Ultrasound-driven mechanosensing and the parameters shaping cellular response in LIPUS

Orthodoxou, Andrea (2026) Ultrasound-driven mechanosensing and the parameters shaping cellular response in LIPUS. PhD thesis, University of Glasgow.

Abstract

Low intensity pulsed ultrasound (LIPUS) therapy is a widely used and approved by regulatory bodies such as the FDA and NICE for bone repair however, reported outcomes remain variable across both clinical and experimental studies. Despite its widespread use, the mechanisms governing cellular responses remain poorly defined, in part due to limitations in in vitro experimental design and in the reporting of ultrasound parameters. Standard cell culture platforms introduce reflections and standing-wave artefacts, resulting in poorly controlled, spatially non-uniform exposure conditions that can also cause heating of the culture plate. Furthermore, commonly reported metrics such as spatial-average temporal-average intensity (ISATA) do not represent the mechanical stimulus experienced at the cell-substrate interface, and the absence of direct in situ field characterisation further limits reproducibility and the interpretation of results.

This thesis addresses these limitations by developing an acoustically and optically transparent cell culture vessel that enables the controlled delivery of ultrasound, fluorescence imaging, and the direct measurement of substrate motion using laser Doppler vibrometry. Ultrasound exposures were systematically varied in pressure, duty cycle, pulse repetition frequency, and temporal envelope, including conditions where ISATA was maintained constant across distinct waveform configurations. This approach allowed the decoupling of acoustic intensity from the resulting mechanical displacement at the cell substrate. Biological responses were assessed using MG63s which is a human-derived osteoblast-like cell line. Cell area, nuclear area, and immunofluorescence quantification of actin and vinculin intensities were used as indicators of the cell mechanosensitive response.

Across all parameter studies, cellular responses were maximised under specific waveform conditions defined by pressure amplitude, duty cycle, and pulse repetition frequency. Exposure conditions matched in ISATA produced different substrate displacements and corresponding cellular responses, demonstrating that ISATA alone is insufficient to describe the mechanical stimulus experienced by the cells. Temporal features of the waveform, particularly strain rate, influenced mechanosensitive outcomes, indicating that the rate of force application is equally important in determining cellular response.

LDV measurements provided frequency-resolved quantification of substrate motion demonstrating nanoscale displacements not only at the 1 MHz carrier frequency, but also at the 1 kHz pulse repetition frequency, with the latter varying significantly across waveform conditions. This work establishes a relevant framework for ultrasound exposure and interpretation, supporting the design of more reproducible and physiologically relevant LIPUS studies.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology; T Technology > TA Engineering (General). Civil engineering (General)
• Colleges/Schools: College of Science and Engineering > School of Engineering
• Supervisor's Name: Mulvana, Dr. Helen and Lucas, Professor Margaret
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-86251
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 Sep 2026 14:45
• Last Modified: 25 Sep 2026 11:04
• Thesis DOI: 10.5525/gla.thesis.86251
• URI: https://theses.gla.ac.uk/id/eprint/86251

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