Ice-Templated and Cross-Linked Xanthan-Based Hydrogels: Towards Tailor-Made Properties

The use of polysaccharides with good film-forming properties in food packaging systems is a promising area of research. Xanthan gum (XG), an extracellular polysaccharide, has many industrial uses, including as a common food additive (E415). It is an effective thickening agent, emulsifier, and stabilizer that prevents ingredients from separating. Nevertheless, XG-based polymer films have some disadvantages, such as poor mechanical properties and high hydrophilic features, which reduce their stability when exposed to moisture and create difficulties in processing and handling. Thus, the objective of this work was to stabilize a XG matrix by cross-linking it with glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, or epichlorohydrin below the freezing point of the reaction mixture. Cryogelation is an ecological, friendly, and versatile method of preparing biomaterials with improved physicochemical properties. Using this technique, XG-based cryogels were successfully prepared in the form of microspheres, monoliths, and films. The XG-based cryogels were characterized by FTIR, SEM, AFM, swelling kinetics, and compressive tests. A heterogeneous morphology with interconnected pores, with an average pore size depending on both the nature of the cross-linker and the cross-linking ratio, was found. The use of a larger amount of cross-linker led to both a much more compact structure of the pore walls and to a significant decrease in the average pore size. The uniaxial compression tests indicated that the XG-based cryogels cross-linked with 1,4-butanediol diglycidyl ether exhibited the best elasticity, sustaining maximum deformations of 97.67%, 90.10%, and 81.80%, respectively.


Introduction
Polysaccharides are attractive and promising natural polymers for the development of sustainable biomaterials with a wide range of applications. Their origins are plant/seed (cellulose, starch, and pectin), bacterial (dextran, xanthan gum, gellan gum, and salecan), seaweeds (alginate, ulvan, and carrageenan), or marine and terrestrial animal by-products (chitin, hyaluronic acid, and chondroitin sulfate) [1][2][3][4][5][6][7][8][9][10]. Polysaccharides possess unique properties including biocompatibility, biodegradability, and lack of toxicity that make them suitable for multiple applications [1][2][3][4][5][6][7][8]. For instance, some polysaccharides with hydrophilic groups (-OH or -COOH), such as dextran, xanthan gum, or alginate, have good water solubility and can form hydrogen bonds with water molecules, which support their suitability as thickening agents to increase the viscosity of the formulation and improve its uniformity [1][2][3]6]. On the other hand, polysaccharides modified with hydrophobic groups, such as fatty acid esters, can be used as emulsifying agents, stabilizing emulsions or suspensions [1][2][3]6]. Similarly, some polysaccharides could be utilized as binding agents to the uniformity of the formulations, while others could be applied as coating agents protecting the bioactive compound from degradation or controlling its release over time [1][2][3]5]. Currently, researchers are exploring different strategies for modifying and Gels 2023, 9,528 3 of 15 to optimize the cross-linking rate to achieve a balance between structural stability and appropriate properties. The optimization of the cross-linking rate depends on several factors, such as the type and concentration of cross-linking agent, the pH and temperature of the reaction, as well as on the duration of the reaction [46]. Different cross-linking agents may have different reaction rates and mechanisms, which can affect the characteristics of the resulting cryogel. By carefully controlling the cross-linking rate during cryogel fabrication, the overall performance and functionality of cryogels can be improved for a wide range of applications, including drug delivery, tissue engineering, biocatalysis, food packaging, and environmental protection. In this regard, the aim of this study was to investigate the chemical cross-linking of XG with glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, or epichlorohydrin under cryogelation conditions. The objectives followed in this work were: (i) to design cross-linked xanthan gum-based materials with improved physicochemical features below the freezing point of the reaction mixture; (ii) to obtain the optimum reaction conditions for preparation of XG-based cryogels by changing the type and amount of cross-linker (glycerol diglycidyl ether (R1), 1,4-butanediol diglycidyl ether (R2), or epichlorohydrin (R3)), XG concentration, and gel preparation temperature; and (iii) to characterize the cross-linked XG-based cryogels using FTIR, SEM, AFM, swelling kinetics, and compressive tests.

Preparation of XG-Based Cryogels as Microspheres, Monoliths, and Films
In the current research, we prepared new cryogel-type biomaterials based on XG using glycerol diglycidyl ether (R1), 1,4-butanediol diglycidyl ether (R2), and epichlorohydrin (R3) as cross-linkers through cryogelation ( Figure 1). The use of different cross-linkers and concentrations allowed us to optimize the degree of cross-linking and tailor the properties of the resulting cryogels. In addition, the preparation of XG-based cryogels in form of microspheres (MS), monoliths (MN), and films (FM) (Figure 2) enabled the evaluation of the effect of cryogelation on different morphologies of the biomaterials. In principle, the synthesis of XG matrices followed the same basic steps, with slight modifications depending on the presentation form of the sample (MS, MN, or FM). The cross-linking reactions took place in an alkaline environment using a 2M solution of sodium hydroxide (NaOH). The first step involved preparing a 1% solution of XG in de- lization or in an oven at 40 °C. Sample code, composition, gel fraction yield (GFY, %), and the type form of XG-based hydrogels are shown in Table 1.   In principle, the synthesis of XG matrices followed the same basic steps, with slight modifications depending on the presentation form of the sample (MS, MN, or FM). The cross-linking reactions took place in an alkaline environment using a 2M solution of sodium hydroxide (NaOH). The first step involved preparing a 1% solution of XG in deionized water. Then, various ratios of cross-linkers (glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, or epichlorohydrin) were added to the XG solution to obtain polymer matrices with different degrees of cross-linking. All stages of the synthesis were carried out in an ice bath to reduce the rate of the cross-linking reaction and maintain the fluidity of the mixture for subsequent processing. The cross-linker ratios used in the study were expressed in volumetric amounts corresponding to 100 mL of XG and NaOH solution mixture ( Table 1). The reaction time in the ice bath is relatively short, typically on the order of minutes, after which the sub-cooled mixture was processed according to the desired form of presentation of the biomaterial, as follows: • To obtain the XG cryogel microspheres, the polymer/cross-linker mixture was introduced into 5 mL syringes equipped with a needle and then dropped into liquid nitrogen (LN, −196 • C). The rapid cooling process in LN causes the droplets to solidify into spherical particles, whose size depends on the diameter of the syringe needle. After removing the microspheres from the LN, they were transferred to a freezer at a temperature of −20 • C for 24 h to complete the cross-linking process ( Figure 2).

•
To obtain XG cryogel monoliths, the polymer/cross-linker mixture was introduced into syringes with a diameter of 4 mm, and then the syringes were transferred to a freezer at a temperature of −20 • C for 24 h. During this time, the cross-linking reaction is completed and XG cryogels of a tubular shape are obtained ( Figure 2).

•
To obtain XG cryogel films, the sub-cooled reaction mixture was poured into Teflon Petri dishes and then transferred to a freezer at a temperature of −20 • C for 24 h ( Figure 2).
After 24 h, all samples were removed from the freezer and transferred in frozen state to lyophilizer for drying. After drying, all samples were purified via extensive washing with distilled water (Figure 2). Finally, the samples were dried again either by lyophilization or in an oven at 40 • C. Sample code, composition, gel fraction yield (GFY, %), and the type form of XG-based hydrogels are shown in Table 1. As shown in Table 1, an increase in cross-linker concentration led to an increase in GFY values, as expected. Furthermore, the GFY values depended on the type of cross-linker and the form of the XG-based cryogels, i.e., microspheres, monoliths, or films. For XG-based cryogels in microsphere form, glycerol diglycidyl ether was the most effective cross-linking agent at a concentration of 7 v/v%, resulting in a GFY value of 87.87%. In the case of XG-based cryogels as monoliths or films, 1,4-butanediol diglycidyl ether was the most efficient cross-linker, conducting to GFY values of 83.4% and 98.8%, respectively. However, the highest GFY values were obtained when XG-based cryogels were prepared as films irrespective of cross-linker type.

FTIR Analysis
To identify the chemical modifications achieved via XG cross-linking, FTIR spectroscopy was used first as a fast characterization tool. The FTIR spectrum of XG powder was compared with the FTIR spectra of XG networks obtained by cross-linking with R1, R2, or R3 (samples MS.R1V3, MS.R2V3, and MS.R3V3, respectively, Figure 3).  In the FTIR spectrum of XG the following absorption peaks are visible: a broad absorption peak at 3560 cm −1 corresponding to the stretching vibration of -OH groups; two peaks at 2920 and 2889 cm −1 assigned to the asymmetric and symmetric vibration of -CH2 groups; two characteristic peaks at 1740 cm −1 and 1634 cm −1 attributed to the stretching of carbonyl (C=O) of the acetyl groups and asymmetrical stretching of C=O group of pyruvate groups, respectively [47][48][49][50]; an absorption peak at 1416 cm −1 corresponding to the symmetric stretching vibration of carboxylate (-COO−) groups [47,51]; the shoulder at 1161 cm −1 and the broad peak at 1020 cm −1 referred to the C-O stretching vibration in secondary alcohol and to the stretching vibration of the C-O-C bridge in anhydroglucose (AGU) units, respectively [23,[48][49][50]. All these peaks were also identified in the FTIR spectra of XG networks obtained via cross-linking (samples MS.R1V3, MS.R2V3, and In the FTIR spectrum of XG the following absorption peaks are visible: a broad absorption peak at 3560 cm −1 corresponding to the stretching vibration of -OH groups; two peaks at 2920 and 2889 cm −1 assigned to the asymmetric and symmetric vibration of -CH 2 groups; two characteristic peaks at 1740 cm −1 and 1634 cm −1 attributed to the stretching of carbonyl (C=O) of the acetyl groups and asymmetrical stretching of C=O group of pyruvate groups, respectively [47][48][49][50]; an absorption peak at 1416 cm −1 corresponding to the symmetric stretching vibration of carboxylate (-COO−) groups [47,51]; the shoulder at 1161 cm −1 and the broad peak at 1020 cm −1 referred to the C-O stretching vibration in secondary alcohol and to the stretching vibration of the C-O-C bridge in anhydroglucose (AGU) units, respectively [23,[48][49][50]. All these peaks were also identified in the FTIR Gels 2023, 9, 528 7 of 15 spectra of XG networks obtained via cross-linking (samples MS.R1V3, MS.R2V3, and MS.R3V3, Figure 3) with some blue-or red-shifts, as follows: the peak at 3560 cm −1 in the FTIR spectrum of XG powder corresponding to the -OH stretching vibration were blue-shifted to 3410, 3439, and 3441 cm −1 in the FTIR spectra of MS.R1V3, MS.R2V3, and MS.R3V3 networks, respectively. The intensity of the band from 1740 cm −1 , characteristic to the stretching of carbonyl (C=O) of the acetyl groups, decreased and experienced a hypsochromic shift to 1724 cm −1 and 1720 cm −1 in the XG cross-linked matrices due to the hydrolysis reaction, which easily occurs in alkaline conditions [52]. The band at 1634 cm −1 corresponding to asymmetrical stretching of C=O group of pyruvate groups was blue-shifted at 1616, 1618, and 1620 cm −1 in the FTIR spectra of MS.R1V3, MS.R2V3, and MS.R3V3 networks, respectively. The characteristic absorption peak at 1020 cm −1 in the FTIR spectrum of XG powder corresponding to the stretching vibration of the C-O-C bridge in AGU units was red-shifted to 1030 cm −1 and 1026 cm −1 in the FTIR spectra of MS.R1V3, MS.R2V3, and MS.R3V3 networks, respectively. In addition, the presence of clear peaks at 1063 and 1061 cm −1 in the FTIR spectra of the XG cross-linked matrices indicates the appearance of supplementary ether (-C-O-C-) and hydroxyl (-OH) groups [23,[48][49][50][51]53]. All these changes support the efficient occurrence of the cross-linking reaction performed with all cross-linkers.

Internal Morphology
The morphology studies of the obtained XG-based networks were carried out by evaluating the porosity, texture, and roughness of the surfaces through SEM (Figures 4 and 5) and AFM ( Figure 6) analysis. MS.R3V3, Figure 3) with some blue-or red-shifts, as follows: the peak at 3560 cm −1 in the FTIR spectrum of XG powder corresponding to the -OH stretching vibration were blue-shifted to 3410, 3439, and 3441 cm −1 in the FTIR spectra of MS.R1V3, MS.R2V3, and MS.R3V3 networks, respectively. The intensity of the band from 1740 cm −1 , characteristic to the stretching of carbonyl (C=O) of the acetyl groups, decreased and experienced a hypsochromic shift to 1724 cm −1 and 1720 cm −1 in the XG cross-linked matrices due to the hydrolysis reaction, which easily occurs in alkaline conditions [52]. The band at 1634 cm −1 corresponding to asymmetrical stretching of C=O group of pyruvate groups was blue-shifted at 1616, 1618, and 1620 cm −1 in the FTIR spectra of MS.R1V3, MS.R2V3, and MS.R3V3 networks, respectively. The characteristic absorption peak at 1020 cm −1 in the FTIR spectrum of XG powder corresponding to the stretching vibration of the C-O-C bridge in AGU units was red-shifted to 1030 cm −1 and 1026 cm −1 in the FTIR spectra of MS.R1V3, MS.R2V3, and MS.R3V3 networks, respectively. In addition, the presence of clear peaks at 1063 and 1061 cm −1 in the FTIR spectra of the XG cross-linked matrices indicates the appearance of supplementary ether (-C-O-C-) and hydroxyl (-OH) groups [23,[48][49][50][51]53]. All these changes support the efficient occurrence of the cross-linking reaction performed with all cross-linkers.

Internal Morphology
The morphology studies of the obtained XG-based networks were carried out by evaluating the porosity, texture, and roughness of the surfaces through SEM (Figures 4  and 5) and AFM (Figure 6) analysis.   MS.R3V3, Figure 3) with some blue-or red-shifts, as follows: the peak at 3560 cm −1 in the FTIR spectrum of XG powder corresponding to the -OH stretching vibration were blue-shifted to 3410, 3439, and 3441 cm −1 in the FTIR spectra of MS.R1V3, MS.R2V3, and MS.R3V3 networks, respectively. The intensity of the band from 1740 cm −1 , characteristic to the stretching of carbonyl (C=O) of the acetyl groups, decreased and experienced a hypsochromic shift to 1724 cm −1 and 1720 cm −1 in the XG cross-linked matrices due to the hydrolysis reaction, which easily occurs in alkaline conditions [52].  [23,[48][49][50][51]53]. All these changes support the efficient occurrence of the cross-linking reaction performed with all cross-linkers.

Internal Morphology
The morphology studies of the obtained XG-based networks were carried out by evaluating the porosity, texture, and roughness of the surfaces through SEM (Figures 4  and 5) and AFM (Figure 6) analysis.     Figure 4 shows a heterogeneous morphology with interconnected pore characteristic of gels obtained via cryogelation. The average pore size depends on both the nature of the cross-linker and the cross-linking ratio. The use of a larger amount of cross-linker leads both to a much more compact structure of the pore walls and to a significant decrease in the average pore size (Figures 4 and 5 make a comparison between samples FM.R1V1 and FM.R1V3, respectively). The morphology of XG-based networks can also be controlled by the form of presentation of the material-MS, MN, or FM ( Figure 5). Thus, if a porous structure is desired, the samples can be synthesized in the form of MS ( Figure 5, sample MS.R1V3), and if a compact structure is desired, XG-based networks can be prepared in the form of films ( Figure 5, sample FM.R1V3). The FM.R1V3 cryogel lacks pores, suggesting that the inherent nature of the cryogel itself is responsible for the absence of pore formation.
The morphology of the XG-based networks obtained in the form of films was also examined by AFM analysis (Figure 6). AFM images were obtained using an SPM Solver PRO-M-type apparatus (NT-MDT Co. Zelenograd, Moscow, Russia) that uses a high-resolution cantilever of the "Golden" type (NSG10/Au/50 silicon coated with a conductive layer of Au). The topographic images ( Figure 6) were obtained in the intermittent scanning regime and were repeated on several areas of the same sample. By analyzing the roughness values in Table 2 as well as the AFM images ( Figure 6A,B), it is observed that the chemically cross-linked films in cryogenic conditions present a structured morphology. The existence of structuring indicates that the orientation of macromolecules in the freezing phase is restricted by the centers of chemical cross-linking due to the decrease of the average free volume of the chain segments. The structured phase is stabilized upon thawing by the existence of the strengthening of physical bonds, so the morphology obtained by the orientation of the macromolecules in the freezing process is partially preserved at room temperature.
The comparative analysis of two films with different degrees of cross-linking (FM.R3V1 and FM.R3V3) shows that in the sample with a lower degree of cross-linking (sample FM.R3V1, cross-linker content of 0.7 v/v%), reduced structuring with a mor-  The morphology of the XG-based networks obtained in the form of films was also examined by AFM analysis (Figure 6). AFM images were obtained using an SPM Solver PRO-M-type apparatus (NT-MDT Co. Zelenograd, Moscow, Russia) that uses a highresolution cantilever of the "Golden" type (NSG10/Au/50 silicon coated with a conductive layer of Au). The topographic images ( Figure 6) were obtained in the intermittent scanning regime and were repeated on several areas of the same sample. By analyzing the roughness values in Table 2 as well as the AFM images ( Figure 6A,B), it is observed that the chemically cross-linked films in cryogenic conditions present a structured morphology. The existence of structuring indicates that the orientation of macromolecules in the freezing phase is restricted by the centers of chemical cross-linking due to the decrease of the average free volume of the chain segments. The structured phase is stabilized upon thawing by the existence of the strengthening of physical bonds, so the morphology obtained by the orientation of the macromolecules in the freezing process is partially preserved at room temperature.
The comparative analysis of two films with different degrees of cross-linking (FM.R3V1 and FM.R3V3) shows that in the sample with a lower degree of cross-linking (sample FM.R3V1, cross-linker content of 0.7 v/v%), reduced structuring with a morphology in the form of rods with random distribution appears. In the sample FM.R3V3, in which the degree of cross-linking is much higher (7 v/v%), an advanced structuring appears Gels 2023, 9, 528 9 of 15 with a floral appearance, and the average surface roughness is almost double that of the sample FM.R3V1 ( Figure 6). The increase in the degree of organizational structuring in the sample with higher cross-linking density shows the cross-linking effect on the polymer by decreasing the molecular mass of the polymer chains between two neighboring crosslinking centers. The decrease in the mobility of the macromolecular chains by increasing the cross-linking density in the FM.R3V3 sample led to a much more complex supramolecular morphology. This effect was determined by the process of orientation and stabilization by physical bonds of the macromolecular chains of XG in freezing conditions with the increase in the density of physical interactions as an effect of the proximity of the chemically cross-linked macromolecular chains.

Mechanical Properties
The strength and uniaxial compression tests on the cryogel samples were performed using Shimadzu mechanical testing equipment (EZ-LX/EZ-SX Series) using the parallel plate measuring system. Wet samples of approximately 5-8 mm length, 6-8 mm width, and 4-5 mm height were placed between the plates, and to ensure complete contact between the sample and the plate surface, an initial force of 0.02 N was applied. The imposed strain rate was 0.2 mm/min, and the applied force was fixed at 100 N. The uniaxial compressive stress (σ, N/m 2 ) and strain (ε) were determined according to a previously presented method [21]. The mechanical properties of XG-based cryogels were evaluated, following the influence of the degree of cross-linking and the type of cross-linker. The stress-strain profiles registered for these biomaterials are shown in Figure 7.

Swelling Properties
The capacity of XG-based polymer networks to swell in aqueous medium was determined by the gravimetric method (Figure 8). The swelling behavior of the cryogels As can be seen from the stress-strain curves registered for the XG-based cryogels, the addition of a higher content of cross-linker induces a stiffening of the matrix due to the creation of more cross-linking points. XG-based cryogels cross-linked with 1,4-butanediol diglycidyl ether (R2) showed the best elasticity, sustaining a maximum deformation of 97.67%, 90.10%, and 81.80%. The cryogels obtained with R2 showed the highest compressive strength with values between 470.58 kPa and 658.84 kPa (Figure 7, Table 3). The modulus of elasticity was calculated from the slope of the linear dependence of the stress-strain curves at a strain between 0 and 10%, and the results obtained are included in Table 3. The values of the modulus of elasticity increased with the increase of the cross-linker content in the samples.

Swelling Properties
The capacity of XG-based polymer networks to swell in aqueous medium was determined by the gravimetric method ( Figure 8). The swelling behavior of the cryogels was evaluated by immersing the dry samples (approx. 0.01 g) in distilled water and weighing them at well-established time intervals.

Swelling Properties
The capacity of XG-based polymer networks to swell in aqueous medium was determined by the gravimetric method ( Figure 8). The swelling behavior of the cryogels was evaluated by immersing the dry samples (approx. 0.01 g) in distilled water and weighing them at well-established time intervals. The swelling kinetics of polymer networks based on XG are influenced by a number of factors, such as chemical composition and types of components; their sensitivity to changes in the analysis environment; the presence of pores, their distribution, and diameter; and the specific contact area with the environment. From the swelling profiles shown in Figure 8, it can be clearly observed that the cross-linking ratio and the presentation form of the XG The swelling kinetics of polymer networks based on XG are influenced by a number of factors, such as chemical composition and types of components; their sensitivity to changes in the analysis environment; the presence of pores, their distribution, and diameter; and the specific contact area with the environment. From the swelling profiles shown in Figure 8, it can be clearly observed that the cross-linking ratio and the presentation form of the XG matrix has a major influence on the water retention capacity. Thus, it was highlighted that with the increase in the amount of cross-linking agent, the polymer matrix absorbs less water as a result of the increase in the cross-linking density. In addition, the glycerol diglycidyl ether cross-linked XG-based polymer networks synthesized as films ( Figure 8C) retained the smallest amount of water due to the much more compact pore wall structure as observed via SEM microscopy analysis (Section 2.3, Figure 5).

Conclusions
Ice-templated hydrogels based on XG were successfully prepared regardless of the cross-linking agent used.
The XG-based materials exhibited a heterogeneous morphology with interconnected pores characteristic of gels obtained by cryogelation; the average pore size depends on both the nature of the cross-linker and the cross-linking ratio.
The influence of both the type and amount of cross-linker and the form of presentation of the matrix (MS, MN, or FM) on the degree and kinetics of swelling was observed; the glycerol diglycidyl ether cross-linked XG-based polymer networks synthesized as films retained the lowest amount of water due to the much more compact pore wall structure.
The addition of a higher content of cross-linker induces a stiffening of the matrix, due to the generation of more cross-linking points. In addition, 1,4-butanediol-diglycidyl-ethercross-linked XG-based polymer networks exhibited the highest elasticity.
Our results highlight that XG-based polymer networks with tailored properties in terms of mechanical strength, porosity, and swelling capacity could be properly developed by conducting the chemical cross-linking by cryogelation approach. This process allows for the preservation of the porous structure, which is advantageous for applications requiring high porosity and interconnected pore networks. The porosity of the XG-based cryogels was adjusted by modifying the nature of the cross-linker and the cross-linking ratio, providing control over the diffusion of substances through the material. This property is particularly relevant for applications like controlled drug delivery systems, where precise regulation of the release kinetics is required. The swelling capacity of the XG-based cryogels was controlled by the degree of cross-linking. This feature is crucial in tissue-engineering applications, as it allows for the absorption of water and nutrients, mimicking the natural environment required for cell growth and tissue regeneration. These materials offer versatility and can be tailored to meet the specific requirements of various applications, ranging from controlled drug delivery and tissue engineering to food packaging and separation processes. Further research and development in this area can lead to advancements in these fields and contribute to the design of novel functional materials.

Synthesis of XG-Based Hydrogels
XG-based hydrogels were prepared as microspheres, monoliths, and films by crosslinking XG with various cross-linkers below the freezing point of the reaction solution at −20 • C. The parameters varied in the preparation of XG cryogels were the type of cross-linker, the concentration of cross-linker, and the shape of the final material. Glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, and epichlorohydrin were used as crosslinkers in a concentration ranging from 0.7 v/v% to 7 v/v% (Table 1). An XG solution with a concentration of 1 wt% was first prepared. After that, each 5 g of XG solution (1%) was mixed with 1 mL of NaOH solution (2M) and the corresponding amount of crosslinker. The mixed solutions were cooled to 0 • C in an ice bath and kept under rigorous stirring about 10 min. Then, the mixtures were processed according to the desired form of presentation of the biomaterial. Thus, to prepare microspheres, the polymer/cross-linker mixture was introduced into 5 mL syringes equipped with a needle and then dropped into liquid nitrogen (LN, −196 • C). To obtain XG cryogel as monoliths, the polymer/cross-linker mixture was introduced into syringes with a diameter of 4 mm, while to synthesize XG cryogel as films, the sub-cooled reaction mixture was poured into Teflon Petri dishes. All samples were transferred to a freezer at a temperature of −20 • C for 24 h to complete the cross-linking irrespective of the shape of the final material. Thereafter, the samples were removed from the freezer and transferred in frozen state to lyophilizer for drying. After drying, all samples were purified by extensive washing with distilled water. Finally, the samples were dried again either via lyophilization or in an oven at 40 • C.

Gel Fraction Yield (GFY, %)
GFY was calculated as ratio between the weight of dried XG hydrogels (W d ) and the weight of all reactants involved in the cross-linking reaction (W m ):

FTIR
The FTIR spectra of XG-based hydrogels were recorded with a Bruker Vertex 70 FTIR spectrophotometer (Bruker, Ettlingen, Germany) in the range 4000-400 cm −1 , at a resolution of 2 cm −1 , using the KBr pellet technique.

Scanning Electron Microscopy (SEM)
The cross-sectional microstructure of the XG-based hydrogels was analyzed with a Quanta 200-FEI-type environmental scanning electron microscope (ESEM) (FEI Company, Hillsboro, OR, USA), at 20 kV in low vacuum mode. To reveal their internal morphology, a sharp blade was used to obtain cross-sections through the monoliths, microspheres, and films. The thickness of XG-based hydrogels as films was evaluated using a Vorel15240 stainless hardened digital caliper [54]. The results were reported as the mean value of five readings from different regions of each film.

Atomic Force Microscopy (AFM)
The 2D and 3D surface images for the XG-based hydrogels were collected using a NTEGRA scanning probe microscope (NT-MDT Spectrum Instruments, Moscow, Russia) in atomic force microscopy (AFM) configuration. Rectangular silicon cantilevers NSG 03 (NT-MDT, Russia) with tips of height aspect ratio were used to scan the film surfaces. The AFM images and the surface parameters were processed and calculated using Nova v.19891 for Solver Software [55].

Mechanical Tests
The uniaxial compression tests were performed on equilibrium-swollen hydrogels. A Shimadzu Testing Machine (EZ-LX/EZ-SX Series, Kyoto, Japan) was used to uniaxially compress the samples cut in form of the plates with a length of 5-8 mm, width of 6-8 mm, and height of 4-5 mm. A force of 100 N and a crosshead speed of 0.2 mm/min were used to analyze all samples. The strain (ε), the elastic modulus (G, kPa), and the compressive nominal stress (σ, kPa) were determined according to previously reported procedures [29,45].

Water Uptake
The swelling kinetics of XG-based hydrogels was gravimetrically studied by soaking a defined amount of dried sample (0.01 g) in 10 mL aqueous solutions. The water uptake (WU, g/g) was calculated as: where W t and W d are the weights of swollen hydrogels at time t and when dried, respectively.