表面微米拓撲結(jié)構(gòu)的構(gòu)建及對血小板和內(nèi)皮細胞的影響
發(fā)布時間：2018-07-25 09:28
【摘要】：心血管植入材料在臨床應用中面臨的最主要的問題是凝血和內(nèi)膜增生,提高這類生物材料的抗凝血和內(nèi)皮化功能被認為是改善其生物相容性的有效途徑。通過材料表面微結(jié)構(gòu)調(diào)控細胞生長行為從而實現(xiàn)生物材料功能化是材料表面改性的一個研究熱點。 本文設(shè)計一種由微米尺度的柱狀結(jié)構(gòu)排列成溝槽結(jié)構(gòu)的復合拓撲結(jié)構(gòu),通過光刻技術(shù)和反應離子深刻蝕技術(shù)在硅基材料表面成功構(gòu)建這種微結(jié)構(gòu),微柱的高度為5μm,直徑為3μm,相鄰微柱間距為2μm,通過微柱的排列,構(gòu)建了9種不同參數(shù)的拓撲結(jié)構(gòu)：微柱隊列寬度分別為18μm、28μm和38μm,微柱隊列間隔寬度分別為10μm、20μm和30μm。通過SEM和表面輪廓儀對樣品表面拓撲結(jié)構(gòu)進行了表征,然后分別進行抗凝血和促內(nèi)皮評價實驗。 在樣品表面進行血小板粘附實驗,通過光鏡、SEM觀測血小板數(shù)量和形態(tài),通過血小板計數(shù)、LDH實驗、GMP-140實驗評價樣品的血液相容性,分析拓撲結(jié)構(gòu)對血小板的影響。結(jié)果表明,本文構(gòu)建的拓撲結(jié)構(gòu)引起了血小板粘附量的增加,血小板主要粘附在拓撲結(jié)構(gòu)中溝槽底面。在微結(jié)構(gòu)化樣品表面上,微柱結(jié)構(gòu)的增加能夠減少血小板的粘附。構(gòu)建的拓撲結(jié)構(gòu)同時抑制了血小板的激活,在本文尺度范圍內(nèi),血小板激活量隨著微柱覆蓋率的增加而減少,隨著溝槽底面寬度增加而增多。在微柱間隙的血小板激活程度較小,不發(fā)生聚集,而在溝槽底面上粘附的血小板激活較嚴重。 在樣品表面進行內(nèi)皮細胞培養(yǎng)實驗,通過光鏡、SEM、熒光染色和CCK-8實驗分析拓撲結(jié)構(gòu)對內(nèi)皮細胞的影響。結(jié)果顯示,在內(nèi)皮細胞培養(yǎng)的初期(4小時),與平板樣品相比,微結(jié)構(gòu)化樣品表面明顯促進了內(nèi)皮細胞的粘附、增殖和鋪展,同時也影響了細胞的形態(tài)和分布。細胞優(yōu)先粘附于微柱頂面,在微柱隊列寬度為18μm和28μm的樣品上內(nèi)皮細胞呈長梭形,當微柱隊列之間間隔寬度為10μm和20μm時,且細胞會“跨越”這一間隔生長,而當這一間隔寬度為30μm時,這種跨越生長的現(xiàn)象不再明顯。隨著內(nèi)皮細胞培養(yǎng)時間的延長(1天和3天),在微柱隊列間隔寬度為20μm和30μm的樣品中,內(nèi)皮細胞開始生長在溝槽底面。微結(jié)構(gòu)化樣品表面對內(nèi)皮細胞形態(tài)有影響,細胞在微結(jié)構(gòu)化樣品上隨著微柱隊列寬度的增加而逐漸由細長的長梭形形態(tài)向圓形轉(zhuǎn)變。在微柱陣列寬度為38μm的樣品上,表面拓撲結(jié)構(gòu)對內(nèi)皮細胞的接觸引導作用較弱。 本研究為通過在材料表面構(gòu)建拓撲結(jié)構(gòu)來實現(xiàn)同時抑制血小板粘附和促進內(nèi)皮細胞生長提供參考,是材料表面改性的一種新思路。
[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.
【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R318.08
本文編號：2143379
[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.
【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R318.08
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