# Biomedical Applications of Polymeric Cryogels
## 2 Preparation and Properties of Supermacroporous Cryogels
### Factors Affecting Properties of Cryogels  
There are many factors affecting properties of cryogels. Temperature at which cryogelation occurs, polymerization time, type of crosslinking, contents of monomers used, and freezing rate are variables determine physical properties of cryogels. [PERSON] et al. have shown the effect of temperature on pore size. According to their study, lower cryogelation temperature results in smaller pore size. Thus, cryogels with different pore size distribution can be generated by changing cryogelation temperature. However, the temperature at which cryogels with maximum pore size are obtained, are preferred [18]. Figure 1: Schematic view of cryogel formation. In a study, 2-hydroxyethyl methacrylate (HEMA)-based cryogel membranes were prepared at different polymerization temperature to observe the effect on the pore size of membranes. They reported smaller pore size formation at lower cryogel temperature [19]. Monomer contents of cryogels and concentrations of reaction constituents significantly affect the cryogelation process. Monomers with lower molecular weights are expected to form larger pores when compared to monomers with larger molecular weights. In addition, increase in the amount of reaction constituents rises the rigidity of cryogels, thickness of cryogel walls and elasticity [20]. Also, rate of cryogelation increases and cryogelation occurs more efficiently with using of more concentrated polymer contents. Water soluble monomers are usually preferred for the preparation of cryogels. HEMA, \(N\)-isopropylacrylamide (NIPA), acrylic acid (AAc), \(N^{\prime}\),\(N^{\prime}\)-dimethylacrylamide (DMA), \(N^{\prime}\),\(N^{\prime}\)-diethylacrylamide (DEA) and acrylamide (AAAm) are some monomer types used for cryogel preparation. Freezing rate is also an important factor for optimal cryogel production process. To produce supermacroporous homogeneous cryogels, the crosslinking speed should be slower than the solvent freezing rate. If the crosslinking speed proceeds faster than the solvent crystallizes, no macro pores will be formed. Therefore, the typical supermacroporous cryogel structure will not occur [21]. Crosslinking degree affects physical properties of cryogels. Stiffness of the cryogel, elasticity and mechanical properties are the factors affected by crosslinking degree. Crosslinking degree also has an influence on swelling degree by affecting pore size with capability of keeping water [22]. \(N^{\prime}\),\(N^{\prime}\)-methylene(bis) acrylamide (MBAA) and poly(ethylene glycolacrylate) have been used as crosslinkers for preparation of different types of cryogels in the literature. Glutaraldehyde is also used for crosslinking of proteins via amine groups of peptide chains. In addition to proteins, glutaraldehyde can crosslink polysaccharides and synthetic polymers such as poly (vinyl alcohol) (PVA) and polyacrylamide in the preparation of cryogels [23]. Cryogels can be classified as supermacroporous hydrogels which are produced by cryogelation. There are some differences between hydrogels and cryogels. To obtain macroporous structure within hydrogels, different pore forming agents which are removed after gelation can be used. Removal of porogens and insufficient interconnectivity are main difficulties of conventional hydrogels in preparation process. Furthermore, chemicals that are used to remove porogens can stay in gel structure and affect binding of target macromolecules or cells. Porogen removal process can be time consuming and creates excessive waste. In addition, porogens which are not removed properly can cause unfavorable effects on target molecules in binding process. If porogens cannot be removed properly, they stay in gel structure and can prevent interconnectivity between macroopres. Thus, non-toxic and easily removable porogens are preferred for producing interconnected macroporous gel structures. At this point, cryogels are attractive options as they are formed by using water as porogen-forming agent.