title: A study of the effects of low intensity pulsed ultrasound on bone cells using controlled in vitro exposure methods
Creators: name: Savva, Jill
Publisher: name: University of Glasgow
Year: 2023

description: Low Intensity Pulsed Ultrasound (LIPUS) is a type of therapeutic ultrasound approved for treatment of non-union fractures since it was found to accelerate healing in a number of in vivo and clinical studies in the 1990’s. However, recent independent clinical trials found no significant healing effects, leading to questions over the effectiveness of the treatment. Many in vitro experiments have not succeeded in finding a clear and consistent mechanism, partly due to the difficulty in designing apparatus to provide adequate control over the LIPUS field applied to cells. This study developed and used controlled in vitro LIPUS exposure methods to investigate the effects of LIPUS fields on bone cells. The field characteristics of LIPUS transducers with operating frequencies of 45 kHz and 1 MHz were investigated by examining manufacturer’s data, measuring the ultrasonic output of a commercial LIPUS transducer and designing and building a new LIPUS transducer based on one of the most common commercial devices. A custom cell culture vessel (the biocell) was developed to allow a cell layer to be grown on a 6 μm-thick membrane and exposed to LIPUS without changing the LIPUS field. The biocell was immersed in a tank of water at a predetermined separation from a LIPUS transducer. Alignment of the transducer and cell layer was controlled via a custom-built positioning system. LIPUS fields experienced by the cell layers were derived from pressure field mapping in a scanning tank, corrected for any transmission loss through the biocell membrane. An acoustic tile placed behind the cell layer minimised reflections. Temperature measurements on the cell growth surface of a biocell mock-up confirmed no significant temperature rise during LIPUS exposures. As the extra-cellular matrix stiffness and topology has a significant effect on cellular responses, a 3D exposure method was also developed by seeding cells on 3D-printed scaffolds. The 3D exposure technique showed promise as a potential method of investigating LIPUS effects on cells in a controlled but more in vivo-like physical environment. The murine preosteoblast cell line MC3T3-E1 was chosen as a suitable bone cell model. Markers were chosen from key studies in the LIPUS literature to assess if the reported cellular responses could be replicated with a controlled exposure system. Cell proliferation was assessed by comparing viable cell counts before and after LIPUS exposure, across the entire growth surface and in pressure bins to assess the effect of pressure amplitude. The expression of protein and genetic markers implicated in mechanotransduction pathways associated with bone growth and mineralisation were also investigated. Initial studies comparing cellular responses to LIPUS at 1 MHz and 45 kHz over a range of pressure amplitudes indicated 45 kHz LIPUS had the least effect on cell counts and PGE2 expression. This led to the hypothesis that the fast rise time of the 1 MHz pressure pulse produced a rapid switch-on of cyclic radiation force, which stimulated cellular mechanotransduction pathways. The fast rise time hypothesis was tested in the Rise Time Study, which compared the effects of exposure to 1 MHz LIPUS with fast and slow rise times. Results of the controlled 2D studies suggested that LIPUS exposure had no significant effect on proliferation and markers associated with mechanotransduction pathways. Effects on mineralisation markers were mixed and likely due to the short-term nature of the study compared with the time period of mineralisation of the MC3T3-E1 cell line. The early mineralisation regulator Runx2 was up-regulated significantly six days post-exposure to fast rise time LIPUS. Runx2 is a key transcription factor whose up-regulation stimulates osteogenic differentiation of Mesenchymal Stem Cells (MSCs) and preosteoblasts, eventually leading to increased mineralisation and hence a healing effect. The result suggests runx2 may be sensitive to ultrasound stimulus alone and, therefore, may be a key marker to explain healing effects of LIPUS. Further study is recommended to repeat and verify the findings.