Cell-culture compatible silk fibroin scaffolds concomitantly patterned by freezing conditions and salt concentration

The morphology of freeze-dried silk fibroin 3D-scaffolds was modified by varying both the NaCl concentration and the freezing temperature of the silk fibroin solution prior to lyophilization. Scanning electron micrographs showed that slow freezing at −22 °C generated sponge-like interconnected porous networks, whereas fast freezing at −73 °C formed stacked leaflet structures. The presence of millimolar NaCl (50–250 mM) increased the porosity of the scaffolds and generated small outgrowths at their surface, depending on the freezing regime. Our results suggest that the morphological differences seen between the materials likely depend on ice and NaCl hydrate crystal nucleation and growth mechanisms. Infrared spectroscopy and X-ray diffraction analyses revealed that the salt concentration and freezing conditions induced no structural changes in fibroin. The seeding of P19 embryonic carcinoma cells showed that the presence of salt and freezing conditions influenced the cell distribution into the scaffolds, with salt addition increasing the access of cells to deeper regions.