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Self-assembled structures primarily arise through enzyme-regulated phenomena in nature under persistent conditions. Enzymatic reactions are one of the main biological processes constructing supramolecular hydrogel networks required for biomedical applications. Such enzymatic processes provide a unique opportunity to integrate hydrogel formation. In most cases, the structure and substrates of hydrogels are adjusted by enzyme catalysis due to enzymes' chemo-, regio- and stereo-selectivity. Such hydrogels processed using various enzyme schemes showed remarkable characteristics as dynamic frames for cells, bioactive molecules, and drugs in tissue engineering, drug delivery, and regenerative medicine. The enzyme-mediated crosslinking hydrogels mimic the extracellular matrices by displaying unique physicochemical properties and functionalities such as water-retention capacity, biodegradability, biocompatibility, biostability, bioactivity, optoelectronic properties, self-healing ability, and shape memory ability. In recent years, many enzymatic systems investigated polymer crosslinking. Herein, we review efficient strategies for enzymatic hydrogelation, including hydrogel synthesis and chemistry, and demonstrate their applicability in biomedical systems. Furthermore, the advantages, challenges, and prospects of enzymatic-crosslinkable hydrogels are discussed. The results of biocompatible hydrogel products show that these crosslinking mechanisms can fulfill requirements for a variety of biomedical applications, including tissue engineering, wound healing, and drug delivery.