Enzyme Bioink for the 3D Printing of Biocatalytic Materials

The field of 3D biofabrication faces major challenges on the road to printing fully functional tissues and organs. One of them is adding functionality to the newly formed tissue for replicating an active biochemical environment. Native extracellular matrices sequester numerous bioactive species, making the microenvironment biochemically active. On the other hand, most 3D-printed constructs have limited activity, serving merely as mechanical scaffolding. Here we demonstrate active scaffolding through the integration of biocatalytic enzymes within the bioink. Enzymes are an attractive class of biocompatible and substrate-specific bioactive agents that can improve tissue regeneration outcomes. However, the difficulty in the application remains in providing enzymes at the targeted site in adequate amounts over an extended time. In this work, a durable biocatalytic active enzyme bioink for 3D extrusion-based bioprinting is developed by covalently attaching the globular enzyme horseradish peroxidase (HRP) to a gelatin methacrylate (Gel-MA) biopolymer scaffold. Upon introducing methacrylate groups on the surface of the enzyme, it undergoes photo-crosslinking in a post-printing step with the methacrylate groups of Gel-MA without compromising its activity. As a result, HRP becomes a fixed part of the hydrogel network and achieves higher stability inside the gel which results in a higher concentration and catalytic activity for a longer time than solely entrapping the protein inside the hydrogel. We also demonstrate the cytocompatibility of this enzyme bioink and show its printing capabilities for precise applications in the field of tissue engineering. Our approach offers a promising solution to enhance the bioactive properties of 3D-printed constructs, representing a critical step towards achieving functional biofabricated tissues. ### Competing Interest Statement The authors have declared no competing interest.