A high frequency ultrasound-based platform to non-destructively quantify geometric, acoustic and mechanical properties of thin, engineered soft connective tissues in vitro.

Assessment of temporal changes in the functional properties of in vitro tissue engineered (TE) constructs is typically done by destructive endpoint analysis, which increases experimental resources, analysis time, and financial burden. To overcome the limitations of existing practices, a novel Modular High Frequency Ultrasound Bulge Testing System (mHFUS-BTS) was designed to perform repeated, non-destructive and non-invasive characterization of cell-seeded biomaterial sheets throughout culture. Quantitative HFUS was validated to accurately measure the acoustic properties (i.e., speed of sound, acoustic impedance) to determine the physical properties (thickness and density) of TE-relevant polymeric scaffolds mounted inside a modified 6-well plate (<2 % mean error for all parameters). Components were interchanged to enable in situ bulge testing to estimate samples' Young's modulus (estimations <5 % mean error). Over an 18-day culture period, the mHFUS-BTS successfully monitored the thickening and softening of cell-seeded electrospun polyurethane polycarbonate scaffolds due to tissue synthesis while cell-free constructs experienced insignificant changes. Sterility was maintained throughout culture, with no effect of HFUS on cell viability or tissue composition compared to non-tested samples. These results demonstrate the mHFUS-BTS can repeatedly assess the physical, acoustic and mechanical properties of engineered tissues in vitro without influencing cell viability or tissue formation.