Silk Hydrogels of Tunable Structure and Viscoelastic Properties using Different Chronological Orders of Genipin and Physical Crosslinking

Catering hydrogel manufacturing process towards defined viscoelastic properties for intended biomedical use is important to hydrogel scaffolding function and cell differentiation. Silk fibroin hydrogels may undergo "physical" crosslinking through β-sheet crystallization during high-pressure carbon dioxide treatment, or covalent "chemical" cross-linking by genipin. We demonstrate here that time-dependent mechanical properties are tunable in silk fibroin hydrogels by altering the chronological order of genipin crosslinking with β-sheet formation. Genipin crosslinking before β-sheet formation affects gelation mechanics through increased molecular weight, affecting gel morphology, and decreasing stiffness response. Alternately, genipin crosslinking after gelation anchored amorphous regions of the protein chain, restricting reptation, and increasing stiffness. These differences are highlighted and validated through dynamic mechanical analysis at large, physiologic strain levels, after incorporation of material characterization at molecular and micron length scales.