Programmable crosslinking strategies for the rational design of engineered protein hydrogels

Protein-based hydrogels have emerged as a versatile class of biomaterials that combine the intrinsic biological functionality of proteins with the structural adaptability of hydrated three-dimensional networks. Their biocompatibility, bioactivity, and extracellular matrix-mimetic characteristics have attracted significant interest in tissue engineering, regenerative medicine, and drug delivery. Advances in protein engineering, synthetic biology, supramolecular chemistry, and biomolecular conjugation have enabled precise control over protein structure, intermolecular interactions, and network organization, transforming proteins into highly programmable building blocks for multifunctional hydrogel systems. This review summarizes the design principles of engineered protein hydrogels, emphasizing the roles of structural motifs, bioactive domains, and stimuli-responsive elements in regulating hydrogel assembly and functionality. Recent developments in physical and chemical crosslinking strategies are highlighted, together with their impacts on network architecture, mechanical performance, degradation behavior, and stimulus responsiveness. Particular attention is given to structure-property-function relationships linking protein design and assembly to hydrogel performance. These advances have facilitated the development of protein hydrogels for tissue regeneration, wound healing, therapeutic delivery, three-dimensional cell culture, and biofabrication, underscoring their promise as next-generation biomaterials.