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.

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Nanocoating of individual mammalian cells with polymer layers has been of increasing interest in biotechnology and biomedical engineering applications. Electrostatic layer-by-layer (LbL) deposition of polyelectrolytes on negatively charged cell surfaces has been utilized for cell nanocoatings using synthetic or natural polymers with a net charge at physiological conditions. Here, our previous synthesis of silk-based ionomers through modification of silk fibroin (SF) with polyglutamate (PG) and polylysine (PL) was exploited for the nanocoating of mammalian cells. SF-PL constructs were cytotoxic to mammalian cells, thus an alternative approach for the synthesis of silk ionomers through carboxylation and amination of regenerated SF chains was utilized. Through the optimization of material properties and composition of incubation buffers, silk ionomers could be electrostatically assembled on the surface of murine fibroblasts and human mesenchymal stem cells (hMSCs) to form nanoscale multilayers without significantly impairing cell viability. The resulting silk-based protein nanoshells were transient and degraded over time, allowing for cell proliferation. The strategies presented here provide a basis for the cytocompatible nanoencapsulation of mammalian cells within silk-based artificial cell walls, with potential benefits for future studies on surface engineering of mammalian cells, as well as for utility in cell therapies, 3D printing, and preservation.

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