Revealing the True Morphological Structure of Macroporous Soft Hydrogels for Tissue Engineering

(1) Background: Macroporous hydrogel scaffolds based on poly [N-(2-hydroxypropyl) methacrylamide] are one of the widely studied biocompatible materials for tissue reparation and regeneration. This study investigated the morphological changes during hydrogel characterization which can significantly influence their future application. (2) Methods: Three types of macroporous soft hydrogels differing in pore size were prepared. The macroporosity was achieved by the addition of sacrificial template particles of sodium chloride of various sizes (0–30, 30–50, and 50–90 µm) to the polymerizing mixture. The 3D structure of the hydrogels was then investigated by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). The SEM was performed with specimens rapidly frozen to various temperatures, while non-frozen gels were visualized with LSCM. (3 and 4) Results and Conclusion: In comparison to LSCM, the SEM images revealed a significant alteration in the mean pore size and appearance of newly formed multiple connections between the pores, depending on the freezing conditions. Additionally, after freezing for SEM, the gel matrix between the pores and the fine pores collapsed. LSCM visualization aided the understanding of the dynamics of pore generation using sodium chloride, providing the direct observation of hydrogel scaffolds with the growing cells. Moreover, the reconstructed confocal z-stacks were a promising tool to quantify the swollen hydrogel volume reconstruction which is not possible with SEM.