*Article* **Preparation and Properties of Egg White Dual Cross-Linked Hydrogel with Potential Application for Bone Tissue Engineering**

**Bingchao Duan \*, Minghui Yang, Quanchao Chao, Lan Wang, Lingli Zhang, Mengxing Gou, Yuling Li, Congjun Liu and Kui Lu \***

> School of Chemical Engineering and Food Science, Zhengzhou University of Technology, Zhengzhou 450044, China

**\*** Correspondence: duanbc100@163.com (B.D.); lukui126@126.com (K.L.)

**Abstract:** In this study, an egg white dual cross-linked hydrogel was developed based on the principle that the external stimulus can denature proteins and cause them to aggregate, forming hydrogel. The sodium hydroxide was used to induce gelation of the egg white protein, subsequently introducing calcium ions to cross-link with protein chains, thereby producing a dual cross-linked hydrogel. The characteristics of the dual cross-linked hydrogels—including the secondary structure, stability, microstructure, swelling performance, texture properties, and biosafety—were investigated to determine the effects of calcium ion on the egg white hydrogel (EWG) and evaluate the potential application in the field of tissue engineering. Results showed that calcium ions could change the β-sheet content of the protein in EWG after soaking it in different concentrations of CaCl2 solution, leading to changes in the hydrogen bonds and the secondary structure of polypeptide chains. It was confirmed that calcium ions promoted the secondary cross-linking of the protein chain, which facilitated polypeptide folding and aggregation, resulting in enhanced stability of the egg white dual cross-linked hydrogel. Furthermore, the swelling capacity of the EWG decreased with increasing concentration of calcium ions, and the texture properties including hardness, cohesiveness and springiness of the hydrogels were improved. In addition, the calcium cross-linked EWG hydrogels exhibited biocompatibility and cell-surface adhesion in vitro. Hence, this work develops a versatile strategy to fabricate dual cross-linked protein hydrogel with biosafety and cell-surface adhesion, and both the strategy and calcium-egg white cross-linked hydrogels have potential for use in bone tissue engineering.

**Keywords:** egg white hydrogel; dual cross-linking; metal ions; secondary structure; biocompatibility
