| Nowadays, peripheral nerve repair remains a common, yet challenging clinical problem. Direct end-to-end suturing is suggested for a short nerve injury. For larger nerve defects or gaps, implantation of a nerve graft is often necessary to bridge the proximal and distal nerve stumps for facilitating nerve regeneration and functional recovery. The typical choice is a nerve autograft that is harvested from another site in the body. However, this recognized"gold standard"technique for peripheral nerve repair is limited by tissue availability, donor-site morbidity, secondary deformities, as well as potential differences in tissue structure and size. Both allografts and xenografts have been attempted, however these techniques are subject to strong immunosuppression and have achieved very poor success. Therefore, much effort has been devoted to seeking promising alternatives to conventional nerve autografts.In recent years, with the rapid development of tissue engineering and neuroscience, using priciples and techniques of tissue engineering to prepare the scaffold which has a specific three-dimensional structure and biological activity then to repair peripheral nerve defect are becoming an innovative research and may be an effective treatment method.Part I: Fabrication and properties of chitosan combined with type I collagen protein for improved nerve tissue-engineering scaffoldsObjective To improve nerve tissue-engineering scaffolds by combining chitosan with type I collagen protein.Methods①Chitosan and type I collagen protein were respectively dissolved in 0.05mol/L acetic acid to get the gelatin.②The scaffolds were freeze-dried and were radiated with ultraviolet rays to make it crosslinked.③The microstructure was observed under scanning electron microscope (SEM) and the pore size, porosity ratio were measured.④In vitro biodegradation rate was measured in 0.01mol/L PBS solution for 30 days.⑤According to the experimental principles of GB/T 16886-ISO 10993 on medical apparatus, the MTT colorimetric assay was adopted and the L929 mouse fibroblasts were used to establish cell proliferation in this study.⑥Mechanical properties of the scaffolds were measured on a universal testing machine equipped with a 50-N load cell.Results All the scaffolds are circular cylinder, the microscopic channels are arranged in parallel manners, and the pore structure becomes uniform with most pore size in the range of 60-130 m; the porosity rate and degradation rate of the scaffolds is 83.30% and 16.95% respectively. The scaffolds have good biocompatibility with L929 cells without exerting any significant cytotoxic effects on the cell phenotype or cell functions and. The mechanical properties test shows that the scaffolds have good tensile strength. Conclusion The improved nerve tissue-engineering scaffolds have good three-dimensional structures and satisfactory biocompatibility, which have the potential to be applied to peripheral nerve tissue engineering.Part II: In vitro evaluation of the biocompatibility of collagen/chitosan biomaterials crosslinked with genipin for peripheral nerve tissue- engineeringObjective To fabricate collagen/chitosan biomaterials crosslinked with genipin (GP) for peripheral nerve tissue-engineering, and compare the biological characteristics with uncross-linking group and glutaraldehyde(GTA) group.Methods①According to the different cross-linking methods, biomaterials were divided into 3 groups:Uncross-linked group, GP group and GTA group.②The microstructure was observed under SEM and the pore size, porosity rate were measured.③Swelling ratio and in vitro degradation rate:The biomaterials were weighed (W0) after crosslinking, then immersed in the culture medium that contained 10 ml aseptic phosphate buffer solution (PBS). The samples were drawn from the culture medium in each group after 24 hours and wiped with filter paper to remove excess liquid and weighed (W1). Swelling ratio(%) = W1 - W0 / W0×100%.The remaining samples were collected from each group after 4, 8, 12 weeks and then weighed (W2) in the same procedure. Degradation rate (%) = W1 - W2 / W1×100%.④Determination of cross-linking index:10 samples were prepared from each group, 5 samples reacted with trinitro-benzen-sulfonic acid (TNBS) and NaHCO3, then they were hydrolyzed with HCl, and extracted with ethyl ether. The absorbance of the diluted solution was measured at 346 nm, so we gain the ATNBS. The other 5 samples were prepared by the same procedure, except for HCl was added before the addition of TNBS, and the absorbance was measured as control (Acontrol). The absorbance after crosslinking:Aafter = ATNBS - Acontrol.⑤Determination of cytotoxicity:According to the experimental principles of GB/T 16886-ISO 10993 on medical apparatus, and two international standard experimental methods were adopted in the study. The L929 fibroblasts were used in this study to establish cell proliferation.⑥The mechanical properties of the samples were measured including compressive strength, tensile strength, breaking elongation and Young's modulus.Results①The biomaterials without any crosslinking were circular cylinder, the microscopic channels were arranged in parallel manners, and the pore sizes of the channels were uniform. The average diameter was 50-120 m;the pore sizes of uncross-linked group remained basically unchanged, GP group and GTA group are smaller than uncross-linked group.②The porosity ratio of GP group and GTA group were higher than uncross-linked group, respectively, but GP group and GTA group weren't significant difference;the swelling ratio of GP group was higher than GTA group, and GTA group was higher than Uncross-linked group.③The crosslinking index of GP group and GTA group were 65% and 86.5%.④GP group and GTA group were put in phosphate buffer solution for 4, 8, 12 weeks, respectively, without significant difference, but uncross-linked group was significant higher than GP group and GTA group.⑤Cell culture in GTA group presented necrosis, while cells cultured in GP group and uncross-linked group grew well, respectively.⑥In the compressive strength test, the compressive strength of the GTA or GP group was significantly lower than that of the uncross-linked group at each strain, the GTA group and the GP group were also observed to be different from each other. In the tensile test, the results showed that the GP group became much softer and more flexible after hydration in PBS than the others. Conclusion In this study, the results have shown that collagen/chitosan scaffolds cross-linked with GP are potential candidates for nerve tissue engineering applications with enhanced biostability and good biocompatibility.
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