Gelatin methacryloyl (GelMA) has emerged as a widely utilized biomaterial in tissue engineering due to its tunable mechanical properties, cell-adhesive motifs, and photo-crosslinkability. However, the physicochemical characteristics and biomedical utility of GelMA hydrogels are greatly influenced by the choice and concentration of photoinitiating systems. Despite increasing acceptance of visible-light and UV-sensitive initiators, a systematic comparative evaluation of their impact on GelMA hydrogel properties has not been studied. In this study, we present the first systematic investigation of how individual photoinitiators, Eosin Y (EY), Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Ruthenium (II) trisbipyridyl chloride ([RuII(bpy)]) (Ru), affect the viscoelastic properties, swelling behavior, degradation kinetics, and cytocompatibility of 5% and 10% (w/v) GelMA hydrogels. Through alteration of photoinitiator concentrations ([EY]: 0.005-0.1 mM, [LAP]: 0.01-0.5% (w/v), [Ru]: 0.02-1 mM) and utilizing consistent light intensity (10 mW/cmat system-specific wavelengths), we identified critical thresholds and plateau behaviors that distinctly influenced the stiffness and integrity of the hydrogels. Our findings revealed that each photoinitiating system exhibits unique advantages and trade-offs. LAP and Ru systems facilitated rapid gelation with easier utilization and were associated with higher swelling and accelerated degradation profiles-features particularly advantageous for applications such as 3D bioprinting and in situ injectable hydrogel systems. However, their atypical behaviors at certain concentrations and light exposure durations highlight the necessity for precise control and further mechanistic exploration. In contrast, EY-mediated hydrogels offered superior stiffness and minimal swelling at optimal concentrations, favoring applications that demand long-term mechanical stability, at the cost of a more complex cross-linking mechanism. Notably, by correlating mechanical and degradation behaviors with NIH-3T3 fibroblast viability, we also assessed biocompatibility window for each concentration of the systems, linking biomaterial performance with biomedical applicability. Overall, our study underlines the importance of tailoring photoinitiator selection and concentration for specific application needs, striking a balance between gelation kinetics, mechanical integrity, degradation behavior, and cytocompatibility. These insights provide a foundational framework for engineering GelMA-based hydrogels, paving the way for reproducible, efficient, targeted biomedical applications. 3 2+ 2
