## 3 Results and Discussion  
It is known that Ch contains amine groups and exhibits typical polyelectrolyte properties [52], while Gel is a polyampholyte containing negative carboxyl and positive amine groups [29]. The third component of the cryogels, namely CS, is an anionic polysaccharide containing sulfo groups [24]. Thus, the formation of a water-stable cryogel with a 3D macroporous structure was due to the ionic interactions of carboxyl and amine groups of Gel, amine groups of Ch, and the sulfo group of CS. The so-formed physical cryogels were further crosslinked using the chemical crosslinker EDS to produce dual crosslinked cryogels (Figure 1). The presence of the functional groups in the cryogels is confirmed by FTIR measurements (Figure 2). To confirm the effect of the EDC crosslinker, a control sample without chemical crosslinking was also measured. The spectra show a band in the range  
Figure 1: Scheme showing the formation of the cryogels. 3000-3600 cm\({}^{-1}\), which is related to the stretching vibrations of the O-H and N-H functional groups (amide A) involved in the intramolecular hydrogen bond between chitosan and gelatin molecules. The bands at 3000-3500 cm\({}^{-1}\) are due to several symmetric and asymmetric stretching vibrations of C-H. Bands at 1636 cm\({}^{-1}\) refer to CO- and CN-amide I. The bands at 1544 cm\({}^{-1}\) refer to the deformation vibrations of NH groups and the stretching vibrations of CN groups (amide II). The absorption of the spectrum in the 1250 cm\({}^{-1}\) range belongs to the stretching vibrations of CN groups (amide III). The main peaks corresponding to amide I, amide II, and amide III are characteristic of Gel, Ch, and CS. The band at 1019 cm\({}^{-1}\) is due to the stretching vibrations of C-O groups. The shift of the peaks in the region of 1406 and 1636 cm\({}^{-1}\) in the control sample without chemical crosslinking relative to those of the cryogels indicates the formation of hydrogen bonds between Ch and Gel. Comparing the spectra of cryogels with and without EDC, one can notice the disappearance of the peak in cryogels (GelChCS10-GelChCS250) in the region of 1406 and a shift of the peak at 1636 cm\({}^{-1}\), indicating the formation of a bond between chitosan and gelatin, while the disappearance of the peak at 1340 cm\({}^{-1}\) indicates the formation of CONH\({}_{2}\) (CS crosslinking with chitosan), which confirms the formation of crosslinked cryogels. To determine the efficiency of chemical crosslinking, the gel content of the cryogels was determined as it affects their physicochemical properties (Table 1). For the GelChCS50 and GelChCS100 cryogels, the gel fraction was maximum, i.e., 82 \(\pm\) 1% and 80 \(\pm\) 2%, respectively. This can be caused by the formation of a stable crosslinked polymer network, in which the sol (soluble part) fraction is present to a lesser extent. This may also be due to the hydrophilic properties of gelatin and the non-dense polymer structure for the rest of the samples. When the cryogels thaw after preparation, macropores are formed in the place of ice crystals while the crosslinked polymer chains act as the pore walls, providing a large surface area for cell attachment and proliferation. Moreover, the more porous the polymer, the better it is for the penetration of fluids and cells. Table 1 shows the density, porosity, and surface area of the cryogels formed at various CS contents. The general trend is that, as the CS concentration increased, the density of the scaffolds also increased while their porosity slightly decreased. Decreasing the pore volume of the scaffolds with increasing CS content could be attributed to the decrease of the freezing temperature of the reaction  
Figure 2: FTIR spectra of GelChCS cryogels. system reducing the amount of ice crystals and hence, the pores. Table 1 shows that all the cryogels had pore volumes around 80%, and hence they are ideal for use in tissue engineering [47].