Study On The Construction Of Collagen-based Scaffold For Dermal Tissue Engineering

The purpose of the work is to construct a collagen-based scaffold for dermal tissue engineering, with respect to the microstructure control, biostability enhancement and combining functions etc. Firstly, the collagen was obtained by the trypsin digestion and acid extraction from bovine tendon. Its purity and structure were characterized by Uv-Vis absorbance spectroscopy, Fourier transform infrared spectroscopy and amino acid analysis. The effects of the freezing temperature, the concentration and pH value of collagen solution, and the freezing time, as well as the addition of chitosan and the cross-linking treatment, on the microstructure of the collagen scaffolds were investigated. The results revealed that the freezing temperature and the pH value of the collagen solution are the primary parameters to control the microstructure. Though the glutaraldehyde (GA) cross-linking changed the surface structure of the scaffolds, no alternation of the three-dimensional porous structure in the internal scaffold was found.In order to enhance the biostability of the collagen-based scaffolds, chitosan or amino acid were added during the cross-linking treatment, which functioned as cross-linking bridges. The in vitro swelling test indicated that the GA treated collagen/chitosan scaffold (Col/Chi-GA) has an enough water-absorbing ability for dermal tissue engineering. By comparing the in vitro biodegradation degree, the denature temperature and the cell-mediated contraction, the biostability of Col/Chi-GA was evaluated. The addition of chitosan can enhance the cross-linking efficiency of GA. Meanwhile, the Col/Chi-GA also possessed a good wet-thermal stability and high ability to resist cell-mediated contraction. With the improvement of the biostability, the Col/Chi-GA still preserved excellent biocompatibility.The effect of different kinds of amino acids on the biostability of collagen-based scaffolds was also studied. The results of in vitro biodegradation test revealed that the collagenase biodegradation degree was greatly decreased when lysine was added, which resulted in a more biological stable scaffold. On the other hand, thebiodegradation degree was accelerated compared with the purely cross-linked when glutamic acid was added, while less influenced by glycin addition. Further investigation revealed that the ratio of NHi/COOH is the key factor to determine the cross-linking efficiency of l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide/ W-hydroxysuccinimide (EDAC/NHS). The optimum biostability of the collagen-based scaffold could be yielded when the ratio of NH2/COOH is 2-14. Meanwhile, the wet-thermal stability and the ability to resist cell-mediated contraction of the collagen/lysine scaffold (Col/Lys) were also investigated. The Col/Lys scaffold showed good cytocompatibility during the cell culture test, where the fibroblasts proliferated very well.A collagen-based scaffold with bioactivity was constructed by using the layer-by-layer self-assembly of chondroitin-4-sulfate (CS) and basic fibroblast growth factor (bFGF). The process of the self-assembly on quartz and collagen film was firstly monitored by the method of Uv-Vis absorbance spectroscopy, contact angle measurement and scanning force microscopy (SFM). The CSftFGF mutilayer exhibited good stability in PBS solution. The release of bFGF is mostly due to the disassembly of the CS/bFGF mutilayer. The fibroblast behaved a higher proliferation rate on the collagen film assembled bFGF. Using the same method, a bioactive collagen scaffold was constructed by bFGF assemble By monitoring the linear increase of Rhodamine-labelled bFGF fluorescence intensity with the assembled layer, the self-assembled process of CS and bFGF on the collagen scaffold was proved. Cell viability test and histological images showed that the assembled bFGF has positive effect on the cell viability. The fibroblasts could infiltrate into the bioactive collagen scaffold and proliferate.To construct an anti-bacteria collagen scaffold, a microcapsule deposited with ciprofloxacin was combine...