*3.2. FT*−*IR Analysis*

The structural characteristics and conformational changes of the hydrogels were evaluated by Fourier transform infrared spectroscopy (FT−IR). As shown in Figure 1c, the absorption peaks at ~3350 and 2953 cm−<sup>1</sup> were assigned to the stretching vibrations of N–H and O–H and of intermolecular hydrogen bonds, respectively [35]. With the increase of calcium concentration, the absorption band at 3436.2 cm−<sup>1</sup> gradually shifted to 3292.3 cm−<sup>1</sup> and the intensity of peaks at 2953 cm−<sup>1</sup> increased significantly, suggesting that the introduction of calcium ions affected the hydrogen bonds between the amino and hydroxyl groups in the polypeptide chain and the protein molecular chain. The typical absorption peaks at 1656 and 1533 cm−<sup>1</sup> were related to the amide I (C=O stretching) and amide II (N–H bending) modes of the protein chain structure, respectively [36]. The intensity changes of the peaks at 1533.5 cm−<sup>1</sup> indicated that the amino and hydrogen bond in the protein molecular were impacted by calcium ions. Previous studies [35] have demonstrated that the amide I band (1700–1600 cm−1) is the most characteristic spectral region related to the secondary structure of proteins and polypeptides, and the secondary structures and conformational changes could be investigated by quantitative analysis of the amide I band. Therefore, the absorption peak of this region was used to quantitatively calculate the specific proportion of each secondary structure (α-helices 1650–1658 cm−1, β-sheets 1640–1610 cm<sup>−</sup>1, β-turns 1700–1660 cm<sup>−</sup>1, random coils 1650–1640 cm−1) [35,37]. The results showed (Table 1 and Figure S1) that the β-sheets content of the hydrogel increased significantly as calcium ion concentration increased up to 1%, and then decreased at higher calcium concentrations. It had been indicated that the structure of β-sheets is prone to protein aggregation and particularly important for the hydrogel formation and stability [37]. The interaction of calcium ions and protein molecules could facilitate the polypeptides to fold the β-sheets; however, the cross-linking between calcium ion and protein chains along with the increasing of calcium ion concentration was dominated and disturbed gradually the hydrogen bonding in β-sheets structures of the protein, leading to the reduce of the β-sheets content in the hydrogels soaking with high concentrations of CaCl2 solution. Meanwhile, the original conformation of EWG0 was changed after introducing calcium ion, resulting in the decrease of β-turns content and the increase of random coil structure. In addition, the changes of the band at 1030–1090 cm−<sup>1</sup> assigned to C–O stretching vibration indicated that the structure of the polypeptide chain was changed by the calcium ions. Together, the results suggested that calcium ions were able to interact with the polypeptides and change secondary structure of the protein in the hydrogel.


**Table 1.** Effect of CaCl2 addition on the secondary structures of egg white protein.

#### *3.3. XRD Analysis and Thermal Stability*

To further study the influence of calcium ions on hydrogel structure, the hydrogels were characterized by XRD, TG and DTG. The XRD spectra of the EWG0, EWG1, EWG2, EWG3 and EWG4 are shown in Figure 2a. The hydrogels exhibited distinct peaks at 2θ = 20◦, which was indicated to the β-sheets secondary structure of the egg white protein. The XRD diffraction intensity of the EWG1 (5885) and EWG2 (5713) was enhanced in comparison with that of EWG0 (5233), and then the intensity decreased along with the increase in the concentration of calcium ions (EWG3:4378; EWG4:3096), confirming that the β-sheets structure in the crystalline region of the protein was disturbed by the interaction of protein with calcium ions [38,39], which was consistent with the FT−IR results.

**Figure 2.** XRD patterns (**a**), TG curves (**b**) and DTG patterns (**c**) of hydrogels.

TG and DTG analyses were applied to investigate the thermal properties of the hydrogels. The TG curves (Figure 2b) revealed that the weight loss of EWG4 (17.3%) was higher than that of EWG0 (9.8%), and the temperature corresponding to the endothermic peak on the DTG curve of the hydrogels cross-linked by calcium ions (EWG1, EWG2, EWG3 and EWG4) (Figure 2c) was increased as compared with EWG0, indicating the water retention capacity and thermal stability of the hydrogel was enhanced after interacting with calcium ion. The second stage of weight loss was mainly due to the breaking of unstable non-covalent bonds of the protein chains and the covalent bonds of the small molecules in the protein backbone. As more calcium ions were added, the hydrogel exhibited an increased degradation temperature and decreased weight loss rate. Meanwhile, the weight loss decreased from 50.767% (EWG0) to 43.926% (EWG4), which was indicative of enhanced thermal stability. The explanation for this pattern is that the interaction of calcium ions with protein promoted secondary cross-linking of the protein chain and increased hydrogel cross-linking density, which could effectively inhibit heat conduction, thereby hindering thermal degradation of protein skeleton, and enhancing the thermal stability of the hydrogel [40,41]. In summary, the results indicated that the dual cross-linked structure involving the calcium ion formed a heat-stable system, which translated into improved stability of the hydrogel.

#### *3.4. Microscopic Examination*

SEM was used to study the microstructure changes of the hydrogels. As shown in Figure 3, EWG0 exhibited a loose and homogeneous three-dimensional structure with

lots of pores. It had been reported that protein molecule chains were able to unfold and rearrange themselves to form an ordered three-dimensional protein network under strong alkaline condition [42]. After soaking in calcium chloride, the hydrogels exhibited inhomogeneous and rough microstructure with the disordered porous structure. When the calcium ion concentration reached 1.0% and 2.0%, the porous structure of EWG3 and EWG4 was significantly reduced, accompanied by the appearance of rough, flat edges and coarse fibers (Figure S2). The results suggested that the interaction with calcium cations induced the secondary crosslinking of the protein chains and improved the degree of crosslinking, leading to the formation of more compact microstructure of the hydrogel [43,44].

**Figure 3.** SEM images of the hydrogels. The scale bar is 10 μm.

#### *3.5. Effect of Calcium Ions on Hydrogel Swelling Performance*

The swelling performance of the hydrogels was strongly associated with hydrophilic groups and the pore network structure of the hydrogel, both of which are key to absorbing water. The swelling behavior of the hydrogel was tested in ultra-pure water. The results (Figure 4) show that the swelling rate increased rapidly with the extension of time and reached the swelling equilibrium after about 4 h. This pattern is common for hydrogels. Compared with the EWG0 group, EWG1, the hydrogel soaked in a low concentration of calcium ions (0.1%) showed a similar swelling rate, whereas EWG2, EWG3 and EWG4 showed gradual decreases with increasing concentration of calcium ions. The formation of the EWG0 hydrogel depended on the physical cross-linking with only weak binding between protein chains, giving water molecules easy access to the hydrogel interior and resulting in high expansibility. Moreover, it was found that the EWG0 hydrogel was gradually degraded and ruptured with extended soaking time, which was because the water molecules destroyed the three-dimensional structure with weak intermolecular forces [45]. It is well known that the degree of cross-linking directly affects the water absorption of a hydrogel [35]. When the egg white hydrogel was soaked in calcium chloride solution, calcium ions interacted with the amino acids of the protein chain to prompt the cross-linking and aggregation of the egg white protein and induce the formation of a tight three-dimensional network, resulting in significant decreases in water absorption and storage capacity of the hydrogels. Moreover, the cross-linked networks with tight structure could resist the destructive infiltration of moisture. The porosities of EWG0, EWG1, EWG2, EWG3 and EWG4 were 97.2 ± 2.34, 96.9 ± 3.16, 93.7 ± 4.24, 91.4 ± 4.94 and 82.4 ± 3.41, respectively (Figure S3). The porosities of the hydrogels decrease with increasing concentration of calcium ions, suggesting that the interaction between calcium ions and protein facilitate the cross-linking and aggregation of the protein chains, leading to the decrease of hydrogel porosity. It was found that the equilibrium swelling ratio of EWG0 was seven times higher than that of EWG4, indicating that the high concentration

of calcium ions could effectively reduce the swelling capacity of the original egg white hydrogel. Therefore, it has great potential for preparing the sensitive double-layer hydrogel actuators using the EWG0 and EWG4 as the humidity responder and humidity inert layer, respectively (Figure S4) [46].

**Figure 4.** (**a**) Swelling kinetics of the hydrogels in distilled water at 37 ◦C; (**b**) equilibrium swelling ratio of the hydrogels in distilled water as a function of CaCl2 concentration. \*\* *p* < 0.01, \*\*\* *p* < 0.001; (**c**) plots of ln(St/S∞) versus lnt; and (**d**) t/St versus t for the hydrogels.

Swelling kinetic of the hydrogels is evaluated by Schott's second-order diffusion kinetic model and Fickian diffusional kinetic model [47,48]. The swelling data achieved from the first 60% of the fractional water uptake are fitted with the following equation to determine water diffusion mechanism of hydrogel samples: ln(St/S∞) = lnk + nlnt, where St and S<sup>∞</sup> are the water uptake at time t and the equilibrium water uptake. The k parameter is a constant of the solvent-polymer system; the n parameter specifies the diffusion mechanism of water molecules. n < 0.5 indicates Fickian diffusion, 0.5 < n < 1 indicates non-Fickian diffusion and n = 1 indicates that the diffusion mechanism is case-II. Figure 4c shows the plots of ln(St/S∞) versus lnt, the slopes and intercepts of the plotted lines could be used to calculate n and k. The values of n for EW0 and EWG1 are close to 1, indicating that the water diffusion mechanism in EWG0 and EWG1 is case-II (relaxationcontrolled) transport. The values of n for EWG2, EWG3 and EWG4 are greater than 0.5, implying the water diffusion mechanisms are non-Fickian diffusion type. The water diffusion mechanism changes of the hydrogels are caused by the secondary cross-linking of the protein chain and the increased crosslinking density of hydrogels, which limit the protein chains relaxation and hinder the diffusion of water [49]. The Schott's secondorder diffusion kinetic model is used to get further information about the swelling rate: t/St = A + Bt, where A = <sup>1</sup> KsS<sup>2</sup> ∞ is the initial swelling rate of the hydrogel and Ks is the swelling rate constant, B = 1/S∞ is the converse of the equilibrium swelling. The plots of t/St versus t are plotted for the hydrogel samples (Figure 4d). The theoretical swelling equilibrium (shown in Table 2) of EWG0, EWG1, EWG2, EWG3 and EWG4 hydrogels are close to their corresponding experimental values. The swelling rate constants (Ks) of EWG3 and EWG4 are higher than that of EWG0, suggesting that the hydrogels with high crosslinking density possess the faster swelling rate and reach the swelling equilibrium in a shorter time, which is consistent with the experimental results.


**Table 2.** Second-order kinetic parameters for hydrogels.

ESR: experimental swelling equilibrium value.

#### *3.6. Effect of Calcium Ions on Hydrogel Texture*

Soaking the egg white hydrogel in different concentrations of calcium ions can change their microstructure, and, thereby, their properties. The textural properties of hardness, cohesiveness and springiness of the hydrogels were tested using a texture analyzer. As shown in Figure 5, the hardness of the hydrogels remained basically unchanged when the concentration of ions was less than 0.5% and when it was significantly enhanced as calcium ion concentration increased. Hardness is related to the structural strength of a hydrogel [50,51], and the overall structure of the protein is changed by the cross-linking of calcium ions with particular amino acids of adjacent peptides, resulting in the enhancement of the hydrogel hardness. Cohesiveness and springiness of the hydrogel were also affected by the addition of calcium. Previous studies [52,53] have shown that the conformation of proteins and polymerized protein chains are affected by divalent metal ions, leading to changes of the texture properties of protein hydrogels. In addition, cohesiveness and elasticity are influenced by the microstructure of the hydrogel. The SEM results showed that the introduction of calcium ions promoted a smaller three-dimensional pore structure and more compact microstructure of the hydrogel, which resulted in the enhancement of the cohesiveness and elasticity of the hydrogels. It could be found that the trends toward less hardness, cohesiveness and springiness of the hydrogels at low calcium concentration that might be because the calcium ions consumed hydroxyl ions, such that the threedimensional network structure of the original hydrogel could not be maintained. As calcium concentration increased, the equilibrium between calcium and hydroxide ions was reached, and the interactions of the redundant calcium with the particular amino acids of the peptide chains gradually dominated and impacted the texture properties of the hydrogels.

**Figure 5.** (**a**) Changes in hardness (**a**), cohesiveness (**b**) and springiness (**c**) of the hydrogels as a function of CaCl2 concentration. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### *3.7. Cytocompatibility*

To investigate the potential of the hydrogels in biomedical applications (e.g., wound healing and bone tissue repair requiring calcium ions [54]), the hydrogels were co-cultured with HEK-293 cells for assessing the cytocompatibility and cell adhesion on the hydrogel. The MTT results (Figure 6a) showed that EWG0 and EWG1 showed cell survival rates similar to the control. With the increase of calcium chloride concentration, the cell survival rates of EWG2, EWG3 and EWG4 groups decreased slightly but remained above 80%, indicating that all hydrogels possessed cytocompatibility. Compared to the egg-white/eggshell-based biomimetic hybrid hydrogels, the cells treated with EWG1 hydrogel for 24 h presented the similar proliferation rate [31], while the EWG2, EWG3 and EWG4 exhibited the lower proliferation rate, indicating that the high concentration calcium ions might be not advantageous to the cell proliferation. In addition, the live/dead cell staining assays (i.e., live cells stained fluorescent green, dead cells stained fluorescent red) were performed to study the cell adhesion and viability on the hydrogel surfaces. The results appear in Figure 6b. HEK-293 cells was able to survival normally and adhere to the EWG4 surface, demonstrating that the cross-linked hydrogels prepared with the highest calcium concentration were non-toxic, cytocompatible and adaptive for cell survival. In conclusion, calcium ion secondary cross-linked egg white gel showed excellent biocompatibility and biosafety. It should have great value for potential applications in the biomedical fields, particularly bone tissue engineering.

**Figure 6.** (**a**) Cell viability of HEK-293 cells on EWG0, EWG1, EWG2, EWG3 and EWG4 after 24 h culturing. (**b**) Live/dead staining florescent photographs of HEK-293 cells loaded with EWG4 for 48 h.

#### **4. Conclusions**

In summary, an egg white dual cross-linked hydrogel was prepared through the induction of sodium hydroxide and the secondary cross-linking of protein chains by calcium ions. Characteristics of the dual cross-linked hydrogel were remarkably affected by the concentrations of calcium ions. The incorporation of calcium ions could benefit thermal stability, swelling rate and texture of the hydrogels, while also reducing swelling capacity. Calcium ions could impact the secondary structure of polypeptide chains and interact with protein chains, leading to more compact microstructure formation of the hydrogels. Remarkably, the egg white dual cross-linked hydrogels exhibited biocompatibility and cell-surface adhesion in vitro, indicating the potential for biomedical application.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14235116/s1, Figure S1. Normalized FT−IR spectra of hydrogels. Each peak represents a different secondary structure. (α-helices 1650–1658 cm<sup>−</sup>1, β-sheets 1640–1610 cm<sup>−</sup>1, β-turns 1700–1660 cm<sup>−</sup>1, random coils 1650–1640 cm−1). Figure S2. SEM images of the hydrogels. The scale bar is 5 μm. Figure S3. The porosities of the hydrogels. Figure S4. The photo of self-bending double layer hydrogel. The upper layer (white) is the hydrogel that soaked in calcium chloride, the lower layer (pale yellow) is the hydrogel that soaked without calcium chloride. The double layer hydrogel exhibited a smaller curvature that could be duo to the inapposite gel thickness.

**Author Contributions:** Formal analysis, investigation, B.D., L.W., L.Z. and M.G.; writing—original draft preparation, B.D., M.Y. and Q.C.; writing—review and editing, Y.L., C.L. and K.L.; resources, B.D. and K.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Basic Research and Applied Basic Research Project of Zhengzhou Science and Technology Bureau (Grant No. zkz202111), the High-level Talent Fund start-up Project of Zhengzhou University of Technology (No. 22078), the National Natural Science Foundation of China (No. 21572046), the Science and Technology Breakthrough Plan of Henan Province (Grant No. 212102310857), the Key Projects of Henan Provincial High School (Grant No. 21B150020, 21A550013, 22B150021), the College Students Innovation and Entrepreneurship Project of Henan Province (No. S202111068004), the College Students Innovation and Entrepreneurship Project of Zhengzhou University of Technology (Preparation and properties of Ion-induced egg white gel).

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

