From Free‐Radical to Radical‐Free: A Paradigm Shift in Light‐Mediated Biofabrication

In recent years, the development of novel photocrosslinking strategies and photoactivatable materials has stimulated widespread use of light-mediated biofabrication techniques. However, despite great progress toward more efficient and biocompatible photochemical strategies, current photoresins still rely on photoinitiators (PIs) producing radical-initiating species to trigger the so-called free-radical crosslinking/polymerization. In the context of bioprinting, where cells are encapsulated in the bioink, the presence of radicals raises concerns of potential cytotoxicity. In this work, a universal, radical-free (RF) photocrosslinking strategy to be used for light-based technologies is presented. Leveraging RF uncaging mechanisms and Michael addition, cell-laden constructs are photocrosslinked by means of one- and two-photon excitation with high biocompatibility. A hydrophilic coumarin-based group is used to cage a universal RF photocrosslinker based on 4-arm-PEG-thiol (PEG4SH). Upon light exposure, thiols are uncaged and react with an alkene counterpart to form a hydrogel. RF photocrosslinker is shown to be highly stable, enabling potential for off-the-shelf products. While PI-based systems cause a strong upregulation of reactive oxygen species (ROS)-associated genes, ROS are not detected in RF photoresins. Finally, optimized RF photoresin is successfully exploited for high resolution two-photon stereolithography (2P-SL) using remarkably low polymer concentration (<1.5%), paving the way for a shift toward radical-free light-based bioprinting.