| BackgroundWound healing is an eternal proposition in the field of burn.Especially for patients with large area burn,wound healing is particularly critical,associated with the survival rate and quality of life directly.Patients with large area naked wound caused by severe stress and excessive consumption,can easily lead to bacterial colonization,causing systemic infection,finally followed with systemic organ injury.As early as possible to heal the wound is the key to the treatment of burn patients,so the selection and development of the wound healing is through the development of the burn,including temporary dressing and permanent dressing.Permanent coverage mainly refers to the dermal substitute and skin grafting,and there are several kinds of grafting that major applied in the treatment of lager area burn patients,including MEEK skin grafting,allogenic mixed skin grafting,micro skin transplantation and cultured epithelial autograft(CEA),however,any kind of skin grafting need temporary dressing appropriate to cover the gap and to ensure permanent covering survival.The development of temporary dressing also played an important role in wound healing.Temporary cover like the traditional gauze,vaseline gauze,and silver-contained wound dressing,also including collagen membrane or collagen scaffold material are widely used.Therefore,the development of wound cover is also an important support to the improvement of the wound healing technology.In the process of large area burn wound healing,each kind of skin grafting always have advantages and disadvantages.Among them,Meek skin grafting is mainly used in the early coverage of the wound after escharectomy,in which the expansion ratio can vary from 1:3 to 1:9,and realized mechanization,but the expansion ratio is also limited.The allogenic mixed skin grafting,skin graft survival rate is stable,but the operation is confused with repeated work and time-consuming.Using micro skin grafting,the autologous skin can be extended to 100 times in theory and 10-20 times in clinical application.However,using CEA,in theory,the autologous skin can be extended 1000 times,but the qualit there are a series of problems such as coverage and directionality y of wound healing is poor.Our study is focuses on the micro skin grafting,the choice of coverage has always been an important factor affecting the survival rate of microskin,high activity of allograft skin is an ideal microskin covering,but lacking source of allograft skin often limit the development of microskin grafting in the large area burn patients.Therefore,it’s key to the future of microskin grafting to searching for a feasible coverage that can simulate the function of allograft skin.In the development of various materials of wound dressing,collagen biomaterial become increasingly widely used in the wound repair field.Collagen is an element component in the process of wound healing,and also play an important roles in the repair of skin and wound.Moreover,the sources of collagen production become more and more,the extraction methods and processing methods become increasingly mature.In the field of wound repair,collagen is widely used as existing products including dermal substitutes and topical dressings.In recent years,more and more fish collagen material from the application in the field of medicine,including hemostatic materials and wound repair,how to use gelatin raw materials have great clinical significance to promote wound healing.The purpose of this study is to find an alternative biological dressing of allogenic skin,combined with the development of fish collagen.Construct a kind of fish collagen membrane bilayer simulating the structure and function of skin,which can be used as the cover of microskin gfafting,providing a resourceful cover choice,so that the microskin gfaft apply more frequently in wound healing of large area burn patients,with higher efficiency.Part1 Preparation of the Fish Collagen Membrane Bilayer(FCM-B)ObjectiveThe preparation of a simulated skin structure,including epidermis and dermis,and Simulation of bilayer skin collagen dressing on the function,can simulate the outer epidermal dense and high toughness with protection and insulation function;the inner loose soft,good adhesion function.The utility model can be used for absorbing the exudate and protecting the wound surface.MethodsThe fish collagen is extracted by acid combined with enzyme digestion and purified from the skin and scales of Tilapia which used to be regarded as food waste.On the basis of the addition of crosslinking agent with no toxicity,the collagen film with obvious difference in physical and chemical properties can be obtained by using different crosslinking methods.The FCM-O is crosslinked by Dehydrothermal treatment,while the FCM-I is obtained by vacuum freeze drying technology.The prepared FCM-O was advanced to the bottom of the FCM-I mold,and then prepared by vacuum freeze-drying.ResultsSuccessfully prepared the bilayer fish collagen membrane,with dense flexible outer layer and loose soft inner layer.It is similar to human skin in appearance and texture.Part 2 Research of the characters of the FCM-BObjectiveVerify the similarity of structure and function between the bilayer fish collagen membrane and the skin,through the structure and physicochemical characteristics test.Methods1.Structure observation1.1 Histological observationUsing FCM-B frozen section,HE staining,Massion staining to observe the structure and the arrangement of collagen,and compared with the normal skin tissue sections to observe the similarity of the structure;1.2 Scanning electron microscopeScanning the FCM-B to observe the structure of collagen membrane,observe the characteristics of each layer,and measure the thickness of each layer.2 Physical properties2.1 mechanical propertiesTake FCM-O,FCM-I and FCM-B dumbbell samples,using the Instron 5567 material testing system(Instron,USA),mechanical testing,index of the young’s modulus,elongation and fracture strength,repeated measurements 3 times.2.2 Water vapor transmission rate(WVTR,Water Vapor Transmission Rate)Obtain FCM-O,FCM-I and FCM-B samples with diameter of 3.5cm,and the results were obtained by using the water vapor transmission rate detector(W3/030,Labthink,China).The measurement was repeated 3 times.2.3 Absorption capacity(C%)The system has the three types of collagen membrane prepared with double distilled water immersion,at the same time the same environment soaked collagen membrane until fully absorb water,through the changes of collagen membrane quality measurements before and after immersion calculation C%.3 Biochemical characteristics3.1BiodegradableThe prepared FCM-B,FCM-O and FCM-I were treated with PBS,the dry weight was calculated before and after soaking,and the degradation time was calculated by t1/2.3.2 CytotoxicityFCM leaching liquor was added according to a certain concentration gradient with human fibroblast cell suspension,using CCK-8 kit,by measuring the absorbance value(OD),relative growth rate calculation of fibroblasts.3.3Statistical methodsThe indicators of FCM(measurement data)results are shown in average and standard deviation;using t test value between the two groups;using oneway-ANOVA analysis in multi group comparison,than using SNK test.Research results:1 Structure ObservationHistological sections showed that FCM-B frozen tissue sections of normal skin and slice has a structural similarity in the bilayer structure and collagen arrangement;electron microscope scanning showed on FCM-B in thickness and porosity is similar to the skin epidermis and dermis.2 Physical propertiesThe mechanical properties of FCM-I are better than FCM-O(p<0.05),and the FCM-B has appropriate mechanical properties.The WVTR of FCM-I was significantly higher than that of FCM-O(p<0.05),and the WTVR of FCM-B was close to the normal skin.The absorption capacity of FCM-I was significantly higher than that of FCM-O(p<0.05).3 Biochemical properties testThe degradation time of FCM-O was about 15 days,T1/2(FCM-I)was about 21 days,and the biodegradation time of FCM-O was significantly longer than that of FCM-I(p<0.05).Cytotoxicity test showed that FCM extract had no significant effect on the growth rate of human fibroblasts.Part 3 The FCM-B covering SD rats microskin transplantationObjectiveIn order to verify the effect of double skin collagen membrane in the process of particle skin transplantation,it can promote the healing of skin wound.MethodsOn the back of the SD rat we cut a full-thickness skin defect wound of diameter 2cm,then removed the skin from skin preparation into particles,and replanted on the wound according to the ratio of 1:10.According to the different covering the rats were divided into control group,allograft group and FCM-B,FCM-1,FCM-O coverage group.In order to observe the effects of different coverage physical effects on the survival of microskin grafting,the wound healing rate was calculated and the shrinkage rate;and made tissue specimens,then did Massron staining and HE staining respectively,to observe the collagen distribution and the epidermal growth,meanwhile did immunohistochemistry related to CD31 antibody.Statistical methodsThe data using oneway-ANOVA analysis in multi group comparison,than using SNK test.ResultsThe early detection of FCM-I and FCM-O of poor wound healing at 4 weeks,FCM-B group and allograft skin covering wound healing coverage group was no significant difference(p>0.05).The rate of wound contraction of allograft skin covering group was lower than that of FCM-B(p<0.05),histological observation showed that the formation of epithelial thickness of FCM-B group was greater than that of allograft skin covering group(p<0.05).ConclusionsThe construction of the bilayer collagen membrane,and skin in the structure of epidermis and dermis with a high degree of similarity,and close to the ideal wound covering requirements in physical and chemical properties,through animal experiments confirmed that FCM-B substitution of skin allografts as microskin covering has a possibility. |
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