Preparation And Characterization Of Acellular Dermal Matrix Based On The Purification Of Papain And The Combined Aqueous Two-phase Method

For patients with large-area full-thickness burns who do not have enough autologous split layer skin for skin grafting,biological skin substitutes can be considered.The current common clinical treatment is autologous skin transplantation.However,insufficient donor skin and damaged areas greatly limit its application.Therefore,biocompatible materials have become more attractive and gradually replaced self-healthy skin,can repair wounds,induce the orderly growth of host fibroblasts and capillaries,make the arrangement of collagen fibers more regular and reduce Local inflammation.At present,the preparation of collagen matrix dressings from pig dermis has become a research hotspot.However,the acellular dermal matrix（ADM）obtained by traditional preparation methods（acid-base method,hypertonic salt method and enzyme-detergent method）has disadvantages such as poor permeability,destruction of collagen structure,and poor biocompatibility,which severely limit its clinical application.The purpose of this study is to find a new and optimal method based on plant enzymes to prepare acellular dermal matrix（ADM）and pre-clinical evaluation to produce biologically important skin substitutes.In this work,the proposed method is based on the use of the following enzymes:papain,papaya lipase（CPL）,and the use of a polymer/saline solution two-phase system for purification.Most of the commercially available papains are crude enzyme products,and the enzyme activity is unstable,and its application in biomedical applications has been greatly restricted.In this paper,a PEG/inorganic salt two-phase system was used,combined with response surface experiment design to optimize the extraction conditions of papain,and the ion exchange method was used to obtain high-purity papain.After optimization,the optimal conditions of the system are:p H7.06,Na₃C₆H₅O₇ mass percentage 18.68%,and PEG6000 mass percentage 18.420%.Under these conditions,the partition coefficient of papain was 2.630%,and the recovery rate of enzyme activity was 76.986%.Corresponding expansion experiments were carried out.Repeated experiments showed good results,which laid the foundation for further factory production.Papaya lipase has not been sold on the market.In this paper,the freeze-dried preparation of papaya lipase was obtained by the phosphate buffer elution method and the influence of different conditions on the hydrolysis activity of papaya lipase was studied.The research results show that the enzyme activity of CPL is 320U/g,and its optimum hydrolysis temperature is about 50°C,and the enzyme activity decreases when it exceeds 50°C.In the temperature range of 25°C-50°C,there is basically no loss of enzyme activity after 6 hours of heat preservation.CPL shows greater enzymatic activity when the p H is 8.These properties provide a theoretical basis for research.On the basis of preliminary experiments,25 of ADM preparation methods based on different enzyme ratios and different times were evaluated.The obtained ADM samples were characterized by scanning electron microscope（SEM）,porosity measurement and water vapor transmission rate test.The results show that the collagen bundles of ADM particles are complete and ordered,with pore diameters ranging from 30μm to 100μm.Detected by Differential Scanning Calorimetry（DSC）,Thermogravimetric Analysis（TGA）and Fourier Infrared Spectroscopy（FTIR）,the results show that when the preparation conditions are the same ratio of papain and papain lipase,and the 5 h processing time is ADM preparation Under these conditions,the collagen structure is complete and the denaturation temperature and degradation temperature have reached high values.Finally,DNA quantitative detection,hemolysis and cytotoxicity test biological evaluation of ADM were carried out.The following conclusions are obtained:Compared with untreated pigskin,the DNA content in the dermal tissue after treatment has changed significantly.It was observed that the DNA content of the decellularized tissues was reduced by about 44.6%,and the enzyme treatment ratios of 50:50 and 75:25 showed the lowest DNA content and had a good effect of removing DNA molecules.Using rabbit ear-derived venous blood to carry out biological hemolysis experiments on different groups of acellular matrix,the hemolysis percentages of the 9 groups of experimental results are all below 5%.Using the CCK-8 method for testing,compared with the control group,the cell survival rates of the experimental group were 87%and 91.2%,respectively,indicating that the material has qualified cytotoxicity.The above results indicate that the material shows good biocompatibility,and the development of this decellularization method to manufacture ADM scaffolds for clinical applications has great potential.