Tissue engineering seeks to address the shortage of donor organs by developing biological substitutes that replicate native tissue function. Hydrogels, due to their high water content, biocompatibility, and ECM-like properties, are widely used as scaffolds in regenerative medicine. In particular, hydrogels based on recombinant spider silk protein (engineered Araneus diadematus fibroin 4) offer tunable mechanical stability and biofunctionality. A key challenge is optimizing the hydrogel network structure influenced by various processing parameters, which is critical for nutrient diffusion, drug delivery, and waste removal in 3D cell cultures.

In this study, we tracked diffusing nanoparticles within spider silk hydrogels using a custom-built fluorescence microscope and Python-based analysis. The particles indicated a well interconnected porous network. Our goal is to understand the observed diffusivity to guide us through an efficient adjustment of processing parameters that will influence the pore structure of the hydrogels. Finally, we optimize the transport properties to find a way to design hydrogels that have optimal conditions for cells. This work supports the development of customizable spider silk-based hydrogel scaffolds for medical applications.