Cryogels: Freezing unveiled by thawing

Cryogels are interconnected supermacroporous gels prepared at sub-zero temperatures having applications in various research fields. The process of cryogelation is ideally thought to take place via following steps: phase separation with ice-crystal formation, cross-linking and polymerization followed by thawing of ice-crystals to form an interconnected porous cryogel network. This phenomenon mostly thought as a theoretical concept has now been revealed here in practical terms via data generated by micro-computed tomography (Micro CT). Micro CT is mainly used for characterizing the gel materials in terms of their physical properties like pore size, porosity, strut size, etc., whereas this work has pioneered its role in elucidating the process of cryogel formation. Most of the concepts developed in the field of science and technology are influenced by naturally occurring phenomenon in one way or the other. The introduction of cryogels in the field of material science as a supermacroporous interconnected porous network also comes from the understanding of the natural phenomenon like permafrost formation. The formation of permafrost occurs in areas where the land remains frozen, interconnected polygonal features are developed at such land surface as a result of repeated freezing and thawing. Based on a similar approach, cryogels are synthesized by a process where the precursor solution of solute dispersed generally in an aqueous solvent is frozen at sub-zero temperature. Over the course of time, the solvent starts freezing and two phases are formed, the frozen phase and the unfrozen-liquid microphase. Solute particles remain restricted to the unfrozen-liquid microphase and start interacting to form polymer. Once the cross-linking has occurred, the cryogel is thawed resulting in the formation of an interconnected porous network with pores surrounded by polymer walls1. The process of cryogel formation, leading to the generation of new material design is a true representation of the materials of today (Fig. 1). Although, the cryogels have been widely used for various applications in fields of biotechnology and bioengineering[2], [3], [4] and [5], yet it is commiserate that the method of cryogel formation is still a hypothesis. This lack of information does not allow to elucidate a true picture of various steps during cryogel formation. To clarify the picture Micro CT was applied for the first time to explain the process of cryogel formation as none of the available methodologies till now have been able to explain this process in detail.Important characteristics of scaffolds determining their architectural and structural details are porosity, pore size, strut thickness, permeability, etc, which can be analysed by different techniques either individually or in combination6. Although, the best alternative is to characterize the scaffold by a single method which is non-destructive yet gives comprehensive information7. Micro CT analysis method in such situations is a better option, which provides a detailed insight into the quantitative and qualitative three dimensional (3-D) morphology of the specimen in a non-destructive manner. In order to study the process of cryogel formation via Micro CT, cryogels were scanned using a high resolution Skyscan 1174 microtomograph for a duration of 11 mins and 34 secs in a consecutive manner. This study was performed via two different methods; one is a direct method of observing cryogel formation at various timepoints during synthesis and another being an indirect method of observing the same phenomenon by studying the thawing behaviour of a completely frozen cryogel at various steps.Polyacrylamide cryogels were used as representative for studying the direct method of cryogel formation since they are very well characterized and most utilized amongst the available cryogels and involve both polymerization and cross-linking. The acrylamide cryogelation process was initiated using radical polymerization with N,N'-methylenebisacrylamide as cross-linker. The precursor solution was kept for incubation in cryostat at −12°C for overall duration of 12 h. The samples were removed at various timepoints, then frozen in liquid nitrogen and further lyophilized to preserve the scaffold structure at particular timepoints; this denoted the thawed stage of cryogel formation. With time, a change in three- dimensional structure and physical parameters such as porosity, object volume and average pore size were observed (Fig. 2). As depicted in Fig. 2, initial timepoints (0 min and 12 min) represent the unlyophilized and lyophilized frozen state of cryogelation process, respectively where the solute is dispersed in the whole solution and ice-crystal formation has not yet begun. At 36 min and 1.8 h ice-crystal formation and polymerization begun simultaneously, while the final timepoints (5 h and 12 h) symbolize the polymerized pore walls (grey region) with the pores indicating the void space (black region) formed by lyophilizing the ice-crystals. The percentage object volume shown by the grey region of the 3-D images represents the area of the cryogel where ice crystals were not present during cryogelation. Initially, the object volume was high (55%), because the polymerization had not yet occurred and the cryogel was present in the form of frozen precursor solution. At later timepoints, the object volume decreased (26%) due to the formation of polymeric walls surrounding the pores. A similar trend was observed for porosity, which increased from 44% to 73% over time, due to ice-crystal formation. Moreover, pore sizes at different timepoints showed a gradual increase from 70 μm to 84 μm with the increase following a different pattern than porosity and object volume.As part of indirect method, chitosan-agarose-gelatin (CAG) cryogels were used for the analysis of pore formation. The rationale for the selection being that these gels are fully characterized and exhibit a uniform pore distribution, however, undergo only cross-linking process and no polymerization. During the study, preformed CAG cryogel scaffolds were saturated in deionised water followed by freezing overnight at −12 °C leading to the freezing of water present in the pores. The scanning of these frozen cryogels was done in a repeated manner for 30 times, constituting a total time of 340 min and 20 sec. During the scanning process the ice-crystals formed in the pores melted down slowly revealing the pores preformed in the cryogel. Variations in the physical parameters like porosity, percent object volume and average pore size were similar when compared to the direct method. A gradual increase or decrease in the physical parameters of the cryogel was observed which reached to standard values after the gel is completely dried which further supports the analysis. The indirect method has the advantage that two phases, i.e., aqueous and polymer phases can be visualized separately at different threshold values which can perhaps be due to the variation in the densities of the two components.Therefore, the cryogelation process which was initially hypothetically understood and not well depicted with a practical illustration of various steps during the process has now become clearer from the current study with Micro CT analysis. The study was performed via two different methods, and the trends obtained for various parameters by both the studies show similarity. This further confirms that the process of cryogelation works invariably without being affected by the monomer/polymer type and the kind of method used for analysis. Instrument CitationSkyscan 1174 microtomography machine Lauda (cryostat) Proline 1840. AcknowledgementsAuthors would like to acknowledge DST-UKIERI award and India-UK Science Bridge programme.