钛氧薄膜表面氨等离子体浸没离子注入以及生物化修饰
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摘要
本文采用非平衡磁控溅射沉积技术在硅基底上制备了钛氧(Ti-O)薄膜,并对其进行氨等离子体浸没离子注入处理,在Ti-O薄膜表面引入氨基基团。采用原子力显微镜(AFM)和X射线光电子能谱(XPS)技术对注入前后的样品进行表面形貌和化学成分的分析,用酸性橙Ⅱ染色法计算注入后Ti-O薄膜表面氨基密度。在此基础上,分别固定以及联合固定细胞外基质成分中的重要生物分子层粘连蛋白(Ln)和纤连蛋白(Fn)。最后通过体外人脐静脉血管内皮细胞(HUVEC)原代培养实验评价未处理的Ti-O薄膜、注入后的Ti-O薄膜以及固定生物分子后的Ti-O薄膜表面的内皮细胞相容性,采用光学显微镜观察细胞形态。
接触角结果显示,注入后的Ti-O薄膜较之未处理的Ti-O薄膜接触角减小,亲水性增加。酸性橙Ⅱ染色法及XPS结果显示经过注入处理后可在Ti-O薄膜表面产生亲水性基团-NH_2。SEM及AFM结果显示注入后的Ti-O薄膜表面粗糙度增加。免疫荧光染色结果显示在经过注入处理的Ti-O薄膜表面成功地分别固定了Ln、Fn,并且Ln和Fn联合固定成功。
体外人脐静脉内皮细胞培养实验结果表明,Ti-O薄膜本身具有一定的内皮细胞亲和性,经过氨等离子注入后的Ti-O薄膜对细胞生长有良好的促进作用,在经过注入处理的Ti-O薄膜表面固定Ln和Fn能进一步促进内皮细胞的生长,且在经过注入处理的Ti-O薄膜表面联合固定层粘连蛋白和纤连蛋白促内皮化效果显著,优于表面固定单种蛋白的方法。
以上试验结果表明,离子注入可以在Ti-O表面引入功能基团氨基,并且同时可以改变薄膜表面的亲水性、粗糙度等性质,可在一定程度上促进内皮细胞的黏附。在具有氨基基团的Ti-O薄膜表面固定Ln和Fn对内皮细胞的生长有良好的促进作用,且在具有氨基基团的Ti-O薄膜表面联合固定层粘连蛋白和纤连蛋白促内皮化效果更为显著。因此,在Ti-O薄膜表面固定多种蛋白是促其表面内皮化的有效方法。
In this work,Ti-O films were synthesized by Unbalance Magnetron Sputtering on silicon substrates,and then treated by plasma immersion ion implantation(PⅢ) using ammonia gases in order to introduce amino group to Ti-O film's surface.The structure and the chemical composition of the surface were analysed by atomic force microscopy(AFM) and X-ray photoelectron spectroscopy(XPS).The density of amino group on the surface of treated Ti-O films can be rationed by Acid OrangeⅡ.After that,laminin(Ln) and fibronectin (Fn) were immobilized to the surface based on the amido group.The morphology of endothelial cells was observed by optical microscopy.In the end,human umbilical vein endothelial cells(HUVEC) primary culture experiments were adopted in vitro to evaluate the compatibility of the endothelial cells on the surface of the untreated Ti-O films、PⅢ-treated Ti-O films and the Ti-O films immobilized by biomolecules.The morphology of endothelial cells was observed by optical microscopy.
The contact angle of PⅢ-treated Ti-O films surface was lower than that of the untreated Ti-O films.It indicates that the wettability of PⅢ-treated Ti-O films is a little higher than the untreated Ti-O films.The Acid OrangeⅡand XPS results show that the functional group of amido has been introduced to the surface of Ti-O film by plasma immersion ion implantation.The results of SEM and AFM suggest that the roughness of the PⅢ-treated Ti-O films surface has been increased.The result of immunofluorescence indicates that laminin and fibronectin has been immobilized to the PⅢ-treated Ti-O film surface.
Endothelial cell culture experiment suggests that,the amino groups are helpful for the adhesion and proliferation of HUVEC.The Laminin and Fibronectin can further enhance HUVEC's adhesion,proliferation,and growth. According to the endothelial cell culture experiment,compared to the PⅢ-treated Ti-O films immobilized with Laminin or Fibronectin only,the PⅢtreated Ti-O films immobilized with both laminin and fibronectin can promote the adhesion and growth of endothelial.
All these results indicated that the ion implantation can introduce functional group onto the surface of Ti-O film to change its properties like wettability and roughness,so it can increase the cell's adhesion.Laminin and fibronectin can be immobilized onto the surface of Ti-O film by amino group.Laminin and fibronectin can promote the adhesion and growth of endothelial cells.This work provide a good way for surface endothelialization by immobilizing laminin and fibronectin onto the surface of PⅢ-treated Ti-O film.
接触角结果显示,注入后的Ti-O薄膜较之未处理的Ti-O薄膜接触角减小,亲水性增加。酸性橙Ⅱ染色法及XPS结果显示经过注入处理后可在Ti-O薄膜表面产生亲水性基团-NH_2。SEM及AFM结果显示注入后的Ti-O薄膜表面粗糙度增加。免疫荧光染色结果显示在经过注入处理的Ti-O薄膜表面成功地分别固定了Ln、Fn,并且Ln和Fn联合固定成功。
体外人脐静脉内皮细胞培养实验结果表明,Ti-O薄膜本身具有一定的内皮细胞亲和性,经过氨等离子注入后的Ti-O薄膜对细胞生长有良好的促进作用,在经过注入处理的Ti-O薄膜表面固定Ln和Fn能进一步促进内皮细胞的生长,且在经过注入处理的Ti-O薄膜表面联合固定层粘连蛋白和纤连蛋白促内皮化效果显著,优于表面固定单种蛋白的方法。
以上试验结果表明,离子注入可以在Ti-O表面引入功能基团氨基,并且同时可以改变薄膜表面的亲水性、粗糙度等性质,可在一定程度上促进内皮细胞的黏附。在具有氨基基团的Ti-O薄膜表面固定Ln和Fn对内皮细胞的生长有良好的促进作用,且在具有氨基基团的Ti-O薄膜表面联合固定层粘连蛋白和纤连蛋白促内皮化效果更为显著。因此,在Ti-O薄膜表面固定多种蛋白是促其表面内皮化的有效方法。
In this work,Ti-O films were synthesized by Unbalance Magnetron Sputtering on silicon substrates,and then treated by plasma immersion ion implantation(PⅢ) using ammonia gases in order to introduce amino group to Ti-O film's surface.The structure and the chemical composition of the surface were analysed by atomic force microscopy(AFM) and X-ray photoelectron spectroscopy(XPS).The density of amino group on the surface of treated Ti-O films can be rationed by Acid OrangeⅡ.After that,laminin(Ln) and fibronectin (Fn) were immobilized to the surface based on the amido group.The morphology of endothelial cells was observed by optical microscopy.In the end,human umbilical vein endothelial cells(HUVEC) primary culture experiments were adopted in vitro to evaluate the compatibility of the endothelial cells on the surface of the untreated Ti-O films、PⅢ-treated Ti-O films and the Ti-O films immobilized by biomolecules.The morphology of endothelial cells was observed by optical microscopy.
The contact angle of PⅢ-treated Ti-O films surface was lower than that of the untreated Ti-O films.It indicates that the wettability of PⅢ-treated Ti-O films is a little higher than the untreated Ti-O films.The Acid OrangeⅡand XPS results show that the functional group of amido has been introduced to the surface of Ti-O film by plasma immersion ion implantation.The results of SEM and AFM suggest that the roughness of the PⅢ-treated Ti-O films surface has been increased.The result of immunofluorescence indicates that laminin and fibronectin has been immobilized to the PⅢ-treated Ti-O film surface.
Endothelial cell culture experiment suggests that,the amino groups are helpful for the adhesion and proliferation of HUVEC.The Laminin and Fibronectin can further enhance HUVEC's adhesion,proliferation,and growth. According to the endothelial cell culture experiment,compared to the PⅢ-treated Ti-O films immobilized with Laminin or Fibronectin only,the PⅢtreated Ti-O films immobilized with both laminin and fibronectin can promote the adhesion and growth of endothelial.
All these results indicated that the ion implantation can introduce functional group onto the surface of Ti-O film to change its properties like wettability and roughness,so it can increase the cell's adhesion.Laminin and fibronectin can be immobilized onto the surface of Ti-O film by amino group.Laminin and fibronectin can promote the adhesion and growth of endothelial cells.This work provide a good way for surface endothelialization by immobilizing laminin and fibronectin onto the surface of PⅢ-treated Ti-O film.
引文
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