Degumming Time Governs Self-Assembled Silk Fibroin Hydrogel Properties through Molecular Weight and Amino Acid Composition.

Silk fibroin hydrogels are advantageous for in vitro tissue platforms, but unpredictable, time-dependent β-sheet formation complicates their utility. Here, we investigate how silk fiber degumming time and fibroin concentration influence inter- and intramolecular interactions and mechanical properties in physically cross-linked Bombyx mori silk fibroin hydrogels. Longer degumming times reduce molecular weight while enriching glycine and alanine residues, which are associated with β-sheet formation. The molecular weight, concentration, and gelation method collectively modulate the gelation kinetics, β-sheet content, and mechanical behavior over time. The spontaneously formed hydrogels show nonmonotonic mechanical property trends, while ultrasonicated silk fibroin hydrogels gel faster but display weaker networks due to a reduction in chain reorganization. Rheological and FTIR analyses reveal how initial protein composition impacts both short-term and long-term hydrogel mechanics. Results connect fibroin processing to material functionality, providing strategies for tuning hydrogel mechanics over time for tissue engineering and disease modeling applications where dynamic mechanical behavior is critical.