【作者】 陈宗刚；

【导师】 卿凤翎； 莫秀梅；

【作者基本信息】 东华大学 ， 纺织化学与染整工程， 2008， 博士

【摘要】 组织工程是一种将细胞生物学和材料学相结合形成的新兴生物医学技术。目的是通过活细胞再生天然组织去代替缺损的组织或器官。方法是在外源性细胞外基质(ECMs)中种植细胞组成复合物,在生物反应器中培养扩增,在体外形成新组织后植入患者体内,与组织整合构建新的组织。作为一种再生治疗,它不存在器官移植中存在的供体短缺和免疫处理等问题,也不存在人造生物材料的生物相容性差的问题。这些优点无疑会使其在21世纪医学发展中的占有重要地位。组织工程的关键也是具有挑战性的一步就是找到合适的外源性细胞外基质来模拟天然组织中的细胞外基质分子的功能。这种外源性细胞外基质就是由生物相容性良好和可生物降解的生物材料制备的三维多孔支架。设计外源性ECMs的最佳方法就是仿生,即模仿天然组织中ECMs分子的结构与功能,将天然ECMs作为支架使细胞聚集而构建组织,控制组织结构并调控细胞表型。本课题就是从仿生角度出发,首次通过对生物相容性良好的天然材料胶原蛋白和壳聚糖的静电纺丝来从组分和结构上仿生天然细胞外基质。首先介绍了静电纺丝的发展、方法、成丝理论、成丝影响因素以及静电纺纤维的广阔应用前景。通过实验为胶原蛋白-壳聚糖静电纺丝找到了合适的溶剂,即体积比为9/1的六氟异丙醇/三氟乙酸混合溶剂,第一次制备出了胶原蛋白-壳聚糖静电纺纳米纤维。研究了一些纺丝参数(纺丝电压、给液速率、纺丝距离),纺丝液浓度和壳聚糖/胶原蛋白不同配比对纺丝结果的影响。发现随着纺丝电压的增大和共混体系中壳聚糖含量的增加,静电纺纳米纤维的直径总的来说呈减小趋势;随着给液速率的增加和纺丝液浓度的增加,静电纺纤维的直径呈增加趋势。但当壳聚糖所占比重过大时,纺丝过程比较难控制。随着纺丝距离的增加,静电纺纳米纤维的直径总的来说略微有些增加。通过实验还收集到了胶原蛋白-壳聚糖共混静电纺纤维单丝及纤维束,这将对进一步的研究静电纺纤维的性能提供很大帮助。对不同壳聚糖含量的胶原蛋白-壳聚糖共混静电纺纤维的性能进行了研究。通过红外光谱分析,发现胶原蛋白和壳聚糖分子间存在一定的相互作用。X射线衍射分析显示,在静电纺丝以后,纤维的物相结构发生变化,静电纺纤维更趋向于无定型态。TG和DSC分析显示了静电纺纤维的热稳定情况,并且DSC分析进一步验证了胶原蛋白和壳聚糖分子间相互作用的存在。通过对纤维单丝和纤维膜力学性能的分析,发现随共混体系中壳聚糖含量的变化,其力学性能也发生变化。纤维单丝的力学性能也进一步验证了胶原蛋白和壳聚糖分子间相互作用的存在。静电纺纤维膜的孔隙率和表面亲水性随共混组分中壳聚糖含量的增加而增加。胶原蛋白-壳聚糖静电纺纳米纤维耐水性差,在水溶液中由于有一定的水溶性而难以保持其纳米纤维的形态,同时,其力学性能也不足够强,为了提高胶原蛋白-壳聚糖静电纺纤维的耐水性及其力学性能,用戊二醛蒸气作交联剂,在密闭干燥器中通过戊二醛蒸气挥发对胶原蛋白-壳聚糖共混静电纺纤维膜进行了交联,并对其性能进行了研究。选择合适的交联时间为2天,扫描电镜观察发现交联后的纤维膜在37℃的去离子水中浸泡4天后依然可以保持良好的纤维形态,交联效果明显。但由于胶原蛋白和壳聚糖的一些主要官能团相同,交联后的红外光谱特征峰位置并没有什么变化,通过红外光谱特征峰观察不出胶原蛋白和壳聚糖分子间交联特征峰的出现。X射线衍射显示,交联之后的胶原蛋白-壳聚糖共混静电纺纤维依然保持一种无定型相。TG和DSC分析显示交联后的纤维膜的热稳定性有一定的提高。研究了交联后纤维膜的干湿态力学性能,并对纤维膜交联前后的力学性能及干湿态的力学性能进行了比较。研究发现,交联以后纤维膜的平均断裂强度都有不同程度的提高;而平均断裂延伸度除了纯胶原蛋白和纯壳聚糖纤维膜的增加之外,共混体系纤维膜的平均断裂延伸度都减小;交联静电纺湿态纤维膜的断裂延伸度都比干态膜的断裂延伸度有不同程度的提高,但平均断裂强度则相反,所有湿态纤维膜的断裂强度比干态膜的断裂强度都有不同程度的降低。细胞与基质材料的相互作用是组织工程研究的一个重要领域。本研究从细胞黏附、铺展、增殖、细胞形态等方面着手,对猪的髋动脉内皮细胞、小鼠的心肌主动脉平滑肌细胞与胶原蛋白-壳聚糖静电纺纤维材料的细胞相容性进行了研究,并和盖玻片和细胞培养板的细胞相容性做了比较,得出以下结论。(1)MTT法测定细胞黏附情况的结果表明,chitosan、ch-co-2-8和ch-co-5-5的细胞黏情况都比较好,细胞黏附量都比细胞培养板高,而ch-co-8-2和collagen则相对差一些。(2)MTT法测定细胞增殖能力的结果表明,无论是对内皮细胞还是平滑肌细胞,也是材料ch-co-2-8、chitosan和ch-co-5-5上面的细胞增殖活力比较强,这些材料上的细胞增殖能力比细胞培养板上的强,而ch-co-8-2和collagen则比较差。(3)SEM显示细胞可以进入纤维膜内部孔隙,进行立体迁移生长。出现以上现象的原因,则可能是因为除了材料的组分对细胞的黏附和增殖有很大的影响外,孔隙率也是一个很重要的因素。同时分析了材料的表面形貌、孔径及孔隙率、表面自由能、表面亲疏水性质、表面电荷性能以及表面生物活性等因素对细胞黏附、铺展和增殖的影响,分析了生物材料与细胞相互作用的复杂性。以上结果显示,胶原蛋白-壳聚糖静电纺纤维是一种细胞相容性良好的生物材料。虽然研究还处于实验阶段,但为组织工程支架材料的制备和选取,进一步开展组织工程化人工器官的研究以及临床应用提供了重要的实验数据和科学依据。更多还原

【Abstract】 Tissue engineering is a newly emerging biomedical technique at the convergence ofcell biology and material science. The objective of tissue engineering is to regeneratenatural tissues from living cells to replace defective or lost tissues and organs. Itstypical method is to produce synthetic tissues by incorporating isolated living cellsinto porous scaffolds and create conditions for cells to proliferate, organize anddevelop into the desired tissues or organs. As a regenerative therapy, tissueengineering avoids the problems associated with organ transplantation, such as donorshortages and permanent immunosuppresive medication, and it does not require theimplantation of artificial biomaterials, which might have poor biocompatibility. Withthese advantages, its medical significance will undoubtedly increase in the 21stcentury. The key and challenge for tissue engineering is how to create excellentartificial ECMs. Here the manual ECMs are three-dimensional biomaterial scaffoldswith excellent biocompatibility and porosity. The functions of biomaterial scaffoldsact as analogues of the natural ECMs found in tissues, which provide information forcells expressing their functions, e.g. adhesion, proliferation, differentiation. Therefore,the aim for biomaterial scaffolds design is to mimic the natural ECMs on both thecomponents and the microstructure.The native extracellular matrix is a molecular complex made up of proteins and polysaccharides and comprises 3-dimensional hierarchical fibrous structures of nanometer scale dimensions. From the point of view of mimic, electrospinning of biocompatible collagen and chitosan blends was studied to biomimic the natural ECMs on both the components and the microstructure for the first time in this paper. At first, the history, the method, the theories, the influencing factors and the applications of electrospinning were introduced. In the experiments, the mixture of 1,1,1,3,3,3 hexafluoro-2-propanol (HFP) and trifluoroacetic acid (TFA) (v/v, 90/10) was found as an appropriate solvent for electrospinning of chitosan and collagen blends, and the collagen-chitosan nanofibers were obtained by electrospinning for the first time. Moreover, the dependence of the fibers diameter on the electrospinning parameters (voltage, feed rate and distance), the concentration of solution and the ratio of chitosan to collagen in the electrospun blends was studied. It was found that the diameter of spun fibers became thick with the increase of the feed rate, the distance and the concentration of solution and became fine with the increase of the voltage and the ratio of chitosan to collagen. The electrospun collagen-chitosan single fibers and fibrous bundles were also collected, which would be benefit to the investigation of fibers properties further.The properties of the electrospun collagen-chitosan nanofibres have also been investigated. Fourier transform infrared spectroscopy (FTIR) of fibers has proved that intermolecular interactions exist between collagen and chitosan. The X-ray diffraction (XRD) has shown collagen, chitosan and their complex fibers give typical amorphous after electrospinning. Thermal properties of fibers have been analyzed by thermogravimetry (TG) and differential scanning calorimentry (DSC) and DSC measurement has also proved the existence of intermolecular interactions between collagen and chitosan. The electrospun collagen-chitosan single fiber and fibrous membrane show different mechanical properties with the difference of chitosan content in the electrospun fibers. The mechanical properties of the single fiber have also proved the existence of intermolecular interactions between collagen and chitosan further. Both the porosity and the surface hydrophilic property of fibrous membrane increase with the increase of chitosan content in electrospun fibers.The electrospun collagen-chitosan nanofibers are so hydrophilic that they can be soluble in water and not keep the fiber form, which can limit its applications. In order to improve both water-resistant ability and mechanical properties of nanofibers, the fibrous membrane was crosslinked by glutaraldehyde (GTA) vapor and time of crosslinking was 2 days. The properties of the crosslinked fibrous membrane were also investigated further. The fibrous form of the membrane has been grossly preserved even after 4 days soaked in 37℃deionized water. FTIR shows that the characteristic absorption bands of the fibers before and after crosslinking do not have obvious difference owing to the same main functional groups of both collagen and chitosan. XRD analyses shows the crosslinked fibers still are amorphous. The thermal properties of the fibers are improved after crosslinking. The average ultimate tensile strength of the collagen-chitosan fibrous membrane has been enhanced, but the average ultimate tensile elongation of the fibrous membrane decreases except for the increase of pure collagen and chitosan fibers. Comparing with the dry crosslinked fibrous membrane, the soaked crosslinked fibrous membrane has better tensile elongation but worse tensile strength.The interaction between cells and artificial ECMs is an important field in tissue engineering research. In this paper, the biocompatibility of the electrospun collagen-chitosan nanofibres was tested by cell culture on the fibrous membrane. The porcine iliac artery endothelial cells and the myocardial artery smooth muscle cells of the mouse were seeded on the electrospun fibrous membrane, the tissue culture plates (TCPs) and the cover slips as controls. Cellular adhesion, cellular proliferation and cellular shape on the fibers were investigated and compared with those on the TCPs and the cover slips. The conclusions can be drawn as follows.(1) MTT measurement shows that the endothelial cells adhesion on the fibers is better than that on both the TCPs and the cover slips when chitosan content is 100%, 20% and 50%, but it is worse at chitosan content of 80% and 0%.(2) MTT measurement indicates that both endothelial cells proliferation and smooth muscle cells proliferation on fibrous membrane are better than that on both the TCPs and the cover slips when chitosan content is 20% 100% and 50%, however, it still is worse at chitosan content of 80% and 0%.(3) SEM measurement shows that cells can migration into the fibrous membrane and grow in the three-dimensional space.Therefore, the ratio of chitosan to collagen will affect cell growth on the electrospun collagen-chitosan fibrous membrane, moreover, the porocity is also an important factor. The effect of other factors on cells growth has been analyzed, such as, the fibers morphologies, the surface free energy, the surface hydrophilic/hydrophobic properties, the surface charge and the surface bioactivity, which means the complexity of the interactions between cells and biomaterials.As a result, electrospun collagen-chitosan fibrous membrane is a promising biomedical material. The research will provide the data and the base for electrospun collagen-chitosan fibrous membrane to be selected as tissue engineering scaffold in clinic.更多还原