High-frequency quantitative ultrasound for the assessment of the acoustic properties of engineered tissues in vitro

Acoustic properties of biomaterials and engineered tissues reflect their structure and cellularity. High-frequency ultrasound (US) can non-invasively characterize and monitor these properties with sub-millimetre resolution. We present an approach to estimate the speed of sound, acoustic impedance, and acoustic attenuation of cell-laden hydrogels that accounts for frequency-dependent effects of attenuation in coupling media, hydrogel thickness, and interfacial transmission/reflection coefficients of US waves, all of which can bias attenuation estimates. Cell-seeded fibrin hydrogel disks were raster-scanned using a 40 MHz US transducer. Thickness, speed of sound, acoustic impedance, and acoustic attenuation coefficients were determined from the difference in the time-of-flight and ratios of the magnitudes of US signals, interfacial transmission/reflection coefficients, and acoustic properties of the coupling media. With this approach, hydrogel thickness was accurately measured by US, with agreement to confocal microscopy (r2 = 0.97). Accurate thickness measurement enabled acoustic property measurements that were inde-pendent of hydrogel thickness, despite up to 60% reduction in thickness due to cell-mediated contrac-tion. Notably, acoustic attenuation coefficients increased with increasing cell concentration (p < 0.001), reflecting hydrogel cellularity independent of contracted hydrogel thickness. This approach enables accu-rate measurement of the intrinsic acoustic properties of biomaterials and engineered tissues to provide new insights into their structure and cellularity. Statement of significance High-frequency ultrasound can measure the acoustic properties of engineered tissues non-invasively and non-destructively with mu m-scale resolution. Acoustic properties, including acoustic attenuation, are re-lated to intrinsic material properties, such as scatterer density. We developed an analytical approach to estimate the acoustic properties of cell-laden hydrogels that accounts for the frequency-dependent ef-fects of attenuation in coupling media, the reflection/transmission of ultrasound waves at the coupling interfaces, and the dependency of measurements on hydrogel thickness. Despite up to 60% reduction in hydrogel thickness due to cell-mediated contraction, our approach enabled measurements of acoustic properties that were substantially independent of thickness. Acoustic attenuation increased significantly with increasing cell concentration (p < 0.001), demonstrating the ability of acoustic attenuation to reflect intrinsic physical properties of engineered tissues. (c) 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.