Synthesis And Biological Properties Of Chitosan-based Scaffolds

Scaffold materials with remarkable properties such as biocompatibility and biodegradable absorption properties were considered to repair bone defects and regenerate bone function.The materials in this dissertation were designed on the basis of both the component and microenvironment of the bone tissue to promote cell growth.Briefly,the structures of chitosan（CS）and gelatin（GEL）were similar to those of the human body’s polysaccharide and collagen,respectively,which could be used to replace the organic component of natural bone.Furthermore,Chitosan could combine gelatin to form an Arg-Gly-Asp（RGD）-like structure to mimic the native extracellular matrix.Hydroxyapatite,as the major mineral constituent of native bone,exhibited excellent osteoconductivity,nonimmunogenicity and bioactivity which promoted cells growth.The electrospinning technique was introduced to prepare nanofiber mat with three-dimensional porous structure,which could promote cell adhesion and proliferation.So,the biomimetic composite scaffolds CS-PEO@GEL core-shell structured nanofibers were firstly prepared by coaxial electrospinning.Hydroxyapatite（HAP）was then deposited on the surface of CS-PEO@GEL nanofibers by wet chemical method.For comparing with core-shell structured CS-PEO@GEL nanofibers,CS-PEO nanofibers,GEL nanofibers,CS-PEO-GEL composite nanofibers were also prepared by single electrospinning,respectively.HAP was then also deposited on the surface of CS-PEO nanofibers,GEL nanofibers,CS-PEO-GEL composite nanofibers,respectively.The results were summarized as follows:1.CS-PEO@GEL nanofibers were prepared by coaxial electrospinning,in which CS-PEO acetic acid solution was used as shell solution and GEL acetic acid solution was used as core solution.The CS-PEO@GEL nanofibers mat formed three-dimensional（3D）porous structure for promoting cells growth.The poor electrospinnability of chitosan was successfully improved by adding gelatin and polyethylene oxide（PEO）.The concentration of chitosan and gelatin had an important influence on the structure of CS-PEO@GEL nanofibers.SEM and TEM showed that CS-PEO@GEL nanofibers with the core of about 100nm and shell of about 150nm were uniform and smooth.The contact angle test showed that the adding of hydrophilic polyoxyethylene and gelatin could effectively improve the hydrophilicity of chitosan.2.The 3%（V/V）glutaraldehyde（GA）solution was chosen to improve the water stability of CS-PEO@GEL nanofibers.The SEM showed that the cross-linked CS-PEO@GEL nanofibers showed better water stability and the three-dimensional porous structure had still been remained even when be kept in water.3.To further enhance osteoblast cell proliferation,hydroxyapatite(Ca₁₀（PO₄）₆（OH）₂,HAP),as the major mineral constituent of native bone,was deposited onto the surface of gelatin-chitosan core-shell structured nanofibers by a wet chemical method.The influence of cycle number,mineralization formwork,temperature and pH value on the mineralization efficiency and structure of the hybrid materials were also studied.The CS-PEO@GEL nanofibers were completely coated by HAP within one cycle and exhibited obvious the three-dimensional porous structure which would be benefit for the growth of cells.When CS-PEO@GEL nanofibers were treated within two cycles,the three-dimensional porous structure of CS-PEO@GEL electrospun mats was destroyed by too much HAP covering.Compared with chitosan nanofiber,gelatin nanofiber and chitosan-gelatin composite nanofibers,gelatin-chitosan core-shell structured nanofibers improved the mineralization efficiency of hydroxyapatite.The XRD pattern and HRTEM indicated that the nanoparticles on the CS-PEO@GEL nanofibers were HAP and could be ascribed to P63/m space group.EDS showed the Ca/P atomic ratio was about 1.64 which was similar to stoichiometric hydroxyapatite(Ca₁₀（PO₄）₆（OH）₂),Ca/P=1.67.4.MG-63 cells were used to evaluate the cytotoxicity assay and the proliferation assay of the CS-PEO nanofibers,HAP@CS-PEO nanofibers,GEL nanofibers,HAP@GEL nanofibers,CS-PEO-GELcompositenanofibers,HAP@CS-PEO-GELcompositenanofibers,CS-PEO@GEL nanofibers and HAP@CS-PEO@GEL nanofibers（C1,C2）.The cells viability of all the samples were all around 80%,which were higher than the standard of cytotoxicity.The results indicated that the materials with low toxicity could be used as preliminary estimate for the proliferation tests of MG-63 on the materials.After MG-63 cells were cultured for 1,3,5,7 days,the cell viability of all nine samples exhibited an increasing in a time dependent manner within seven days,which indicated that MG-63 cells cultured on the materials were successfully attached and grown.It also indicated that all the materials exhibited better biocompatibility,and were suitable for cells growth.The cell viability of CS-PEO-GEL composite nanofibers and CS-PEO@GEL nanofibers were higher than CS-PEO nanofibers and GEL nanofibers.Compared with CS-PEO-GEL composite nanofibers,CS-PEO@GEL core-shell structured nanofibers in which CS served as the shell absorbed the cells more easily and promoted the cells growth.So the cells viabilities of CS-PEO@GEL nanofiberes were a bit higher than CS-PEO-GEL composite nanofibers.When HAP was deposited on the natural polymer nanofibers（CS-PEO,GEL,CS-PEO-GEL,CS-PEO@GEL）,the cell viability of samples were higher than those of natural polymer nanofibers,which could be ascribed to the presence of HAP.High porosity in scaffolds could improve cells adhesion by increasing the surface area.More importantly,the porous structure could promote cell proliferation,migration,revascularization and new bone formation.All in all,hydroxyapatite deposited on the gelatin-chitosan core-shell structured nanofibers had the best osteogenic property.The biomimetic composite scaffolds could be suggested as a promising material for bone tissue engineering.