表面微米拓扑结构的构建及对血小板和内皮细胞的影响

发布时间：2018-07-25 09:28

【摘要】：心血管植入材料在临床应用中面临的最主要的问题是凝血和内膜增生,提高这类生物材料的抗凝血和内皮化功能被认为是改善其生物相容性的有效途径。通过材料表面微结构调控细胞生长行为从而实现生物材料功能化是材料表面改性的一个研究热点。本文设计一种由微米尺度的柱状结构排列成沟槽结构的复合拓扑结构,通过光刻技术和反应离子深刻蚀技术在硅基材料表面成功构建这种微结构,微柱的高度为5μm,直径为3μm,相邻微柱间距为2μm,通过微柱的排列,构建了9种不同参数的拓扑结构：微柱队列宽度分别为18μm、28μm和38μm,微柱队列间隔宽度分别为10μm、20μm和30μm。通过SEM和表面轮廓仪对样品表面拓扑结构进行了表征,然后分别进行抗凝血和促内皮评价实验。在样品表面进行血小板粘附实验,通过光镜、SEM观测血小板数量和形态,通过血小板计数、LDH实验、GMP-140实验评价样品的血液相容性,分析拓扑结构对血小板的影响。结果表明,本文构建的拓扑结构引起了血小板粘附量的增加,血小板主要粘附在拓扑结构中沟槽底面。在微结构化样品表面上,微柱结构的增加能够减少血小板的粘附。构建的拓扑结构同时抑制了血小板的激活,在本文尺度范围内,血小板激活量随着微柱覆盖率的增加而减少,随着沟槽底面宽度增加而增多。在微柱间隙的血小板激活程度较小,不发生聚集,而在沟槽底面上粘附的血小板激活较严重。在样品表面进行内皮细胞培养实验,通过光镜、SEM、荧光染色和CCK-8实验分析拓扑结构对内皮细胞的影响。结果显示,在内皮细胞培养的初期(4小时),与平板样品相比,微结构化样品表面明显促进了内皮细胞的粘附、增殖和铺展,同时也影响了细胞的形态和分布。细胞优先粘附于微柱顶面,在微柱队列宽度为18μm和28μm的样品上内皮细胞呈长梭形,当微柱队列之间间隔宽度为10μm和20μm时,且细胞会“跨越”这一间隔生长,而当这一间隔宽度为30μm时,这种跨越生长的现象不再明显。随着内皮细胞培养时间的延长(1天和3天),在微柱队列间隔宽度为20μm和30μm的样品中,内皮细胞开始生长在沟槽底面。微结构化样品表面对内皮细胞形态有影响,细胞在微结构化样品上随着微柱队列宽度的增加而逐渐由细长的长梭形形态向圆形转变。在微柱阵列宽度为38μm的样品上,表面拓扑结构对内皮细胞的接触引导作用较弱。本研究为通过在材料表面构建拓扑结构来实现同时抑制血小板粘附和促进内皮细胞生长提供参考,是材料表面改性的一种新思路。
[Abstract]:Coagulation and intimal hyperplasia are the main problems in the clinical application of cardiovascular implants. Improving the anticoagulant and endothelial functions of these biomaterials is considered to be an effective way to improve their biocompatibility. The functionalization of biomaterials through the regulation of cell growth behavior by the surface microstructure of materials is a hot topic in the study of material surface modification. In this paper, we design a composite topological structure which is arranged into groove structure by columnar structure of micron scale. The microstructures are successfully constructed on the surface of silicon-based materials by photolithography and reactive ion etching techniques. The microcolumn is 5 渭 m in height, 3 渭 m in diameter and 2 渭 m in the distance between the adjacent microcolumns. Through the arrangement of the microcolumns, the topological structures of 9 different parameters are constructed: the widths of the microcolumn queue are 18 渭 m ~ 28 渭 m and 38 渭 m respectively, and the spacing width of the microcolumn queue is 10 渭 m ~ 20 渭 m and 30 渭 m respectively. The surface topologies of the samples were characterized by SEM and surface profilometer, and then the anticoagulant and endothelium-promoting evaluation experiments were carried out. Platelet adhesion experiments were carried out on the surface of the samples. The number and morphology of platelets were observed by light microscope SEM. The blood compatibility of the samples was evaluated by platelet count and LDH test and the effect of topological structure on platelets was analyzed. The results show that the proposed topology leads to the increase of platelet adhesion, and the platelet adheres to the bottom of the groove in the topology. On the surface of microstructured samples, the increase of microcolumn structure can reduce platelet adhesion. The constructed topology also inhibited platelet activation. In the scale of this paper, the platelet activation decreased with the increase of microcolumn coverage and increased with the increase of groove bottom width. The platelet activation in the microcolumn space was small and no aggregation occurred, but the platelet adhesion on the bottom of the groove was more serious. The endothelial cells were cultured on the surface of the samples. The effects of topological structure on the endothelial cells were analyzed by light microscopy, fluorescence staining and CCK-8 experiments. The results showed that in the early stage of endothelial cell culture (4 hours), the surface of microstructured samples significantly promoted the adhesion, proliferation and spread of endothelial cells, and also affected the morphology and distribution of endothelial cells. The endothelial cells were attached to the apical surface of the microcolumn, and the endothelial cells were fusiform on the samples with the width of 18 渭 m and 28 渭 m. When the spacing between the columns was 10 渭 m and 20 渭 m, the cells would grow "across" this interval. However, when the interval width is 30 渭 m, the phenomenon of spanning growth is no longer obvious. With the increase of the culture time of endothelial cells (1 and 3 days), the endothelial cells began to grow on the bottom of the grooves in the samples with 20 渭 m and 30 渭 m spaced between the microcolumn cohorts. The morphology of endothelial cells was affected by the surface of the microstructured samples. With the increase of the queue width of the microcolumns, the cells gradually changed from the slender fusiform to the circular shape on the microstructured samples. In the sample with the width of 38 渭 m of the microcolumn array, the surface topology has a weak contact guidance effect on the endothelial cells. This study provides a reference for the suppression of platelet adhesion and the promotion of endothelial cell growth through the construction of topological structures on the surface of materials, which is a new way of material surface modification.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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