Preparation And Characterization Of New Type Collagen Dermal Scaffold

The establishment of skin tissue engineering supplied a new clue and efficient method to improve the wound healing quality,solve the shortage of skin source and so on.As the extracellular matrix,dermal scaffold played an importing role in constructing artificial composite skin,it provides suitable environment for cells to adhere,vegetate,transfer,multiply and differentiate,was always the research keystone of skin tissue engineering.Resent years,using pig skin to prepare acellular dermal matrix(ADM) and used as dermal scaffold to construct artificial composite skin had become the research hotspot.Whereas,because the limitation of ideas and the defects of preparing technology,ADM had many shortcomings,such as strong immunogenicity,bad permeability,low biological activity and so on.To overcome the shortcomings of ADM,a new type collagen dermal scaffold, which was different form current ADM and had natural matrix net structure,was prepared by treating suckling pigskin using new preparation technics and various chemical,biological and physical comprehensive methods.The study of the thesis was guided by the principle of tissue engineering and on the base of previous research, focused on the effect of the sequence of cross-linking and alkali treatment,GA cross-linking and enzyme treatment on the performance of dermal scaffold.The collagen purity was also determined in the thesis.Through above studies,the preparation technique and collagen purity determination method of new type collagen dermal scaffold was optimized,and the major performance of the scaffold was characterized.The main research contents of the thesis were listed as follow:(1) The effect of sequence of GA cross-linking & alkali treatment,GA cross-linking and enzyme treatment on the performance of dermal scaffold was studied,the results showed that:Scaffold prepared by the sequence of GA cross-linking firstly,latter alkali treatment had better porosity and water vapor permeability then the other one, so this sequence was adopted in the thesis;the tensile-strength,shrinkage temperature and in vitro degradation time of the scaffold was improved as GA dosage was increased,and GA cross-linking had little influence on the porosity,permeability and hydrophilicity of the scaffold;The porosity and permeability of the scaffold was improved with the increase of the enzyme dosage and the prolong of the action time.(2) The collagen purity of the new type collagen dermal scaffold was determined using colorimetry method and the condition of the hydrolysis was optimized.The results showed that:the optimal hydrolysis condition was that 10mL 6mol/L HCl was added to 50mg dried scaffold,then hydrolyzing for 32h at 100℃;the method has good repeatability.The collagen purity of the dried scaffolds was 90%,which showed that epidermis,fattiness and interfibdllary substance were removed almost completely.(3) According to the principle of tissue engineering and the research results of(1), (2)combining with the advantages of existing materials and the requirements of ideal dermal scaffold,a new type collagen dermal scaffold was prepared,and its main performance including physical and chemical performance,biocompatibility were studied systematically.The scaffold surface was smooth and glossy,and the fibers woven closely,before GA treatment was milk white,when treated by GA,it became light yellow.The results of HE observation showed that cells in the dermal had been removed completely.SEM results illustrated that there were a lot of pores at different sizes on the surface,and the pores got through into the inner;Collagen fiber bundles maintains its original structure and be loose appropriately,and the scaffold has rich internal porous structure;The pH value was about 6.5～7.5,which was accorded with body fluid's pH value;The tensile-strength was between 11.56～11.56MPa,which demonstrates that it has good tensile strength;The porosity was about 70%and the water vapor permeability was around 2900 g·m^-2·24h^-1,which was suitable for the recovery of wound;The degradation time was between 15h and 70h,and can be adjusted by changing GA treatment process to prepare scaffolds with different degradation speed.The results of MTT method showed that its cytotoxicity was first grade,which was in accord with the cytotoxicity request of human biological material; The cell culture results indicated that it had good cytocompatibility and can effectively promote cells to adhere,proliferate and growth,which made good foundation for its further study and application in clinic.The new type collagen dermal scaffold,whose interfibrillary substance among the dermis,such as epidermis,fat cell,mucoprotein ect.,was removed to weaken the antigen,and cross-linked by GA.It has high permeability,suitable degradation time, good tensile-strength,low immunogenicity and good biocompatibility,basically meets the requirements of ideal dermal scaffold,is expected to be used as complex transplant dermal scaffolds and used to construct tissue engineering skin.