Tailoring Gellan Gum Spongy-Like Hydrogels' Microstructure by Controlling Freezing Parameters

Gellan gum (GG) spongy-like hydrogels have been explored for different tissue engineering (TE) applications owing to their highly attractive hydrogel-like features, and improved mechanical resilience and cell performance. Although the whole process for the preparation of these materials is well-defined, we hypothesized that variations occurring during the freezing step lead to batch-to-batch discrepancies. Aiming to address this issue, two freezing devices were tested, to prepare GG spongy-like hydrogels in a more reproducible way. The cooling and freezing rates, the nucleation time and temperature, and the end freezing time were determined at different freezing temperatures (-20, -80, and -210 °C). The efficacy of the devices was assessed by analyzing the physicochemical, mechanical, and biological properties of different formulations. The cooling rate and freezing rate varied between 0.1 and 128 °C/min, depending on the temperature used and the device. The properties of spongy-like hydrogels prepared with the tested devices showed lower standard deviation in comparison to those prepared with the standard process, due to the slower freezing rate of the hydrogels. However, with this method, mean pore size was significantly lower than that with the standard method. Cell entrapment, adhesion, and viability were not affected as demonstrated with human dermal fibroblasts. This work confirmed that batch-to-batch variations are mostly due to the freezing step and that the tested devices allow fine tuning of the scaffolds' structure and properties.