Scalable one-step synthesis of gelatin-dithiolane for neural tissue engineering

Protein-based hydrogels crosslinked using dithiolanes provide a promising viscoelastic matrix for soft tissue engineering and regenerative medicine including the neural niches due to their inherent biocompatibility, bioactivity, and adaptable extracellular matrix (ECM)-like viscoelastic behavior. Recently, we developed gelatin-dithiolane (GelDT) as a new class of ECM-mimicking viscoelastic hydrogels that displayed multi-functional properties, stimuli responsiveness and enabled independent tuning of the stiffness and matrix stress relaxation rate to precisely tune the matrix for improved cellular functions. However, the synthesis of GelDT remained laborious and inefficient. Herein, we report a scalable, one-step synthesis of GelDT that enables precise control over dithiolane functionalization (3-97%) using a carbonate-bicarbonate buffer system under mild aqueous conditions, while reducing organic solvent consumption from liters to the milliliters scale and eliminating the use of reducing agents. GelDT hydrogels obtained using the new synthesis route exhibit high stability (weeks), tunable stiffness, shear thinning, and self-healing properties essential for minimally invasive delivery. Additionally, pre-gelation tuning via physiochemical crosslinking allowed the fabrication of GelDT hydrogels at a remarkably low gelatin concentration (1.5% w/v) while ensuring fast gelation. The GelDT hydrogel supported the high viability and metabolic activity of encapsulated human iPSC-derived neural progenitor cell (NPC) spheroids. The GelDT hydrogel maintained NPC stemness (SOX2+, Ki-67+) and facilitated successful neuronal differentiation (MAP2+) in 3D culture. This work establishes a scalable, cytocompatible platform for producing dynamic protein-based hydrogels for regenerative medicine.