Biofunctionalization of silk biomaterial surfaces with extracellular matrix (ECM) molecules, cell binding peptides, or growth factors is important in a range of applications, including tissue engineering and development of implantable medical devices. Passive adsorption is the most common way to immobilize molecules of interest on preformed silk biomaterials but can lead to random molecular orientations and displacement from the surface, limiting their applications. Herein, we developed techniques for covalent immobilization of biomolecules using enzyme- or photoinitiated formation of dityrosine bonds between the molecule of interest and silk. Using recombinantly expressed domain V of the human basement membrane proteoglycan perlecan (rDV) as a model molecule, we demonstrated that rDV can be covalently immobilized via dityrosine cross-linking without the need to modify rDV or silk biomaterials. Dityrosine-based immobilization resulted in a different molecular orientation to passively absorbed rDV with less C- and N-terminal region exposure on the surface. Dityrosine-based immobilization supported functional rDV immobilization where immobilized rDV supported endothelial cell adhesion, spreading, migration, and proliferation. These results demonstrate the utility of dityrosine-based cross-linking in covalent immobilization of tyrosine-containing molecules on silk biomaterials in the absence of chemical modification, adding a simple and accessible technique to the silk biofunctionalization toolbox.

Human tissues and organs exhibit complex hierarchical and gradient structures that are essential to their function and should be recapitulated within biomaterial scaffolds targeting their regeneration. Unidirectional freezing, an ice templating technique where ice acts as a porogen, is uniquely suited to recapitulating the architectural anisotropy, gradients, and hierarchical transitions of human tissues, but ice templating of polymeric systems, including silk fibroin, remains less well understood than their colloidal counterparts. To address this, a versatile and accessible freezing setup for silk that allows tuning of freezing parameters including the polymer cooling rate (30 °C min−1 to 2 °C min−1) and ice solidification velocity (2.5 to 0.6 mm min−1) using liquid nitrogen, is developed. Real time visual and thermal monitoring of the freezing process for multiple silk concentrations (2–10% wt/v) and material states (liquid, hydrogels) is performed and the conditions are correlated with pore morphology. Unprecedented control over pore size (100–90 000 µm2) and pore morphology (cellular–lamellar), consistent pore alignment, and generation of gradient porosity in silk scaffolds are demonstrated. For the first time, impact of shear thinning behavior of silk in ice crystal formation is demonstrated, showing non‐linear and complex freezing phenomena in silk.