【作者】 罗彤；

【导师】 高润霖； 阮英茆； 乔树宾；

【作者基本信息】 中国协和医科大学 ， 内科学， 2008， 博士

【摘要】 研究背景药物洗脱支架（DES）是目前冠心病介入治疗的重要进展之一。第一代DES通过包被于金属支架表面的不可降解的聚合物涂层携带药物,药物从聚合物中缓慢释放,抑制局部平滑肌细胞增殖,从而使支架内再狭窄发生率显著降至10%以内。但与裸金属支架（BMS）相比,DES轻度但有统计学意义地增加了晚期支架血栓的发生率,这可能与不可降解的涂层在局部造成持续炎症反应和过敏反应有关。为避免不可降解的聚合物涂层永久残留于血管壁中所引起的不利作用,部分新一代的DES采用了以聚乳酸（PLA）类材料为代表的生物可降解的聚合物涂层。目前已有采用PLA涂层的DES(如Excel支架、BioMATRIX^TM支架、Nobori^TM支架等)上市或开展了临床研究,显示有良好的安全性和有效性。不仅如此,为了进一步避免永久残留于血管壁中的金属支架对血管壁的长期异物刺激,发展全降解的DES使其在完成防止血管弹性回缩和药物洗脱的历史使命后从血管壁中被完全降解吸收,是新型DES发展的趋势之一。其中镁合金可降解裸金属支架已进行了初步临床试验,但尚无镁合金DES的研究报告。目的为开发采用新型可降解聚合物涂层的国产DES,本研究探索了生物可降解聚乳酸聚羟基乙酸共聚物（PLGA）和聚左旋乳酸（PLLA）涂层携载雷帕霉素的新型DES的可行性,置入健康小型猪冠状动脉后观察其生物安全性和对新生内膜增生的抑制作用,并与采用不可降解聚合物涂层的第一代DES进行对比。另外,本研究还对镁合金PLA涂层雷帕霉素洗脱支架（SES）的可行性进行了初步探索。方法为评价PLGA涂层SES的效果,4种支架被置入健康小型猪的冠状动脉:316L不锈钢BMS（316L）、L605钴基合金BMS（L605）、不带雷帕霉素的PLGA涂层L605支架（PLGA）和带雷帕霉素的PLGA涂层L605支架（PLGA SES）。试验支架由天健医疗科技（苏州）有限公司提供。PLGA SES的雷帕霉素量为120μg/cm²,体外试验显示雷帕霉素在70天时完全洗脱,涂层约在80～90天时完全降解。术后4周、12周和26周时将动物处死进行组织病理学和组织形态学分析,测量支架上内膜厚度、支架间内膜厚度、新生内膜面积和面积狭窄百分比,通过扫描电镜（SEM）观察内皮愈合情况。此外我们还测量了支架段冠状动脉管壁炎症面积,并采用双变量相关分析（Spearman法）和偏相关分析研究了冠状动脉炎症与新生内膜增生程度的关系。为评价PLLA涂层SES的效果,4种支架被置入健康小型猪的冠状动脉:316L不锈钢BMS（316L）、L605钴基合金BMS（L605（2））、不带雷帕霉素的PLLA涂层L605支架（PLLA）和带雷帕霉素的PLLA涂层L605支架（PLLASES）。试验支架由万瑞飞鸿（北京）医疗器材有限公司提供。PLLASES的雷帕霉素量为140±20μg/cm²,体外试验显示雷帕霉素在60天时完全洗脱,涂层约在60天时完全降解。4周时将动物处死进行组织病理学和组织形态学分析,SEM观察内皮愈合情况。将上述2种采用可降解聚合物涂层的SES与同期完成的火鸟支架（Firebird,微创医疗器械（上海）有限公司）的结果进行对比分析。最后,为初步观察全降解的镁合金PLA涂层SES的效果,3种支架被置入健康小型猪的冠状动脉:AZ31B镁合金BMS（BMS）、不带雷帕霉素的PLA涂层AZ31B支架（PLA）和带雷帕霉素的PLA涂层AZ31B支架（SES）,每种4枚支架。试验支架由中国科学院金属研究所提供。4周时将动物处死进行组织病理学和组织形态学分析。结果一、PLGA涂层SES的可行性研究:4周时,SEM观察显示PLGA SES组的多数标本内皮细胞覆盖面积约占总内膜面积的75-100%。与316L、L605和PLGA组相比,PLGA SES组的支架上内膜厚度（0.06mm对0.41mm、0.30mm和0.24mm,P均＜0.05）、支架间内膜厚度（0.06mm对0.29mm、0.33mm和0.28mm,P均＜0.05）、新生内膜面积（0.64mm²对2.09 mm²、2.11 mm²和1.42 mm²,P均＜0.05）和面积狭窄百分比（13.85%对47.27%、46.80%和33.80%,P均＜0.05）均显著小于各对照组,而各对照组间无统计学差异。PLGA SES组的支架段冠状动脉炎症显著轻于各对照组。双变量相关性分析显示,冠状动脉炎症面积与新生内膜面积显著正相关（相关系数0.719,P＜0.001）,在控制支架种类、损伤指数和外弹力板围绕面积后,偏相关分析显示两者依然正相关（相关系数0.498,P=0.01）。PLGA SES组的心肌、肝、脾、肺、肾的病理观察未见明显异常,血化验未见与支架相关的有意义的改变。12周时SEM显示PLGA SES组均有完整的内皮细胞覆盖。其内膜增生有所进展但仍无支架内狭窄发生,与4周时相比支架间内膜厚度（0.10mm对0.06mm,P=0.036）增加有统计学差异,而支架上内膜厚度（0.15mm对0.06mm,P=0.304）、新生内膜面积（0.91mm²对0.64mm²,P=0.054）和面积狭窄百分比（30.01%对13.85%,P=0.076）增加未达到统计学差异。26周时,SEM显示PLGA SES组均有完整的内皮细胞覆盖。2例无明显炎症反应的标本内膜增生程度与4周时相似,而另2例有中度炎症反应的标本形成支架内狭窄。二、PLLA涂层SES的可行性研究:4周时,SEM显示PLLASES组内皮细胞覆盖面积约占总内膜面积的50-80%。PLLA SES组与316L组相比支架上内膜厚度减少了76%（0.10mm对0.41mm,P=0.004）,支架间内膜厚度减少了72%（0.08mm对0.29mm,P=0.002）,新生内膜面积减少了62%（0.79mm²对2.09mm²,P=0.001）,面积狭窄百分比也显著降低（21.22%对47.27%,P=0.008）。而PLLA SES组与L605组和PLLA组之间无明显差异。PLLA组有2例支架段冠状动脉发生中度炎症反应,而PLLASES组和L605组均无明显炎症反应。PLLA SES组的心肌、肝、脾、肺、肾的病理观察未见明显异常,血化验未见与支架相关的有意义的改变。三、PLGA SES、PLLA SES与火鸟支架的对比研究:4周时,3组的内皮愈合情况相似,均无支架内狭窄发生。PLGA SES的支架上内膜厚度（0.06mm对0.12mm,P=0.146）、支架间内膜厚度（0.06mm对0.16mm,P=0.075）和面积狭窄百分比（13.85%对22.81%,P=0.274）较火鸟组有减少趋势但未达到统计学显著性,而新生内膜面积（0.64mm²对1.38mm²,P=0.011）显著小于火鸟组。PLLA SES的支架上内膜厚度（0.10mm对0.12mm,P=0.234）、支架间内膜厚度（0.08mm对0.16mm,P=0.115）和面积狭窄百分比（21.22%对22.81%,P=0.756）与火鸟组无统计学差异,新生内膜面积则有小于火鸟组的趋势（0.79mm²对1.38mm²,P=0.052）。PLGA SES和PLLA SES的支架段冠状动脉炎症显著轻于火鸟支架。各组的心肌、肝、脾、肺、肾的病理观察均未见明显异常,血化验均未见与支架相关的有意义的改变。四、镁合金PLA涂层SES可行性的初步研究:在支架置入术后第7天就观察到支架解体,4周时部分支架仅见残渣甚至完全吸收消失。PLA和SES组支架段冠状动脉有明显炎症反应,管腔严重狭窄。结论1、以L605钴铬合金为骨架、采用生物可降解PLGA涂层的雷帕霉素洗脱支架可有效抑制健康小型猪冠状动脉支架置入术后4周和12周的内膜增生,并有良好的组织相容性和血液相容性;4周时内皮细胞覆盖面积约占总内膜面积的75%以上,12周时有完整的内皮覆盖;对主要脏器的功能、结构无不良影响,提示有较好的临床应用前景。2、在国内首次报道病理所见,支架段冠状动脉管壁的炎症面积大小与新生内膜增生的程度显著正相关,显示炎症是促进支架术后新生内膜增生的重要因素。3、以L605钴铬合金为骨架、采用生物可降解PLLA涂层携带雷帕霉素的新型药物洗脱支架可有效抑制健康小型猪冠状动脉支架置入术后4周时的内膜增生,有良好的组织相容性和血液相容性;4周时内皮细胞覆盖面积约占总内膜面积的50-80%;并对主要脏器的功能、结构无不良影响。4、在国内首次在动物模型上比较了可降解与不可降解聚合物涂层雷帕霉素洗脱支架对抑制新生内膜增生的作用及血管壁炎症反应的情况,结果表明与采用不可降解的EVAC聚合物涂层的火鸟支架进行对比,PLGA涂层雷帕霉素洗脱支架和PLLA涂层雷帕霉素洗脱支架抑制新生内膜的增生的作用至少相似,并且炎症反应更轻。5、在国内上首次探索了镁合金药物洗脱支架的可行性,结果提示PLA涂层镁合金雷帕霉素洗脱支架的腐蚀吸收速度过快、支撑力不足、组织相容性欠佳,尚需继续研究加以改进。更多还原

【Abstract】 The Feasibility Studies of New Types of Cobalt Alloy Biodegradable Polymer Coating Sirolimus-eluting Stents And Magnesium Alloy Biodegradable Polymer Coating Sirolimus-eluting StentBackgroundThe treatment of coronary artery disease has been revolutionized by drug-eluting stents（DES）.Unlike the Bare metal stents（BMS）,DES containing sirolimus or paclitaxel reduce the rate of restenosis to less than 10%due to their antiproliferative effect on the vascular neointima in the coronary artery.But Permanent polymers used in first generation of DES may trigger chronic inflammation and hypersensitivity reactions,which may contribute to the increased risk of late thrombosis and rebound restenosis.Some of new generation DES use bioabsorbable or biodegradable polymer coating on the metallic stent struts to minimize these risks,such as the Excel stent,BioMATRIX^TM stent and the Nobori^TM stent.Moreover,totally biodegradable stents including magnesium alloy stent,which will be completely absorbed in the arterial wall after doing their jobs to prevent recoil and elute drugs,are being developed to avoid the long-term stimulation to the vessel wall caused by the durable stent struts. ObjectivesIn order to research and develop new types of biodegradable polymer coating DES in China,the safety and efficacy of polylactic acid-co-polyglycolic acid（PLGA） coating SES and poly-L-lactic acid （PLLA） coating SES on inhibiting the neointimal proliferation in miniswine coronary model were investigated,and compared with that of durable polymer coating SES.The feasibility of a magnesium biodegradable polymer coating SES was also studied.MethodsTo evaluate a biodegradable PLGA coating SES,4 types of stents were implanted in minipig coronary arteries using coronary angiography to optimize stent apposition:316L stainless steel bare metal stents（316L）; L605 cobalt chromium bare metal stents（L605）;PLGA polymer coated L605 stents（PLGA）;and L605 stents coated with PLGA containing sirolimus（PLGA SES）.The dose density of sirolimus was 120μg/cm². The stents were manufactured by TianJian Medical（Suzhou） Co.,Ltd. Vitro studies demonstrated that the drug was completely eluted in 70 days and polymer degradation was completed in 80-90 days.Animals underwent angiographic restudy and were then sacrificed at 4 weeks,12 weeks and 26 weeks,respectively,for histopathologic and histomorphometric examinations including neointimal thickness over the struts,neointimal thickness between the struts,neointimal area and percentage area stenosis.The endothelialization was assessed by scanning electron microscope（SEM）.The correlation between inflammation and neointimal area was also analyzed.To evaluate a biodegradable PLLA coating SES,4 types of stents were implanted in minipig coronary arteries:316L stainless steel bare metal stents（316L）;L605 cobalt chromium bare metal stents（L605（2））; PLLA polymer coated L605 stents（PLLA）;and L605 stents coated with PLLA containing sirolimus（PLLA SES）.The dose density of sirolimus was 140±20μg/cm².The stents were manufactured by WanRuiFeiHong Medical（Beijing） Co.,Ltd.Vitro studies demonstrated that the drug was completely eluted and polymer was completely degradated in 60 days. Animals were sacrificed following angiographic restudy at 4 weeks for histopathologic and histomorphometric examinations.The endothelialization was assessed by SEM.The 2 biodegradable polymer coating SESs were also compared with Firebird（MicroPort Medical（Shanghai） Co.,Ltd.）,which was a durable EVAC coating SES.For the preliminary observation of a biodegradable magnesium alloy PLA coating SES,2 AZ31B magnesium alloy stents,2 magnesium alloy stents coated with PLA and 2 magnesium PLA coating SESs were implanted in minipig coronary arteries.The stents were offered by Metal Institute,Chinese Academy of Sciences.And histopathologic examination was conducted at 4 weeks after implantation.ResultsAt 4 weeks after implantation,SEM observation found that the luminal surface is about 75%to 100%endothelialised in PLGA SES group.As compared with 316L,L605 and PLGA groups,PLGA SES had significantly lower neointimal thickness over the struts（0.06mm vs. 0.32mm,0.30mm and 0.21mm,respectively,P＜0.05）,neointimal thickness between the struts（0.06mm vs.0.24mm、0.28mm and 0.29mm, respectively,P＜0.05）,neointimal area（0.59mm² vs.2.04 mm²,1.91 mm² and 1.54 mm²,respectively,P＜0.05） and percentage area stenosis （13.85%vs.42.41%,45.63%and 33.79%,respectively,P＜0.05）, while there were no statistical differences among the 3 controls.No in-stent stenosis was found in PLGA SES group.Bivariate correlations analysis showed there was statistically positive correlation between inflammatory area and neointimal area（p＜0.001,correlation coefficient=0.719）.When stent type,mean injury score and EEL area were controlled,partial correlations analysis showed there was still statistically positive correlation between inflammatory area and neointimal area（p=0.01,correlation coefficient=0.498）.In the segments of PLGA SES implantation,no inflammatory reaction was noted,and no abnormal changes in histopathologic examinations of cardiac muscle, liver,spleen,lung and kidney tissues and in blood sample tests were observed in PLGA SES group.At 12 weeks after PLGA SES implantation,the luminal surface is fully endothelialised.As compared with the results of 4 weeks,neointimal thickness between the struts was significantly increased（0.10mm vs. 0.06mm,P=0.036）,while there were no significant differences statistically between neointimal thickness over the struts（0.15mm vs. 0.06mm,P=0.304）,neointimal area（0.91mm² vs.0.64mm²,P=0.054） and percentage area stenosis（30.01%vs.13.85%,P=0.076）,respectively.At 26 weeks after PLGA SES implantation,the luminal surface is fully endothelialised and the neointimal proliferation was similar to the result of 4 weeks in 2 vessels without inflammation,while in-stent stenosis was found in the other 2 vessels with obvious inflammation.At 4 weeks after implantation,SEM observation found that the luminal surface is about 50%to 80%endothelialised in PLLA SES group. As compared with 316L group,PLLA SES had 76%reduction of neointimal thickness over the struts（0.10mm vs.0.41mm,P=0.004）,72% reduction of neointimal thickness between the struts（0.08mm vs.0.29mm, P=0.002）,62%reduction of neointimal area（0.79mm² vs.2.09mm², P=0.001）and significant reduction of percentage area stenosis（21.22%vs. 47.27%,P=0.008）,respectively.There were no significant differences statistically between PLLA SES and L605（2）,and no in-stent stenosis was found in both groups.In the segments of stents implantation,no inflammatory reaction was noted in L605（2） and PLLA SES groups, while moderate inflammatory reaction was observed in 2 arteries in PLLA group.No abnormal changes in histopathologic examinations of cardiac muscle,liver,spleen,lung and kidney tissues and in blood sample tests were found in the PLLA SES group.At 4 weeks after implantation,as compared with Firebird stent, PLGA SES had a tendency to have lower neointimal thickness over the struts（0.06mm vs.0.12mm,P=0.146）,neointimal thickness between the struts（0.06mm vs.0.16mm,P=0.075） and percentage area stenosis （13.85%vs.22.81%,P=0.274）,however,there were no significant differences statistically.The neointimal area was significantly reduced in PLGA SES than in Firebird stent group（0.64mm2 vs.1.38mm2,P= 0.011） There were no significant differences between PLLA SES and Firebird stent in neointimal thickness over the struts（0.10mm vs.0.12mm, P=0.234）,neointimal thickness between the struts（0.08mm vs.0.16mm, P=0.115）,neointimal area（0.79mm² vs.1.38mm²,P=0.052） and percentage area stenosis（21.22%vs.22.81%,P=0.756）.No in-stent stenosis was found in any group and neointimal healing was similar in the 3 SES groups.Inflammatory response in the PLGA SES and PLLA SES groups were significantly reduced,as compared with Firebird group. In the preliminary study on the biodegradable magnesium alloy PLA coating SES,stent disintegration was observed even at 1 week after implantation.At 4 weeks,only stent debris remained in the arterial wall in some samples,and complete degradation was found in 2 stents.Serious inflammatory response and in-stent stenosis were observed in both PLA and SES groups.ConclusionsIn the present study,we found that:1.The biodegradable PLGA coating SES had good biocompatibility and could significantly inhibit the neointimal proliferation 4 and 12 weeks after stent implantation in porcine coronary artery model.At 4 weeks,the luminal surface is 75%to 100%endothelialised in most vessels,and endothelialization was completed at 12 weeks.2.Our study was the first pathologic observation in China to find that there was statistically positive correlation between inflammatory area and neointimal area.Inflammation might promote neointimal proliferation after coronary stent implantation.3.The biodegradable PLLA coating SES had good biocompatibility and could significantly inhibit the neointimal proliferation 4 weeks after stent implantation in porcine coronary artery model.At 4 weeks,the luminal surface is 50%to 80%endothelialised. 4.We conducted the first study in China to compare the inflammatory reaction and the inhibition of neointimal proliferation between biodegradable polymer coating SES and permanent polymer coating SES in animal model.The results showed that the efficacy on inhibiting neointimal hyperplasia of PLGA coating SES and PLLA coating SES was at least similar to that of Firebird stent,and inflammatory reaction in arterial wall was significantly reduced in the 2 biodegradable polymer coating SES groups.5.We conducted the first study to explore the feasibility of magnesium alloy drug-eluting stent,and found that the biodegradable magnesium PLA coating SES in this study couldn’t satisfy the clinical need yet due to its fast degradation,poor mechanical strength and inacceptable biocompatibility.Further studies to improve its mechanical and chemical features are needed.更多还原