Dynamic Gelatin Hydrogels Crosslinked by Dithiolane-Norbornene Click Chemistry

Hydrogels prepared from gelatin are ideal for mimicking the extracellular matrix (ECM) owing to their inherent cell-adhesive and protease-labile peptide sequences. While gelatin is highly water-soluble, it does not form the triple-helical structure. As a result, physically crosslinked gelatin-based hydrogels are only stable at low temperatures, precluding their use in 3D cell culture. Gelatin-methacryloyl (GelMA) and gelatin-norbornene (GelNB) have been developed to enable the stable crosslinking of gelatin-based hydrogels via chain-growth or step-growth photopolymerization. However, most gelatin-based hydrogels lack dynamically tunable properties unless macromers with dynamically crosslinkable motifs are used. Here, we integrate GelNB with dithiolane-containing crosslinker poly(ethylene glycol)-tetra-lipoic acid (PEG4LA)-for modular photo-crosslinking of GelNB into hydrogels under cytocompatible light exposure (365 nm, 5 mW/cm²) with a low photoinitiator concentration (1 mm LAP). Even under these mild reaction conditions, the stiffness of GelNB/PEG4LA hydrogels could be dynamically tuned by inducing dithiolane ring-opening via secondary light exposure, thereby creating dynamic and cytocompatible hydrogels suitable for in situ encapsulation, culture, and differentiation of human induced pluripotent stem cells (hiPSCs).