| Collagen is a very important fibrin that is mainly distributed in mammals.Because of its low immunogenicity and good biocompatibility,it has been widely used in the fields of,for instance,food,cosmetics,medicine,and biological materials.In this thesis,pig skin is selected as the raw material to study the preparation method of pig acellular dermal matrix material.The preparation process is optimized through orthogonal experiment design,and the performances of the optimized pig acellular dermal matrix materials were systematically characterized.The porcine acellular dermal matrix and bovine type I collagen-based scaffold were further compounded by using the crosslinking reagent glutaraldehyde,the performances of the composite scaffold are characterized.The composite scaffold is subjected to biological evaluation.The main research contents are as follows:1.Using market-purchased fresh pig skin as the raw material,pig acellular dermal matrix material is prepared by alkaline method,the material-to-liquid ratio,Na OH,Triton X-100,Temperature,decellularization time,and the number of fluid exchanges are selected as factors to design an orthogonal experiment.The results shows that the optimal process parameters of the preparation of porcine acellular dermal matrix material are: the material-to-liquid ratio is 15:1,the mass fraction of Na OH is 2%,the mass fraction of Triton X-100 is 2%,the temperature is 25 °C,and the time is 4 hours in total.The number of fluid changes is 4 times.The materials prepared under these conditions have good biocompatibility.The results of DAPI staining shows that only sporadic blue fluorescent spots are observed,indicating that most of the cells have been removed.2.In order to investigate the performances of the prepared porcine acellular dermal matrix material,the porcine acellular dermal matrix material is characterized by scanning electron microscopy(SEM),infrared spectroscopy(FT-IR),tensile strength,thermogravimetric analysis and cell proliferation test.The results shows that the material had a honeycomb porous network structure with good connectivity between pores;the tensile strength test result is 2.06 ± 0.48 MPa;FT-IR results shows that the material had collagen amides A,B,I,II and III characteristic peaks,indicating that the internal structure is not damaged during the decellularization process.The results of thermogravimetric analysis shows that even heated to 900 °C,some of the material remained,indicating that it has good thermal stability.L929 mouse fibroblasts are inoculated on the material.After a period of cultivation,it is found that the cells could proliferate rapidly on the material,demonstrating the good biocompatibility.3.So as to prepare a porcine acellular dermal matrix/collagen composite scaffold,different concentrations of collagen solutions were explored.By referring to the dissolution of bovine type I collagen,1% collagen solution cannot be formed,5%collagen solution cannot be dissolved,and 3% collagen solution is fully dissolved and formed a uniform gel.After freeze-drying,the honeycomb porous structures inside were observed.The pores showed comparable size and good connectivity.By designing an orthogonal experiment and using mechanical properties as evaluation indicators,the final mass ratio of porcine acellular dermal matrix and bovine type I collagen is 1:6,the concentration of cross-linking agent glutaraldehyde is 0.20%,the cross-linking temperature is 37 °C,and the cross-linking The time of 60 min is the optimal preparation condition.4.In order to investigate the performances of the porcine acellular dermal matrix/collagen composite scaffold,scanning electron microscopy(SEM),infrared spectroscopy(FT-IR),and measurements of tensile strength,water absorption performance,water vapor transmission rate,porosity,thermogravimetric analysis,and in vitro degradation rate are performed,and the results are compared with the traditional bovine type I collagen scaffold.The results shows that the internal structure of the composite scaffold is three-dimensional porous structure as observed by SEM;FT-IR showed that the composite scaffold retained the internal structure of collagen,and the characteristic peak of glutaraldehyde is detected,confirming the successful crosslinking;the shrinkage temperature of the bovine type I collagen-based scaffold is 70-72 ℃,and the shrinkage temperature of the composite collagen scaffold is 78-79 ℃;the tensile strength of the collagen-based scaffold is 1.56 ± 0.08 MPa,the tensile strength of the composite scaffold is 2.76 ± 0.06 MPa;the water absorption multiple of the collagen-based scaffold is 28.6 ± 0.57 times,and the water absorption multiple of the composite scaffold is 31.3 ± 1.01 times;the WVTR of the collagen-based scaffold is 859.53 g/ m2/day,the WVTR of the composite scaffold is 942.75 g/m2/day;the porosity of the collagen-based scaffold is 60.3 ± 2.0%,and the porosity of the composite scaffold was 65.6 ± 1.2%;the denaturation temperature of the bovine type I collagenbased scaffold is 85.12 ℃,and the denaturation temperature of the composite collagen scaffold is 94.24 ℃;the collagen-based scaffold reaches the maximum degradation rate at 301 °C,The composite scaffold reaches the maximum degradation rate at 320 °C;the in vitro degradation rate of the collagen-based scaffold is 64.7 ± 0.6%,and the in vitro degradation rate of the composite scaffold is 45.6 ± 1.4%.Compared with bovine type I collagen-based scaffolds,cross-linked composite collagen scaffolds show advantages in all aspects of performance characterization.5.So as to carry out biological evaluation of the porcine acellular dermal matrix/collagen composite scaffold,the biocompatibility of the composite scaffold is verified through cytotoxicity test and cell proliferation experiment.The results shows that the cell survival rate of the composite scaffold was ≥ 80%.According to the relationship between the relative cell proliferation rate and the cytotoxicity classification in the US Pharmacopoeia,the composite scaffold is demonstrated to be non-cytotoxic;L929 mouse fibroblasts are seeded on the composite scaffold and proliferated in the early stage.The cell proliferation rate is lower than that of the collagen-based scaffold at the initial stage,and turned to be higher than that of the collagen-based scaffold at the later stage,showing an acceleration trend over cell proliferation. |
PDF Full Text Request