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Preparation and Properties Study of Recombinant Human CollagenⅢ Tissue Engineering Scaffold

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Tutor: FanYanRong
School: Nanjing University of Technology and Engineering
Course: Biochemical Engineering
Keywords: recombinant human collagen Ⅲ,chitosan,high density fermentation,vacuum freeze dr
CLC: R318.08
Type: Master's thesis
Year:  2014
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Abstract:
Collagen has become conventional carrier material for tissue engineering due to its good biological properties. The recombinant human collagen used in this study was pro-duced by genetic engineering method to design the collagen protein gene sequences, and then has the characteristics of human collagen protein through translation process. Compared with the collagen from animal body, the recombinant human collagen has the advantages of hydrophilic, no virus risk, and easy to industrial production. Which indicates a promising application in tissue engineering. However, the single component as a biological scaffold can not meet the demand of scaffold materials, we use recombinant human collagen III combining with chitosan through glutaraldehyde cross-linking to prepare recombinant human collagen/chitosan three-dimensional sponge scaffolds, and preliminarily studied scaffold physicochemical properties and cellular compatibility. The results are as follows.1. This paper firstly using Pichia pastoris gene engineering bacteria to produce recom-binant human collagen (RHSC) through high density fermentation, then using SDS-PAGE gel electrophoresis, mass spectrometry, infrared spectrum method to analyze protein, found that RHSC was a kind of extracellular product, its monomer molecular weight was about56kD, polymer molecular weight was two times of single, its primary structure coincided with the designed objective gene, and the structure of RHSC was similar to Sigma type III collagen.2. Explore the preparation technology of recombinant human collagen III/chitosan scaffolds. Recombinant human collagen III and Chitosan were mixed by mass ratio of5:1,3:1,1:1,1:3,1:5. And then glutaraldehyde was added for crosslinking, followed by vacuum free drying. The scaffolds were observed for the morphology, pore size, porosity, water absorption rate, degradation rate, thermal decomposition temperature, physical and chemical properties. Results showed the pore size of these scaffolds were among20-50um; the overall porosity were above93%; the degradation performance was significantly improved at the addition of chitosan. Comprehensive various physical and chemical properties as a result, the scaffold prepared at ratio1:1showed uniform pore size distrib-ution, high cross-linking degree, suitable water absorption and degradation rate, and its thermal decomposition temperature of249.29℃, which conformed to the basic require-ments of biological materials. 3. With different concentrations of glutaraldehyde to prepare scaffolds, we found that the composite scaffolds with0.05%glutaraldehyde showed a uniform distribution of poro-us structure, rich distribution of pore, high cross-linking degree, good stability inphysiolo-gical saline. Therefore, a final concentration of0.05%glutaraldehyde as crosslinkingagent was optimal to prepare recombinant human collagen Ⅲ/chitosan scaffolds.4. Prepare the liquid immersion of recombinant human collagen III/chitosan scaffold prepared at1:1ratio,0.05%glutaraldehyde, and preliminarily discussed its compatibility on human skin fibroblasts by AO/EB staining, MTT experiment. Found that its cytotoxic-ity was small, since the scaffold extract not affect cell growth activity, which reflecting good biocompatibility, to be applied in the skin tissue engineering.
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