Experimental Study On The Effect Of Trypsin Enzyme And Hypertonic Saline On Dermal Acellular Matrix

In recent years,the skin damage repair has become a medical and health problem that needs to be solved clinically.As a skin substitute,acellular matrix has been gradually studied by researchers due to its advantages including promoting seed cell adhesion and proliferation,maintaining dryness,regulating differentiation,saving senescent stem cell,angiogenesis,and rich in growth factors.At present,there are many fabrication methods of acellular matrix,but related properties evaluation were not systematic and comprehensive.To address the above problems,the study used gamma ray sterilization + 0.05% trypsin/EDTA decellularization,peracetic acid 0.2% PAA/4% ethanol solution(hereinafter referred to as peracetic acid)sterilization + hypertonic salt decellularization to prepare acellular matrix respectively.Then analyzed the physical,chemical and biological properties of the treated matrix systematically and comprehensively,then compared the results with normal skin tissues to study the effect of Trypsin enzyme and hypertonic saline on the performance of the dermal acellular matrix.The main content and conclusions of this article are concluded as follows:(1)The human fibroblasts(HFB)were successfully extracted by enzymatic digestion and cultured to 4-6 generations and then subjected to Vimentin immunofluorescence staining identification.The preparation of decellularized scaffolds was set as group A(8 cases)as the control group and immersed in PBS,group B(8 cases)was sterilized by gamma radiation +0.05% trypsin/EDTA decellularization,group C(8 cases)was sterilized by gamma ray,and group D(8 cases)was sterilized by peracetic acid(0.2% PAA/4% ethanol solution)+ hypertonic salt decellularization,respectively.(2)For the effect of Trypsin enzymatic decellularization on the acellular matrix of the dermis,Phys-chemical and biological properties of the three groups of materials A,B,and C were separated tested.Scanning Electron Microscope(SEM)and Atomic Force Microscope(AFM)were used to observe the micro-topography(local height and roughness)of the matrix,the results indicated that the enzyme treatment had not destroyed the internal micro-topography of the matrix;Porosity testing showed that enzymes had not significant effect for increasing porosity;Mechanical properties results showed that the compressive elastic modulus of the acellular dermal matrix after enzyme treatment was close to normal skin.The chemical properties study detected the total content of collagen,the ratio of collagen I/III,fibronectin,elastin,and p H,which indicated that the enzyme treatment had no obvious effect on the chemical properties of the substrate.The biological characteristics test found that the enzyme had a significant effect on cell removal,but no effect on cell activity,and can reduce the toxic effect of gamma ray sterilization on cells,which could lead to cells migrate into the matrix rapidly.and growth into the matrix.(3)For the effects of peracetic acid and hypertonic saline on the performance of acellular matrix,the physical,chemical and biological properties of the matrix were also explored tested.Results showed that treatment with hypertonic saline was beneficial to increase the porosity of the matrix and had no statistically significant effect on the compressive elastic modulus of the matrix.The effect of hypertonic saline on the total content of collagen and the ratio of collagenⅠ/Ⅲ was statistically significant compared with control group,however,the effect on fibronectin retention is better.In addition,Hypertonic saline has a poor effect on cell removal,and the treated matrix had a certain toxic effect on the cells,which was not benefit to allow cells grow into the matrix.the growth of cells into the matrix.Conclusion:Comparing the performance of decellularized matrix fabricated by Trypsin enzyme and hypertonic saline,found that the fibronectin retention effect of hypertonic saline decellularized matrix was better than that of enzyme decellularized matrix,while the apparent density and decellularization effect of enzyme decellularized matrix were all better than that of hypertonic salt decellularization.The destruction of collagen components was lower than that of hypertonic saline decellularization,and no cytotoxicity were detected,and the cells are prefer to grow into the inside of the matrix.The experimental results showed that the acellular matrix prepared byγ-ray sterilization and 0.05% Trypsin enzyme/EDTA decellularization can be used for construction of tissue-engineered skin.It can provide a reference for the preparation and mounting of heterogeneous dermal acellular matrix,and also applied in electrospinning or 3D printed tissue engineering skin scaffolds provide physical and chemical parameters.