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Abstract

Hydrogels composed of natural or engineered proteins have gained significant attention in biomedical research due to their biocompatibility, tunability, and structural similarity to native extracellular matrices. This thesis applies two distinct crosslinking strategies to create recombinant protein-based hydrogels designed for cell encapsulation and artificial cornea applications. The overall objective was to develop fully bio-derived, self-assembling hydrogel systems that are mechanically robust, cytocompatible, and free from conventional synthetic crosslinkers, which might compromise biocompatibility. The first hydrogel system employs covalent crosslinking through SpyTag/SpyCatcher chemistry, coupled with a redox-responsive self-assembly mechanism based on protein fragment reconstitution. By integrating split-domain reconstitution with irreversible SpyTag/SpyCatcher complex formation, self-assembling hydrogels were developed that can undergo gel–sol transitions under reducing conditions. These engineered hydrogels mimic key features of the extracellular matrix, expanding their potential for biomedical applications, particularly cell encapsulation. The combined design enabled tunable gelation kinetics and mechanical stiffness, as confirmed by rheological analysis. The hydrogels supported high mammalian cell viability in both 2D and 3D cultures, with over 90% viability retained after release triggered by elevated glutathione levels in a reducing environment. These properties highlight the system’s suitability for encapsulation platforms and dynamic biological environments. The second hydrogel system utilizes a crosslinking strategy involving a denatured protein assembly with cysteine-containing variants of recombinant protein constructs. Upon denaturation, buried cysteine residues become accessible and form intermolecular disulfide bonds. During renaturation, physical chain entanglements are introduced, reinforcing the network without the need for synthetic initiators. The resulting hydrogels were optically transparent across the visible spectrum, with refractive indices between 1.36 and 1.41, closely matching that of the native human cornea. Mechanical characterization confirmed sufficient stiffness and suturability, while primary human corneal epithelial cells cultured on the hydrogels maintained >90% viability over seven days, supporting their potential for ocular tissue integration and mimicry. Together, these studies demonstrate how distinct crosslinking strategies can be optimized through recombinant protein design to create fully protein-based, cytocompatible hydrogels. The materials developed here serve as a foundation for future applications in cell encapsulation, soft tissue scaffolds, and artificial cornea development.