Study On Preparation And Promotion Of Wound Healing Of Ovalbumin/Gelatin Composite Hydrogel

Ovalbumin（OVA）accounts for about 54%of the total protein in egg whites.At present,the research and application of OVA mainly focus on emulsifying properties,gel properties or foaming properties.However,in addition to the above food-related functional properties,OVA also has the function of accelerating hemoglobin synthesis,promoting metabolism and growth,and accelerating wound healing.Therefore,using OVA as substrate to develop new biological materials is of great significance for the high-value utilization of egg white proteins.In this study,OVA derived from egg white was used as raw material to construct a series of ovalbumin composite hydrogels.The effects of different protein ratios on the microstructure,gel properties and biocompatibility of hydrogels were explored.Meanwhile,the therapeutic effects of ovalbumin based hydrogels on different types of wounds were systematically studied.This study aims to provide new research ideas and theoretical basis for the application of protein-based hydrogels in the field of biomedicine.The primary research contents and results are as follows:（1）The thiolated ovalbumin（t OVA）was obtained by chemical modification method.Then,the SDS-PAGE,free sulfhydryl group determination and internal fluorescence spectrum detection were used to characterize the t OVA.Ultimately,the amount of modifier added was determined to be 50×mole excessive.The thiolated gelatin（t Gel）was prepared by the same modification method as ovalbumin.The thiolated t OVA and t Gel were obtained by chemical modification.The t OVA/t Gel@Ag hydrogels were prepared according to the coordination principle,which named O10,O7G3,O5G5,O3G7and G10 respectively.Fourier transform infrared（FTIR）spectra confirmed that the sulfhydryl group coordinated with silver ions to form-SAg bond.A reasonable ratio of t OVA and t Gel makes hydrogels with orderly three-dimensional structure,compact and uniform pore size and typical gel characteristics.X-ray diffraction（XRD）results showed that the silver ions were evenly dispersed in the hydrogel network structure and well bonded with the protein.The antibacterial results of t OVA/t Gel@Ag hydrogels were consistent with the cumulative release of silver ions.O3G7 hydrogel has the best antibacterial effect（the diameters of antibacterial circles against Escherichia coli and Staphylococcus aureus were 10.67 mm and 10.5 mm,respectively）.Acridine orange/ethidium bromide（AO/EB）staining and CCK-8 toxicity test proved that the hydrogels had no cytotoxicity to L929 cells.In addition,the hemolysis rates of t OVA/t Gel@Ag hydrogels were below 5%.The results of L929 cells and hemolysis tests indicate that t OVA/t Gel@Ag hydrogels have good in vitro compatibility.（2）The full-thickness wound model on the dorsum of male mouse was established,and t OVA/t Gel@Ag hydrogels was applied to acute wound treatment.The pictures of wound and wound healing rate at different time points showed that on day 9 after wound modeling,the wound healing rate of O5G5 and O3G7 group was higher than that of the control group,reaching 92.79%and 96.23%,respectively.At the same time,the increase of total protein（TP）content indicated that the hydrogels promoted the growth of granulation tissue.According to the determination of HYP,O3G7 hydrogel had significantly increase collagen content,which is consistent with Masson staining and immunohistochemical analysis of col-Ⅰ.Hematoxylin and eosin（H&E）staining results showed that hair follicle tissue and intact epidermis could be observed in O3G7 group on day 9.The results of immunofluorescence and immunohistochemistry showed that hydrogel treatment reduced the expression of tumor necrosis factor-α（56.87%of the control group）and up-regulated the expression of transforming growth factor-β（1.29times of the control group）.These results indicate that O3G7 hydrogel had a significant promoting effect on acute wound healing.In addition,histopathological sections of the main organs of mice showed no pathological damage or morphological abnormalities in the organs after hydrogel treatment,indicating that hydrogel would not cause acute toxicity to mice.This study proves that hydrogels prepared from food-derived proteins would be promising candidate materials in the field of bio-engineering..（3）Among many types of modern wound dressings,hydrogels have distinct advantages in the treatment of local scald.In order to further evaluate the effect of O3G7on burn wound,deepⅡdegree burn models were established on the dorsum skin of mouse.The experimental results showed that O3G7 hydrogel could accelerate the burn wound healing.After 14 days,the burn wound healing rate reached 97.30%.At different time points(3^rd,7^th and 14^th day),wound tissue was collected for TP and HYP content detection,H&E staining,masson’s trichrome（MT）staining,immunofluorescence analysis and immunohistochemical analysis.The results show that O3G7 hydrogel increases the content of TP and HYP,effectively prevent the expansion of burn wounds caused by infection in the early phase.In addition,O3G7 up-regulated the expression of VEGF and down-regulated the expression of CD68.Therefore,this chapter provides theoretical guidance for the preparation of multifunctional protein-based hydrogels.O3G7hydrogel could inhibit infection,promote collagen formation,accelerate angiogenesis and reepithelialization,and contribute to faster repair of the burn wounds.