Scalable One-Step Synthesis of Gelatin-Dithiolane for Neural Tissue Engineering

Abstract

Protein-based hydrogels crosslinked using dithiolanes provide a promising viscoelastic matrix for soft tissue engineering and regenerative medicine including neural niche 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 hydrogel that displayed multi-functional properties, stimuli responsiveness and enabled independent tuning of 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 robust one-pot synthesis of GelDT that enables precise control over dithiolane functionalization (3–97%) using a carbonate–bicarbonate buffer system under mild aqueous conditions, while reducing the organic solvent consumption from liters to few milliliters scale and eliminating the use of reducing agents. The 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 physio-chemical crosslinking allowed fabrication of GelDT hydrogels at 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.