Protective Effects Of New Glycyrrhizin-based Hydrogel Dressings On Acute Wounds

Objective The untimely treatment of acute wounds will increase the risk of body infection,and has become a common problem bothering emergency surgeons.The purpose of this study was to design a hydrogel dressing that could immediately and effectively protect the acute defect wounds on the body surface in the emergency treatment of multiple injuries,and to deeply explore its gelling mechanism,micro-morphology,mechanical properties and biological activity.Methods（1）After oxidation by NaIO₄,glycyrrhizinic acid（GA）was fully reacted with carboxymethyl chitosan（CMCS）to determine the optimal combination of oxidation degree and crosslinking concentration by determining the gelling result and gelling time.（2）The material properties of the hydrogel were characterized by Fourier transform infrared spectroscopy,scanning electron microscope（SEM）,and advanced rotational rheometer.（3）After L929 mouse fibroblasts were co-cultured with the hydrogel extract for a period of time,the biocompatibility of the hydrogel was detected by living/dead cell staining and cell counting kit-8（CCK-8）.（4）The antibacterial activity of the hydrogel against staphylococcus aureus（S.aureus）and escherichia coli（E.coli）was tested by the inhibition zone method and OD value counting method.（5）Sprague-Dawley（SD）rat liver injury model was used to detect the hemostatic time and bleeding volume of the hydrogel.（6）Twenty-four SD rats were randomly divided into a control group（not covered）,a positive control group（Alginate dressing for clinical use）,a CMCS dressing group and an OGA-CMCS hydrogel dressing group,with six rats in each group.After the rat model of full-thickness skin defect was established,the wound closure of each treatment group at a specific time point was recorded by photographing,and the rats were sacrificed in batches on the 7th and 14th days,and the fresh wound tissue was removed.（7）The morphological changes of new tissues after treatment were observed by hematoxylin-eosin（HE）and Masson staining.（8）The expressions of platelet-endothelial cell adhesion molecule-1（CD31）andα-smooth muscle actin（α-SMA）in the neonatal tissue of wounds in each treatment group were measured by immunofluorescence double staining.（9）The expression levels of macrophage markers inducible nitric oxide synthase（iNOS）,CD68 and CD163 in the wound neogenesis tissues of all the treatment groups were detected by immunofluorescence staining.（10）The expression levels of inflammatory cytokines interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）,interleukin-4（IL-4）and interleukin-10（IL-10）in the new tissues of wounds in each treatment group were detected by enzyme-linked immunosorbent assay（ELISA）.Results（1）After screening of the prepared OGA-CMCS hydrogels,three groups of hydrogels with the molar ratio of NaIO₄/GA of 1:1,1.5:1,and 2:1 at the concentration of4%OGA+6%CMCS were selected for subsequent experiments.（2）Material characterization showed that a new absorption peak appeared in OGA-CMCS hydrogel at the wavelength of 1599 cm^-1,and a uniform porous structure was observed inside.The hydrogel had low swelling rate,good rheological properties,self-healing ability and injectability.（3）In vitro cell experiments showed that the cell viability of the three groups of hydrogel extracts after 48 h co-culture with L929 mouse fibroblasts was up to 90%,showing the excellent biocompatibility of hydrogel.（4）The antibacterial experiment showed that OGA-CMCS hydrogel had obvious antibacterial effect on S.aureus and E.coli when incubated with bacteria for 12 h.（5）The hemostatic time and bleeding volume of OGA-CMCS hydrogel in vivo were significantly lower than those of the blank group and the gauze treatment group（P<0.05）.（6）Compared with other treatment groups,the OGA-CMCS hydrogel dressing group had a more significant effect in promoting wound closure（P<0.05）.（7）Compared with other treatment groups,the collagen volume fraction on the 7th day after surgery in the OGA-CMCS hydrogel dressing group was increased（P<0.05）.On the 14th day after surgery,the thickness of granulation tissue was higher than that of control group（P<0.01）,and the volume fraction of collagen was significantly increased as compared with those of control group and Alginate dressing group（P<0.01）.（8）On the 14th day after surgery,the fluorescence densities of CD31 andα-SMA in the wound nascent tissues of OGA-CMCS hydrogel dressing group were higher than those of other treatment groups（P<0.05）.（9）On the 7th day after surgery,the average optical density of iNOS in the OGA-CMCS hydrogel dressing group was significantly lower than that in the control group and Alginate dressing group（P<0.01）.The percentage of CD163/CD68 positive cells was significantly increased in comparison with other treatment groups（P<0.01）.（10）Compared with the control group,the expression levels of pro-inflammatory factors IL-6 and TNF-αin the OGA-CMCS hydrogel dressing group were significantly reduced（P<0.001）,while the expression levels of anti-inflammatory factors IL-4 and IL-10 were significantly increased（P<0.001）.Conclusion（1）The oxidized GA and CMCS can form a gel system through schiff base reaction,and the hydrogel has the advantages of high gelling speed and uniform internal pores.（2）OGA-CMCS hydrogel have good adhesion,viscoelasticity,swelling property,self-healing property and injectability.（3）OGA-CMCS hydrogel has excellent biocompatibility,antibacterial property and rapid hemostatic property.（4）OGA-CMCS hydrogel dressing could effectively promote the rapid closure of the wound surface with acute defects on the body surface,accelerate the growth of granulation tissue and collagen deposition,induce angiogenesis,promote the polarization process of macrophages from the pro-inflammatory（M1 type）to the anti-inflammatory（M2 type）in the wound surface,and inhibit the expression of inflammatory factors in the wound surface.Therefore,in this study,a novel glycyrrhizic acid-based hydrogel dressing was prepared for the first time,which provided a new idea and experimental basis for the emergency treatment of acute wounds on the body surface.