**1. Introduction**

Natural macromolecules are a kind of polymer, existing in plants and animals, including the human body. They include polysaccharides, peptides, polynucleotides, and polyesters [1]. Natural polymers are an abundant resource for applications in the food and medical industries, due to that they have many advantages such as good biocompatibility, biodegradability, non-toxicity, and sustainability [2,3]. Hydrogel is a kind of three-dimensional network material. Hydrogels prepared from natural polymers should inherit the advantages of natural macromolecules, such as biocompatibility and biodegradability [4]. Thus, they are attracting attention particularly in biotechnology for uses such as drug delivery [5], biological sensing [6], wound dressing [7], and desalination [8,9].

Protein is a kind of natural polymer, abundant in nature, and with great prospects in polymer research [10,11]. In the field of polymer material science, the development of protein hydrogels is an important direction of current research [12–14]. Protein hydrogels retain a three-dimensional network structure similar to the extracellular matrix of animal tissue, featuring high water content. Hydrogels based on the proteins collagen [15], fibrin [16], elastin [17], and silk [18] have exhibited superior characteristics as compared

**Citation:** Duan, B.; Yang, M.; Chao, Q.; Wang, L.; Zhang, L.; Gou, M.; Li, Y.; Liu, C.; Lu, K. Preparation and Properties of Egg White Dual Cross-Linked Hydrogel with Potential Application for Bone Tissue Engineering. *Polymers* **2022**, *14*, 5116. https://doi.org/10.3390/polym 14235116

Academic Editors: Jingpeng Li, Yun Lu and Huiqing Wang

Received: 26 September 2022 Accepted: 19 November 2022 Published: 24 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to synthetic polymer-based hydrogels, such as biocompatibility, biodegradability, and low immunogenicity. Thus, these natural hydrogels are being extensively used for tissue engineering of bone and cartilage and in other biomedical application [19–22].

Egg white protein has a variety of functional characteristics, such as gelation, water holding capacity, foaming and emulsification. It is important in food manufacturing because it can improve the functionality, texture and flavor of food products [23]. Under heating, freezing, high pressure, extreme acid and alkali, ions, enzyme and other treatments, egg white protein is able to coagulate and form hydrogel with a three-dimensional network structure, which can strongly affect the structures, senses and flavors of egg white protein products [24,25]. The formation of protein hydrogels can be attributed to the formation of hydrogen bonds, disulfide bonds and electrostatic interaction, which induce the aggregation of protein molecules [26]. Proteins undergo denaturation in response to certain external stimuli (e.g., physical factors such as heating, ultraviolet light and pressure or pH, metal ions), and denaturation is one of the important mechanisms in the formation of protein hydrogels. The gelation of egg white protein involves multiple processes, including denaturation, aggregation and formation of gel network, and the gelling properties mainly depend on the medium conditions such as pH, ionic strength and salt type [27]. The interaction and the molecular conformation of protein chains will change during protein hydrogel formation. Studies have revealed that metal ions are able to impact the intermolecular and intramolecular interactions of protein chains and the conformation of protein molecules, further affecting the characteristics of protein gels [28,29].

In recent years, hydrogel materials have been extensively used in bone tissue engineering [30,31]; however, the application of protein hydrogels in bone tissue restoration and bone scaffold are severely limited due to their poor mechanical property [32]. Currently, tough and strong hydrogels are achieved by building dual network structures, compositing inorganic nanoparticles and introducing conductive materials and fibrous networks, improving the mechanical properties and the bone tissue repair ability of hydrogels [32]. Traditional hydrogels are fabricated through a single cross-linked mode, resulting in the lack of the energy dissipation pathways. Meanwhile, the dual crosslinking can improve the intermolecular interaction and cross-linking density in the hydrogel, providing an effective way to dissipate energy and increase the mechanical strength [33]. In this study, we prepared egg white hydrogel (EWG) according to the principle that proteins could be denatured by strong alkali, subsequently introducing calcium ions to induce aggregation and cross-linking with protein chains, thereby producing a dual cross-linked hydrogel (Figure 1a). Moreover, the secondary structure, stability, microstructure, swelling performance, texture and biocompatibility of the obtained hydrogels were investigated to research the effect of calcium ion on EWG.

**Figure 1.** (**a**) Schematic of the EWG hydrogel preparation process; (**b**) photos of the hydrogel immersed in different concentrations of CaCl2 solution. The number represents the concentration of CaCl2 solution; (**c**) FT−IR spectra of the hydrogels.

#### **2. Materials and Methods**

#### *2.1. Materials*

Sodium hydroxide (NaOH) and calcium chloride (CaCl2) were provided by Aladdin (Shanghai, China). Fresh eggs were purchased from a supermarket. The egg white was carefully separated from the yolk and then kept in a sealed chamber at 4 ◦C until further use. The Dulbecco's Modified Eagle's Medium (DMEM; glutamine, high glucose), fetal bovine serum (FBS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and the cell staining agent (Calcein AM and PI) were obtained from Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China. All other chemicals were of analytical grade and used without further purification.

#### *2.2. Preparation of the Egg White Hydrogel*

NaOH solution (25 mg/mL) was added dropwise into the egg white (3:7 volume ratio) at room temperature with gentle stirring, then the solution was transferred to a mold (60 mm × 15 mm × 40 mm) and kept stationary 4 ◦C to release any air bubble present in the solution. After gelling, the egg white hydrogel was obtained and washed thoroughly with pure water to remove the residual sodium hydroxide.

CaCl2 was dissolved in deionized water to obtain the sample solution at different concentrations. The egg white hydrogel samples (the samples were prepared in the size of 15 mm × 15 mm × 50 mm with a knife) were immersed in CaCl2 solution (0.1%, 0.5%, 1%, 2%, 3% *w*/*v*), respectively, for 2 h at room temperature. After soaking, the hydrogel samples were taken and washed with pure water to remove the calcium chloride on the surface. The hydrogel samples soaked by pure water and CaCl2 solution by 0.1%, 0.5%, 1% and 2% (*w*/*v*) were coded as EWG0, EWG1, EWG2, EWG3 and EWG4, respectively.

#### *2.3. Characterization*

The wet hydrogels were frozen in liquid nitrogen and snapped immediately, then freeze-dried using a vacuum freezing dryer (LEG-10C, Sihuan Furui Technology Development Co., Ltd., Hong Kong, China). The freeze-drying conditions were as follows: vacuum: 1Pa; cryo-temperature: −70 ◦C; material temperature: −50 ◦C; duration of drying: 24 h. The fracture sections of the freeze-dried samples were sputtered with gold for scanning electron microscopy (SEM, Zeiss, SIGMA, Roedermark, Germany) analysis.

The hydrogel samples were cut into particle-like size after being freeze-dried and vacuum-dried for 2 h at 60 ◦C. Then samples were transfer to a mortar and grind to a fine powder for measurements. The structural changes of the hydrogels were characterized by Fourier transform infrared spectroscopy (FT−IR, Spectrum3, Perkin Elmer, Waltham, MA, USA), X-ray Diffraction Analysis (XRD, Regaku ultima IV, Japan). The data of amide I band (1700–1600 cm<sup>−</sup>1) of FT-IR spectrum were analyzed by PeakFit software.

#### *2.4. Swelling Tests*

The thermal stability of the hydrogels was investigated by thermogravimetric analysis (TG, Discovery TGA 550, New Castle, DE, USA). The analysis was performed from room temperature to 800 ◦C with a heating rate of 15 ◦C/min in air atmosphere.

The swelling ratios of the hydrogels in pure water were tested through the gravimetric method. The freeze-dried hydrogels were immersed into pure water at 37 ◦C for modelling the body temperatures. Then the water in the surface of the hydrogels was gently wiped, and the weight of the samples was registered at predefined period. The swelling ratio (SR) was calculated as SR = (Ws − Wd)/Wd, where the Ws and Wd are the weight of the swollen and dried hydrogel, respectively. Similarly, the porosity ((Ws − Wd)/Ws) of different hydrogels after achieving swelling equilibrium was determined by using the identical measurement method of the mass swelling ratio [34].

#### *2.5. Texture Tests*

Texture analysis was performed using a texture analyzer (TA, BROOKFIELD CT3, USA) at room temperature. The hydrogel samples were prepared in the size of 10 mm × 10 mm × 15 mm with a knife, and the cylindrical probe TA4/1000 cylinder was selected for measurement. The clipped samples were compressed twice at 1 mm/s to 50% of their original height. The results were calculated with Texture Expert version 1.22 (Stable Micro Systems, Surrey, UK). All of these steps were performed six times.

#### *2.6. Cell Experiments*

HEK293 cells (human embryonic kidney-293 cells) were obtained from the China Center for Typical Culture Collection and cultured at 37 ◦C in a 5% CO2 incubator. The culture medium containing 89.20% DMEM with 98 μg/mL penicillin/Streptomycin, 9.80% fetal bovine serum was changed every 2 days.

The cytotoxicity of the hydrogels was determined via an MTT assay. The hydrogel samples (100 mg) were sterilized under a UV lamp for 24 h, and then placed in DMEM containing 9.80% fetal bovine serum for 24 h at 37 ◦C to prepare the hydrogel extract solution. Thereafter, the resulting extracts were filtered using a 0.22-mm syringe for MTT tests. HEK293 cells (5 × 103 cells/well) were incubated in 96-wells plates for 12 h, and the extract solution of different hydrogels was added for 24 h incubation. Then the medium was aspirated and 10 μL of MTT (5 mg/mL) was added to each well for another 4-h incubation. After the culture medium was removed, 200 μL of DMSO was added in each well. After gently shaking for few minutes, the plates were carried out to measure the absorbance at 492 nm using a microplate reader (SparkTM 10M, Tecan). The cell viability was calculated based on the equation: Cell viability (%) = (the absorbance of sample-treated group/the absorbance of PBS-treated group) × 100%.

The live/dead cell staining assays were performed to evaluate the cell adhesion on the hydrogel surfaces and the biocompatibility. The hydrogel EWG4 sample with 10 mm diameter and a thickness of about 0.1 cm was sterilized under a UV lamp for 24 h, and then transferred to the bottom of 24-well plastic culture plates. HEK293 cells were seed to 24-well plastic culture plates on the sample (5 × <sup>10</sup><sup>4</sup> cells/well) for 48 h incubation. Then, the samples were washed by physiological saline and stained by the cell staining agent (Calcein AM and PI) for 30 min at 37 ◦C. Thereafter, the samples were washed again for three times with physiological saline and then observed using a fluorescence microscope (Olympus BX51, Olympus Corporation, Tokyo, Japan).

#### *2.7. Statistical Analysis*

All measurements were repeated at least three times independently and expressed as mean ± standard deviation (SD). All values reported in this study are expressed as mean ± standard deviation, and *p* < 0.05 (\*), *p* < 0.01 (\*\*) and *p* < 0.001 (\*\*\*) signify significant and extremely significant differences, respectively.

#### **3. Results and Discussion**

#### *3.1. Preparation and Morphology Analysis of the EWG Hydrogel*

There are a variety of proteins in fresh egg white solution. In the liquid state, these proteins are stabilized via electrostatic interaction, hydrogen bonds and thiol ester bonds between protein chains. However, when heated or exposed to high pressure, alkali or acid, these chains are induced to unfold and rearrange into various forms. In this work, the egg white hydrogel was prepared by alkali induction and it was transparent, light yellow, smooth and ductile. Then, the egg white hydrogel was soaked in calcium chloride solution with different concentrations in order to induce secondary cross-linking between the calcium ions and the egg white hydrogel through the interaction of calcium ions with the particular amino acids on the polypeptide chain. As shown in Figure 1b, the hydrogel samples remained solid without breaking or decomposition after soaking in calcium chloride solution for 2 h. With increasing concentration, more calcium ions gradually infiltrated into the hydrogel, leading to deeper color and decreased transparency of the hydrogels.
