to explore the effect of kHz ultrasound on endothelial cell behaviour.
Methods: Human umbilical vein endothelial cells (HUVECs, Promocell, Germany) cultured in supplemented endothelial growth medium (PromoCell, Germany) were seeded in a 35mm Petri dish. A DuoSon US therapy device (SRA Developments Ltd, Ashburton, UK) treated cells with 45kHz continuous wave US for 5 mins at varying intensities (10, 25 & 75mW/cm2). Effects on cell number and viability were analysed 24h and 72h post-treatment using trypan blue viability analysis. Semi-quantitative reverse transcription polymerase chain reaction (sqRT PCR) identified expression levels of endothelial cell marker genes and angiogenesis-related genes including vascular endothelial growth factor A (VEGFA) and its corresponding receptor (VEGFR2).
Results: Cells counted 24h after US treatment showed a modest increase in cell number when compared with controls. After 72h, a statistically significant increase in cell number was seen in all US-treated groups. Ultrasonic intensities of 25mW/cm2 showed the greatest increase in cell number, 27% higher than controls. Cell viability was unaffected after all US treatment. VEGFA expression was elevated in all ultrasonic treated groups after 72h; 25mW/cm2 resulted in the highest expression increase. VEGFR2 expression increased after 25 & 75mW/cm2 treatment compared with controls and 10mw/cm2 exposure.
Conclusions: 45kHz continuous wave US affected endothelial cell numbers and gene expression in a dose-dependent manner. Therefore the use of low frequency ultrasonic surgical devices may influence angiogenesis and subsequent wound healing during and after hard and soft tissue cutting procedures.
Acknowledgements: this project is funded by EPSRC EP/R045291/1 grant.
Methods: Human umbilical vein endothelial cells (HUVECs, Promocell, Germany) cultured in supplemented endothelial growth medium (PromoCell, Germany) were seeded in a 35mm Petri dish. A DuoSon US therapy device (SRA Developments Ltd, Ashburton, UK) treated cells with 45kHz continuous wave US for 5 mins at varying intensities (10, 25 & 75mW/cm2). Effects on cell number and viability were analysed 24h and 72h post-treatment using trypan blue viability analysis. Semi-quantitative reverse transcription polymerase chain reaction (sqRT PCR) identified expression levels of endothelial cell marker genes and angiogenesis-related genes including vascular endothelial growth factor A (VEGFA) and its corresponding receptor (VEGFR2).
Results: Cells counted 24h after US treatment showed a modest increase in cell number when compared with controls. After 72h, a statistically significant increase in cell number was seen in all US-treated groups. Ultrasonic intensities of 25mW/cm2 showed the greatest increase in cell number, 27% higher than controls. Cell viability was unaffected after all US treatment. VEGFA expression was elevated in all ultrasonic treated groups after 72h; 25mW/cm2 resulted in the highest expression increase. VEGFR2 expression increased after 25 & 75mW/cm2 treatment compared with controls and 10mw/cm2 exposure.
Conclusions: 45kHz continuous wave US affected endothelial cell numbers and gene expression in a dose-dependent manner. Therefore the use of low frequency ultrasonic surgical devices may influence angiogenesis and subsequent wound healing during and after hard and soft tissue cutting procedures.
Acknowledgements: this project is funded by EPSRC EP/R045291/1 grant.