Mechanobiology is of central importance in regenerative medicine. The passive mechanical properties of the extracellular matrix are well known to influence cell functions. Similarly, active mechanical stimulation can potentiate specific cellular responses, and recently the influence of the viscoelastic properties of the matrix have become clear. While the field has made great progress in understanding how cells sense and respond to mechanical cues, there is an important need for improved methods to study mechanobiology at the cellular scale.

We applied two complementary ultrasound techniques to characterize a range of hydrogel extracellular matrices over time. Spectral ultrasound imaging (SUSI) was used to obtain high resolution, quantitative, spatiotemporal information about the composition of samples, while dual-mode ultrasound elastography (DUE) was used to measure mechanical properties.

SUSI generated 3D grayscale images of construct morphology at sub-millimeter resolution. Spectral analysis of the backscattered radio frequency ultrasound signals provided system-independent, quantitative assessment of the constructs based on the midband fit and slope of the linearized RF spectrum. SUSI was further used to monitor mineralization of collagen constructs by immersion in simulated body fluid, and to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. Spectral analysis revealed the diameter, concentration, and acoustic attenuation of scatterers within the constructs, and color-coded parameter maps tracked their spatiotemporal variation.

DUE uses high frequency focused ultrasound to induce compression in a sample and interleaved ultrasound imaging to measure the resulting deformation. DUE was applied to noninvasively perform creep testing on a range of engineered constructs. The technique provided spatial and temporal mapping of local and bulk displacements and strains at high resolution, showing differences in characteristic parameters within the construct volume. Burger’s lumped parameter model was further used to extract viscoelastic parameters describing material behavior.

In summary, SUSI and DUE have been shown to provide quantitative information on the composition and mechanical properties of 3D engineered tissues at sub-millimeter resolution. Importantly, both techniques are non-invasive and applicable to studying the development of mineralizing extracellular matrices over time, including sub-surface properties. These advanced ultrasound techniques therefore present a valuable tool for longitudinal monitoring of engineered tissue development.