高抗凝、可诱导再生小口径人工血管的研究
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摘要
目的血管疾病是世界上发病率最高的疾病之一,而血管移植术是其目前主要的治疗手段。临床上应用的血管移植术材料主要来源为自体血管、异体血管和人工材料。目前,大、中口径人工血管移植应用效果较好,而小口径移植血管(直径<6mm)易发生吻合口内膜增生以及血栓形成,远期通畅率不理想。血管组织工程技术为血管移植术所需材料、特别是小口径血管,提供了新途径。但目前,构建小口径组织工程化血管仍不尽如人意,如在人工血管内壁种植血管内皮细胞周期较长,短期内不能形成完整内膜,无法满足临床需求,而且同样存在远期通畅率不理想等问题。为此本课题将新鲜牛心包用于体外实验研究,将猪来源小口径血管用于体内实验研究,应用组织工程中的脱细胞技术和化学交联技术进行预处理,再进行肝素结合以及负载生长因子等改性处理,以期短期高效地制备出具有抗凝血活性、可诱导自体内皮再生的小口径人工血管。
方法(一)材料前期处理：反复冻融+TritonX-100联合作用对新鲜牛心包膜进行脱细胞处理,行HE染色后光镜下观察脱细胞效果；采用碳化二亚胺(EDC)作为交联剂,交联脱细胞牛心包膜,并通过生物力学检测、体外胶原酶降解实验以及变性温度测试考察交联程度。(二)材料表面肝素化处理,采用三种途径负载肝素：(1)EDC共价键合肝素；(2)聚乙烯亚胺(PEI, MW=1800D, 10kD,20kD)离子结合肝素；(3) EDC-PEI共价-离子联合负载肝素,即在EDC共价键合肝素基础上再采用PEI离子结合肝素。采用电感耦合等离子体质谱仪(ICP-MS)和红外光谱评价材料负载肝素情况,采用接触角测量仪评价材料表面的亲疏水性,检测肝素化材料的含水量,并通过溶血试验、血小板粘附实验、抗凝血时间、复钙时间及细胞毒性试验评价材料的血液相容性及细胞相容性。(三)肝素化材料负载血管内皮细胞生长因子(VEGF165)实验,分别考察EDC共价键合肝素、PEI离子结合肝素以及共价-离子联合肝素化材料对碘标记VEGF165(125I-VEGF165)的控制释放能力,并进行促血管内皮细胞增殖实验。(四)将筛选出的最佳肝素化方式运用到小口径异种血管的表面改性,通过对犬实施血管置换手术观察这种小口径异种血管的可行性。
结果(一)光镜下观察发现,材料中的细胞脱除彻底,细胞外基质形态良好,结构完整,排列有序,表明反复冻融+TritonX-100联合作用是一种有效、经济、快捷的脱细胞方法。脱细胞基质经EDC交联处理后,力学性能及变性温度较新鲜牛心包显著提高且具有良好的抗降解性能。
(二)PEI离子结合肝素量与结合层数呈正相关,可通过调节结合层数达到所需肝素量要求,而EDC共价键合肝素量为定值,与PEI离子结合肝素层数达到18层左右时的肝素量相当。此外,采用三种分子量的PEI分别进行离子结合15层肝素处理,含水量及接触角测试结果表明PEI1800D处理组含水能力和亲水性能均优于PEI10kD、PEI20kD处理组；溶血试验中,PEI1800D溶血率为2.66%,PEI10kD溶血率为5.02%,PEI20kD溶血率为5.58%,表明PEI1800D溶血试验合格。通过上述实验筛选出PEI1800D作为阳离子剂进行后续实验。分别对交联对照组、EDC共价键合肝素组、PEI1800D离子结合肝素组、EDC-PEI1800D联合负载肝素组进行溶血试验、血小板粘附、凝血时间、复钙时间测试,发现EDC共价键合肝素组溶血试验不合格；EDC-PEI1800D联合负载肝素组抗凝血效果最佳,经过长时间生理盐水浸泡(15d),该组抗凝血性能保持最好。细胞毒性试验中,PEI1800D离子结合肝素组为0.5级,EDC-PEI1800D联合负载肝素组为1.0级,细胞毒性试验合格,优于EDC共价键合肝素组的1.3级。
(三)肝素化材料对VEGF165控释实验结果显示,EDC共价键合肝素对VEGF165的控释力优于PEI1800D离子结合肝素组,验证了共价键合稳定性高于离子结合方式,因此,采用EDC-PEI联合负载肝素方式可以弥补单纯使用离子结合肝素稳定性差的缺点。内皮细胞增殖实验中,EDC-PEI1800D联合负载肝素结合VEGF165组促细胞增殖力最好。
(四)结合VEGF165的EDC-PEI1800D联合肝素化小口径血管移植后,短期观察通畅效果良好,需要进一步长期观察。
结论EDC-PEI1800D联合负载肝素结合VEGF165方式具有良好的抗凝血性、促血管内皮细胞增殖能力,同时具有一定的降解性能和适当的力学性能,制备周期短,冻干灭菌后可室温长期保存,为小口径人工血管尽快适应临床要求提供了新的血管制备途径。
Objective:Treatment of obstructive atherosclerotic disease, which is a major cause of mortality and morbidity in the world, may involve replacement and bypassing of affected arteries using autologous, allogenic and synthetic vascular grafts. Although vascular grafts have been used successfully to replace large-diameter blood vessels, the long term patency of small-diameter (<6mm) vascular grafts is still disappointing, primarily due to stenosis and thrombus formation. Vascular tissue engineering has provided new approaches for implantation, especially small-diameter vascular grafts. Vascular grafts with a confluent lining of endothelial cells can be obtained after expansion of cell numbers by cell culture in vitro. Drawbacks of this approach are the time interval between the need of an endothelialized vascular graft and its availability, as well as the increased risk of bacterial infection, limiting its use to non-emergency situations. In this study, decellularized fresh specimens of bovine pericardiums in vitro and pig small-diameter blood vessels in vivo were studied with respect to crosslinking, heparinization and vascular endothelial cell growth factor (VEGF) binding and release, in order to produce high-performance small-diameter artificial blood vessels with highly anticoagulant property and ability to induce regeneration in short time.
Methods:1 Matrix preparation:Fresh specimens of bovine pericardiums were treated by repeatedly freezing and thawing+TritonX-100. Tissue samples were then observed by HE staining to confirm the removal of cells. Acellular tissue samples were crosslinked by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS),and the content of crosslink was detected by mechanical properties, in vitro degradation by collagenase and the shrinkage temperature indicating the resistance against thermal denaturation.
2 Heparin immobilization,3 methods:(1) Heparinized EDC treated acellular tissue samples; (2) Heparinized poly(ethyleneimine) (PEI, MW=1800D, 10kD,20kD) treated acellular tissue samples; (3) Heparinized EDC-PEI treated acellular tissue samples. Determination of immobilized heparin was tested by ICP-MS and FTIR; the surface wettability was tested by contact angle; the moisture uptake was analyzed; cell and blood compatibility were evaluated by cytotoxic test, hemolysis test, platelet attachment test, PT/APTT and recalcification time.
3 VEGF165 labeling and binding:VEGF165 was labeled with 125I in comparison of EDC treated group, PEI treated group and EDC-PEI treated group in order to observe the binding and release of VEGF165. Proliferation of human umbilical vein endothelial cells was tested.
4 Small-diameter vascular xenografts were modified by the best heparinization way and implanted in canine left carotid artery to evaluate the feasibility.
Results:1 Histological analysis revealed that repeatedly freezing and thawing +TritonX-100 resulted into a complete loss of cellular structures and no loss or disruption of extracellular matrix. This suggested that it was an effective, economic, fast decellularization method. Mechanical properties and heat shrinkage temperature were greater in matrix crosslinked by EDC than that in fresh specimens of bovine pericardiums, and had the perfect anti-degradeation function.
2 Amount of heparin with PEI was increasing by multilayers. Different multilayers would produce different amount of heparin. Heparin immobilized by EDC equaled to heparin produced by 18 multilayers. Heparin was treated by 15 multilayers with 3 MW of PEI, moisture uptake and contact angle tests revealed that wettability and hydrophilia abiltity were greater in PEI1800D treatment group than that in PEI10kD and PEI20kD group. In hemolysis test, hemolysis ratio was 2.66% in PEI1800D treatment group,5.02% in PEIlOkD treatment group,5.58% in PEI20kD treatment group. These revealed that only PEI1800D treatment group was qualification. Hemolysis test, platelet attachment, PT/APTT and recalcification time were tested in control group, EDC-Hep group, PEI1800D-Hep group and EDC-PEI1800D-Hep group, confirmed that anticoagulant properties were the best in EDC-PEI1800D-Hep group during immersed in saline for 15 days. In cytotoxic test, PEI1800D-Hep group was 0.5 degree, EDC-PEI1800D-Hep group was 1.0 degree, EDC-Hep group was 1.3 degree. These suggested that PEI1800D-Hep group and EDC-PEI1800D-Hep group were better than EDC-Hep group.
3 Through the results of binding and release of VEGF165 from heparized materials, we could make conclusion that controlled release of VEGF165 from EDC-Hep group was better than that of PEI-Hep group, which confirmed the stability of heparin was better in crosslinking. Therefore, the stability of heparin in EDC-PEI1800D-Hep group was satisfactory too. Proliferation of human umbilical vein endothelial cells in EDC-PEI1800D-Hep group was the best.
4 EDC-PEI1800D-Hep grafts binding VEGF165 were implanted in canine left carotid artery. All grafts were not occluded in short time. We should observe continuously.
Conclusions:Fresh exogenic tissue, binding heparin and VEGF165 with EDC-PEI1800D, had better anticoagulant properties, proliferation of vascular endothelial cells, mechanical properties and anti-degradeation function. Period of preparation was short, and we could make conservation after freezing and sterilization for a long time. These results demonstrated that these small-diameter artificial blood vessels with highly anticoagulant property and ability to induce regeneration would be applied in clinical settings in future.
方法(一)材料前期处理：反复冻融+TritonX-100联合作用对新鲜牛心包膜进行脱细胞处理,行HE染色后光镜下观察脱细胞效果；采用碳化二亚胺(EDC)作为交联剂,交联脱细胞牛心包膜,并通过生物力学检测、体外胶原酶降解实验以及变性温度测试考察交联程度。(二)材料表面肝素化处理,采用三种途径负载肝素：(1)EDC共价键合肝素；(2)聚乙烯亚胺(PEI, MW=1800D, 10kD,20kD)离子结合肝素；(3) EDC-PEI共价-离子联合负载肝素,即在EDC共价键合肝素基础上再采用PEI离子结合肝素。采用电感耦合等离子体质谱仪(ICP-MS)和红外光谱评价材料负载肝素情况,采用接触角测量仪评价材料表面的亲疏水性,检测肝素化材料的含水量,并通过溶血试验、血小板粘附实验、抗凝血时间、复钙时间及细胞毒性试验评价材料的血液相容性及细胞相容性。(三)肝素化材料负载血管内皮细胞生长因子(VEGF165)实验,分别考察EDC共价键合肝素、PEI离子结合肝素以及共价-离子联合肝素化材料对碘标记VEGF165(125I-VEGF165)的控制释放能力,并进行促血管内皮细胞增殖实验。(四)将筛选出的最佳肝素化方式运用到小口径异种血管的表面改性,通过对犬实施血管置换手术观察这种小口径异种血管的可行性。
结果(一)光镜下观察发现,材料中的细胞脱除彻底,细胞外基质形态良好,结构完整,排列有序,表明反复冻融+TritonX-100联合作用是一种有效、经济、快捷的脱细胞方法。脱细胞基质经EDC交联处理后,力学性能及变性温度较新鲜牛心包显著提高且具有良好的抗降解性能。
(二)PEI离子结合肝素量与结合层数呈正相关,可通过调节结合层数达到所需肝素量要求,而EDC共价键合肝素量为定值,与PEI离子结合肝素层数达到18层左右时的肝素量相当。此外,采用三种分子量的PEI分别进行离子结合15层肝素处理,含水量及接触角测试结果表明PEI1800D处理组含水能力和亲水性能均优于PEI10kD、PEI20kD处理组；溶血试验中,PEI1800D溶血率为2.66%,PEI10kD溶血率为5.02%,PEI20kD溶血率为5.58%,表明PEI1800D溶血试验合格。通过上述实验筛选出PEI1800D作为阳离子剂进行后续实验。分别对交联对照组、EDC共价键合肝素组、PEI1800D离子结合肝素组、EDC-PEI1800D联合负载肝素组进行溶血试验、血小板粘附、凝血时间、复钙时间测试,发现EDC共价键合肝素组溶血试验不合格；EDC-PEI1800D联合负载肝素组抗凝血效果最佳,经过长时间生理盐水浸泡(15d),该组抗凝血性能保持最好。细胞毒性试验中,PEI1800D离子结合肝素组为0.5级,EDC-PEI1800D联合负载肝素组为1.0级,细胞毒性试验合格,优于EDC共价键合肝素组的1.3级。
(三)肝素化材料对VEGF165控释实验结果显示,EDC共价键合肝素对VEGF165的控释力优于PEI1800D离子结合肝素组,验证了共价键合稳定性高于离子结合方式,因此,采用EDC-PEI联合负载肝素方式可以弥补单纯使用离子结合肝素稳定性差的缺点。内皮细胞增殖实验中,EDC-PEI1800D联合负载肝素结合VEGF165组促细胞增殖力最好。
(四)结合VEGF165的EDC-PEI1800D联合肝素化小口径血管移植后,短期观察通畅效果良好,需要进一步长期观察。
结论EDC-PEI1800D联合负载肝素结合VEGF165方式具有良好的抗凝血性、促血管内皮细胞增殖能力,同时具有一定的降解性能和适当的力学性能,制备周期短,冻干灭菌后可室温长期保存,为小口径人工血管尽快适应临床要求提供了新的血管制备途径。
Objective:Treatment of obstructive atherosclerotic disease, which is a major cause of mortality and morbidity in the world, may involve replacement and bypassing of affected arteries using autologous, allogenic and synthetic vascular grafts. Although vascular grafts have been used successfully to replace large-diameter blood vessels, the long term patency of small-diameter (<6mm) vascular grafts is still disappointing, primarily due to stenosis and thrombus formation. Vascular tissue engineering has provided new approaches for implantation, especially small-diameter vascular grafts. Vascular grafts with a confluent lining of endothelial cells can be obtained after expansion of cell numbers by cell culture in vitro. Drawbacks of this approach are the time interval between the need of an endothelialized vascular graft and its availability, as well as the increased risk of bacterial infection, limiting its use to non-emergency situations. In this study, decellularized fresh specimens of bovine pericardiums in vitro and pig small-diameter blood vessels in vivo were studied with respect to crosslinking, heparinization and vascular endothelial cell growth factor (VEGF) binding and release, in order to produce high-performance small-diameter artificial blood vessels with highly anticoagulant property and ability to induce regeneration in short time.
Methods:1 Matrix preparation:Fresh specimens of bovine pericardiums were treated by repeatedly freezing and thawing+TritonX-100. Tissue samples were then observed by HE staining to confirm the removal of cells. Acellular tissue samples were crosslinked by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS),and the content of crosslink was detected by mechanical properties, in vitro degradation by collagenase and the shrinkage temperature indicating the resistance against thermal denaturation.
2 Heparin immobilization,3 methods:(1) Heparinized EDC treated acellular tissue samples; (2) Heparinized poly(ethyleneimine) (PEI, MW=1800D, 10kD,20kD) treated acellular tissue samples; (3) Heparinized EDC-PEI treated acellular tissue samples. Determination of immobilized heparin was tested by ICP-MS and FTIR; the surface wettability was tested by contact angle; the moisture uptake was analyzed; cell and blood compatibility were evaluated by cytotoxic test, hemolysis test, platelet attachment test, PT/APTT and recalcification time.
3 VEGF165 labeling and binding:VEGF165 was labeled with 125I in comparison of EDC treated group, PEI treated group and EDC-PEI treated group in order to observe the binding and release of VEGF165. Proliferation of human umbilical vein endothelial cells was tested.
4 Small-diameter vascular xenografts were modified by the best heparinization way and implanted in canine left carotid artery to evaluate the feasibility.
Results:1 Histological analysis revealed that repeatedly freezing and thawing +TritonX-100 resulted into a complete loss of cellular structures and no loss or disruption of extracellular matrix. This suggested that it was an effective, economic, fast decellularization method. Mechanical properties and heat shrinkage temperature were greater in matrix crosslinked by EDC than that in fresh specimens of bovine pericardiums, and had the perfect anti-degradeation function.
2 Amount of heparin with PEI was increasing by multilayers. Different multilayers would produce different amount of heparin. Heparin immobilized by EDC equaled to heparin produced by 18 multilayers. Heparin was treated by 15 multilayers with 3 MW of PEI, moisture uptake and contact angle tests revealed that wettability and hydrophilia abiltity were greater in PEI1800D treatment group than that in PEI10kD and PEI20kD group. In hemolysis test, hemolysis ratio was 2.66% in PEI1800D treatment group,5.02% in PEIlOkD treatment group,5.58% in PEI20kD treatment group. These revealed that only PEI1800D treatment group was qualification. Hemolysis test, platelet attachment, PT/APTT and recalcification time were tested in control group, EDC-Hep group, PEI1800D-Hep group and EDC-PEI1800D-Hep group, confirmed that anticoagulant properties were the best in EDC-PEI1800D-Hep group during immersed in saline for 15 days. In cytotoxic test, PEI1800D-Hep group was 0.5 degree, EDC-PEI1800D-Hep group was 1.0 degree, EDC-Hep group was 1.3 degree. These suggested that PEI1800D-Hep group and EDC-PEI1800D-Hep group were better than EDC-Hep group.
3 Through the results of binding and release of VEGF165 from heparized materials, we could make conclusion that controlled release of VEGF165 from EDC-Hep group was better than that of PEI-Hep group, which confirmed the stability of heparin was better in crosslinking. Therefore, the stability of heparin in EDC-PEI1800D-Hep group was satisfactory too. Proliferation of human umbilical vein endothelial cells in EDC-PEI1800D-Hep group was the best.
4 EDC-PEI1800D-Hep grafts binding VEGF165 were implanted in canine left carotid artery. All grafts were not occluded in short time. We should observe continuously.
Conclusions:Fresh exogenic tissue, binding heparin and VEGF165 with EDC-PEI1800D, had better anticoagulant properties, proliferation of vascular endothelial cells, mechanical properties and anti-degradeation function. Period of preparation was short, and we could make conservation after freezing and sterilization for a long time. These results demonstrated that these small-diameter artificial blood vessels with highly anticoagulant property and ability to induce regeneration would be applied in clinical settings in future.
引文
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