Currently, no solutions with the capacity for growth and adaptation exist for replacing heart valves with congenital defects. Tissue engineered heart valves (TEHVs) offer a biological and potentially permanent replacement solution for pediatric patients. However, this potential has yet to be realized:​ existing TEHV designs poorly mimic the anisotropic mechanical properties of their healthy native counterparts leading to poor biomechanical function. In vitro mechanical conditioning bioreactors have been developed to accelerate tissue production and tune the mechanical properties of TEHVs. However, bioreactors capable of altering the anisotropic properties of engineered constructs are limited to small-scale setups. Furthermore, many mechanical conditioning bioreactors are incapable of non-destructively assessing a construct’s geometric and mechanical properties in vitro. As such, these platforms provide no insight as to whether target properties are being achieved during culture.

A bioreactor was designed to biaxially stretch thin, biomaterial sheets in vitro. Each stretch axis was independently controlled to modulate the strain anisotropy generated. The bioreactor biaxially stretched sheets as large as 70 x 40 mm2, uniformly applied strain >70% of the stretched area and generated strain amplitudes up to 40%. In vitro, the device could generate different anisotropic strain protocols as cells seeded on the sheet aligned towards the vector-sum stretch direction of each protocol. Next, the Modular High Frequency Ultrasound (HFUS) Bulge Testing system (mHFUS-BTS) was developed. The mHFUS-BTS was evaluated using a smaller scaled, modified 6-well plate to enable higher throughput. The HFUS system measured the cell-seeded sheets’ thickness, density and acoustic properties while the bulge testing system estimated the sample’s Young’s modulus in vitro. Lastly, the bioreactor was adapted for compatibility with the mHFUS-BTS. The modifications enabled HFUS measurements of the samples inside the bioreactor and bulge testing to occur. Furthermore, these modifications reduced the amount of sample material used, applied stretch more uniformly to mechanically anisotropic materials, and maintained the same stretching capabilities as the initial design. From this thesis, two platforms were established for developing in vitro protocols to better engineer pediatric TEHVs that mimic their healthy native counterparts in properties and function.