Riboflavin : an endogenous photo-initiator for GelMA/PEG-SH hydrogels as scaffold for spinal cord injury treatment

Spinal cord injury (SCI) is traumatic, physiologically debilitating, and remains incurable. Gelatin methacrylate (GelMA) is a widely used hydrogel showing potential as a scaffold for SCI-related neurite regeneration. Common visible light photo-initiators (PIs) such as ruthenium/sodium persulfate and Eosin Y systems are exogenous to the spinal cord and exhibit concentration-dependent cytotoxicity in vitro. Riboflavin (vitamin B2) is a potential biocompatible, blue-light-activated alternative; as an endogenous cofactor within the spinal cord, it supports metabolic pathways essential for myelin maintenance. However, because crosslinking requires concentrations substantially higher than physiological levels, its cytocompatibility is evaluated experimentally here. Riboflavin photo-crosslinking typically takes minutes but can be reduced to under a minute through thiol-ene click chemistry using a thiol-functionalized (-SH) polymer such as polyethylene glycol (PEG). Here, we assessed the cytocompatibility of individual hydrogel components in the media of SH-SY5Y neuroblastoma monolayers. We then identified a blue light illumination time non-cytotoxic to SH-SY5Y cells and induced no qualitative impairment to neurite outgrowth from rat cortical explants encapsulated in UltiMatrix™. Rheology showed for the first time that riboflavin alone can crosslink a hybrid GelMA/PEG-SH hydrogel, and that its stiffness (G') is tunable by varying riboflavin concentration, polymer molar ratio, and light crosslinking time. Finally, we examined how tuning polymer molar ratio and total hydrogel concentration affects SH-SY5Y viability when seeded on swollen hydrogels. The results indicate that: a) 4armPEG-SH-2k is toxic above 0.25% w/v (per ISO 10993-5, <80% viability); b) 120 s of 455 nm light (100 mW/cm²) was cyto-compatible and induced no qualitative impairment to neurite outgrowth; c) GelMA/PEG-SH hydrogels can be riboflavin-crosslinked with tunable stiffness to a target G' range of 100-200 Pa; and d) molar ratio affects cell viability more than total polymer concentration (6 to 10% range). Hydrogel stiffness alone did not fully account for the cell viability improvement trends. This suggests factors beyond bulk mechanics, which may involve biochemical and cell-material interactions, drive the cellular response. Overall, this work demonstrates for the first time the feasibility of using riboflavin to crosslink and tune a hybrid GelMA/PEG-SH hydrogel, supporting its further development as a candidate platform for SCI tissue engineering.