钛表面肝素和纤维连接蛋白微图形的构建及其对血小板和内皮细胞行为的影响
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摘要
对心血管人工植入材料而言,内膜增生和血栓的形成是其植入后面临的主要问题。通过表面改性技术促进内皮细胞粘附的同时减少血小板的粘附能有效提高材料的生物相容性,其中表面图形化技术正成为研究热点。
本文采用软刻蚀技术中的微转移模塑技术在预处理的钛(Ti)表面构建仿生生物大分子微图形。钛的预处理包括NaOH碱活化和3-氨丙基三乙氧基硅烷(APTE)偶联处理,通过扫描电子显微镜(SEM)、接触角测量仪、傅里叶红外变换光谱分析(FTIR)和氨基定量的方法对预处理的钛表面进行理化表征。聚二甲基硅氧烷(PDMS)印章由浇铸法制备,运用其压印具有阻抗蛋白和细胞粘附的单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC),而后组装具有抗凝作用的肝素(Hep)和促进内皮细胞粘附的纤维连接蛋白(Fn)混合物(Hep-Fn),构成iPEG-SC/Hep-Fn微图形。通过SEM、接触角测量仪、FTIR、Hep及Fn的染色对制备的Hep-Fn微图形进行表征。在此基础上进行体外血小板粘附实验和内皮细胞培养实验,研究图形对血小板及内皮细胞行为的影响。
SEM、接触角、FTIR、光镜及荧光显微镜结果显示：碱活化后的钛表面变粗糙,亲水性加强,表面出现羟基的吸收峰；硅烷偶联后的钛表面粗糙度进一步加大,疏水性增大,FTIR检测有CH2的吸收峰,氨基在表面的密度约为51nmol/cm2。图形化的样品表面能看到清晰的条纹图形,FTIR出现mPEG-SC、Hep和Fn的特征吸收峰。Hep和Fn的染色进一步证实mPEG-SC的生物惰性,Hep和Fn只分布在没有mPEG-SC的图形区域。
血小板粘附实验表明：平板Ti和Hep-Fn表面血小板布满整个表面,血小板伸出伪足,没有团聚。由于肝素的抗凝作用,Hep-Fn表面血小板粘附数量少于Ti。nPEG-SC平板表面几乎没有血小板粘附；图形样品表面血小板选择性分布在Hep-Fn条纹区域,与平板的Hep-Fn相比能进一步减少血小板粘附数量。不同尺寸的图形样品Hep-Fn的有效面积不同致使血小板的粘附数量有所不同。激活的血小板在数量和分布上和粘附有着同样的趋势,图形化样品表面激活的血小板数量比平板的Ti和Hep-Fn样品少。APTT结果显示平板和图形的Hep-Fn没有明显延长APTT时间,但也没有加剧凝血时间,具有—定抗凝作用。
内皮细胞粘附结果显示：平板的Hep-Fn表面与钛相比,能促进内皮细胞的粘附、铺展、增殖；mPEG-SC表面几乎没有内皮细胞粘附。图形化表面内皮细胞都沿着Hep-Fn图形方向分布,不同尺寸的图形对内皮细胞的取向角、长宽比、形态指数等均有不同影响：大于细胞尺寸的图形使细胞呈2个或多个并行排列,有利于细胞铺展和增殖；与细胞尺寸相近的图形能够使细胞成单细胞阵列分布,拉长并产生引导作用。
以上结果表明：具有生物功能的mPEG-SC/Hep-Fn微图形能调节血小板和内皮细胞分布的同时,降低血小板粘附并调节内皮细胞粘附、生长和增殖,有效提高了钛金属的生物相容性。
For cardiovascular implants, neointimal hyperplasia and thrombus are the major problems in their application. Promoting endothelial cell adhesion or reducing platelet adhesion through surface modification technology can effectively improve the biocompatibility of the materials, and surface patterning technology has becoming popular.
In this paper, microtransfer molding technology (μTM), one method of soft lithography was used on titanium (Ti) surface after pretreatment to fabricate bionic biological molecules micropatterns. The pre-modified of Ti surface including NaOH alkali activation and3-aminopropyl triethoxysilane (APTE) treatment, and its surface characteristics observed by scanning electron microscopy (SEM), contact angle measurement, infrared spectrum analysis (FTIR) and amino quantitative method. Polydimethylsiloxane (PDMS) was prepared by the casting method, and then used it to imprint a-Methoxy-poly(ethylene glycol)-ω-succinimidyl carbonate (mPEG-SC) on the pre-modified Ti surface, which could resist proteins and cells ahhesion. After that the mixture of heparin and fibronectin, which have the properties of anticoagulation and promotion of endothelial cell adhesion, respectively, was assembled to fabricate the micropattern of mPEG-SC/Hep-Fn. SEM, contact angle measurement, FTIR, Hep and Fn staining method were used to examine the surface morphology and chemical composition of the mPEG-SC/Hep-Fn micropattem. On this basis, platelet adhesion and endothelial cell culture experiments in vitro were carried out to study the effect of mPEG-SC/Hep-Fn micropattem on the behavior of platelets and endothelial cells.
SEM, contact angle, FTIR, light microscopy and fluorescence microscopy results showed that: on alkali activated titanium surface, the roughness and surface hydrophilic were increased,-OH absorption peak was detected. On silane coupling Ti surface, the roughness was further increased, hydrophobicity was increased, CH2absorption peak was detected and the amino density of NH2was about51nmol/cn2. Clear stripes could be observed on the micropattem sample surface. FTIR showed the absorption peaks of mPEG-SC, Hep and Fn. And the Hep and Fn staining further confirmed the biological inertness of mPEG-SC, Hep and Fn was only distributed in the regions not coated with mPEG-SC.
Platelet adhesion experiments showed that:on Ti and Hep-Fn surface, platelets covered the entire surface with extended pseudopods, but did not aggregation. As the anticoagulant effect of heparin, the numbers of platelets adherent on Hep-Fn surface was less than Ti. On mPEG-SC surface, there was almost no platelet adhesion; on micropaterned sample surface, platelet selective distributed on Hep-Fn stripe region, further reduce platelet adhesion amount compared to nonpatterned Hep-Fn surface. Different number of platelet adhesion due to different sizes of Hep-Fn on micropatterned sample. The amount and distribution of activated platelets were similar with the results of total platelet adhesion. APTT results showed that both nonpatterned and micropatterned Hep-Fn did not significant prolongation of APTT time, but they also did not aggravate clotting time, which may had anticoagulant effect.
EC adhesion results showed that: nonpatterned Hep-Fn surface could promote EC adhesion, spreading and proliferation compared to titanium. The mPEG-SC surface almost had no endothelial cell adhesion. ECs distributed along with the Hep-Fn direction on the patterned sample surfaces. The different sizes of micropatterns had different effects on the orientation angle, ratio of length to width as well as shape index of EC:there could be two or more ECs parallel arranged on the micropatterns with greater size than EC, which could increase cell spreading and proliferation; and there could only be a single cell distribution on the micropatterns with similar size to EC, which could elongate and has guiding function to EC.
The above results suggest that:the biological functions of mPEG-SC/Hep-Fn micropattern could regulate the distribution of platelet and endothelial cell, at the same time reduce platelet adhesion and regulate the adhesion, growth and proliferation of EC. And this may effectively improve the biocompatibility of Ti.
本文采用软刻蚀技术中的微转移模塑技术在预处理的钛(Ti)表面构建仿生生物大分子微图形。钛的预处理包括NaOH碱活化和3-氨丙基三乙氧基硅烷(APTE)偶联处理,通过扫描电子显微镜(SEM)、接触角测量仪、傅里叶红外变换光谱分析(FTIR)和氨基定量的方法对预处理的钛表面进行理化表征。聚二甲基硅氧烷(PDMS)印章由浇铸法制备,运用其压印具有阻抗蛋白和细胞粘附的单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(mPEG-SC),而后组装具有抗凝作用的肝素(Hep)和促进内皮细胞粘附的纤维连接蛋白(Fn)混合物(Hep-Fn),构成iPEG-SC/Hep-Fn微图形。通过SEM、接触角测量仪、FTIR、Hep及Fn的染色对制备的Hep-Fn微图形进行表征。在此基础上进行体外血小板粘附实验和内皮细胞培养实验,研究图形对血小板及内皮细胞行为的影响。
SEM、接触角、FTIR、光镜及荧光显微镜结果显示：碱活化后的钛表面变粗糙,亲水性加强,表面出现羟基的吸收峰；硅烷偶联后的钛表面粗糙度进一步加大,疏水性增大,FTIR检测有CH2的吸收峰,氨基在表面的密度约为51nmol/cm2。图形化的样品表面能看到清晰的条纹图形,FTIR出现mPEG-SC、Hep和Fn的特征吸收峰。Hep和Fn的染色进一步证实mPEG-SC的生物惰性,Hep和Fn只分布在没有mPEG-SC的图形区域。
血小板粘附实验表明：平板Ti和Hep-Fn表面血小板布满整个表面,血小板伸出伪足,没有团聚。由于肝素的抗凝作用,Hep-Fn表面血小板粘附数量少于Ti。nPEG-SC平板表面几乎没有血小板粘附；图形样品表面血小板选择性分布在Hep-Fn条纹区域,与平板的Hep-Fn相比能进一步减少血小板粘附数量。不同尺寸的图形样品Hep-Fn的有效面积不同致使血小板的粘附数量有所不同。激活的血小板在数量和分布上和粘附有着同样的趋势,图形化样品表面激活的血小板数量比平板的Ti和Hep-Fn样品少。APTT结果显示平板和图形的Hep-Fn没有明显延长APTT时间,但也没有加剧凝血时间,具有—定抗凝作用。
内皮细胞粘附结果显示：平板的Hep-Fn表面与钛相比,能促进内皮细胞的粘附、铺展、增殖；mPEG-SC表面几乎没有内皮细胞粘附。图形化表面内皮细胞都沿着Hep-Fn图形方向分布,不同尺寸的图形对内皮细胞的取向角、长宽比、形态指数等均有不同影响：大于细胞尺寸的图形使细胞呈2个或多个并行排列,有利于细胞铺展和增殖；与细胞尺寸相近的图形能够使细胞成单细胞阵列分布,拉长并产生引导作用。
以上结果表明：具有生物功能的mPEG-SC/Hep-Fn微图形能调节血小板和内皮细胞分布的同时,降低血小板粘附并调节内皮细胞粘附、生长和增殖,有效提高了钛金属的生物相容性。
For cardiovascular implants, neointimal hyperplasia and thrombus are the major problems in their application. Promoting endothelial cell adhesion or reducing platelet adhesion through surface modification technology can effectively improve the biocompatibility of the materials, and surface patterning technology has becoming popular.
In this paper, microtransfer molding technology (μTM), one method of soft lithography was used on titanium (Ti) surface after pretreatment to fabricate bionic biological molecules micropatterns. The pre-modified of Ti surface including NaOH alkali activation and3-aminopropyl triethoxysilane (APTE) treatment, and its surface characteristics observed by scanning electron microscopy (SEM), contact angle measurement, infrared spectrum analysis (FTIR) and amino quantitative method. Polydimethylsiloxane (PDMS) was prepared by the casting method, and then used it to imprint a-Methoxy-poly(ethylene glycol)-ω-succinimidyl carbonate (mPEG-SC) on the pre-modified Ti surface, which could resist proteins and cells ahhesion. After that the mixture of heparin and fibronectin, which have the properties of anticoagulation and promotion of endothelial cell adhesion, respectively, was assembled to fabricate the micropattern of mPEG-SC/Hep-Fn. SEM, contact angle measurement, FTIR, Hep and Fn staining method were used to examine the surface morphology and chemical composition of the mPEG-SC/Hep-Fn micropattem. On this basis, platelet adhesion and endothelial cell culture experiments in vitro were carried out to study the effect of mPEG-SC/Hep-Fn micropattem on the behavior of platelets and endothelial cells.
SEM, contact angle, FTIR, light microscopy and fluorescence microscopy results showed that: on alkali activated titanium surface, the roughness and surface hydrophilic were increased,-OH absorption peak was detected. On silane coupling Ti surface, the roughness was further increased, hydrophobicity was increased, CH2absorption peak was detected and the amino density of NH2was about51nmol/cn2. Clear stripes could be observed on the micropattem sample surface. FTIR showed the absorption peaks of mPEG-SC, Hep and Fn. And the Hep and Fn staining further confirmed the biological inertness of mPEG-SC, Hep and Fn was only distributed in the regions not coated with mPEG-SC.
Platelet adhesion experiments showed that:on Ti and Hep-Fn surface, platelets covered the entire surface with extended pseudopods, but did not aggregation. As the anticoagulant effect of heparin, the numbers of platelets adherent on Hep-Fn surface was less than Ti. On mPEG-SC surface, there was almost no platelet adhesion; on micropaterned sample surface, platelet selective distributed on Hep-Fn stripe region, further reduce platelet adhesion amount compared to nonpatterned Hep-Fn surface. Different number of platelet adhesion due to different sizes of Hep-Fn on micropatterned sample. The amount and distribution of activated platelets were similar with the results of total platelet adhesion. APTT results showed that both nonpatterned and micropatterned Hep-Fn did not significant prolongation of APTT time, but they also did not aggravate clotting time, which may had anticoagulant effect.
EC adhesion results showed that: nonpatterned Hep-Fn surface could promote EC adhesion, spreading and proliferation compared to titanium. The mPEG-SC surface almost had no endothelial cell adhesion. ECs distributed along with the Hep-Fn direction on the patterned sample surfaces. The different sizes of micropatterns had different effects on the orientation angle, ratio of length to width as well as shape index of EC:there could be two or more ECs parallel arranged on the micropatterns with greater size than EC, which could increase cell spreading and proliferation; and there could only be a single cell distribution on the micropatterns with similar size to EC, which could elongate and has guiding function to EC.
The above results suggest that:the biological functions of mPEG-SC/Hep-Fn micropattern could regulate the distribution of platelet and endothelial cell, at the same time reduce platelet adhesion and regulate the adhesion, growth and proliferation of EC. And this may effectively improve the biocompatibility of Ti.
引文
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