An ultra-high-speed imaging study of ultrasound-mediated mechanotransduction

Abstract

Ultrasonic surgical cutting tools are an emerging and increasingly popular technology in the surgical arena. They utilise ultrasonic vibrations to cut biological tissues with enhanced precision compared with traditional devices, resulting in reduced post-operative healing times. However, an in-depth understanding of how these tools elicit these advantages, and the way in which biological tissues and cells respond to this high-frequency vibration, is largely unexplored. This knowledge gap is largely due to difficulties in producing in vitro protocols to controllably apply clinically relevant ultrasonic stimulation onto cells, which prevents recreation, and thus studying, of the cutting site environment. The development of a method that enables such an investigation could provide valuable insight into the cell response to ultrasonic stimulation and therefore result in a deeper understanding of these surgical tools. Furthermore, ultrasound has been known to stimulate therapeutic biological effects through mechanobiological pathways, particularly in fracture healing. As one of the main uses of these cutting tools is in bone tissues, the possibility of combining precise cutting with regenerative stimulation could result in a surgical device that stimulates healing post-incision. However, investigating the interface of these capabilities first requires a method to controllably study appropriate stimuli using an in vitro model. The work presented in this thesis addresses the aforementioned gap in the literature through the development of a novel method, namely the image-based ultrasonic cell shaking (IBUCS) test, which enables investigation into the ultrasound-cell interaction from a mechanobiological perspective. This protocol utilises an ultra-high-speed (UHS) camera attached to a microscope to effectively obtain high magnification, high temporal resolution images of substrate-driven cell deformation at 20 kHz ultrasonic frequency. To first address the mechanical aspect of the cell response, digital image correlation (DIC) was performed to quantify deformation and explore cell mechanics in the ultrasonic regime. From this analysis, single cells and monolayers were found to have statistically similar levels of deformation, at amplitudes significantly larger than that of the substrate eliciting the stimulus. Additionally, investigation into the impact of increasing inertial stimulation on cell response revealed strain amplitude values in the region of 10% resulted in cell detachment from the substrate. The biological aspect of the mechanobiological response was then investigated by translating components of the IBUCS test to aseptic conditions. Viability tests revealed cell health was not affected by strain amplitudes of 0.00025 and 0.0005 after up to 30 seconds of stimulation, despite temperature increases of approximately 12◦C. Following this, mineralisation staining revealed a significant increase in calcium, and thus osteogenic differentiation after 40% excitation compared with the control group. However, qPCR analysis revealed no significant difference in differentiation and mechanotransductive related gene expression. In summary, a novel platform has been developed that elicits pre-characterised, substrate-driven ultrasonic deformation onto cells, enabling investigation into the interaction from both a mechanical and biological perspective. This method will allow for investigation and understanding of the interaction between ultrasonic surgical cutting tools and biological cells, with the objective of optimising cutting and implementing therapeutic effects.

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Identifiers

ORCID for Miranda Ballard: orcid.org/0009-0000-4497-004X

ORCID for Philippa Reed: orcid.org/0000-0002-2258-0347

ORCID for Alex Marek: orcid.org/0000-0002-2254-3773

ORCID for Fabrice Pierron: orcid.org/0000-0003-2813-4994

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