Abstract
Despite extensive research, current methods for creating three-dimensional (3D) silk fibroin (SF) scaffolds lack control over molecular rearrangement, particularly in the formation of β-sheet nanocrystals that severely embrittle SF, as well as hierarchical fiber organization at both micro- and macroscale. Here, we introduce a fabrication process based on electrowriting of aqueous SF solutions followed by post-processing using an aqueous solution of sodium dihydrogen phosphate (NaH2PO4). This approach enables gelation of SF chains via controlled β-sheet formation and partial conservation of compliant random coil structures. Moreover, this process allows for precise architecture control in microfiber scaffolds, enabling the creation of 3D flat and tubular macro-geometries with square-based and crosshatch microarchitectures, featuring inter-fiber distances of 400 μm and ∼97% open porosity. Remarkably, the crosslinked printed structures demonstrated a balanced coexistence of β-sheet and random coil conformations, which is uncommon for organic solvent-based crosslinking methods. This synergy of printing and post-processing yielded stable scaffolds with high compliance (modulus = 0.5-15 MPa) and the ability to support elastic cyclic loading up to 20% deformation. Furthermore, the printed constructs supported in vitro adherence and growth of human renal epithelial and endothelial cells with viability above 95%. These cells formed homogeneous monolayers that aligned with the fiber direction and deposited type-IV collagen as a specific marker of healthy extracellular matrix, indicating that both cell types attach, proliferate, and organize their own microenvironment within the SF scaffolds. These findings represent a significant development in fabricating organized stable SF scaffolds with unique microfiber structures and mechanical and biological properties that make them highly promising for tissue engineering applications.