医用涤纶材料抗凝及促内皮化表面改性研究
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摘要
合成医用高分子材料被广泛的应用于包括心血管人工器官在内的人工器官植入类的材料中。然而当高分子材料与血液接触时,在界面上会形成一系列的复杂的相互作用,导致凝血反应和血栓的形成,从而限制了高分子材料在血液接触材料上的进一步应用。生物材料表面覆盖内皮细胞层是改善材料血液相容性的理想方法,但直接把内皮细胞种植在基质材料表面不仅增殖速度慢,而且由于细胞与材料表面之间的作用力比较小,使得通常在短的时间内就容易脱落分离。
因此,本研究将具有强细胞亲合力的天然大分子如胶原蛋白、明胶等粘附蛋白通过共价键结合于医用涤纶PET材料表面,以加快内皮细胞的粘附与生长。但这些粘附蛋白不仅是细胞粘附的优良介质,也有利于血小板的粘附和血栓的形成。血小板的粘附往往在材料与血液接触的瞬间即发生,而细胞的粘附则需要较长的时间,这样可能在材料表面还未覆盖一层内皮细胞之前,已经形成了血栓。为阻止这种状况的发生,我们在涤纶材料表面共固定抗凝生物分子肝素(白蛋白)和促内皮生物分子胶原(明胶),以期获得同时具有良好的抗凝血性能和促内皮细胞生长的医用涤纶表面。
本文通过X射线光电子能谱XPS分析了改性前后材料表面的组成成分的变化。在肝素—胶原(PET-CL-Hep)及肝素—明胶(PET-Gel-Hep)共固定的表面,不仅出现的N1s峰,还出现了较为明显的S2s,S2p峰,根据XPS高分辨谱计算可得PET-CL-Hep和PET-Gel-Hep的硫元素含量分别为1.55%和2.18%,这说明了肝素已与胶原或明胶共固定于材料表面。同时在白蛋白—胶原(PET-CL-BSA)和白蛋白—明胶(PET-Gel-BSA)的表面,氮元素的含量分别比仅固定胶原(PET-CL)和仅固定明胶(PET-Gel)的氮含量高4.74%和5.64%,这也提示了白蛋白与胶原或明胶分子的在PET表面的混合有效固定。接触角测量与表面能计算的结果表明肝素、白蛋白与胶原或明胶共固定改性后的材料表面接触角减小,表面能增加,材料表面的亲水性较好。
采用甲苯胺蓝染色法测得接枝丙烯酸的材料表面的羧基密度为5.29×10~(-9)mol/cm~2,采用天狼猩红染色法得到PET-CL表面的胶原含量为3.14μg/cm~2,PET-Gel表面的明胶为2.02μg/cm~2。利用甲苯胺蓝及正己烷萃取法可得PET-CL-Hep表面的肝素含量为1.73μg/cm~2,PET-Gel-Hep表面的肝素1.91/μg/cm~2。
体外血小板粘附及血小板乳酸脱氢酶(LDH)检测血小板数量的实验一致证明,肝素或白蛋白与胶原或明胶的共固定,使PET表面的抗凝血性能有所改善。LDH实验测得PET-CL-Hep、PET-Gel-Hep、PET-CL-BSA、PET-Gel-BSA表面的血小板黏附率分别为12.83%、17.24%、10.53%和12.19%,与未改性的涤纶表面血小板26.21%,PET-Gel 40.38%和PET-CL47.21%的黏附率相比,血小板粘附数量明显减少,血小板激活程度减轻。PET-CL-Hep和PET-Gel-Hep的APTT检测时间为68.9s和65.5,比未改性的涤纶表面延长9.7s和6.3s,比原血浆延长14.9s和11.5s。活化的部分凝血活酶时间(APTT)检测表面肝素与胶原或明胶的共固定可抑制内源性凝血系统的激活。采用酶联免疫法定量评价不同改性材料表面纤维蛋白原的变性情况。PET-CL-Hep、PET-Gel-Hep、PET-CL-BSA、PET-Gel-BSA的表面纤维蛋白原变性量分别为25.32%、22.48%、19.20%、20.59%,而空白样品的纤维蛋白原变性量为35.57%,这说明肝素(白蛋白)与胶原(明胶)的共固定,可减少纤维蛋白原的变性暴露出与血小板结合,从而减少了血小板在材料表面的聚集。上述一系列评价的结果均证明肝素(白蛋白)与胶原(明胶)的共固定,可改善PET的抗凝血性能。
通过体外内皮细胞粘附及细胞Alamar Blue实验,定量表征内皮细胞在PET材料的黏附率、增殖率、细胞活性,并通过光学显微镜观察材料表面的细胞形态变化。在细胞培养的第120h,未改性涤纶表面的细胞数为4.88×10~4cells/cm~2,细胞活性为158.6%,而PET-CL-Hep,PET-Gel-Hep,PET-CL-BSA,PET-Gel-BSA表面的细胞数量分别为9.37,9.73,10.66,9.07(×10~4cells/cm~2),细胞活性依次为202.3%,205.9%,214.8%,199.4%。上述结果表明在医用涤纶表面,通过肝素或白蛋白与胶原或明胶的共固定可改善材料表面的促内皮细胞生长性能。
In recent years, synthetic polymers are widely used in biomedical materials for artificial organs including cardiovascular artificial organs. However, once blood contact with polymers, complicated interaction will happen on the interface so as to form coagulating reaction and thrombus. In that case, the farther application of synthetic polymers in blood-contacting devices was restricted. It is believed that the endothelialization of the blood contacting device surface may overcome the problem. Therefore, a surface that enhances cell-material interaction and promotes the endothelialization at a shorter time is necessary.
In our research, natural proteins like gelatin and collagen molecules were immobilized onto the biomedical poly(ethylene terephthalate) (PET, dacron) surfaces. However, the immobilization of gelatin or collagen on the surface of materials may accelerate the aggregation of the platelets before the formation of endothelial layer. Hence, surface anticoagulative modification before endothelialization is necessary. Our study focuses on the co-immobilization of heparin (or albumin) and collagen (or gelatin) molecules on the PET surface which can construct the anticoagulant surface before the endothelium formation and also accelerate the endothelialization of polymer surface.
The surface constitution of the modified PET is determined by X-ray photoelectron spectroscopy (XPS). According to the resurts of XPS, The detection of S2p, S2s might be in virtue of the sulfonic groups of the heparin chemically attached to the membrane surfaces, which demonstrates that of the molecules of heparin and collagen or gelatin co-immobilized on the PET surfaces. Comparing to the only collagen and gelatin immobilized surface, the nitrogen contents increase 4.74% and 5.64% on the surface of albumin-collagen (PET-CL-BSA) and albumin-gelatin (PET-Gel-BSA) co-immobilized PET film surface. This proves that albumin combined with collagen or gelatin molecules are effectively co-immobilized onto the PET film surfaces. The results of static contact angle indicate the hydrophilicity of the modified PET surface is more remarkably improved than that of the unmodified PET surface.
The determination of carboxyl group on the acrylic acid grafted PET surface is 5.29×10~(-9)mol/cm~2. According to the protein measurement, the concentration of gelatin immobilized on only gelatin-PET (PET-Gel) is 2.02μg/cm~2, while the concertration of collagen immobilized on only collagen-PET (PET-CL) is 3.14μg/cm~2. The quantification of heparin on heparin-collagen co-immobilized surface (PET-CL-Hep) is 1.73μg/cm~2, and heparin on heparin-gelatin co-immobilized surface(PET-CL-Hep) is 1.91μg/cm~2.
The results of lactate dehydrogenase(LDH) and the platelet adhesion test in vitro all indicat that the co-immobilization of heparin or albumin with collagen or gelatin molecules could improve the anticoagulation. The percentage of platelet adhered on the surface of PET-CL-Hep is 12.83%, PET-Gel-Hep 17.24%, PET-CL-BSA 10.53%, and PET-Gel-BSA 12.19%, thus the percentages of adhered platelet decreased significantly compared to those of untreated PET (26.21%) and the PET-Gel surface (40.38%) , the PET-CL surface (47.21%). The results of coagulant factor tests reveal that the activated partially thromboplastion time (APTT) of PET-CL-Hep is 68.9s and PET-Gel-Hep is 65.5s, which is longer than those of the virgin PET (59.2s) and the plasma (54s). The change of APTT indicates that the modified PET films can suppress intrinsic coagulant system. The number of denatured fibrinogen on different modified surfaces is also determined by enzyme linked immunosorbent assay (ELISA). The resulte shows that the proption of denatured fibrinogen on PET-CL-Hep is 25.32%, on PET-Gel-Hep is 22.48%, on PET-CL-BSA is 19.20%, and on PET-Gel-Hep is 20.59%, which is decreased comparing the unmodified PET (35.57%). All show that after the co-immobilization of heparin (albumin) and collagen (gelatin), the anticoagulation of the film surfaces has improved.
The results of endothelial cells culture in vitro shows significant difference in the behavior of cell adhesion and proliferation among different materials. PET-CL-Hep and PET-Gel-Hep films support cell attachment and proliferation more than unmodified film surfaces. The ECs on the PET-CL-BSA and PET-Gel-BSA can spread and proliferate. Quantitative analysis of cell adhesion is performed for these different modified and untreated PET surfaces. For the 120 hour cultured samples, the number of cells on PET-CL-Hep, PET-Gel-Hep, PET-CL-BSA, PET-Gel-BSA are 9.37, 9.73, 10.66,9.07(×10~4cells/cm~2), and the activity of cell is 202.3%, 205.9%, 214.8%, 199.4%. This is in accordance with the optical microscopy images, which suggests that cell adhesion onto PET is favoured by surface co-immobilization of heparin (albumin) with collagen (gelatin).
因此,本研究将具有强细胞亲合力的天然大分子如胶原蛋白、明胶等粘附蛋白通过共价键结合于医用涤纶PET材料表面,以加快内皮细胞的粘附与生长。但这些粘附蛋白不仅是细胞粘附的优良介质,也有利于血小板的粘附和血栓的形成。血小板的粘附往往在材料与血液接触的瞬间即发生,而细胞的粘附则需要较长的时间,这样可能在材料表面还未覆盖一层内皮细胞之前,已经形成了血栓。为阻止这种状况的发生,我们在涤纶材料表面共固定抗凝生物分子肝素(白蛋白)和促内皮生物分子胶原(明胶),以期获得同时具有良好的抗凝血性能和促内皮细胞生长的医用涤纶表面。
本文通过X射线光电子能谱XPS分析了改性前后材料表面的组成成分的变化。在肝素—胶原(PET-CL-Hep)及肝素—明胶(PET-Gel-Hep)共固定的表面,不仅出现的N1s峰,还出现了较为明显的S2s,S2p峰,根据XPS高分辨谱计算可得PET-CL-Hep和PET-Gel-Hep的硫元素含量分别为1.55%和2.18%,这说明了肝素已与胶原或明胶共固定于材料表面。同时在白蛋白—胶原(PET-CL-BSA)和白蛋白—明胶(PET-Gel-BSA)的表面,氮元素的含量分别比仅固定胶原(PET-CL)和仅固定明胶(PET-Gel)的氮含量高4.74%和5.64%,这也提示了白蛋白与胶原或明胶分子的在PET表面的混合有效固定。接触角测量与表面能计算的结果表明肝素、白蛋白与胶原或明胶共固定改性后的材料表面接触角减小,表面能增加,材料表面的亲水性较好。
采用甲苯胺蓝染色法测得接枝丙烯酸的材料表面的羧基密度为5.29×10~(-9)mol/cm~2,采用天狼猩红染色法得到PET-CL表面的胶原含量为3.14μg/cm~2,PET-Gel表面的明胶为2.02μg/cm~2。利用甲苯胺蓝及正己烷萃取法可得PET-CL-Hep表面的肝素含量为1.73μg/cm~2,PET-Gel-Hep表面的肝素1.91/μg/cm~2。
体外血小板粘附及血小板乳酸脱氢酶(LDH)检测血小板数量的实验一致证明,肝素或白蛋白与胶原或明胶的共固定,使PET表面的抗凝血性能有所改善。LDH实验测得PET-CL-Hep、PET-Gel-Hep、PET-CL-BSA、PET-Gel-BSA表面的血小板黏附率分别为12.83%、17.24%、10.53%和12.19%,与未改性的涤纶表面血小板26.21%,PET-Gel 40.38%和PET-CL47.21%的黏附率相比,血小板粘附数量明显减少,血小板激活程度减轻。PET-CL-Hep和PET-Gel-Hep的APTT检测时间为68.9s和65.5,比未改性的涤纶表面延长9.7s和6.3s,比原血浆延长14.9s和11.5s。活化的部分凝血活酶时间(APTT)检测表面肝素与胶原或明胶的共固定可抑制内源性凝血系统的激活。采用酶联免疫法定量评价不同改性材料表面纤维蛋白原的变性情况。PET-CL-Hep、PET-Gel-Hep、PET-CL-BSA、PET-Gel-BSA的表面纤维蛋白原变性量分别为25.32%、22.48%、19.20%、20.59%,而空白样品的纤维蛋白原变性量为35.57%,这说明肝素(白蛋白)与胶原(明胶)的共固定,可减少纤维蛋白原的变性暴露出与血小板结合,从而减少了血小板在材料表面的聚集。上述一系列评价的结果均证明肝素(白蛋白)与胶原(明胶)的共固定,可改善PET的抗凝血性能。
通过体外内皮细胞粘附及细胞Alamar Blue实验,定量表征内皮细胞在PET材料的黏附率、增殖率、细胞活性,并通过光学显微镜观察材料表面的细胞形态变化。在细胞培养的第120h,未改性涤纶表面的细胞数为4.88×10~4cells/cm~2,细胞活性为158.6%,而PET-CL-Hep,PET-Gel-Hep,PET-CL-BSA,PET-Gel-BSA表面的细胞数量分别为9.37,9.73,10.66,9.07(×10~4cells/cm~2),细胞活性依次为202.3%,205.9%,214.8%,199.4%。上述结果表明在医用涤纶表面,通过肝素或白蛋白与胶原或明胶的共固定可改善材料表面的促内皮细胞生长性能。
In recent years, synthetic polymers are widely used in biomedical materials for artificial organs including cardiovascular artificial organs. However, once blood contact with polymers, complicated interaction will happen on the interface so as to form coagulating reaction and thrombus. In that case, the farther application of synthetic polymers in blood-contacting devices was restricted. It is believed that the endothelialization of the blood contacting device surface may overcome the problem. Therefore, a surface that enhances cell-material interaction and promotes the endothelialization at a shorter time is necessary.
In our research, natural proteins like gelatin and collagen molecules were immobilized onto the biomedical poly(ethylene terephthalate) (PET, dacron) surfaces. However, the immobilization of gelatin or collagen on the surface of materials may accelerate the aggregation of the platelets before the formation of endothelial layer. Hence, surface anticoagulative modification before endothelialization is necessary. Our study focuses on the co-immobilization of heparin (or albumin) and collagen (or gelatin) molecules on the PET surface which can construct the anticoagulant surface before the endothelium formation and also accelerate the endothelialization of polymer surface.
The surface constitution of the modified PET is determined by X-ray photoelectron spectroscopy (XPS). According to the resurts of XPS, The detection of S2p, S2s might be in virtue of the sulfonic groups of the heparin chemically attached to the membrane surfaces, which demonstrates that of the molecules of heparin and collagen or gelatin co-immobilized on the PET surfaces. Comparing to the only collagen and gelatin immobilized surface, the nitrogen contents increase 4.74% and 5.64% on the surface of albumin-collagen (PET-CL-BSA) and albumin-gelatin (PET-Gel-BSA) co-immobilized PET film surface. This proves that albumin combined with collagen or gelatin molecules are effectively co-immobilized onto the PET film surfaces. The results of static contact angle indicate the hydrophilicity of the modified PET surface is more remarkably improved than that of the unmodified PET surface.
The determination of carboxyl group on the acrylic acid grafted PET surface is 5.29×10~(-9)mol/cm~2. According to the protein measurement, the concentration of gelatin immobilized on only gelatin-PET (PET-Gel) is 2.02μg/cm~2, while the concertration of collagen immobilized on only collagen-PET (PET-CL) is 3.14μg/cm~2. The quantification of heparin on heparin-collagen co-immobilized surface (PET-CL-Hep) is 1.73μg/cm~2, and heparin on heparin-gelatin co-immobilized surface(PET-CL-Hep) is 1.91μg/cm~2.
The results of lactate dehydrogenase(LDH) and the platelet adhesion test in vitro all indicat that the co-immobilization of heparin or albumin with collagen or gelatin molecules could improve the anticoagulation. The percentage of platelet adhered on the surface of PET-CL-Hep is 12.83%, PET-Gel-Hep 17.24%, PET-CL-BSA 10.53%, and PET-Gel-BSA 12.19%, thus the percentages of adhered platelet decreased significantly compared to those of untreated PET (26.21%) and the PET-Gel surface (40.38%) , the PET-CL surface (47.21%). The results of coagulant factor tests reveal that the activated partially thromboplastion time (APTT) of PET-CL-Hep is 68.9s and PET-Gel-Hep is 65.5s, which is longer than those of the virgin PET (59.2s) and the plasma (54s). The change of APTT indicates that the modified PET films can suppress intrinsic coagulant system. The number of denatured fibrinogen on different modified surfaces is also determined by enzyme linked immunosorbent assay (ELISA). The resulte shows that the proption of denatured fibrinogen on PET-CL-Hep is 25.32%, on PET-Gel-Hep is 22.48%, on PET-CL-BSA is 19.20%, and on PET-Gel-Hep is 20.59%, which is decreased comparing the unmodified PET (35.57%). All show that after the co-immobilization of heparin (albumin) and collagen (gelatin), the anticoagulation of the film surfaces has improved.
The results of endothelial cells culture in vitro shows significant difference in the behavior of cell adhesion and proliferation among different materials. PET-CL-Hep and PET-Gel-Hep films support cell attachment and proliferation more than unmodified film surfaces. The ECs on the PET-CL-BSA and PET-Gel-BSA can spread and proliferate. Quantitative analysis of cell adhesion is performed for these different modified and untreated PET surfaces. For the 120 hour cultured samples, the number of cells on PET-CL-Hep, PET-Gel-Hep, PET-CL-BSA, PET-Gel-BSA are 9.37, 9.73, 10.66,9.07(×10~4cells/cm~2), and the activity of cell is 202.3%, 205.9%, 214.8%, 199.4%. This is in accordance with the optical microscopy images, which suggests that cell adhesion onto PET is favoured by surface co-immobilization of heparin (albumin) with collagen (gelatin).
引文
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