Enzyme-functionalized Biomaterials for the 3D Bioprinting

3D bioprinting has emerged with tremendous attention as a new biotechnological tool. This technology allows for precise 3D fabrication of functional, complex dimensional tissue-engineered scaffolds in a layer-by-layer fashion. One of the major challenges is to develop materials for bioinks, supporting tissue functions. However, entrapped bioagents quickly diffuse out of the matrix potentially limiting their effectiveness. The objective of this thesis is to develop bioinks using enzymes as functional bioactive compounds for 3D bioprinting and integrate them into a bioink polymer matrix in a durable way for controlled long-lasting applications. In Chapter 1 an overview of the literature on 3D bioprinting for tissue engineering and recent advancements in functional bioinks and their methods. Chapter 2 describes the development of an enzyme-functionalized bioink material for extrusion-based 3D bioprinting using horseradish peroxidase as a model enzyme. By chemically modifying the enzyme with methacrylate groups, it can be attached to a Gel-MA bioink scaffold during photo-crosslinking. This provided a controlled catalytic activity inside the gel over a continuous time frame. The ink shows good printability and cytocompatible properties. In the following Chapters 3 and 4, the versatility of this enzyme bioink system was demonstrated using other enzymes with different functionalities that were explored in more detail. Developing an oxygen-generating enzyme bioink using the enzyme catalase (Chapter 3) as an integral part of the scaffold enabled a controlled supply of oxygen. By depletion of H2O2, the catalase-containing bioink showed precise oxygen generation inside the matrix. Additionally, the use of the antibacterial enzyme lysozyme (Chapter 4) highlighted the development of an antibacterial enzyme bioink which improved the long-term antibacterial properties of 3D printed structures. Finally, in Chapter 5, an alternative type of chemistry, the thiol-ene reaction allows higher crosslinking efficiency with only a small ...