Elucidating the Chemistry Behind Thiol-Clickable GelAGE Hydrogels for 3D Culture Applications

This study elucidates the molecular mechanism and reaction chemistry of a thiol-clickable gelatin hydrogel platform (GelAGE) designed to emulate articular cartilage mechanics for osteoarthritis (OA) research. Gelatin type A was functionalized with allyl glycidyl ether (AGE) and crosslinked with dithiothreitol (DTT) via thiol-ene photopolymerization, producing hydrogels with tunable stiffness (19–142 kPa). NMR spectroscopy revealed that functionalization proceeds predominantly through lysine residues under mild alkaline conditions and extends to arginine residues at higher pH (~11), yielding double-substituted sites and enhanced crosslinking density. Increased allyl concentration and reaction duration correlated with higher degrees of functionalization and mechanical strength. The hydrogel stiffness range overlaps physiologic cartilage (100–3490 kPa), validating GelAGE as a modular platform for 3D chondrocyte culture and OA disease modeling.