Dynamic by Design: Advancing Biomaterials to Further Mimic Native Tissues in 3D and 4D Volumetric Biofabrication

The limited ability of animal models to predict human biological responses remains a significant obstacle in drug development and regenerative medicine. Despite decades of research and substantial investment, around 90% of drugs still fail during clinical trials, mainly due to interspecies genetic and physiological differences. In response, biofabrication technologies have emerged as promising tools for creating more human-relevant in vitro models. However, the field still faces critical challenges in replicating functional human tissues, particularly due to a lack of dynamic, biofunctional materials that can support long-term, complex tissue structures. This thesis focuses on the development of advanced biomaterials that allow for 4D biofabrication, where materials are not only three-dimensional but also respond dynamically over time. Specifically, it presents a suite of chemically defined hydrogels tailored for volumetric bioprinting and post-printing modification. These materials were designed to mimic the native extracellular matrix while enabling spatiotemporal control over their biochemical and mechanical properties. One core innovation is the application of volumetric photografting, a method that enables the precise, light-mediated incorporation of bioactive molecules within 3D hydrogels. This technique was demonstrated to preserve the biological functionality of molecules, such as vascular endothelial growth factor (VEGF), thereby enhancing both cellular coverage and depth of invasion in engineered tissues on gelatin-norbornene hydrogels. To expand the accessibility of this method, a modular additive, AddGraft, was developed. AddGraft is a heterobifunctional crosslinker that can be incorporated into a wide range of acrylated hydrogels, enabling 4D manipulation without requiring chemical modification of the base polymer. This tool allowed localised stiffening and chemical patterning of cell-laden hydrogels in a fully biocompatible manner, significantly broadening the applicability of 4D photografting. Another novel strategy introduced in this work addresses a persistent limitation in 3D printing: resolution. A temperature-responsive material system was created to fabricate cell-laden channels that initially allow for easy cell seeding at larger diameters and are then thermally shrunk to physiological scale. This approach preserves cell viability and structure while enabling higher resolution than current bioprinting methods typically allow. Furthermore, to better mimic the mechanical and biochemical cues found in living tissues, a hybrid supramolecular hydrogel, named HybriGel, was developed. This material integrates reversible physical interactions with covalent crosslinking to balance structural fidelity and biological responsiveness. It enables high-resolution printing of robust 3D constructs, allowing cells to migrate through the matrix, a feature previously only achievable in softer, less stable gels. HybriGel supported enhanced proliferation, T-cell migration, and organoid formation. When combined with AddGraft, HybriGel forms a powerful platform for spatiotemporally controlled biofabrication, allowing modulation of both mechanical and biochemical signals within a single system. This synergy offers a new level of design flexibility in tissue engineering. This work redefines biomaterials not as passive scaffolds, but as active and responsive components that interact dynamically with their cellular environment. These advances pave the way for more functional, adaptable, and human-relevant in vitro tissue models, offering viable alternatives to animal testing and accelerating the development of personalised therapies and tissue regeneration strategies.