| Composite hydrogel dressing has attracted wide attention as a treatment method for skin burns.Hydrogel is a three-dimensional network structure formed through physical blending of several polymers,which can form stable moist environment in burn wounds,prevent bacterial infection,and effectively promote wound healing.This study aims at developing a composite plant essential oil hydrogel with excellent antibacterial properties.It has significant bacteriostatic effect against Gram-positive bacteria and negative bacteria,stable material structure,good biocompatibility,appropriate properties and can effectively promote the repair of burn wounds.In this study,a series of plant essential oil hydrogels were prepared by physical crosslinking plant essential oils(EEO,CEO and GEO)with carboxymethyl chitosan(CMC)/carbomer(CBM)hydrogel matrix.The antibacterial activity,composition,structure and physicochemical properties of the complex plant essential oil hydrogels were examined.The cell viability and cell proliferation ability of the hydrogel were determined by in vivo cell experiments,and the wound healing activity was further verified by the second degree burn model.The specific research results are as follows:(1)Screening of antimicrobial additivesThe screening results showed that plant essential oil(EEO,GEO and CEO)had significant antibacterial activity against E.coli and S.aureus.Then CBM/CMC,CBM/CMC/EEO,CBM/CMC/GEO and CBM/CMC/CEO were prepared in fixed proportion,and the antibacterial activity of four composite hydrogels was further analyzed by turbidimetric method,bacteriostatic circle method and microbial challenge test.The results showed that CBM/CMC/EEO,CBM/CMC/GEO and CBM/CMC/CEO were a kind of complex plant essential oil hydrogels with homogeneous composition,stable structure and good antibacterial activity.The turbidimetric method showed that all hydrogels bacteriostatic rate to S.aureus was more than 38.41+0.21%,and the bacteriostatic rate on E.coli was more than 18.21+1.20%,which could be used for further performance study.(2)Composition analysis of complex plant essential oil hydrogelsThe molecular weights of CMC and CBM were determined by gel permeation chromatography(GPC).The results showed that the molecular weight of CMC was mainly in two parts,1.96k Da and 195.74k Da,and the molecular weight of CBM was mainly concentrated in 15.85k Da and 1894.76k Da two parts.The carboxylation degree of CMC is 73.73%determined by point-titration method.GC-MS results showed that the main components of GEO,CEO and EEO were 1,3-cyclohexadiene and cyclohexene;anethole andα-pinene;eucalyptol and limonene,respectively,which are consistent with the components of essential oils in the existing research.(3)Characterization and properties of complex plant essential oil hydrogelsFour kinds of composite hydrogels composed of CBM/CMC,CBM/CMC/EEO,CBM/CMC/GEO and CBM/CMC/CEO with uniform composition and stable properties were prepared,and the hydrogels characterization and properties were studied.Fourier transform infrared spectroscopy(FT-IR)results indicating that EEO,GEO and CEO was evenly mixed into the CBM/CMC hydrogel matrix.Scanning electron microscopy(SEM)results showed that the complex plant essential oil hydrogels had a rough three-dimensional network structure,which was conducive to cell attachment and wound repair than CBM/CMC hydrogel matrix.The results of X ray diffraction(XRD)showed that the polymer chains of the composite plant essential oil hydrogel were intertwined,and there was no internal ordered crystal structure.The rheological properties of the complex plant essential oil hydrogels showed that the addition of plant essential oil lead to decrease of the storage modulus(G‘)and loss modulus(G’’)of the CBM/CMC hydrogel matrix,and CBM/CMC/EEO hydrogel had the lowest G‘and G’’,which had better mobility and was more conducive to its adhesion on the wound surface.The test results of viscosity,swelling property,water holding capacity and water vapor transmission showed that the CBM/CMC/EEO hydrogel had the best vapor permeability,viscosity,moderate swelling properties and reasonable range of water loss rate compared with CBM/CMC/GEO and CBM/CMC/CEO hydrogels.Its viscosity,swelling rate,water loss rate and water vapor transmission rate were 24966 Pa.s,262.04%,30%and 2.23 g/m~2/day,respectively.The stability test further confirmed the stability of all hydrogels prepared in this experiment,which can tolerate high speed low speed centrifugation,high temperature and low temperature and repeated freezing and thawing.(4)In vivo cell experiment of complex plant essential oil hydrogelUsing L929cell in vivo cell experiment,MTT assay was used to detect the cytotoxicity of the complex plant essential oil hydrogels.The experimental results showed that the concentration of compound plant essential oil hydrogel extract could promote the proliferation of skin fibroblasts in the range of 1-1000 g/m L and had no cytotoxicity.When the concentration increased to 10000 g/m L,the survival rate of cell was still over 92%.The results of cell scratch test showed that CBM/CMC/EEO composite plant oil hydrogel induced the proliferation of L929 cells more significantly.After 24 hours,the cell coverage rate reached 59.83%,and the coverage rate reached100%after 48 hours,which was significantly higher than other hydrogels(CBM/CMC,CBM/CMC/CEO and CBM/CMC/GEO).(5)In vitro burn experiment of complex plant essential oil hydrogelIn this study,mice burn model was constructed to further investigate the burn healing activity of CBM/CMC/EEO hydrogel(Gel group).The wound healing process was observed through the wound healing rate and pathological section.There was no significant difference in wound healing rate between PC group and gel group,which was much higher than NC group.The results of H&E and Masson staining showed that the complete epithelial cells and dermal collagen fibers were formed in PC group and gel group on the 7th day,and arranged orderly,accompanied by the appearance of new skin accessory organs.On the 21st day,the skin of PC group was slightly abnormal,while the skin tissue section of Gel group was no different from that of normal mice.The results of biochemical analysis of burn tissue in mice showed that the activities of SOD and GSH-Px were increased,the antioxidant level of tissue cells was increased,and the MDA level was decreased in PC and gel groups,that is,the degree of lipid peroxidation was significantly decreased,and the degree of cell damage was weakened.The expression of cytokines in burn tissues of mice was detected by Elisa.The inflammatory cytokines(IL-6 and TNF-α)in group Gel decreased with time to normal level,and the growth factors(TGF-β,VEGF,EGF)increased continuously with time.It was further confirmed that CBM/CMC/EEO hydrogel could treat wound healing by regulating the level of proinflammatory and growth promoting cytokines in wound healing process.In summary,CBM/CMC/EEO composite plant essential oil hydrogel has stable structure,which can effectively inhibit the antibacterial activity and promote the proliferation of skin cells.It is an ideal hydrogel dressing for treating burn wounds. |
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