Resonant Acoustic Rheometry for High-Throughput, Non-Contact Mechanical Characterization of Viscoelastic Biomaterials

Abstract

Mechanical testing of viscoelastic biomaterials is of critical importance in biomedical engineering, enabling basic research into the role of the extracellular matrix, investigatory and diagnostic testing of tissues and biofluids, and the development and characterization of tissue engineered therapeutics. Conventional material testing approaches used for soft biomaterials generally require force application through direct contact with a sample, leading to potential contamination and damage, and thereby limiting these approaches to end-point measurements. To overcome these limitations, we have developed a new measurement technique, Resonant Acoustic Rheometry (RAR), which enables high-throughput, quantitative, and non-contact viscoelastic characterization of biomaterials, soft tissues, and biological fluids. RAR uses ultrasonic pulses to both generate microscale perturbations and measure the resulting resonant oscillations at the surface of soft materials using standard labware. Resonant oscillatory properties obtained from the frequency spectra of the surface oscillations, including the resonant frequency and the damping coefficient, are used to quantify material properties such as shear modulus, shear viscosity, and surface tension in both viscoelastic solids and liquids. We developed a prototype RAR system and tested it on a range of soft biomaterials, with shear moduli ranging from under 100 Pa to over 50 kPa, including fibrin, gelatin, and polyethylene glycol (PEG). Shear moduli measured using RAR were validated both computationally using finite element analysis and experimentally using conventional shear rheometry, with excellent linear correlation in measured elasticity between techniques (R2 > 0.95). By performing parallel RAR experiments using microwells of different sizes, we verified that resonant oscillatory behaviors could be used to quantify the intrinsic viscoelastic properties of a material. We also demonstrated the rapid, non-contact monitoring of changes in material properties over a variety of temporal scales, ranging from processes occurring on the order of milliseconds to those occurring over hours and days. High temporal resolution RAR measurements, with sampling intervals as low as 0.2 seconds, were used to characterize the gelation process. Characteristic features of the resonant surface waves during phase transition were applied to identify the gel point for various hydrogels. High sample throughput was demonstrated by performing longitudinal RAR testing to explore the impact of hydrogel polymer and crosslinker concentration on both reaction kinetics and final mechanical properties in full factorial experiments consisting of over 15,000 unique measurements. We were able to identify individual effects of design parameters as well as interactions that led to unexpected mechanical properties, demonstrating the importance of combinatorial methods and high-throughput mechanical characterization in material design. These studies demonstrate that RAR can rapidly and accurately assess the mechanical properties of soft viscoelastic biomaterials. The measurements generated are analogous to those produced using conventional mechanical testing, and RAR is further capable of longitudinal viscoelastic studies over time. RAR applies automation in both data collection and analysis, allowing high throughput measurement of an array of samples without contact or the need for manual intervention. Furthermore, RAR uses standard microwell plates, which simplifies sample preparation and handling. The viscoelastic properties of soft biomaterials are relevant in a wide range of applications, including for clinical diagnostic assays and the development of hydrogel materials for regenerative medicine. RAR represents a fast, accurate, and cost-effective method for materials characterization in these applications.