Biomimetic Engineering of Fibrillar Extracellular Matrix for Developing Novel Cardiac Cellular Constructs

Abstract

In biological tissues, the extracellular matrix (ECM) regulates tissue-specific functions and biological processes, including functional development and disease progression. In the human heart, the structural anisotropy of the ECM plays a pivotal role in forming functional cardiac syncytia by directing cardiomyocyte alignment and facilitating coordinated electrophysiological and contractile activity. Replicating this anisotropic architecture is essential for building advanced ex vivo models of human heart tissues that mimic the native structure-function relationship and enabling more accurate studies of cardiac physiology, disease modeling, and drug screening. The motivation for this work stems from the need for a more natural, protein-based tissue substrate that incorporates the biochemical and biophysical advantages of native fibrillar proteins, such as cell-ECM interaction, mechanical extensibility and adaptability, and support for anisotropic cellular organization. This dissertation leverages recent advances in engineered ECM (EECM) of fibrillar fibronectin to investigate how the biochemical and biophysical properties of EECM influence cellular organization and functional outcomes in tissue systems. Building upon prior work on hydrodynamically induced FN fibrillogenesis to create protein-based, fibrillar EECM, a new methodology was developed to enable the assembly of anisotropic fibronectin structures by modulating the intermolecular interactions that define the native structure of FN within a controlled shear environment. This process replicates the essential structural features of native tissue architecture and offers reproducibility and scalability for diverse tissue engineering applications. Cellular structural analyses revealed that the fabricated EECM supports anisotropic cell alignment, a key determinant of functional tissue organization in in vivo cardiac tissues. Engineered cardiac tissues (ECTs) formed on fibrillar EECMs demonstrated alignment-dependent electrophysiology, characterized by intercellular calcium transient behavior. ECTs exhibited significantly enhanced functional properties compared to monolayer controls cultured on Matrigel-coated surfaces. These improvements are attributed to the extensible and fibrillar nature of the EECM, which not only provides the functional advantages of natural protein composition but also supports dynamic adaptation to contraction, enabling more physiologically relevant tissue behavior. To evaluate their functional utility, cardiac tissues were benchmarked against established conventional in vitro culture models, highlighting superior functional metrics, including calcium transient amplitude, conduction velocity, and upstroke slope. Furthermore, these tissues demonstrated effective excitation-mechanical coupling, making them amenable for use in drug screening applications to evaluate the effects of drugs on contractile strength, beat rate, calcium transient duration, and conduction velocity. This advancement addresses critical gaps in replicating the structure-function relationship of native myocardium using protein-based tissue substrates, offering significant potential for drug screening, disease modeling, and regenerative medicine. Complementary studies in adipocyte differentiation provide additional insights into the significance of using native protein-based tissue substrates as exemplified by EECM. The analysis of differentiated adipocytes in cell-remodelable and degradable EECM microenvironments revealed intrinsic remodeling behaviors and varying degrees of adipogenesis in response to biomechanical and chemical cues. These findings further underscore the critical role of EECM design and media formulation in regulating tissue-specific behavior. Together, this work establishes a framework for the rational design of ECM platforms to promote functional outcomes across diverse tissue types.