Modular Enzyme-Responsive Polysaccharide-Based Hydrogels for Biofabrication

Abstract

Engineered human tissue and disease models can decrease the cost and time of developing new drugs and treatments, facilitate personalized medicine, and eliminate the need for animal models that poorly represent the human body and are ethically problematic. However, the current conventional cell expansion methods using 2D culture flasks cannot enable the development of such complex multi-cellular 3D models. In general, hydrogels are considered promising materials that can make the biofabrication of tissue models possible. Hydrogels are highly hydrated materials comprised of either synthetic or naturally derived polymers, or a combination of both, and can form an environment mimicking the biomacromolecular network surrounding cells in the body. This network of biopolymers, known as extracellular matrix (ECM), is comprised of proteins such as collagen, laminin, fibronectin, and polysaccharides such as hyaluronan (HA), heparan, keratan, and chondroitin sulfate. The design of hydrogels representing the physical and biochemical properties of the ECM and which can be used for biofabrication is challenging but of increasing interest due to the rapid progress in the development of 3D and 4D bioprinting techniques. As the ECM properties differ between various tissues and disease conditions and change over time, a dynamic modular hydrogel system is needed to that can be optimized for each cell and tissue type. This thesis aims to develop modular enzyme-responsive polysaccharide-based hydrogels for 3D cell culture and biofabrication. The natural polysaccharides, hyaluronic acid (HA) and alginate (Alg) were used as the main backbone in the hydrogels developed in this thesis. HA was modified by conjugating bicyclo[6.1.0]non-4-yne (BCN) to the backbone to form HA-BCN-based hydrogels by a bioorthogonal strain-promoted alkyne-azide cycloaddition. The click reaction between BCN and azide groups allowed for modulating the biochemical and mechanical properties of the HA-BCN hydrogels. HA-BCN was further decorated with peptides to explore peptide folding and dimerizationmediated dynamic cross-linking and biofunctionalization. This system was further used to explore possibilities to dynamically alter the properties of 3D bioprinted structures, mimicking the biomineralization process in bone tissue. In a different study, a tumor model comprising fibroblast and breast cancer cells (MCF7) was bioprinted using HA-BCN cross-linked by matrix metalloporotease (MMP) cleavable and PEG-diazide MMP-resistant crosslinkers, demonstrating the synergistic relationship between hydrogel degradability and cancer cell growth, intensified by the presence of fibroblasts. The possibility of incorporating a conductive module into this hydrogel system was explored using the enzyme-assisted polymerization of ETE-S to form an