Mechanisms of pore formation in hydrogel scaffolds textured by freeze-drying

Abstract

Whereas freeze-drying is a widely used method to produce porous hydrogel scaffolds, the mechanisms of pore formation involved in this process remained poorly characterized. To explore this, we focused on a cross-linked polysaccharide-based hydrogel developed for bone tissue engineering. Scaffolds were first swollen in 0.025% NaCl then freeze-dried at low cooling rate, i.e. −0.1 °C min−1, and finally swollen in aqueous solvents of increasing ionic strength. We found that scaffold’s porous structure is strongly conditioned by the nucleation of ice. Electron cryo-microscopy of frozen scaffolds demonstrates that each pore results from the growth of one to a few ice grains. Most crystals were formed by secondary nucleation since very few nucleating sites were initially present in each scaffold (0.1 nuclei cm−3 °C−1). The polymer chains are rejected in the intergranular space and form a macro-network. Its characteristic length scale coincides with the ice grain size (160 μm) and is several orders of magnitude greater than the mesh size (90 nm) of the cross-linked network. After sublimation, the ice grains are replaced by macro-pores of 280 μm mean size and the resulting dry structure is highly porous, i.e. 93%, as measured by high-resolution X-ray tomography. In the swollen state, the scaffold mean pore size decreases in aqueous solvent of increasing ionic strength (120 µm in 0.025% NaCl and 54 µm in DBPS) but the porosity remains the same, i.e. 29% regardless of the solvent. Finally, cell seeding of dried scaffolds demonstrates that the pores are adequately interconnected to allow homogenous cell distribution. Statement of SignificanceThe fabrication of hydrogel scaffolds is an important research area in tissue engineering. Hydrogels are textured to provide a 3D-framework that is favorable for cell proliferation and/or differentiation. Optimum hydrogel pore size depends on its biological application. Producing porous hydrogels is commonly achieved through freeze-drying. However, the mechanisms of pore formation remain to be fully understood. We carefully analyzed scaffolds of a cross-linked polysaccharide-based hydrogel developed for bone tissue engineering, using state-of-the-art microscopic techniques. Our experimental results evidenced the shaping of hydrogel during the freezing step, through a specific ice-templating mechanism. These findings will guide the strategies for controlling the porous structure of hydrogel scaffolds.