One-Step FRESH Bioprinting of Low-Viscosity Silk Fibroin Inks

Silk fibroin (SF) is an attractive material for composing bioinks suitable for three-dimensional (3D) bioprinting. However, the low viscosity of SF solutions obtained through common dissolution methods limits 3D-bioprinting applications without the addition of thickeners or partial gelation beforehand. Here, we report a method of 3D bioprinting low-viscosity SF solutions without additives. We combined a method of freeform reversible embedding of suspended hydrogels, known as the FRESH method, with horseradish peroxidase-catalyzed cross-linking. Using this method, we successfully fabricated 3D SF hydrogel constructs from low-viscosity SF ink (10% w/w, 50 mPa s at 1 s^-1 shear rate), which does not yield 3D constructs when printed onto a plate in air. Studies using mouse fibroblasts confirmed that the printing process was cell-friendly. Additionally, cells enclosed in printed SF hydrogel constructs maintained > 90% viability for 11 days of culture. These results demonstrate that the 3D bioprinting technique developed in this study enables new 3D bioprinting applications using SF inks and thus has a great potential to contribute to tissue engineering and regenerative medicine.