Engineered Tough Silk Hydrogels through Assembling β‑Sheet Rich Nanofibers Based on a Solvent Replacement Strategy

β-Sheet rich silk nanofiber hydrogels are suitable scaffolds in tissue regeneration and carriers for various drugs. However, unsatisfactory mechanical performance limits its applications. Here, insight into the silk nanofibers stimulates the remodeling of previous solvent systems to actively regulate the assembly of silk nanofibers. Formic acid, a solvent of regenerated silk fibroin, is used to shield the charge repulsion of silk nanofibers to facilitate the nanofiber assembly under concentrated solutions. Formic acid was replaced with water to solidify the assembly, which induced the formation of a tough hydrogel. The hydrogels generated with this process possessed a modulus of 5.88 ± 0.82 MPa, ultimate stress of 1.55 ± 0.06 MPa, and toughness of 0.85 ± 0.03 MJ m^–3, superior to those of previous silk hydrogels prepared through complex cross-linking processes. Benefiting from the dense gel network and high β-sheet content, these silk nanofiber hydrogels had good stability and antiswelling ability. The modulus could be modulated via changing the silk nanofiber concentration to provide differentiation signals to stem cells. Improved mechanical and bioactive properties with these hydrogels suggest utility in biomedical and engineering fields. More importantly, our present study reveals that the in-depth understanding of silk nanofibers could infuse power into traditional fabrication systems to achieve more high performance biomaterials, which is seldom considered in silk material studies.