Biomimetic Photo-reversible Hydrogels for 3D Bioprinting in Living Systems

Hydrogels have become increasingly important in Tissue Engineering for their viscoelastic properties and inherent biocompatibility with living tissues. Their 3D networks replicate the physical traits of the ECM, making them good candidates for soft tissue applications. However, conventional hydrogels often lack control and adaptability. This has led to the development of 'smart’ or stimuli-responsive hydrogels that can change their properties in response to specific triggers. Light is especially appealing as a non-invasive trigger capable of inducing reversible changes with high spatial and temporal precision. This Thesis describes the design of biomimetic photo-reversible hydrogels to create tunable hydrogels for both in vitro and in vivo applications. We envisioned the use of coumarin photo-reversible crosslinkers (CMMC, HCCA) conjugated to different backbones (PEG, HA, dECM) to enable reversible [2πs+2πs] cycloaddition at orthogonal wavelengths without requiring photoinitiators. Hydrogels can be crosslinked, selectively de-crosslinked, and re-crosslinked for precise microenvironment spatiotemporal control, even directly within living systems. Initially, we engineered CMMC-PEG that can reversibly respond to single-photon UV and multi-photon NIR light exposure. Hydrogels were incorporated into a microfluidic device as an active compartment designed to generate asymmetric biochemical stimuli and interfere with developmental processes. This was confirmed using fluorescent diffusion tests on an ex ovo chicken embryo model of neural tube morphogenesis. Additionally, by utilizing micropatterned CMMC-PEG hydrogels, we successfully produced elongated human neural organoids that better mimic neural tube structures. In vivo, HCC-8armPEG was precisely crosslinked into elastic, spring-like force sensors (iMesh) directly within the developing neural tube of chicken embryos. These hydrogel sensors adhered to the closing neural folds, enabling the quantification of tissue biomechanics during morphogenesis. Then, we designed hyaluronic acid hydrogels functionalized with coumarin derivatives, yielding scaffolds with tunable stiffness and reversible photochemistry that are compatible with neural stem cells and brain organoids. We used CMMC-HA in vivo to develop intravital reverse 3D bioprinting (iR3D) within the mouse visual cortex, a subtractive technique that enabled localized removal of custom shapes inside the hydrogel matrix with high spatial fidelity. Human cortical organoids (hCBO) were embedded within CMMC-HA + Matrigel™ (HA+Mtr) hydrogels, then crosslinked into a surgically damaged brain area, and iR3D was used to de-crosslink microchannels to connect hCBOs to the surrounding host tissue. Finally, we functionalized decellularized ECM from porcine tissues, obtaining CMMC-dECM hydrogels that crosslink when exposed to light while retaining key matrix structural proteins, as shown by proteomic analysis. They also supported the growth of cyst-like organoid growth in vitro, offering a potential Matrigel™ substitute. This Thesis establishes coumarin-functionalized hydrogels as a versatile platform with adjustable, reversible, and biomimetic features. Thanks to a light-controlled, non-toxic system that can adapt to various backbones, coumarin-functionalized hydrogels show the potential to connect in vitro and in vivo applications by enabling dynamic control over the cellular microenvironment across multiple scales. Future work should expand the platform's generalizability, enhance biological validation, and simplify the technology, allowing these hydrogels to evolve into reliable tools for biomedical research, allowing for the customization of culture environments based on the changing needs of biological systems or the development of new therapies.