Silk fibroin (SF) was enzymatically crosslinked with tyramine-substituted silk fibroin (SF-TA) or gelatin (G-TA) to fabricate hybrid hydrogels with tunable gelation kinetics, mechanical properties and bioactivity. Horseradish peroxidase (HRP)/hydrogen peroxide (H 2 O 2 ) mediated crosslinking of SF in physiological buffers results in slow gelation and limited mechanical properties. Moreover, SF lacks cell attachment sequences, leading to poor cell-material interactions. These shortcomings can limit the uses of enzymatically crosslinked silk hydrogels in injectable tissue fillings, 3D bioprinting or cell microencapsulation, where rapid gelation and high bioactivity are desired. Here SF/SF-TA and SF/G-TA composite hydrogels were characterized for hydrogel properties and the influence of conjugated cyclic arginine-glycine-aspartic acid (RGD) peptide or G-TA content on bioactivity was explored. Both SF-TA and G-TA significantly increased gelation kinetics, improved mechanical properties and delayed enzymatic degradation in a concentrationdependent manner. β-sheet formation and hydrogel stiffening were accelerated by SF-TA content but delayed by G-TA. Both cyclic RGD and G-TA significantly improved morphology and metabolic activity of human mesenchymal stem cells (hMSCs) cultured on or encapsulated in composite hydrogels. The hydrogel formulations introduced in this study provide improved control of gel formation and properties, along with biocompatible systems that can be utilized in tissue engineering and cell delivery applications.

We report a fundamental study on effect of different silk degumming time on biomaterial properties of enzymatically crosslinked hydrogels.

Here, the Fenton reaction is used to prepare silk hydrogels through oxidation of tyrosine residues in silk fibroin, leading to dityrosine crosslinking. At pH 5.7, gelation occurs rapidly within 30 s, and the resultant opaque gels show soft properties with a storage modulus of ~100 Pa. The addition of ascorbic acid to the Fenton reaction increases the dityrosine bonds in the hydrogels but has little effect on the rheological or mechanical properties. The results indicate that Fe(III) ions significantly This article is protected by copyright. All rights reserved. 2 interacted with silk fibroin during the Fenton reaction, most likely binding to sites such as tyrosine, glutamate, and aspartate residues, triggering the formation of β-sheet structures that may impede dityrosine bond formation due to steric hindrance. The use of an iron chelator or the operation of the Fenton reaction at pH 9.2 enables control over the interaction of Fe(III) ions with silk fibroin, achieving a hydrogel with improved optical properties and enhanced dityrosine bond formation.Hydrogels prepared by the Fenton reaction are cytocompatible as L929 mouse fibroblasts remain viable and are proliferative when seeded on the hydrogels. The results offer a useful approach to generate chemically crosslinked silk fibroin hydrogels without the use of enzyme-catalyzed reactions for biomedical applications.