| BackgroundArtificial dermal can effectively repair wounds that damage the dermis,promote wound healing,and improve the function and appearance after the wound is healed.At present,the most representative artificial dermal is double layers,which composed of a collagen layer that acts as a scaffold for skin tissue growth and a silica gel layer that has a barrier function.In the early stage after the transplantation of artificial dermal,due to the inability of the artificial dermal to establish a satisfactory blood supply with the wound,its anti-infective ability is weaker.As bacteria proliferate on the wound surface,collagenase is secreted,and after degradation,the collagen of the artificial dermal becomes a good medium for bacterial proliferation,which in turn causing an increased infection rate.Once infection occurs after transplantation,not only will it cause a failure in transplantation,but it may also cause sepsis,and threaten the life of the patient.Numerous anti-infective measures such as thorough debridement before transplantation,frequent change of wound dressings after transplantation and systemic use of antibiotics,etc.are used in the prevention of post-artificial dermal transplantation infection,although affected,there is still a high rate of infection post-transplantation.A large number of studies have integrated various growth factors into artificial dermal to promote wound healing.Learn from this,we hope to develop an artificial dermal with built-in anti-infective ability that can provide new ways of methods to address the problem of infection after artificial dermal transplantation,and will expand the application scope of artificial dermal,and providing more options for the treatment of wounds.This study use the Lando?double layer artificial dermis as a basic artificial dermal,select the appropriate clinical anti infective agents,modify the anti infective ingredients of artificial dermal,and verify its continuous anti infection ability and the effect of promoting wound healing.Part 1:Selection of antibacterial components that endow artificial dermal with persistent anti infection abilityObjective:An anti-infective component with strong bacteriostatic abilities was selected to be used as the component which endow artificial dermal with persistent anti infection ability for subsequent research.Methods:Clinical wound disinfectants were selected as possible options for anti-bacterial components.The disinfectants selected for this study included:iodophor,chlorhexidine,bromo-geramine,polyhexamethylene biguanide?PHMB?,compound lysozyme and nitrofurazone.Microdilution broth method was used to assess the minimum inhibitory concentration?MIC?and minimum bactericidal concentration?MBC?of the aforementioned disinfectants against standard strains and 20 clinical strains of bacteria commonly seen in wounds?Staphylococcus aureus,Acinetobacter baumannii,Klebsiella pneumoniae?.The MIC and MBC were compared against the dilution multiples MICdilutionilution and MBCdilutionilution of the disinfectants.By comparing the MIC and MBC of a single disinfectant against clinical strains and standard strains,clinical strains that have either MICs or MBCs or both greater than those of the standard strains were considered as strains with reduced susceptibility to the said disinfectant.The susceptibility of the clinical strains to each disinfectant was preliminarily determined by calculating the ratio of the strains with reduced susceptibility.The disinfectant components with smaller MICdilutionilution and MBCdilutionilution and higher sensitivity in clinical strains were selected as candidate anti-bacterial components in this study,and are used to prepare artificial dermal that possess anti-infective ability for the next stage of the study.Results:The MICdilutionilution of toiodophor,chlorhexidine,bromo-geramine,PHMB,composite lysozyme and nitrofurazone against Staphylococcus aureus relative to the commonly used concentrations were 2-5,2-6,2-6,2-7,2-6,2-1,respectively;the MBCdilutionilution of the above disinfectants relative to the commonly used concentrations were 2-5,2-5,2-6,2-7,2-6,-?MBC>the concentration commonly used for wounds?,respectively;the proportions of strains with reduced susceptibility were 30%,30%,55%,5%,5%,50%,respectively.The MICdilutionilution of toiodophor,chlorhexidine,bromo-geramine,PHMB,composite lysozyme and nitrofurazone against Acinetobacter baumanni relative to the commonly used concentrations were2-4,2-4,2-5,2-5,2-2,2-1,respectively;the MBCdilutionilution of the above disinfectants relative to the commonly used concentrations were2-3,2-4,2-4,2-5,2-1,-?MBC>the concentration commonly used for wounds?,respectively;the proportions of strains with reduced susceptibility were 40%,25%,60%,10%,45%,55%,respectively.The MICdilutionilution of toiodophor,chlorhexidine,bromo-geramine,PHMB,composite lysozyme and nitrofurazone against Klebsiella pneumoniae relative to the commonly used concentrations were 2-4,2-6,2-6,2-7,2-2,2-1,respectively;the MBCdilutionilution of the above disinfectants relative to the commonly used concentrations were 2-4,2-5,2-6,2-7,2-1,-?MBC>the concentration commonly used for wounds?,respectively;the proportions of strains with reduced susceptibility were 35%,20%,40%,10%,40%,45%,respectively.Conclusion:PHMB has a lower MICdilutionilution and MBCdilution,and has a high sensitivity in clinical strains,thus was selected as the anti-bacterial component for subsequent experiments.Part 2:Preparation of artificial dermal with continuous anti-infective ability and testing of characteristicsObjective:PHMB was used as the anti-bacterial component.The immersion method,microsphere method and sandwich-type method were considered in the selection of a method that will facilitate the preparation of an artificial dermal with continuous anti-infective ability.The duration of continuous anti-infective ability and the characteristics of the experimental samples were tested.Methods:The artificial dermal was processed according to the following:1.Soaked in a PHMB solution at concentrations of 0.1%,0.5%,1%and 2%,respectively for 24 hours;2.Embeded by gelatin microspheres with PHMB concentrations of 0.1%,0.5%,1%and 2%respectively,they were then prepared into gelatin microspheres with diameters of 50?m and 100?m,respectively,after immersion for 24 hours,the gelatin microspheres were absorbed by the artificial dermal substitute 3.PHMB at concentrations of 0.1%,0.5%,1%,and 2%was prepared into hydrogels,and added in-between the silica gel layer and collagen layer of the artificial dermal.The artificial dermal with anti-infective ability were made into round shape with 5mm in diameter,and placed in culture plates that were dabbedwithStaphylococcusaureus,Acinetobacterbaumanni,Klebsiella pneumoniaestandard strains,which were changed 1/d.The size of bacterial inhibition ring was recorded,and the duration of the sustainable formation of bacterial inhibition ring of each method was analyzed.Experimental samples with longer durations of anti-infective ability were selected for testing of characteristics.In vitro cytotoxicity experiment:Artificial dermal without the addition of anti-bacterial components were used as a control.The influence on the subculture of the fibroblasts and vascular endothelial cells were determined on the 3rd,7th,14th,and 21st day of the experiment,and the survival rates were compared.In vitro natural degradation experiment observed degradation under natural conditions.In vivo implantation experiment:subcutaneous implantation in rats,to observe the general reaction of the implantation site,and record the conditions of in vivo degradation.The experimental samples and artificial dermal substitutes without the addition of the anti-bacterial components were observed under a scanning electron microscope?SEM?,the differences between the two were compared.Results:Experimental samples with different concentrations prepared using the immersion method had shorter continuous anti-bacterial ability durations,which were not last to 1d.Artificial dermal substitute immersed in1%or 2%PHMB of 50?m microsphere could achieve the longest continuous anti-bacterial duration,which is 1d.Sandwich-type method which add PHMB at a concentration of 1%or 2%have the durations of the continuous anti-bacterial ability for Staphylococcus aureus,Acinetobacter baumannii and Klebsiella pneumonia standard strains were 8 days,7days,and 7 days,respectively.In summary,the addition of PHMB hydrogel at the concentration level of 1%into the artificial dermal substitute could achieve a longer continuous anti-bacterial duration.Experimental results for cytotoxicity shows that artificial dermal with or without anti-bacterial components did not show a significant difference in the survival rate of fibroblasts and vascular endothelial cells,P>0.05.In vitro natural degradation experiments show that the hydrogel has a lower degradation rate.On the 6th week,the degradation rate is 27.388±1.943%.In vivo implantation experiment shows that the implantation site exhibited no obvious inflammatory response,with a satisfactory growth of the incisional wound.The degradation rate on the 3rd week was 23.648±2.336%.SEM results show that after adding the hydrogel layer,the structure of the collagen layer did not have obvious changes,and PHMB distribution could be observed inside the pores of the collagen layer.Conclusion:Inserting a hydrogel layer containing 1%PHMB between the collagen layer and silica gel layer of the artificial dermal can effectively provide the artificial dermal substitute with a continuous anti-infective ability,which lasts for a longer duration and can satisfy clinical needs.Furthermore,it has low biotoxicity,and does not change the original structure of the collagen layer of the artificial dermal substitute.Part 3:Validation of the in vivo effects of artificial dermal substitute with anti-infective abilityObjective:To validate the effects of the artificial dermal substrate containing 1%PHMB hydrogel on wound healing and its anti-infective effects.Methods:Clean wound transplantation experiment:40 SD rats were used for establishment of full-layer wound models.The experimental animals were divided into 2groups,namely,Artificial Dermis Group?ADG?and anti-infection Artificial Dermis Group?AADG?.The wounds in groups were given different treatments:no treatment,covered with artificial dermal substitute,covered with a hydrogel layer containing 1%PHMB,and covered with artificial dermis with anti-infective ability.The wound dressings were changed on the 3rd,7th,14th,and 21st day,and wound healing conditions were observed.New infection rate during each wound dressing change was computed according to general observations.Wound tissues were taken,pathological sectioning for HE staining and CD31immunohistochemistry were conducted,the Micro-vascular density?MVD?was calculated,and wound tissue bacterial quantification experiment was conducted to analyze the bacterial content of the wound.Infected wound transplantation experiment:Twenty SD rats were randomly divided into ADG and AADG groups,with 10 rats in each group.Infection wound model was created on the backs of the rats.ADG was covered with artificial dermis,and AADG was covered with artificial dermis with continuous anti-infective ability.The wound dressings were changed on the 3rd,7th,14th,and 21st day,and wound infection conditions were observed,and wound infection rate was calculated.All the rats were sacrificed on the 21st day,and wound tissue quantitative bacterial were analysised.Continuous exposure to infective environment after transplantation:Twenty SD rats were used for establishment of full-layer wound models,and were randomly divided into ADG and AADG groups,with 10 rats in each group.These wounds were covered with artificial dermis and artificial dermis with continuous anti-infective ability,respectively.On each day,1 mL of Acinetobacter baumannii standard strain suspended solution was dripped outside the silica gel layer.The wound dressings were changed on the 3rd,6th,9th,and12th day,and the infection rates in the two groups were calculated.Results:Clean wound transplantation experiment:On the 7th day,during the changing of the wound dressing,ADG had 1 infected wound,new infection rate is6.67%?1:15?.After the infected wounds were excluded,the bacterial content of uninfected wounds of each group was assessed.On the 3rd,7th,and 14th day,the wound tissue bacterial contents of AADG wound were significantly less than those of ADG,P<0.05.ADG and AADG were similar with regards to general performance and HE staining.The MVD of AADG on the 3rd day after wound creation was less than that of ADG,P<0.05,but did not show significant differences in the later period,P>0.05.Infected wound transplantation experiment:After transplantation to the infected wound,all wound infection symptoms in ADG were not relieved,the infection rate was 100%.There were 7wound infection symptoms that disappeared in AADG,infection rate was 30%,which was significantly lower than ADG,P<0.05.21 d after injury,whether infected wounds or no-infected wounds,the bacterial content of infected wounds in AADG were significantly lower than ADG,P<0.05.Continuous exposure to infective environment after transplantation experiment:On the 6th day,all wounds in ADG were infected.On the 12th day,all wounds in AADG were infected.The infection rates of the two groups were significantly different on the 3rd and 6th day,P<0.05.Conclusion:Artificial Dermis with anti-infective ability do not significantly influence wound healing,and can effectively control infection after transplantation to the infected wound,and have certain inhibitory ability against factors that can cause wound infection after transplantation. |
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