| Purpose of study:As the first protective organ of the human body,skin is highly susceptible to damage,which brings pain to patients on the one hand and increases the burden of national health insurance on the other.However,the traditional wound dressings,mainly gauze,bandages and tapes,are cheap but single in nature and easily cause secondary damage to the wound tissue,making it difficult to adapt to the complex wound environment.Therefore,there is still a lack of a wound dressing with simple preparation,low price and stable properties.The stable oxidation level in the wound is significant for wound healing,once the redox imbalance caused by excessive accumulation of reactive oxygen species in the wound due to various internal and external factors,it will aggravate the wound infection and seriously affect the wound healing.Although the existing wound dressings have strong antibacterial ability,most of them are prone to drug resistance because they carry antibiotics.So,the prevention of bacterial infections while avoiding the development of organismal resistance while avoiding excessive accumulation of reactive oxygen species in wounds is a priority direction for wound dressings.Based on this,a tannic acid of gallnut/hyaluronic acid/graphene(TA/HA/G)electrospinning fiber film was prepared by electrospinning technique.And it was investigated whether the electrospinning fiber film has the ability to maintain the redox balance of wound tissue as well as and antibacterial.Then,the ability of electrospinning fiber film dressing to accelerate infected wound healing was evaluated by constructing a mouse infected wound model.which is intended for subsequent clinical studies and eventual extension application to provide the basis and confidence for subsequent clinical research and eventual application.Research methods:(1)It is based on the theory of "Jun-Chen-Zuo-Shi" of Chinese medicine,whereby three materials,namely pentosidine tannic acid(The main therapeutic effect through antioxidant and antibacterial,as Jun),graphene(Assisting tannic acid of gallnut through physical antimicrobial,as Chen)and hyaluronic acid(Healing wounds in other ways by mimicking the extracellular matrix,as Zuo),are prepared into a stable initial electrospinning fiber film under a high voltage electric field by electrospinning(The ability to direct the active ingredients directly to the wound,as Shi)technology.The absorption peaks of TA/HA/G electrospinning fiber films containing different concentrations of pentosidine tannic acid were determined by monitoring TMB-ox.And further the changes in absorption spectra of TMB-ox at the absorption peak for 8 min were obtained to detect the POD simulation effect of TA/HA/G electrospinning fiber films containing different concentrations of tannic acid of gallnut.The SOD mimetic effect of each electrospinning fiber film dressing mentioned above was also detected by using a superoxide dismutase assay kit.The ·OH degradation ability of each of the above electrospinning fiber films was subsequently tested using a hydroxyl radical assay kit.Finally,the TA/HA/G electrospinning fiber film dressing with the best antioxidant performance was clarified according to the above antioxidant experiments,and then the optical density at 490 nm of this electrospinning fiber film dressing after co-culture with NIH-3T3 cells and H2O2 was measured by MTT method to detect its CAT simulation effect,and the level of ROS in NIH-3T3 cells was measured by fluorescence microscopy and flow cytometry.(2)The morphological characteristics of TA/HA/G electrospinning fiber film with optimal antioxidant properties were analyzed by electron scanning microscopy,and the mechanical properties of this electrospinning fiber film were analyzed by universal electronic material testing machine,and the water absorption,water retention and degradation properties of this electrospinning fiber film were further investigated.(3)Staphylococcus aureus was used as the test strain to test the antibacterial activity of TA/HA/G electrospinning fiber films.In addition,the MTT method was used to measure the optical density at 490 nm of different electrospinning fiber films and the control group after co-culture with NIH-3T3 cells to detect cell viability as a means to assess the biocompatibility of this electrospinning fiber films.(4)A mouse skin injury infection model was established using Staphylococcus aureus as the target strain,and the wound changes in mice treated with electrospinning fiber film with optima]antioxidant properties and the control group were studied,as well as the wound area was imaged and measured,further calculating the wound closure rate.Wound tissues were collected on day 14 for HE staining and Masson staining to observe the histopathological changes of wounds,and IL-6 aggregation in wound sections on day 14 was observed by immunohistochemical analysis.The results of the study:(1)Stable quality TA/HA/G electrospinning fiber films were obtained.The characteristic absorption peaks of each experimental and control group were found at 652 nm in the POD simulation effect,and the absorbance values of TA/HA/G electrospinning fiber films showed a time-and tannic acid of gallnut concentration-dependent increase.The inhibition of superoxide anion(·O2-)by electrospinning fiber films in the SOD simulation effect increased with the increase of tannic acid of gallnut concentration.In ·OH scavenging experiments,the ability of electrospinning fiber films to scavenge ·OH similarly showed an accompanying increase in of tannic acid of gallnut concentration.The final TA/HA/G electrospinning fiber film containing 3mg/ml tannic acid of gallnut showed the highest POD,SOD-like antioxidant properties and·OH scavenging ability.In the CAT simulation effect,the H2O2 solution of TA/HA/G electrospinning fiber films containing 3mg/ml tannic acid of gallnut showed statistically significant differences compared to the H2O2 solution alone at 100,250,500,and 1000 mM H2O2 concentrations.In the cytofluorimetric assay,the green fluorescence signal was barely visible in NIH-3T3 cells with H2O2 solution containing this TA/HA/G electrospinning fiber film compared with H2O2 solution alone,similar to the fluorescence signal of the control group without H2O2 solution.(2)Morphological analysis revealed that the TA/HA/G electrospinning fiber film containing 3mg/ml tannic acid of gallnut consisted of irregular,continuous,and smooth surface fiber structure with an average diameter of 1.38 ± 0.62μm.In the mechanical property test,no fracture was observed in each group of materials during the compression behavior up to 90%.With increasing compression displacement,this TA/HA/G electrospinning fiber film group was able to withstand greater compression strength than the rest of the groups,with a maximum compression strength of 19.59 KPa when the displacement reached 1.19 mm.In the 48-hour moisture absorption performance study,the water absorption rate of this TA/HA/G electrospinning fiber film dressing was always higher than the rest of the dressing groups and the control group,with a water absorption rate of 83.10±1.23%.In terms of moisture retention performance at 48 hours,the water retention rate of this TA/HA/G electrospinning fiber film dressing was always higher than the rest of the dressing group and the control group,with a water retention rate of 20.11 ± 1.25%at the 48th hour still.After 12 days of degradation,the degradation rate of this TA/HA/G electrospinning fiber film dressing was always higher than that of the rest of the dressing group and the control group,in which the degradation rate was as high as 54.16±2.42%at day 12,which was statistically significantly different from the rest of the two groups(3)Bacterial colonies were barely visible in the TA/HA/G electrospinning fiber film group containing 3 mg/ml tannic acid of gallnut on LB agar plates,similar to the number of colonies in the positive control group with added antibiotics,while the hyaluronic acid/graphene(HA/G)electrospinning fiber film group had fewer colonies than the hyaluronic acid(HA)alone electrospinning fiber film group.the bactericidal rate in this TA/HA/G electrospinning fiber film group was 96.74±2.57%,which was statistically different from the HA electrospinning fiber film group and HA/G electrospinning fiber film group,and almost similar to the 96.44±1.70%of the control group.In the biocompatibility experiments,the absorbance values of the HA electrospinning fiber film group,the HA/G electrospinning fiber film group and the TA/HA/G electrospinning fiber film group containing 3 mg/ml pentosidine tannic acid were statistically significantly different compared to the control group at the 12,24 and 36 h time points,and the absorbance values of the TA/HA/G electrospinning fiber film group were consistently higher than those of the remaining groups at these three time points.(4)The wound crust shedding and new skin formation were faster in the TA/HA/G electrospun film group of mice with 3 mg/ml pentosidine tannic acid than in all other groups.The wound area was statistically different on day 14 with 1.12 ± 0.54 mm2,as well as the wound closure rate of 97.91 ± 1.15%compared to the control and HA electrospinning film groups.Masson trichrome staining showed more collagen fiber deposition in the wound of TA/HA/G electrospinning fiber film group.Moreover,IL-6 pro-inflammatory factor was observed to be less distributed in the TA/HA/G electrospinning fiber film groups in immunohistochemistry.Research conclusion:As per the concept of "Jun-Chen-Zuo-Shi" formula of traditional Chinese medicine.The TA/HA/G electrospinning fiber film has antioxidant effect and ·OH scavenging activity similar to natural antioxidant enzymes,and has excellent mechanical properties,water retention,water absorption and degradation,as well as outstanding antibacterial activity and good biocompatibility to reduce wound inflammation while accelerating wound healing.Given its good wound healing ability,it has the potential to become a new wound healing dressing and offers the possibility for later clinical studies. |
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