Engineering of Functional, Striated Muscle Tissues with Controllable 3D Architectures Using a Novel, Thermoresponsive, Nanofabricated Substratum

Abstract

Most tissues in the human body demonstrate multiscale organization, from extracellular matrix (ECM) structure, to cell morphologies, to overall tissue architecture. Further, in the cases of cardiac and skeletal muscle, tissue structure is critical to appropriate tissue function. However, current tissue engineering methods lack the ability to properly recreate scaffold-free, cell dense tissues with physiological structures. A platform which could engineer 3D tissues with controllable architectures could thus enable the study of more complex biological phenomenon, such as the effect of tissue structure on skeletal muscle development and engineered cardiac tissue function. For this reason, we developed a simple, yet versatile platform combining a thermoresponsive nanofabricated substratum (TNFS) incorporating nanotopographical cues and a gel casting method for the fabrication of scaffold-free 3D tissues with controllable architectures. The developed TNFS could be engineered with a variety of nanotopographies and thus cell monolayer structures which can be spontaneously detached via a change in culture temperature. The detached, nanoengineered cell sheets can then be stacked using our gel casting method to engineer specifically structured, 3D tissues. To this end, we first used the developed TNFS to engineer organized myoblast tissues with specific tissue architectures to demonstrate proof of concept engineering of 3D tissues with layer-by-layer architectural control. We found that using the gel casting method and TNFS, individual aligned myoblast sheets can be stacked into trilayer tissues and maintain individual layer alignment and stacked layer angles without reorganization between the individual sheets, whereas unpatterned controls demonstrated reorganization and layer mixing. We then utilized our developed platform to analyze the effects of engineered myoblast tissue structures on myoblast fusion and subsequent myotube morphology and alignment. We found that parallel-aligned myoblast bilayers could differentiate into aligned myotube sheets in a single layer, however orthogonally-oriented myoblast bilayers lost structural organization during differentiation. Additionally, transferred ECM and tissue structure from the TNFS could provide sufficient alignment cues to allow for the formation of aligned muscle tissue in a 3D microenvironment similar to that of the myofiber niche. Finally, we utilized our developed platform to engineer multilayered human cardiac tissues. We first found that by incorporating a vascular cell population capable of producing ECM, aligned human induced Pluripotent Stem (iPS) cell-derived cardiac sheets could be detached and stacked together. We then engineered 4-layer, aligned and helical cardiac tissues as microscale models of physiologically-structured myocardium. Aligned and helical 3D tissues demonstrated different contractile profiles, such as linear and spiraling, and also demonstrated improved contractile function over unpatterned controls, with aligned 3D cardiac tissues demonstrating the largest contractile magnitudes and velocities. These findings highlight the importance of tissue structure on cardiac function, and can be utilized in future works to engineer structured cardiac organoids for eventual in vitro whole-organ experiments. Taken together, these works present a novel platform which can be utilized for a variety of studies to engineer complex cell microenvironments and tissue architectures.