熔融离心纺丝聚乳酸纤维复合明胶支架的制备及骨组织工程中的应用

发布时间：2018-05-07 02:36

本文选题：熔融纺丝 + 骨组织工程支架　；参考：《吉林大学》2014年博士论文

【摘要】：长期以来，生物可降解高分子材料应用于骨组织工程一直是组织修复的热点，多种高分子材料组织工程支架材料被不断开发出来。在组织工程支架材料设计与制备中，良好的孔隙结构对骨修复效果十分重要，研究证明：一方面，在多孔材料中可见新生骨形成而在无孔粒子材料中未见骨形成[1]；一方面，当孔隙率和孔隙结构在一定范围内增加时对细胞增殖、粘附、长入以及支架血管化有着显著地促进作用[2-5]。目前，多孔的生物可降解高分子组织工程支架因其具有可控的孔隙率和孔径、良好的力学强度和加工性能等优点，已经成为组织工程支架研究的主流。但目前以传统粒子沥滤支架为代表的海绵状多孔支架仍存在一些缺点，如孔隙之间连通不佳，易于出现闭孔和微孔连接等，在一定程度上限制了组织长入和营养物质交换，不利于骨组织修复。而纤维性多孔支架虽然解决了海绵状多孔支架的孔隙间连通性问题，但却存在其他不足，具体来说：以静电纺丝纤维毡为代表的纳米级纤维支架，其具有良好的抗菌性、可控性和利于细胞增殖和分化等优点，但无法提供足够的厚度以及利于细胞长入的孔隙直径，难以满足治疗大段骨缺损的需求；以快速成型法为代表制备的微米级纤维支架，虽然解决了静电纺丝纤维毡的厚度和孔隙直径问题，但由于缺乏纳米级和亚纳米级纤维结构，对细胞功能的促进作用不甚理想。为此，本研究采用一种新型的熔融离心纺丝方法，制备得到棉花样、直径分布广泛（100nm-40μm）的聚乳酸无纺纤维，并采用有机溶剂气体表面粘连方法将该种纤维制备成为新型的纤维组织工程支架，，以期克服传统海绵状多孔支架和纤维性支架的局限。然后利用明胶表面涂覆的方法对新型纤维支架进行修饰，使其从结构和生物活性上均更加接近天然细胞外基质，从而获得更加智能化的组织工程支架。首先，明确熔融离心纺丝方法制备聚乳酸纤维所需的适宜条件即温度和转速，接下来对不同原材料和转速所制备的聚乳酸无纺纤维进行扫描电镜、直径分布、力学强度、热力学性质、结晶度以及初步的体外生物相容性和功能分析；然后，明确有机溶剂气体表面粘连方法制备聚乳酸纤维支架的所需的适宜条件即粘联作用时间和所需纤维的最佳密度，然后对不同密度纤维制备的单纯聚乳酸纤维组织工程支架进行力学性能、孔隙率、扫描电镜、初步的体外生物相容性和功能分析，并通过表面明胶涂覆方法对聚乳酸纤维支架进行改性修饰，对改性修饰后支架进行了扫描电镜和体外矿化能力研究；最后，将制备所得的聚乳酸纤维复合明胶支架进行兔桡骨缺损修复实验，检验其骨缺损修复能力。通过上述研究，对新型的熔融离心纺丝方法制备聚乳酸纤维及支架应用于临床提供依据，期望开发出可应用于临床的骨组织工程材料。第一部分：熔融离心纺丝聚乳酸纤维的制备、表征以及生物相容性研究。通过多次试验，得到聚乳酸（PLLA，粘均分子量：90595）进行熔融离心纺丝的适宜温度为：旋碟中心220℃、旋碟边缘180℃；能够得到成形纤维的最低转速和最高转速分别为300rpm和500rpm，而制备纤维产量最大的转速为900rpm，得到初次纺丝纤维三种。以制备所得纤维分别作为原材料，按照各自的制备转速进行二次纺丝，得到二次纺丝纤维三种。利用扫描电镜、直径分布、力学强度、热力学性质、结晶度、热力学降解、X线衍射和红外衍射等对所制备的六种不同聚乳酸纤维进行表征。将小鼠胚胎成骨细胞前体（MC3T3-E1）细胞种植于六种纤维和静电纺丝膜上，利用MTT法检测不同材料在1d、3d和7d对成骨增殖的影响，利用扫描电镜法观察不同材料在1d、3d、7d和14d对细胞形态和长入能力的影响，并利用六种纤维和静电纺丝纤维毡的24h材料浸提液，行MTT检测，检验材料的急性细胞毒性。结果显示：熔融离心纺丝所获得聚乳酸无纺纤维拥有棉花样、直径分布较宽的三维立体结构；其理化性质可根据不同的原材料和转速进行调整；相对于静电纺丝纤维毡，熔融离心纺丝纤维其细胞生物相容性更好。第二部分：熔融离心纺丝聚乳酸纤维复合明胶支架的制备、表征以及体外相容性实验。采用三氯甲烷气体作为粘连剂，利用溶剂气体表面粘连法成功制备了聚乳酸纤维支架，其最佳粘连时间为90min，并明确支架成形所需最低的纤维密度为0.1g/cm3，此外还制备了密度为0.15g/cm3以及0.2g/cm3的支架，利用力学强度、孔隙率、扫描电镜等对三种不同密度的纤维支架进行了表征，并将小鼠胚胎成骨细胞前体（MC3T3-E1）细胞种植于0.15g/cm3纤维支架上，利用扫描电镜法观察密度为0.15g/cm3纤维支架在7d时对细胞长入能力的影响。将密度为0.15g/cm3的纤维支架进行明胶表面涂覆修饰，制备得到0.1%、0.5%、1%和10%浓度明胶表面涂覆修饰的复合支架。利用扫描电镜法观察改性后支架的表面形态和体外矿化后沉积物表面形态，利用矿化前后质量改变、沉积物X线衍射以及等离子体发射光谱等方法对复合支架进行表征。结果显示：采用三氯甲烷气体作为粘连剂，粘连作用60-90min后可成功制备出聚乳酸纤维支架；其力学强度及孔隙率等性质可根据粘连时间和纤维密度等条件进行人为调节；密度为0.15g/cm3的单纯聚乳酸纤维支架具有良好的力学强度和孔隙结构，并且利于细胞的长入；利用0.5%浓度明胶对支架进行表面修饰后可获得孔隙率和体外矿化能力良好的类细胞外基质支架。第三部分：熔融离心纺丝聚乳酸纤维复合明胶支架的兔桡骨缺损修复实验。根据前期工作，选择力学强度、孔隙结构和矿化能力性能较好的聚乳酸/明胶复合支架进行兔桡骨缺损修复试验，分别于2week、4week、6week、9week和12week进行大体观察和X线观察。结果显示：兔桡骨缺损修复效果：聚乳酸纤维/明胶复合支架10%羟基磷灰石/聚乳酸粒子粒滤支架单纯聚乳酸纤维支架单纯聚乳酸粒子沥滤支架商品化纳米羟基磷灰石/聚酰胺复合支架空白对照组。
[Abstract]:For a long time, biodegradable polymer materials used in bone tissue engineering have always been the hot spots of tissue repair. A variety of polymer scaffold materials have been developed. In the design and preparation of tissue engineering scaffold materials, good pore structure is very important to the effect of bone repair. New bone is found in the material and no [1] is found in the porous material. On the one hand, when the porosity and pore structure increase in a certain range, the cell proliferation, adhesion, long entry and stent vascularization have a significant effect on [2-5].. The advantages of controlled porosity and pore size, good mechanical strength and processing performance have become the mainstream in the research of tissue engineering scaffolds. However, there are still some shortcomings in the spongy porous scaffolds represented by the traditional particle leaching support, such as the poor connectivity between the pores, the easy out of the obturator and the microporous connection and so on, to a certain extent. The tissue long entry and the exchange of nutrients are not conducive to the repair of bone tissue. While the fibrous porous scaffold solves the problem of the connectivity between the pores of the spongy porous scaffold, but there are other deficiencies. Specifically, the nanoscale fiber scaffold, represented by the electrospun fiber felt, has good antibacterial, controllability and benefit. Cell proliferation and differentiation, but can not provide enough thickness and the pore diameter that is beneficial to cell growth. It is difficult to meet the need for the treatment of large bone defects. Micrometer fiber scaffolds, represented by rapid prototyping, have solved the problem of the thickness and pore diameter of the electrospun fiber felt, but due to the lack of nanoscale and subgrade. Nano fiber structure is not ideal for promoting the function of cell. Therefore, a new type of melt centrifugal spinning method was used to prepare a cotton like fiber with a wide diameter (100nm-40 mu m) in diameter, and a new fiber tissue was prepared by the method of organic solvent gas surface bonding. In order to overcome the limitations of the traditional spongy porous scaffold and fibrous scaffold, the scaffold is used to modify the new type of scaffold with gelatin surface coating to make it closer to the natural extracellular matrix from the structure and biological activity, so as to obtain a more intelligent tissue engineering scaffold.
First, the suitable conditions for the preparation of polylactic acid fibers were defined as temperature and rotational speed. Then, the scanning electron microscopy, diameter distribution, mechanical strength, thermodynamic properties, crystallinity, and preliminary biocompatibility and functional analysis of polylactic acid non woven fibers prepared by different raw materials and rotational speeds were followed. The optimum conditions required for the preparation of polylactic acid fiber scaffolds by the organic solvent gas surface adhesion method are the optimal density of the adhesion time and the required fiber, and the mechanical properties, porosity, scanning electron microscopy, and primary biocompatibility and work in vitro are then carried out for the simple polylactic tissue engineering scaffolds prepared by different density fibers. The modified polylactic acid fiber scaffold was modified by surface gelatin coating method, and the modified scaffold was studied by scanning electron microscope and in vitro mineralization ability. Finally, the prepared poly (lactic acid) composite gelatin scaffold was used to repair the radius defect of rabbit and to test the repair ability of bone defect. The application of the new method of melting centrifugal spinning to the preparation of polylactic acid fibers and scaffolds is provided for clinical application of bone tissue engineering materials.
The first part: the preparation, characterization and biocompatibility study of the melt centrifuged poly (lactic acid) fiber. Through several experiments, the optimum temperature of poly lactic acid (PLLA, viscosity average molecular weight: 90595) for melting centrifuge spinning is: 220 centigrade and 180 centigrade at the edge of rotating disc; the minimum speed and maximum speed of the forming fiber can be obtained. Not 300rpm and 500rpm, and the maximum speed of fiber production is 900rpm, and the first spinning fiber three kinds of fibers are obtained. The fibers are prepared as raw materials and spinning two times according to their respective rotational speed, and three kinds of spinning fibers are obtained. Scanning electron microscope, diameter distribution, mechanical strength, thermodynamic properties, crystallinity and thermodynamics are used. Six kinds of poly (lactic acid) fibers were characterized by degradation, X-ray diffraction and infrared diffraction. The mouse embryonic osteoblast precursor (MC3T3-E1) cells were planted on six kinds of fibers and electrospun membranes. The effects of different materials on the osteogenesis of 1D, 3D and 7d were detected by MTT, and the different materials were observed by scanning electron microscopy in 1D, 3D, 7. The effects of D and 14d on the cell morphology and the ability to grow, and using the 24h extracts of six kinds of fibers and electrospun fiber felt, were used to test the acute cytotoxicity of the material by MTT test. The results showed that the poly lactic acid non spun fiber obtained by molten centrifugal spinning has a three dimensional structure with a wide diameter distribution of cotton and its physical and chemical properties. It can be adjusted according to different raw materials and rotational speed. Compared with the electrospun fiber mat, the melt centrifugal spinning fiber has better cell biocompatibility.
The second part: the preparation, characterization and in vitro compatibility of the melt centrifuged poly (lactic acid) fiber composite gelatin scaffold. Using trichloromethane gas as an adhesion agent, the polylactic acid fiber scaffold was successfully prepared by the solvent gas surface adhesion method. The optimum adhesion time was 90min, and the minimum fiber density needed for the stent formation was clearly defined. For 0.1g/cm3, the scaffolds with density of 0.15g/cm3 and 0.2g/cm3 were also prepared. Three kinds of fiber scaffolds with different densities were characterized by mechanical strength, porosity and scanning electron microscope, and the mouse embryonic osteoblast precursor (MC3T3-E1) cells were planted on the 0.15g/cm3 fibrous scaffold. The density was observed by scanning electron microscope (0.15g/cm3). The effect of fiber scaffold on the cell growth ability at 7d. The surface coating of 0.1%, 0.5%, 1% and 10% gelatin surfaces was prepared by coating gelatin surface with the density of 0.15g/cm3 fiber scaffold. The surface morphology of the modified stent and the surface morphology after the mineralization were observed by scanning electron microscope. The composite scaffolds were characterized by quality changes before and after mineralization, X-ray diffraction of sediments and plasma emission spectroscopy. The results showed that chloroform gas was used as adhesion agent, after adhesion of 60-90min, the polylactic acid fiber scaffold could be successfully prepared. The mechanical strength and porosity could be based on the adhesion time and fiber. Density and other conditions are adjusted artificially; the simple polylactic acid fiber scaffold with a density of 0.15g/cm3 has good mechanical strength and pore structure, and is beneficial to the growth of the cell. After the surface modification of the scaffold with 0.5% concentration gelatin, the porosity and good extracorporeal mineralization ability of the extracellular matrix scaffold can be obtained.
The third part: a rabbit radial defect repair experiment with a melt centrifuged poly (lactic acid) fiber composite gelatin scaffold. According to the earlier work, a polylactic acid / gelatin composite scaffold with better mechanical strength, pore structure and mineralizing ability was selected to repair the radius defect of rabbit, and the general observation was made in 2week, 4week, 6week, 9week and 12week, respectively. The results showed that the repair effect of rabbit radial defect: polylactic fiber / gelatin composite scaffold 10% hydroxyapatite / polylactic acid particle filter scaffold simple polylactic acid scaffold for pure polylactic acid leaching stent commercial nano hydroxyapatite / polyamide composite scaffold in empty white control group.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08

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