【作者】 王小梅；

【导师】 陈云； 汪晖；

【作者基本信息】 武汉大学 ， 生理学， 2011， 博士

【摘要】 抗凝血材料是生物材料的重要组成部分,被广泛应用于与人体血液和组织相接触的医疗器械或组织工程化材料上,例如人工心脏瓣膜、血液透析系统、体外循环系统、血管栓塞剂、心脏起搏器、人工血管、血管支架、介入导管、外科手术线等；这类与血液直接接触的材料不仅要求具有组织相容性,不会对生物体组织引起炎症,而且要求具有血液相容性,能够抗血栓,不会在材料表面发生凝血现象,还要求具有与人体组织相似的弹性、延展性及良好的耐疲劳性等生物力学性能。因此,研制一种无需注射抗凝剂、抗血栓作用强而致凝作用弱、生物相容性和力学性能俱佳的医用抗凝血生物材料具有重要的理论意义和应用价值。肝素作为临床上最常用的抗凝剂,其主要给药途径为静脉注射,存在全身用药、用量大、易引起出血、血小板减少、过敏等副作用,而肝素化生物材料是减少这些副作用最有效的途径,肝素化材料也是最主要的抗凝血材料。本论文拟以醋酸为主要溶剂体系,以壳聚糖和大豆蛋白质这两种具有良好生物相容性和生物降解性的天然高分子为主要原料,制备不同组成和结构的壳聚糖/大豆蛋白质复合材料；利用肝素分子与蛋白质、壳聚糖分子间的物理和化学作用,将肝素结合到材料上,制备肝素化生物材料。通过不同的肝素化工艺来控制肝素在材料上的分布和结合程度。通过体外和体内实验,系统评价肝素化生物材料的血液和组织相容性及其抗凝血功能,揭示影响其性能和功能的规律,以期获得新型肝素化生物材料。本工作的主要内容包括以下几个方面：(1)壳聚糖/大豆蛋白质复合膜的制备及其生物学性能评价：以壳聚糖和大豆蛋白质为原料,以醋酸水溶液为溶剂,通过共混和流延法制备了一系列壳聚糖/大豆蛋白质复合膜。通过扫描电镜观察、红外光谱分析、x-射线衍射分析、水接触角测试、力学性能测试、体外细胞培养及动物体内埋植实验等方法研究了不同组成的壳聚糖/大豆蛋白质复合膜的结构与性能。结果表明,随着复合膜中大豆蛋白质含量的增加,复合膜的结晶度降低,亲水性增加。在大豆蛋白质含量为10%时力学性能最高。大豆蛋白质的存在,具有促进细胞增殖,提高细胞相容性、组织相容性和生物降解性的作用。因此,可以通过调节大豆蛋白质的含量来调控复合材料的微观结构、力学性能和生物学性能。(2)表面肝素化壳聚糖/大豆蛋白质复合膜的制备及抗凝血功能研究：以EDC为交联剂,将肝素接枝到壳聚糖/大豆蛋白质复合膜表面,获得表面肝素化壳聚糖/大豆蛋白质复合膜。通过甲苯胺蓝法、红外光谱分析、静态水接触角测试、力学性能测试等方法评价了表面肝素化壳聚糖/大豆蛋白质复合膜的结构和物理化学性能；通过体外细胞培养和抗凝血试验评价了表面肝素化复合材料的细胞相容性和血液相容性。结果表明,经表面肝素化的壳聚糖/大豆蛋白质复合膜的亲水性能明显提高,但力学性能有所降低,表面肝素密度在0.59～1.12μg/cm2之间；体外细胞培养实验结果显示,肝素化壳聚糖/大豆蛋白质复合膜能促进细胞粘附和生长,具有良好的细胞相容性；抗凝血试验结果显示,表面肝素化显著地减少了血小板的粘附,显著延长了复钙时间(表面肝素化组338 s-352s,未肝素化组112s-201 s1,抑制了血栓的形成,而且几乎没有溶血发生,表现出较好的血液相容性。(3)共混法制备肝素化壳聚糖/大豆蛋白质复合膜及其抗凝血性能：采用共混法制备了一系列交联型(EDC为交联剂)和未交联型肝素化壳聚糖/大豆蛋白质复合膜。通过甲苯胺蓝法、红外光谱分析、静态水接触角测试、力学性能测试等方法评价了肝素化壳聚糖/大豆蛋白质复合膜的结构和物理化学性能；通过体外细胞培养和抗凝血试验评价了肝素化复合材料的细胞相容性和血液相容性。结果表明,交联型肝素化复合膜比未交联型以及表面肝素化复合膜的肝素结合量大(交联型组1.24～2.47μg/cm2；未交联型组0.67～1.29μg/cm2；表面肝素化组0.59～1.12μg/cm2),具有更好的力学性能,其亲水性能也较好；体外细胞培养实验结果显示,交联型肝素化壳聚糖/大豆蛋白质复合膜能促进细胞生长和增殖,具有良好的细胞相容性；抗凝血试验结果显示,交联型肝素化有效地减少了血小板的粘附,显著延长了复钙时间((交联型组903s-1179 s；未交联型组292s-306 s；表面肝素化组338 s-352s,未肝素化组112s-201 s),抑制了血栓的形成,而且几乎没有溶血发生,表现出良好的血液相容性。(4)壳聚糖/大豆蛋白质复合海绵的制备及其生物学性能评价：采用冷冻干燥法制备了一系列壳聚糖/大豆蛋白质复合海绵。通过红外光谱分析、X-衍射分析及扫描电镜观察等方法研究大豆蛋白质含量对复合海绵结构与性能的影响；通过体外细胞培养实验和体内植入实验综合评价了壳聚糖/大豆蛋白质复合海绵的生物相容性和生物降解性。结果表明,这种复合海绵材料具有三维多孔结构。体外细胞培养实验显示,复合海绵材料中所含的大豆蛋白质能为细胞生长提供营养成分,促进细胞在复合海绵表面及内部粘附和生长,提高细胞的增殖活性。动物体内埋植实验结果表明,壳聚糖/大豆蛋白质复合海绵材料具有良好的组织相容性和降解性,大豆蛋白质促进其降解,多孔结构有利于组织进入也促进其降解。因此复合海绵的降解速度快于相对应的复合膜。由此,壳聚糖/大豆蛋白质复合海绵具有良好的生物相容性和降解性,具有作为组织工程支架材料的潜力。(5)壳聚糖涂层纤维素/大豆蛋白质复合膜的制备及其细胞相容性和血液相容性研究：以氢氧化钠/尿素溶液为共溶剂,制备一系列纤维素/大豆蛋白质复合膜,然后将较低分子量壳聚糖(50000 Da)涂层在纤维素/大豆蛋白质复合膜表面。经红外光谱分析、X-射线衍射分析、扫描电镜观察、水接触角测试以及力学性能检测等评价壳聚糖涂层对于复合材料的结构和性能的影响；通过体外细胞培养和抗凝血试验评价了壳聚糖涂层复合材料的细胞相容性和血液相容性。结果表明,壳聚糖涂层粘附在多孔的原始纤维素/大豆蛋白质复合膜表面,并且形成了一层光滑平整的表面。力学性能测试表明,壳聚糖涂层增强了复合膜的力学性能,尤其是湿态条件下的力学性能。细胞培养实验和MTT测试结果显示,壳聚糖涂层改变了纤维素/大豆蛋白质复合膜的组分和微观结构,并且减缓了大豆蛋白质在细胞培养介质中的释放速度,能够促进细胞在复合膜表面粘附,提高了细胞增殖活性。血液相容性评价结果表明,壳聚糖涂层有效地减少了血小板的粘附,延长了复钙时间,降低了溶血率。因此,壳聚糖表面涂层是提高和改善纤维素/大豆蛋白质复合膜力学性能、细胞相容性以及血液相容性的一种简便而行之有效的方法。综上所述,本工作制备了不同系列的壳聚糖/大豆蛋白质复合膜、肝素化壳聚糖/大豆蛋白质复合膜以及壳聚糖/大豆蛋白质复合海绵,通过高分子化学与物理方法表征了各类复合膜与复合海绵的结构与理化性能,通过体外细胞培养和体内植入实验综合评价了复合膜和复合海绵的细胞相容性与组织相容性,通过抗凝血实验评价了其血液相容性,从而获得了具有良好细胞相容性、组织相容性和抗凝血功能的壳聚糖/大豆蛋白质复合材料,为其应用提供了理论依据和实验数据。更多还原

【Abstract】 Anticoagulant material, an important part of biological materials, which has been widely used as medical device and tissue engeering materials contacted with blood, such as artificial heart valve, hemodialysis system, extracorporeal circulation system, blood vessels embolic agents, heart pacemakers, artificial vascular, stents, interventional catheter, surgical line. The materials which directly contact with blood should have not only histocompatibility without inflammation, but also have good blood compatibility such as antithrombotic, without clotting phenomenon. In addition, it is required that the materials’mechanical properties such as elasticity and ductility are similar with human tissue and favorable fatiguedurability. Therefore, it is importantly to develop biomedical anticoagulant materials which have strong antithrombotic ability, good biocompatibility and mechanical properties without injecting anticoagulants as a substitutes.Heparin is the most commonly clinlic anticoagulation drug. The mostly delivery method is intravenous injection. However, it caused some side-effects such as dosage, bleeding, thrombocytopenia, allergies. Heparinization is one of the most effective ways that reduce the side effects which direct injection of heparin caused. And heparinized materials are the uppermost anticogulant materials.In this work, acetic acid was used as the co-solvent and two kinds of natural polymers, chitosan and soy protein, were used as main raw materials to prepare chitosan/soy protein composites with different composition and structure. The heparinized biomaterials were prepared with the physical or chemical interaction between heparin molecules and protein molecules, as well as chitosan molecules. The distribution and binding degree of heparin ware controlled by different heparinization technologies. The biological characteristics of the chitosan/soy protein composites including blood compability and histocompatibility were systematically evaluated by in vitro testing and in vivo animal experiments. Meanwhile, the factors affected on the properties and function of the new heparinized biomaterials were revealed. The main contents are as following: (1) Preparation and characterization of chitosan/soy protein composite membrane materials:A series of chitosan/soy protein composite membrane was prepared by blending and casting using chitosan and soy protein isolate (SPI) as the raw materials and acetic acid aqueous solution as co-solvent. The effects of SPI content on the structure and properties of the final chitosan/soy protein composite membranes were investigated by scanning electron microscope (SEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), water contact angle, tensile testing, in vitro cell culture and in vivo implantation experiments. The results revealed that the crystallinity decreased and the hydrophilicity of the composite membranes increased with the increase of SPI content. It was found that the mechanical properties of chitosan/soy protein composite membranes were excellent when SPI content was 10%. SPI had obvious promoted cell proliferation and improved cytocompatibility, tissue compatibility and biodegradability of the membranes. Therefore, it could regulate the microstructure, mechanical properties and biological properties of the materials by adjusting SPI content.(2) Preparation, characterization and hemocompability evaluation of surface-heparinized chitosan/soy protein composite membranes:Using EDC as cross-linker, heparin was grafted on to the surface of chitosan/soy protein composite membranes for hepariniztion. The structure and properties of the surface-heparinized chitosan/soy protein composite membranes were investigated by assay with toluidine blue, attenuated total reflection-fourier transforms infrared spectrometry, water contact angle test and tensile testing; The cytocompability and hemocompability of surface-heparinzed chitosan/soy protein composite membranes in vitro cell culture and anticoagulation experiments. The results revealed that the hydrophilicity of chitosan/soy protein composite membranes were remarkably improved by suface-grafting of heparin, but the mechanical properties decreased and the content of immobiled-heparin was 0.59～1.12μg/cm2. Moreover, the heparinized membranes could improve of growth of L929 cells and showed good cytocompability. Surface-heparinization could effectively reduce platelet adhesion, extend the plasma recalcification time (surface-heparinization groups 338 s～352 s, non-heparinization groups 112 s～201 s), curb thrombosis and reduce the hemolysis rate. It indicated that the surface-heparinized membranes have good blood compatibility.(3) Preparation, characterization and hemocompability evaluation of heparinized chitosan/soy protein composite membranes:A series of heparinized chitosan/soy protein composite membranes were prepared by blending, with or without cross-linking. The structure and properties of the heparinized chitosan/soy protein composite membranes were investigated by assay with toluidine blue, Attenuated total reflection-Fourier transforms infrared spectrometry, water contact angle test and tensile testing; The cytocompability and hemocompability of the heparinized chitosan/soy protein composite membranes were evaluated by in vitro cell culture and anticoagulation experiments. The results revealed that the amount of immobilized-heparin of the cross-linking heparinized chitosan/soy protein composite membranes was higher than that on the non-crosslinking heparinized and surface-heparinized chitosan/soy protein composite membranes (crosslinking heparinized groups 1.24～2.47μg/cm, non-crosslinking heparinized groups 0.67-1.29μg/cm2, surface-heparinization groups 0.59～1.12μg/cm2). The cross-linking heparinized membranes were more hydrophilic than the non-crosslinking heparinized membranes, and showed the mechanical properties than he non-crosslinking heparinized and surface-heparinized membranes. Moreover, the cross-linking heparinized membranes could improve the proliferation of L929 cells, and showed good cytocompability. Cross-linking heparinization can effectively reduce platelet adhesion, extended the plasma recalcification time (crosslinking heparinized groups 903 s～1179 s, non-crosslinking heparinized groups 292 s～306 s, surface-heparinization groups 338 s～352 s, non-heparinization groups 112 s～201 s), curb thrombosis and reduce the hemolysis rate. The cross-linking heparinized membranes showed better blood compatibility than the non-crosslinking heparinized membranes and surface-heparinized membranes.(4) The chitosan/soy protein sponges were fabricated in order to enhance the biodegradability of chitosan-based composites. A series of chitosan/soy protein isolate sponges were prepared by freeze-drying process. The effects of soy protein content on the structure and properties of the sponges were investigated by FT-IR, XRD and SEM. The biocompatibility and biodegradability of chitosan/soy protein sponges were evaluated by in vitro cell culture and in vivo implantation experiments. The results showed that the chitosan/soy protein sponges exhibited uniform three-dimensional porous structure, and the pore structure of the chitosan/SPI sponges can keep well with an increase in soy protein content. The in vitro cell culture process showed that the incorporation of soy protein isolate in the composite sponges could provide nutrients for cell growth, which was beneficial to the cell’s growth and spread on the sponges, as well as to the enhancement of cell viability during cell culture. The sponges implanted into rats showed good biocompatibility and biodegradability. The biodegradation rate of the chitosan/soy protein sponges was higher than the corresponding membranes, which is due to the incorporation of SPI, the three-dimensional porous structure. Therefore, chitosan/soy protein sponges may be used as cell scaffolds with good biodegradability and biodegradability.(5) Preparation and characterization of chitosan-coated cellulose/soy protein composite membrane with improved physical properties and hemocompability:A series of cellulose/soy protein membranes was prepared using NaOH/urea solution as co-solvent, then chitosan (50000Da) was coated on the surface of cellulose/soy protein membranes. The original cellulose/soy protein composite membrane and chitosan-coated cellulose/soy protein composite membrane were characterized by FT-IR, XRD, SEM, water contact angle testing, and tensile testing. Chitosan-coated cellulose/SPI membranes had smoother surface microstructure and enhanced mechanical properties as compared with the corresponding cellulose/SPI membranes. The cytocompatibility and hemocompatibility of cellulose/SPI membranes and coated cellulose/SPI membranes were evaluated by cell culture, MTT assay, in vitro platelet adhesion testing, plasma recalcification time measurement, and hemolysis assay. The higher cell adherence and improved cytocompatibility of chitosan-coated cellulose/SPI membranes were mainly ascribed to the coated chitosan and the altered surface microstructure of cellulose/SPI membranes. Chitosan-coated cellulose/SPI membranes also showed lower platelet adhesion, longer PRT, and a lower hemolysis rate, all resulting from the good hemocompatibility of chitosan and the smoother membrane surface after chitosan coating. Undoubtedly, surface coating with chitosan improved the microstructure, mechanical properties, cytocompatibility, and hemocompatibility, thus widening the possible range of applications of cellulose/soy protein-based biomaterials.In conclusion, several series of chitosan/soy protein membranes, heparinized chitosan/SPI membranes and chitosan/soy protein composite sponges were successfully prepared in this work. The structure and properties of the membranes and sponges were characterized by methods of polymer chemistry and physics. The cytocompatibility and tissue compatibility of the biocomposites were evaluated by in vitro cell culture and in vivo animal experiments. And the hemocompatibility was evaluated by anticoagulation experiments. Thereby it obtained the chitosan/soy protein composite biomaterials that had good cytocompatibility, tissue compatibility and good hemocompatibility, and provided the theory basis and the experimental data for potential applications.更多还原