Ti表面构建Ⅳ型胶原/肝素抗凝促内皮双功能层的研究与探索
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摘要
针对临床上常见的血栓、再狭窄等并发症,心血管支架材料表面修饰已从单分子、单功能研究逐渐转向多分子、多功能的设计。基于内皮细胞层的天然抗凝功能及支架材料表面内皮化的理念,兼具抗凝与促内皮双功能的表面微环境设计与构建成为新的研究热点。但是,微环境设计中,不同功能生物分子的选用、不同功能生物分子之间的作用发挥以及制约与平衡等,是支架材料表面微环境构建中面临并急需解决的问题。本文以起抗凝作用的肝素为目标分子,结合有促内皮化功能的Ⅳ型胶原,在Ti基材料表面进行了抗凝/促内皮双功能微环境的构建、表征、优化以及系列体内外生物相容性评价。
首先,在碱活化后富含羟基活性基团的负电荷Ti基材料表面,通过聚赖氨酸有机过渡层或硅烷化的方法引入大量氨基基团。在此基础上,构建Ⅳ型胶原／肝素生物修饰层。利用FTIRR、SEM、 AFM、水接触角测试、免疫荧光染色、QCM-D等方法对生物修饰层进行的定性和定量表征,结果表明,肝素和Ⅳ型胶原能够通过层层静电自组装的方式固定到Ti表面。通过优化得出,在肝素浓度为5mg/mL条件下,适宜的工艺参数为：以聚赖氨酸构建氨基化表面、Ⅳ型胶原浓度为25μg/mL、自组装双层的数目为15个、肝素与Ⅳ型胶原的反应时间分别为6分钟和25分钟。通过体外静态实验发现,生物修饰层具有一定抗凝血性能并能够促进内皮细胞与内皮祖细胞的粘附、生长、增殖以及内皮细胞的迁移,细胞相容性较好。但是,体外动态血液实验显示,生物修饰层的血液相容性表现较差,容易形成血栓,体现了肝素与Ⅳ型胶原两种不同功能分子不同环境下的竞争与平衡。动物体内系列实验结果表明,生物修饰层表面可以促进内膜新生,内皮化程度较大,但新生内膜较厚。这可能与生物修饰层表面Ⅳ型胶原对于细胞无选择性有关。
接着,本文对两种不同功能生物分子的相互作用、竞争与平衡机制进行的研究发现,在抗凝血与促凝血的竞争方面,肝素主要是通过内源性凝血途径抑制凝血酶防止血栓形成,以及同种电荷排斥的作用减少血小板的粘附,肝素还可以减少早期血小板膜表面糖蛋白GPⅡb/Ⅲa的表达。而Ⅳ型胶原通过引起凝血因子Ⅻ的自我激活启始内源性凝血途径,通过胶原蛋白序列PGQOGVMGF、GFOGER、(GPO)x与血小板受体血管性血友病因子(von Willebrand Factor, VWF)的A3区(VWF-A3),整合素α2β1和糖蛋白VI(Glycoprotein VI, GpVI)结合,促进血小板的粘附和激活,进而引起纤维蛋白原变性形成纤维蛋白,促进血栓的发生。在促内皮化与抑制内皮化的竞争方面,Ⅳ型胶原主要通过肽链上的氨基酸残基构成的CB3(IV)片段与内皮细胞表面的整合素受体α1β1和α2β1结合,促进细胞的增殖。而肝素则通过糖胺抑制选择性蛋白激酶C (PKC)介导的ODC基因表达来抑制内皮细胞的增殖。另外,Ⅳ型胶原表面的正电荷,肝素表面的负电荷,二者对于表面有负电荷的内皮细胞也具有相反的作用。最终,肝素与Ⅳ型胶原主要在内源性凝血途径、血小板粘附以及内皮细胞粘附与增殖等方面进行生物功能竞争,进而达到一个相对平衡的状态。
鉴于Ⅳ型胶原存在条件下,也会促进平滑肌细胞的生长,考虑选择具有内皮细胞选择性粘附的GREDVY多肽分子,结合肝素,在聚赖氨酸引入氨基的表面构建GREDVY多肽/肝素生物修饰层。同样,利用FTIR、AFM、水接触角测试、免疫荧光染色等方法对生物修饰层进行的定性定量表征,结果表明,肝素和GREDVY多肽能够通过静电结合作用共同固定到Ti表面。利用血小板粘附与激活实验、APTT测试、细胞培养实验、内皮细胞与血小板的竞争性粘附实验、内皮细胞与平滑肌细胞的竞争性粘附实验等体外静态评价手段进行血液相容性与细胞相容性评价,结果表明,在具备抗凝血及促内皮细胞粘附作用的同时,肝素与GREDVY多肽构建的生物修饰层表现出显著抑制平滑肌粘附与增殖的功能。
总之,使用不同功能的生物分子进行双功能或多功能生物修饰层的构建具有可行性,生物修饰层外层两种功能生物分子的交叉分布,为双功能或多功能的实现提供了可能。但是多功能的实现并非几种功能的简单叠加,而是存在着竞争与平衡。这种竞争与平衡关系会随着外界环境的变化有所调整,从而通过新一轮的竞争实现新的平衡。本文获得的进展对血液接触材料的表面微环境构建中生物分子的选用与设计提供了重要参考,为心血管支架材料表面多功能微环境构建开拓了新的思路。
Owing to the emergence of stent thrombosis (ST), in-stent restenosis (ISR) and other clinical complications after the stent implantation, the modification of cardiovascular stent surface have gradually changed from single-molecule, single-functional designs to multi-molecule, multi-functional ones. Based on the theories that endothelial cell layer has good property of natural anti-coagulation and endothelialization on material's surface is absolutely necessary for the cardiovascular stent, the surface modification pursuing both the anti-coagulation function and promoting endothelialization micro-environment becomes a new hotspot. However, there still exist some problems that need to resolve, for example, the design of the micro-environment including the selection of different functional biomolecules, the restriction and equilibrium between two functional biomolecules. In this paper, heparin, acting as anticoagulant, was used as the target molecule to construct a bifunctional micro-environment of anticoagulation/promoting endothelialization on the Ti-based material surface, combining with the promoting endothelialization molecule type IV collagen. Series of characterization tests, optimization as well as in vitro and in vivo animal tests were carried out to evaluate the properties of the multi-functional layer.
Firstly, negatively charged surface is produced with hydroxyl reactive groups on the Ti-based materials after alkaline activation, then large number of amino groups are introduced to the surface by assembling a polylysine media layer or silanizing with APTES. Then the biological modification layer, type IV collagen/heparin biological modification layer, was obtained based on the former layer through coating two different molecules, type IV collagen and heparin. Some quantitative and qualitative characterizations, such as FTIR, SEM, AFM, the water contact angle test, the immunofluorescence staining and quartz crystal microbalance with dissipation (QCM-D) were carried out, it was indicated that heparin and type IV collagen can be fixed on the Ti-surface through layer-by-layer electrostatic self-assembly. The experimental parameters results of process optimization indicate that the optimum parameter are assembling a polylysine media layer, the heparin concentration is5mg/mL, the concentration of type IV collagen is25μg/mL, the optimal number of double layer is15, and the best reaction time of heparin and type IV collagen is6minutes and25minutes respectively. The in vitro static results show that the bionic layer has anticoagulant properties and good cytocompatibility, on which ECs and EPCs could be promoted to adhere, grow, proliferate and migrate. The in vitro dynamic blood tests show that the bionic layer has a poor performance of hemocompatibility, on which thrombosis can easily happen, indicating there exist the competition and balance effects between heparin and type Ⅳ collagen with two different functions in different environment. The in vivo animal experiment results manifest that the bionic surface can encourage new endometrial growth which has more endothelialization but thicker endothelial neointimal, this maybe related to that IV collagen belongs to non-selective biomolecule and could promote the migration and proliferation of both smooth muscle cells and other cells.
Moreover, the interaction and the competition-equilibrium mechanisms of heparin and type IV collagen were also studied. It was found that during the competition of anticoagulant and procoagulant, heparin prevent thrombosis mainly via endogenous coagulation pathway, and reduce platelet binding through the charge repulsion effect. In addition, the heparin also can inhibit the expression of GPⅡ b/Ⅲa on the surface of the prophase platelets. While the type Ⅳ collagen play a role on promoting clotting factor XII self-activating then resulting in starting extrinsic coagulation pathways, and binding with the platelet receptors VWF-A3, α2β1and Glycoprotein Ⅵ through collagen sequences PGQOGVMGF, GFOGER and (GPO)x leading to facilitating platelet adhesion and activated, thereby causing the degeneration of fibrinogen to fibrin. In the competition of inhibiting and promoting endothelialization, fragment3(CB3(Ⅳ)) on type IV collagen, constituted of amino acid residues of peptide chain, show the advantage that could promote the cell proliferation by binding with the integrate receptors α1β1and α2β1on the endothelial cell surface. On the contrary, heparin represents a role of inhibiting the proliferation of endothelial cells through the ODC gene expression mediated by the select protein kinase C (PKC) which could be prevented by glycosaminoglycan on the heparin. In addition, compared to type IV collagen with the positive charges, the heparin with the negative charges possesses opposite effect on the endothelial cells which bear negative charges on the surface. Ultimately, heparin and type IV collagen could achieve a relative equilibrium condition with the competitions during the extrinsic coagulation pathway, platelet adhesion and endothelial cell adhesion and proliferation.
Finally, due to the type IV collagen in this work also promotes the smooth muscle cells adhesion and growth, GREDVY polypeptide which has the endothelial cells selectivity adhesion property was chosen to construct the GREDVY polypeptide/heparin bionic layer. FTIR, AFM, the water contact angle test, and the immunofluorescence staining etc. were also used to characterize the GREDVY polypeptide/heparin bionic layer. It was shown that heparin and GREDVY polypeptide can be jointly fixed by electrostatic binding on Ti. The in vitro static experiments, including the platelet adhesion experiments, APTT test, cell culture experiments, competitive binding experiments of endothelial cells and platelets, were used for hemocompatibility and cytocompatibility contrastive evaluation, and the evaluation of competitive adhesion and growth experiments of endothelial cells and smooth muscle cells on Ti and GREDVY polypeptide/heparin bionic layer. The results showed that GREDVY polypeptide/heparin bionic layer not only represents a good hemocompatibility and promoting endothelial cells adhesion, but also inhibits smooth muscle cells adhesion and proliferation obviously.
In short, it is feasible to construct a dual-function or multifunction surface using the biological molecules with different functions. The cross-distribution of the two biomolecules on the outer layer may provide the realization of the dual-function or multifunction. But the implementation of the multi-function is not a simple sum of several functions, there is a competition and balance. The competition and balanced relationship will be adjusted with the changes of external biological environments, and a new balance can be achieved after a new round of competition. Progress in this work provides an important reference for surface microenvironment building on the artificial materials contact the blood, including the selection and design of biological molecules, which opens up new ideas for the construction of multi-function micro-environment on the cardiovascular scaffold surface.
首先,在碱活化后富含羟基活性基团的负电荷Ti基材料表面,通过聚赖氨酸有机过渡层或硅烷化的方法引入大量氨基基团。在此基础上,构建Ⅳ型胶原／肝素生物修饰层。利用FTIRR、SEM、 AFM、水接触角测试、免疫荧光染色、QCM-D等方法对生物修饰层进行的定性和定量表征,结果表明,肝素和Ⅳ型胶原能够通过层层静电自组装的方式固定到Ti表面。通过优化得出,在肝素浓度为5mg/mL条件下,适宜的工艺参数为：以聚赖氨酸构建氨基化表面、Ⅳ型胶原浓度为25μg/mL、自组装双层的数目为15个、肝素与Ⅳ型胶原的反应时间分别为6分钟和25分钟。通过体外静态实验发现,生物修饰层具有一定抗凝血性能并能够促进内皮细胞与内皮祖细胞的粘附、生长、增殖以及内皮细胞的迁移,细胞相容性较好。但是,体外动态血液实验显示,生物修饰层的血液相容性表现较差,容易形成血栓,体现了肝素与Ⅳ型胶原两种不同功能分子不同环境下的竞争与平衡。动物体内系列实验结果表明,生物修饰层表面可以促进内膜新生,内皮化程度较大,但新生内膜较厚。这可能与生物修饰层表面Ⅳ型胶原对于细胞无选择性有关。
接着,本文对两种不同功能生物分子的相互作用、竞争与平衡机制进行的研究发现,在抗凝血与促凝血的竞争方面,肝素主要是通过内源性凝血途径抑制凝血酶防止血栓形成,以及同种电荷排斥的作用减少血小板的粘附,肝素还可以减少早期血小板膜表面糖蛋白GPⅡb/Ⅲa的表达。而Ⅳ型胶原通过引起凝血因子Ⅻ的自我激活启始内源性凝血途径,通过胶原蛋白序列PGQOGVMGF、GFOGER、(GPO)x与血小板受体血管性血友病因子(von Willebrand Factor, VWF)的A3区(VWF-A3),整合素α2β1和糖蛋白VI(Glycoprotein VI, GpVI)结合,促进血小板的粘附和激活,进而引起纤维蛋白原变性形成纤维蛋白,促进血栓的发生。在促内皮化与抑制内皮化的竞争方面,Ⅳ型胶原主要通过肽链上的氨基酸残基构成的CB3(IV)片段与内皮细胞表面的整合素受体α1β1和α2β1结合,促进细胞的增殖。而肝素则通过糖胺抑制选择性蛋白激酶C (PKC)介导的ODC基因表达来抑制内皮细胞的增殖。另外,Ⅳ型胶原表面的正电荷,肝素表面的负电荷,二者对于表面有负电荷的内皮细胞也具有相反的作用。最终,肝素与Ⅳ型胶原主要在内源性凝血途径、血小板粘附以及内皮细胞粘附与增殖等方面进行生物功能竞争,进而达到一个相对平衡的状态。
鉴于Ⅳ型胶原存在条件下,也会促进平滑肌细胞的生长,考虑选择具有内皮细胞选择性粘附的GREDVY多肽分子,结合肝素,在聚赖氨酸引入氨基的表面构建GREDVY多肽/肝素生物修饰层。同样,利用FTIR、AFM、水接触角测试、免疫荧光染色等方法对生物修饰层进行的定性定量表征,结果表明,肝素和GREDVY多肽能够通过静电结合作用共同固定到Ti表面。利用血小板粘附与激活实验、APTT测试、细胞培养实验、内皮细胞与血小板的竞争性粘附实验、内皮细胞与平滑肌细胞的竞争性粘附实验等体外静态评价手段进行血液相容性与细胞相容性评价,结果表明,在具备抗凝血及促内皮细胞粘附作用的同时,肝素与GREDVY多肽构建的生物修饰层表现出显著抑制平滑肌粘附与增殖的功能。
总之,使用不同功能的生物分子进行双功能或多功能生物修饰层的构建具有可行性,生物修饰层外层两种功能生物分子的交叉分布,为双功能或多功能的实现提供了可能。但是多功能的实现并非几种功能的简单叠加,而是存在着竞争与平衡。这种竞争与平衡关系会随着外界环境的变化有所调整,从而通过新一轮的竞争实现新的平衡。本文获得的进展对血液接触材料的表面微环境构建中生物分子的选用与设计提供了重要参考,为心血管支架材料表面多功能微环境构建开拓了新的思路。
Owing to the emergence of stent thrombosis (ST), in-stent restenosis (ISR) and other clinical complications after the stent implantation, the modification of cardiovascular stent surface have gradually changed from single-molecule, single-functional designs to multi-molecule, multi-functional ones. Based on the theories that endothelial cell layer has good property of natural anti-coagulation and endothelialization on material's surface is absolutely necessary for the cardiovascular stent, the surface modification pursuing both the anti-coagulation function and promoting endothelialization micro-environment becomes a new hotspot. However, there still exist some problems that need to resolve, for example, the design of the micro-environment including the selection of different functional biomolecules, the restriction and equilibrium between two functional biomolecules. In this paper, heparin, acting as anticoagulant, was used as the target molecule to construct a bifunctional micro-environment of anticoagulation/promoting endothelialization on the Ti-based material surface, combining with the promoting endothelialization molecule type IV collagen. Series of characterization tests, optimization as well as in vitro and in vivo animal tests were carried out to evaluate the properties of the multi-functional layer.
Firstly, negatively charged surface is produced with hydroxyl reactive groups on the Ti-based materials after alkaline activation, then large number of amino groups are introduced to the surface by assembling a polylysine media layer or silanizing with APTES. Then the biological modification layer, type IV collagen/heparin biological modification layer, was obtained based on the former layer through coating two different molecules, type IV collagen and heparin. Some quantitative and qualitative characterizations, such as FTIR, SEM, AFM, the water contact angle test, the immunofluorescence staining and quartz crystal microbalance with dissipation (QCM-D) were carried out, it was indicated that heparin and type IV collagen can be fixed on the Ti-surface through layer-by-layer electrostatic self-assembly. The experimental parameters results of process optimization indicate that the optimum parameter are assembling a polylysine media layer, the heparin concentration is5mg/mL, the concentration of type IV collagen is25μg/mL, the optimal number of double layer is15, and the best reaction time of heparin and type IV collagen is6minutes and25minutes respectively. The in vitro static results show that the bionic layer has anticoagulant properties and good cytocompatibility, on which ECs and EPCs could be promoted to adhere, grow, proliferate and migrate. The in vitro dynamic blood tests show that the bionic layer has a poor performance of hemocompatibility, on which thrombosis can easily happen, indicating there exist the competition and balance effects between heparin and type Ⅳ collagen with two different functions in different environment. The in vivo animal experiment results manifest that the bionic surface can encourage new endometrial growth which has more endothelialization but thicker endothelial neointimal, this maybe related to that IV collagen belongs to non-selective biomolecule and could promote the migration and proliferation of both smooth muscle cells and other cells.
Moreover, the interaction and the competition-equilibrium mechanisms of heparin and type IV collagen were also studied. It was found that during the competition of anticoagulant and procoagulant, heparin prevent thrombosis mainly via endogenous coagulation pathway, and reduce platelet binding through the charge repulsion effect. In addition, the heparin also can inhibit the expression of GPⅡ b/Ⅲa on the surface of the prophase platelets. While the type Ⅳ collagen play a role on promoting clotting factor XII self-activating then resulting in starting extrinsic coagulation pathways, and binding with the platelet receptors VWF-A3, α2β1and Glycoprotein Ⅵ through collagen sequences PGQOGVMGF, GFOGER and (GPO)x leading to facilitating platelet adhesion and activated, thereby causing the degeneration of fibrinogen to fibrin. In the competition of inhibiting and promoting endothelialization, fragment3(CB3(Ⅳ)) on type IV collagen, constituted of amino acid residues of peptide chain, show the advantage that could promote the cell proliferation by binding with the integrate receptors α1β1and α2β1on the endothelial cell surface. On the contrary, heparin represents a role of inhibiting the proliferation of endothelial cells through the ODC gene expression mediated by the select protein kinase C (PKC) which could be prevented by glycosaminoglycan on the heparin. In addition, compared to type IV collagen with the positive charges, the heparin with the negative charges possesses opposite effect on the endothelial cells which bear negative charges on the surface. Ultimately, heparin and type IV collagen could achieve a relative equilibrium condition with the competitions during the extrinsic coagulation pathway, platelet adhesion and endothelial cell adhesion and proliferation.
Finally, due to the type IV collagen in this work also promotes the smooth muscle cells adhesion and growth, GREDVY polypeptide which has the endothelial cells selectivity adhesion property was chosen to construct the GREDVY polypeptide/heparin bionic layer. FTIR, AFM, the water contact angle test, and the immunofluorescence staining etc. were also used to characterize the GREDVY polypeptide/heparin bionic layer. It was shown that heparin and GREDVY polypeptide can be jointly fixed by electrostatic binding on Ti. The in vitro static experiments, including the platelet adhesion experiments, APTT test, cell culture experiments, competitive binding experiments of endothelial cells and platelets, were used for hemocompatibility and cytocompatibility contrastive evaluation, and the evaluation of competitive adhesion and growth experiments of endothelial cells and smooth muscle cells on Ti and GREDVY polypeptide/heparin bionic layer. The results showed that GREDVY polypeptide/heparin bionic layer not only represents a good hemocompatibility and promoting endothelial cells adhesion, but also inhibits smooth muscle cells adhesion and proliferation obviously.
In short, it is feasible to construct a dual-function or multifunction surface using the biological molecules with different functions. The cross-distribution of the two biomolecules on the outer layer may provide the realization of the dual-function or multifunction. But the implementation of the multi-function is not a simple sum of several functions, there is a competition and balance. The competition and balanced relationship will be adjusted with the changes of external biological environments, and a new balance can be achieved after a new round of competition. Progress in this work provides an important reference for surface microenvironment building on the artificial materials contact the blood, including the selection and design of biological molecules, which opens up new ideas for the construction of multi-function micro-environment on the cardiovascular scaffold surface.
引文
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