| Bone tissue engineering is used to repair the tissue lesions or defect in the bone through application of life science and engineering basic principle and technology. This is basic idea:Specific the osteoblast grow in scaffold materials which have certain biological characteristics and can be absorbed gradually in the human body. Osteoblast and scaffold materials are formed biological material compound and then the compound are implantated in human body for tissue defect. Scaffold materials gradually been degradation and absorbed by human body followed by cell proliferation and constantly secretion matrix. Bone tissue is finally formed in a new and original function and morphology.In recent years, Bone tissue engineering is the most popular areas. It has great advantages in solving traumatic, infection, tumor by reason of bone defect. Three key elements of bone tissue engineering includes seed cells, grow factor and scaffold materials,and the scaffold materials is undoubtedly the key of bone tissue engineering.It will not only be seed cells and growth factors transported to a defect area,but also provide supporting function of the new bone tissue. Now, scaffold materials research is more and more in the bone tissue engineering, especially composite scaffold materials, chitosan/silk fibroin as a new scaffold materials become a hot spot in the bone tissue engineering.Now, all kinds of materials are used for bone tissue engineering, and its has advantages and drawbacks. But composite material are most commonly used in the clinical applications and laboratory research. Silk fibroin(SF) and chitosan(CS) are natural polymer materials with a good biological characteristics and unique mechanical properties.They become more and more popular in tissue engineering field in recent years.In this experiment, we prepared the blend of SF and CS as a scaffold in the different proportion through freeze-drying merger chemical crosslinking methods. The purpose was to find ideal scaffold materials which was suitable for the growth of osteoblast in the way of exploring and analyzing the material structure for characterization. In order to carry on cytology in vitro test research to lay a solid foundation for bone defect restoration and reconstruction in the near future.Chapter one Preparation and Characterization of porous silk fibroin/chitosan scaffoldsObjectsThis paper aims to explore and analyze the material structure for characterization on the basis of making a variety of compound scaffolds with different proportion of silk fibroin and chitosan.Methods2%of silk fibroin solution was made by dissolution from CaCl2solvent systems (CaCl2:C2H5OH:H2O=1:2:8(mole ratio)), filtration, concentration and purification. Dissolve Chitosan in acetic acid solution to prepare3%chitosan-acetic acid solution. Blend the Silk fibroin (SF) and Chitosan (CS) in a freeze-drying and chemical crosslinking methods approach in different proportion to get different scaffold materials. Then observed the morphology of the SF/CS scaffolds under a scanning electron microscopy, and evaluate the characterization in the ways of FTIR,EDS XRD.ResultsMore stable scaffolds were created with the method of blending, and modifying each other. The40%SF-60%CS group proves the preferred ratio of cell scaffold material due to its better aperture(>90%) for the growth of osteoblasts. The experiment also explains that complex internal structure was changed through FTIR, EDS, XRD,and further confirmed that the complex is stable and successful.Chapter two Physicochemical property and Degradation behaviors of porous silk fibroin/chitosan scaffoldsObjectsThe components of the best ratio was screened through the water absorption and swelling ration.At last, degradation test aims to observe the best components whether to adapt to the biological materials with the well degradation rate in the degradation of the body.MethodsAccording to the first part, a variety of compound scaffolds with different proportion of silk fibroin and chitosan were prepared. Water absorption was calculated through the percentage, which is the comparison between the weight of solid scaffold materials submerged in water and dry weight. Swelling ratio was calculated through the volume change between dry state and wet condition. The best group were found through the comprehensive above test results. Finally,Silk fibroin/chitosan scaffold were researched in the degradation rate and the pH change in degradation liquid into the simulated body fluid(SBF), lasting64days.ResultsEach quality of silk fibroin/chitosan scaffold materials has good water absorption. Statistical analysis results show that there was no significant difference between groups (P>0.05). But the swelling ratio roughly was in reducing along with the increase of silk fibroin content. Swelling rate close to80%in100%CS group had significant difference between the other the groups (P<0.05). However,20%SF-80%CS group and40%SF-60%CS group were slightly higher than the60%SF-40%CS group and80%SF-20%CS group, there were differences (P0.05). But between20%SF-80%CS group and40%SF-60%CS group were not statistically significant (P>0.05). Comprehensive above the experimental results,40%SF-60%CS group was filtered for optimal combination.40%SF-60%CS group of degradation test in vitro lasted for a period of64days. The results showed that the decomposition rate was relatively stable. The decomposition rate became significant in the first15days, and scaffold materials lost14.43%of the original weight. In the next21days, the decomposition rate slowed, but the decomposition rate was more slowly in the later28days. At last,scaffold materials only lost18.25%of the original weight until64days. The pH value of degradation liquid had been in7.42-7.62narrow range after more than two months of degradation time, and close to neutral. All these results laid to the foundation for the application of scaffold materials in body.Chapter three Biocompatibility and Osteogenesis of porous silk fibroin/chitosan scaffoldsObjectsMG-63Cells were grown in40%SF-60%CS scaffold to explore biocompatibility and osteogenesis in vitro.MethodsBecause pure silk fibroin scaffold materials (100%SF) was particularly dissolved in aqueous solution because of flimsiness. So the experimental group:pure CS group (100%CS) and40%SF-60%CS group. Blank control group:glass slides group.Firstly, MG-63Cells were grown in40%SF-60%CS scaffold.To observe the MG-63growth in the scaffolds though Hoechst fluorescence staining and to study the biocompatibility by the way of calculating adhesion rate and proliferation rate.Lastly,to explore osteogenesis by means of forming mineralization nodules and promoting MG-63cells secreting alkaline phosphatase(ALP) activity. Factorial analysis of variance was used to determine the significance of differences, and One-Way ANOVA was used for each time period in order to compare between groups, P<0.05was considered a statistically significant difference.ResultsThe MG-63cells were in good growing condition on the support. After inoculating for6hours, the cells started to adhere to pore wall of the supporting material. After24hours, the majority of cells have been stuck. After3days, cells have stuck tightly together, and all the pore wall and pore space of the supporting material have been filled with cells.Early results for adhesion rate showed that two experimental groups were significantly difference better than the blank control group(P<0.05). The number of cells on scaffolds was increased in1h,3h,6h. Two experimental groups are statistically significant difference in1h and3h(P<0.05).But in6h, two experimental groups of adhesion rate were close to75%,there were no significant difference (P>0.05).The MG-63cell proliferation rate of two experimental groups were significantly higher than the blank control group. In the1d and5d, two experimental groups were no statistically significant difference (P>0.05). Howerer two experimental groups were statistically significant difference (P<0.05) in the3d.Compared with blank control,two experimental groups promoted MG-63cells to secrete alkaline phosphatase(ALP)(P<0.05). In all time points, two experimental groups compared and found that the differences was significant(P<0.05),and in which40%SF-60%CS group was higher than100%CS group.The result of hoechst fluorescence staining show that MG-63cells had good activity and the proliferation ability in40%SF-60%CS group scaffold. We found that whether cells secreted matrix or finally reunited,40%SF-60%CS group were better than100%CS group in the process of forming mineralization nodules.Conclusions1. To blending2%SF with3%CS in order to preparate scaffolds in the different proportion though the method of freeze drying and chemical crosslinking. We found that microstructure of SF/CS scaffolds were changed through the FLIR and XRD. Blending CS, silk fibroin in the form of unstablea-helix structure/no rules curly structure turned to βfolded structure, and crystallization degree was enhanced. Especially40%SF-60%CS group was to be the first choice because of the osteoblasts growth and suitabled for the better aperture and the stability of structure. 2. Each quality of silk fibroin/chitosan scaffold materials has good water absorption. But the swelling ratio roughly was in reducing along with the increase of silk fibroin content. Comprehensive above the experimental results,40%SF-60%CS group was filtered for optimal combination.40%SF-60%CS group of degradation test in vitro lasted for a period of64days. The results showed that the decomposition rate was relatively stable and pH value of degradation liquid was remaind neutral.3.40%SF-60%CS group scaffold was used for early adhesion, growth,and proliferation to MG-63cells. After modificated each other,40%SF-60%CS group scaffold had good biocompatibility and better cells affinity, and could promote the osteoblast biological function. The most important is that Osteoblasts could be secrete cells matrix.In a word,40%SF-60%CS group scaffold is a good bone tissue engineering materials, and can be used for the research and clinical work in bone tissue engineering in the future. |
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