Interplay between crosslinking and ice nucleation controls the porous structure of freeze-dried hydrogel scaffolds

Freeze-drying is a process of choice to texture hydrogel scaffolds with pores formed by an ice-templating mechanism. Using state-of-the-art microscopies (cryo-EBSD, µCT, CLSM), this work evidences and quantifies the effect of crosslinking and ice nucleation temperature on the porous structure of thin hydrogel scaffolds freeze-dried at a low cooling rate. We focused on a polysaccharide-based hydrogel and developed specific protocols to monitor or trigger ice nucleation for this study. At a fixed number of intermolecular crosslinks per primary molecule (p=5), the mean pore size in the dry state decreases linearly from 240 to 170 µm, when ice nucleation temperature decreases from -6°C to -18°C. When ice nucleation temperature is fixed at -10°C, the mean pore size decreases from 250 to 150 µm, as the crosslinking degree increases from p=3 to p=7. Scaffold infiltration ability was quantified with synthetic microspheres. The seeding efficiency was assessed with MC3T3-E1 individual cells and HepaRGTM spheroids. These data collapse into a single master curve that exhibits a sharp transition from 100% to 0%-efficiency as the entity diameter approaches the mean pore size in the dry state. Altogether, we can thus precisely tune the porosity of these 3D materials of interest for 3D cell culture and cGMP production for tissue engineering.