鈦表面肝素和纖維連接蛋白微圖形的構建及其對血小板和內(nèi)皮細胞行為的影響
發(fā)布時間：2018-10-12 11:57
【摘要】：對心血管人工植入材料而言,內(nèi)膜增生和血栓的形成是其植入后面臨的主要問題。通過表面改性技術促進內(nèi)皮細胞粘附的同時減少血小板的粘附能有效提高材料的生物相容性,其中表面圖形化技術正成為研究熱點。 本文采用軟刻蝕技術中的微轉移模塑技術在預處理的鈦(Ti)表面構建仿生生物大分子微圖形。鈦的預處理包括NaOH堿活化和3-氨丙基三乙氧基硅烷(APTE)偶聯(lián)處理,通過掃描電子顯微鏡(SEM)、接觸角測量儀、傅里葉紅外變換光譜分析(FTIR)和氨基定量的方法對預處理的鈦表面進行理化表征。聚二甲基硅氧烷(PDMS)印章由澆鑄法制備,運用其壓印具有阻抗蛋白和細胞粘附的單甲氧基聚乙二醇琥珀酰亞胺碳酸酯(mPEG-SC),而后組裝具有抗凝作用的肝素(Hep)和促進內(nèi)皮細胞粘附的纖維連接蛋白(Fn)混合物(Hep-Fn),構成iPEG-SC/Hep-Fn微圖形。通過SEM、接觸角測量儀、FTIR、Hep及Fn的染色對制備的Hep-Fn微圖形進行表征。在此基礎上進行體外血小板粘附實驗和內(nèi)皮細胞培養(yǎng)實驗,研究圖形對血小板及內(nèi)皮細胞行為的影響。 SEM、接觸角、FTIR、光鏡及熒光顯微鏡結果顯示：堿活化后的鈦表面變粗糙,親水性加強,表面出現(xiàn)羥基的吸收峰；硅烷偶聯(lián)后的鈦表面粗糙度進一步加大,疏水性增大,FTIR檢測有CH2的吸收峰,氨基在表面的密度約為51nmol/cm2。圖形化的樣品表面能看到清晰的條紋圖形,FTIR出現(xiàn)mPEG-SC、Hep和Fn的特征吸收峰。Hep和Fn的染色進一步證實mPEG-SC的生物惰性,Hep和Fn只分布在沒有mPEG-SC的圖形區(qū)域。 血小板粘附實驗表明：平板Ti和Hep-Fn表面血小板布滿整個表面,血小板伸出偽足,沒有團聚。由于肝素的抗凝作用,Hep-Fn表面血小板粘附數(shù)量少于Ti。nPEG-SC平板表面幾乎沒有血小板粘附；圖形樣品表面血小板選擇性分布在Hep-Fn條紋區(qū)域,與平板的Hep-Fn相比能進一步減少血小板粘附數(shù)量。不同尺寸的圖形樣品Hep-Fn的有效面積不同致使血小板的粘附數(shù)量有所不同。激活的血小板在數(shù)量和分布上和粘附有著同樣的趨勢,圖形化樣品表面激活的血小板數(shù)量比平板的Ti和Hep-Fn樣品少。APTT結果顯示平板和圖形的Hep-Fn沒有明顯延長APTT時間,但也沒有加劇凝血時間,具有—定抗凝作用。 內(nèi)皮細胞粘附結果顯示：平板的Hep-Fn表面與鈦相比,能促進內(nèi)皮細胞的粘附、鋪展、增殖；mPEG-SC表面幾乎沒有內(nèi)皮細胞粘附。圖形化表面內(nèi)皮細胞都沿著Hep-Fn圖形方向分布,不同尺寸的圖形對內(nèi)皮細胞的取向角、長寬比、形態(tài)指數(shù)等均有不同影響：大于細胞尺寸的圖形使細胞呈2個或多個并行排列,有利于細胞鋪展和增殖；與細胞尺寸相近的圖形能夠使細胞成單細胞陣列分布,拉長并產(chǎn)生引導作用。 以上結果表明：具有生物功能的mPEG-SC/Hep-Fn微圖形能調節(jié)血小板和內(nèi)皮細胞分布的同時,降低血小板粘附并調節(jié)內(nèi)皮細胞粘附、生長和增殖,有效提高了鈦金屬的生物相容性。
[Abstract]:Intimal hyperplasia and thrombosis are the main problems after implantation of artificial cardiovascular materials. Surface modification technology can promote endothelial cell adhesion and reduce platelet adhesion, which can effectively improve the biocompatibility of materials, among which surface graphics technology is becoming a hot topic. In this paper, bionic macromolecular micrographics were constructed on the surface of pretreated titanium (Ti) by microtransfer molding in soft etching. The pretreatment of titanium includes activation of NaOH base and (APTE) coupling treatment of 3-aminopropyl triethoxy silane. The contact angle of titanium is measured by scanning electron microscope (SEM),). The surface of titanium was characterized by (FTIR) and amino quantitative analysis by Fourier transform infrared spectroscopy (FTIR). Poly (dimethylsiloxane) (PDMS) seal was prepared by casting method. After imprinting monomethoxy polyethylene glycol succinimide carbonate (mPEG-SC) with impedance protein and cell adhesion, heparin (Hep) with anticoagulant activity and fibronectin (Fn) mixture (Hep-Fn), which promoted endothelial cell adhesion, were assembled to form iPEG-SC/Hep-Fn micrograph. The Hep-Fn micrographs were characterized by SEM, contact angle measuring instrument, FTIR,Hep and Fn staining. On this basis, platelet adhesion test and endothelial cell culture were carried out in vitro. SEM, contact angle, FTIR, light microscope and fluorescence microscope showed that the surface of titanium after alkali activation became rough, hydrophilic was strengthened, and hydroxyl absorption peak appeared on the surface. After silane coupling, the surface roughness and hydrophobicity of titanium were further increased. The absorption peak of CH2 was detected by FTIR, and the density of amino on the surface was about 51 nmol / cm ~ 2. A clear pattern of stripe can be seen on the surface of a graphical sample, and the characteristic absorption peaks of mPEG-SC,Hep and Fn appear in FTIR. The staining of Hep and Fn further confirms the biological inertia of mPEG-SC. Hep and Fn are only distributed in the graphical region without mPEG-SC. Platelet adhesion test showed that platelets covered the whole surface of Ti and Hep-Fn, and platelets extended pseudopodia without agglomeration. Because of the anticoagulant effect of heparin, the number of platelet adhesion on Hep-Fn surface was less than that on Ti.nPEG-SC plate surface. Compared with plate Hep-Fn, platelet adhesion was further reduced. The number of platelet adhesion varies with the difference of the effective area of Hep-Fn in different size graphic samples. The number and distribution of activated platelets showed the same trend as that of adhesion. The number of activated platelets on the surface of graphical samples was less than that of Ti and Hep-Fn samples. The results of APTT showed that the Hep-Fn of plate and pattern did not prolong the APTT time obviously. But also did not aggravate the coagulation time, with-anticoagulant effect. The results of endothelial cell adhesion showed that the surface of Hep-Fn on the plate could promote the adhesion, spread and proliferation of endothelial cells compared with titanium, and there was almost no adhesion of endothelial cells on the surface of mPEG-SC. The morphologic surface endothelial cells are distributed along the Hep-Fn pattern. The shapes of different sizes have different effects on the orientation angle, aspect ratio and morphological index of the endothelial cells. The shapes larger than the cell size cause the cells to be arranged in two or more parallel. The pattern similar to the cell size can make the cell into a single cell array distribution, elongate and produce a leading role. The results show that the biofunctional mPEG-SC/Hep-Fn micrograph can regulate the distribution of platelet and endothelial cells, decrease platelet adhesion and regulate the adhesion, growth and proliferation of endothelial cells, and effectively improve the biocompatibility of titanium.
【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：2266023
[Abstract]:Intimal hyperplasia and thrombosis are the main problems after implantation of artificial cardiovascular materials. Surface modification technology can promote endothelial cell adhesion and reduce platelet adhesion, which can effectively improve the biocompatibility of materials, among which surface graphics technology is becoming a hot topic. In this paper, bionic macromolecular micrographics were constructed on the surface of pretreated titanium (Ti) by microtransfer molding in soft etching. The pretreatment of titanium includes activation of NaOH base and (APTE) coupling treatment of 3-aminopropyl triethoxy silane. The contact angle of titanium is measured by scanning electron microscope (SEM),). The surface of titanium was characterized by (FTIR) and amino quantitative analysis by Fourier transform infrared spectroscopy (FTIR). Poly (dimethylsiloxane) (PDMS) seal was prepared by casting method. After imprinting monomethoxy polyethylene glycol succinimide carbonate (mPEG-SC) with impedance protein and cell adhesion, heparin (Hep) with anticoagulant activity and fibronectin (Fn) mixture (Hep-Fn), which promoted endothelial cell adhesion, were assembled to form iPEG-SC/Hep-Fn micrograph. The Hep-Fn micrographs were characterized by SEM, contact angle measuring instrument, FTIR,Hep and Fn staining. On this basis, platelet adhesion test and endothelial cell culture were carried out in vitro. SEM, contact angle, FTIR, light microscope and fluorescence microscope showed that the surface of titanium after alkali activation became rough, hydrophilic was strengthened, and hydroxyl absorption peak appeared on the surface. After silane coupling, the surface roughness and hydrophobicity of titanium were further increased. The absorption peak of CH2 was detected by FTIR, and the density of amino on the surface was about 51 nmol / cm ~ 2. A clear pattern of stripe can be seen on the surface of a graphical sample, and the characteristic absorption peaks of mPEG-SC,Hep and Fn appear in FTIR. The staining of Hep and Fn further confirms the biological inertia of mPEG-SC. Hep and Fn are only distributed in the graphical region without mPEG-SC. Platelet adhesion test showed that platelets covered the whole surface of Ti and Hep-Fn, and platelets extended pseudopodia without agglomeration. Because of the anticoagulant effect of heparin, the number of platelet adhesion on Hep-Fn surface was less than that on Ti.nPEG-SC plate surface. Compared with plate Hep-Fn, platelet adhesion was further reduced. The number of platelet adhesion varies with the difference of the effective area of Hep-Fn in different size graphic samples. The number and distribution of activated platelets showed the same trend as that of adhesion. The number of activated platelets on the surface of graphical samples was less than that of Ti and Hep-Fn samples. The results of APTT showed that the Hep-Fn of plate and pattern did not prolong the APTT time obviously. But also did not aggravate the coagulation time, with-anticoagulant effect. The results of endothelial cell adhesion showed that the surface of Hep-Fn on the plate could promote the adhesion, spread and proliferation of endothelial cells compared with titanium, and there was almost no adhesion of endothelial cells on the surface of mPEG-SC. The morphologic surface endothelial cells are distributed along the Hep-Fn pattern. The shapes of different sizes have different effects on the orientation angle, aspect ratio and morphological index of the endothelial cells. The shapes larger than the cell size cause the cells to be arranged in two or more parallel. The pattern similar to the cell size can make the cell into a single cell array distribution, elongate and produce a leading role. The results show that the biofunctional mPEG-SC/Hep-Fn micrograph can regulate the distribution of platelet and endothelial cells, decrease platelet adhesion and regulate the adhesion, growth and proliferation of endothelial cells, and effectively improve the biocompatibility of titanium.
【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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