Design considerations for photoinitiator selection in cell-laden gelatin methacryloyl hydrogels

Light-assisted bioprinting of protein-derived hydrogels has been widely used for tissue engineering and regenerative medicine. The practical challenges of the photoinitiators (PIs) are often overlooked in using photo-crosslinkable bioinks for in situ and in vitro applications. A higher concentration of PI is believed to increase the network density of a hydrogel thus reducing its mass transfer capacity, but PI can form reactive oxygen species (ROS) and cause unwanted side reactions around biological compartments. This study systematically investigates the role of ROS generation on mesenchymal stem cells encapsulated in gelatin-methacryloyl hydrogels when using type I PIs-e.g. lithium phenyl(2,4,6-trimethyl-benzoyl)phosphinate and 2-hydroxy-1-(4-hydroxyethyl-phenyl)-2-methyl-1-propanone, and type II PI-e.g. Eosin Y. The results reveal that higher concentrations of type I PIs provide a higher elastic modulus at the expense of enhanced ROS generation and a proportional decrease in viability. We report a novel hydrogel system with minimal PI loading where a reduction in elastic modulus is accompanied by a simultaneous decrease in pore size and ROS level leading to a significant increase in stem cell viability over one week of in vitro culture. In contrast, the type II PI reveals a moderate fluctuation of elastic modulus over a range of PI concentration correlated to fluctuations in ROS generation. Monitoring ROS level variations enables evaluation of each PI's impact on cell response, providing a strategy for the biofabrication of cell-laden constructs. This framework can inform the rational design of photo-crosslinkable hydrogels for light-assisted bioprinting and in situ crosslinking applications in regenerative medicine.