人造纤维织物外支架—牛颈静脉带瓣管道复合体的制备研究
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摘要
研究背景:
目前,血管替代主要有三类材料。第一类是人造血管,制作材料以涤纶、聚四氟乙烯等应用较广,其优点主要是可选择性强,口径尺寸齐全,机械强度高可承受高血压。缺点是材料本身无法参与机体代谢,异物长期存在,腔内外细胞无法透过人造血管壁生长更替,内膜增厚明显,长期有使用寿命限制;本身是异物,易感染等等;第二类是自体血管,如大隐静脉、胸廓内动脉、挠动脉、胃网膜右动脉等,优点是组织相容性好,无排斥反应,缺点是受口径尺寸限制大,取材创伤大。中动脉以上口径没有可以用于替代的自体动脉血管来源。静脉替代动脉时无法长时间承受高压,发生曲张变形、瘤变,引起血流动力学改变,易导致血栓形成,或是增厚狭窄发生阻塞等。没有合适的肺动脉替代血管;第三类是生物工程血管,如人类脐静脉,异种材料如牛颈静脉带瓣管道以及以合成高分子材料骨架上种植内皮细胞体外培养血管等等。组织工程方法处理后的血管经过降低抗原性处理后排斥反应轻微,有天然胶原蛋白等结构作为基质,可以由机体代谢替换。所以组织工程血管理论上兼顾前两类血管的优点,如可选择口径尺寸限制少,可以参与机体代谢,有一定生长变化范围,可以早期内皮化等等。但是正因为可以参与组织代谢和降解,去抗原性处理本身也可导致管壁结构破坏,机械强度差或不稳定是其最大的问题。同时处理过程也较复杂等等。显然,第三类也就是生物工程血管是解决肺动脉替代瓶颈问题的合适材料,有比之其他两类代血管材料更加广阔的应用前景。
牛颈静脉带瓣管道是一种新型的肺动脉血管重建材料,主要应用于右室与肺动脉连接发育不良的复杂先天性心脏病的外科手术治疗。上世纪90年代国外开展牛颈静脉带瓣管道研究并应用于临床。牛颈静脉带瓣管道具有取材方便、类似人的肺动脉瓣结构的瓣膜,抗返流性能强,血管直径跨度范围大,更适合于临床婴幼儿病例。临床上已经有牛颈静脉带瓣管道产品,系采用常规生物组织交联剂戊二醛固定制备而成。
目前,肺动脉血管替代产品完全由国外公司垄断,价格高昂。单一血管材料价格相当于其他住院费用总和,病人负担沉重。临床应用迫切需要改良的、价格相对低廉的、有良好流体力学性能且组织相容性好的血管替代品种。国内吴忠仕等为了提高牛颈静脉带瓣管道的抗钙化性能,采用去细胞结合光氧化反应法制备的牛颈静脉带瓣管道已经进行了大量的实验研究并应用临床。
经过去细胞和光氧化处理的牛颈静脉带瓣管道在临床上取得了良好效果。但是,机械性能、防渗性能等等仍存在一些不足之处。比如在当肺动脉压力较高时,手术时发现牛颈静脉管道有一定的扩张变形,可能引起血流动力学的紊乱,有引发血栓形成、管道瘤样扩张、钙化等潜在可能,术后复查彩超常见到带瓣管道瓣膜返流的情况,以上原因多归于牛颈静脉壁本身以及处理过程导致的弹力蛋白和胶原蛋白不足。那么,如何在尽可能完全去除异体血管材料抗原性的同时保证其有足够的机械性能,或是使得静脉材料代替动脉时可以承受较高的血压,是扩大生物工程血管应用范围的一条正确思维路线。通过化学药品处理(交联)组织血管的机械强度足够,然而弹性不足,钙化严重、细胞毒性大等问题却成了最大制约。本课题试图通过纤维织物包裹、给予替代管道以支撑,防止其过度扩张,起到弥补弹力蛋白和胶原蛋白等结构力量不足的作用。这样,去细胞等酶解处理可以进一步降低移植物抗原性至适当程度,这样就减少了过度处理导致替代材料结构破坏、机械支持力不足的顾虑。
人造纤维支架一生物血管组合与同是人造纤维纺织而成的、目前广泛使用的”人造血管”不同之处在于,细胞可以自由地通过前者网眼的间隙内外双方向生长,代谢更替天然蛋白基质;而人造血管不能透过水分,也不能代谢,血管内外无法进行组织交换,内膜增生最终导致管腔狭窄闭塞。
本文将用来包裹组织工程血管的纤维织物称为“纤维织物外支架”,简称“外支架”。有外支架支持的组织工程血管称为“外支架生物血管复合体”,简称“复合体”。
第一章人造纤维织物支架与牛颈静脉带瓣管道复合体的制备和体外机械性能测试
目的:
利用人造纤维外支架提高牛颈静脉的机械性能,改进牛颈静脉的组织工程处理方法。
方法和材料:
人造纤维织物外支架的制备实验选择了尼龙和涤纶(聚酯)纤维作为外支架纤维材料。编链针编到所需长度以后,把编链最后一针和头端连接起来,然后按空隙大小的不同,编一定长度的编链网孔,如此反复直至编完。
细胞成分去除完全的牛颈静脉管道的制备包括:(1)牛颈静脉取材、(2)均按照三步法去细胞、(3)光氧化进一步处理。
评价指标:(1)大体观察牛颈静脉管壁的颜色,柔韧性,厚薄,及瓣膜结构的变化等。(2)显微镜观察新鲜、光氧化、去细胞和去细胞结合光氧化组管壁和瓣膜细胞排布、胶原和弹力膜等排布变化。结果:在20mmHg下带有人造织物外支架的牛颈静脉没有出现扩张。同样,在30mmHg和40mmHg压力下,复合体的膨胀形变仍不明显,均在10%以内。压力在60mmHg时,膨胀维持在10%左右。而单纯的牛颈静脉在20mmHg时膨胀率已经达到15%左右,在30,40,60mmHg压力下分别膨胀约42%,53%和75%。两组统计学上有明显差异。血管瓣膜返流量测定结果显示,在20mmHg,30mmHg压力下带有人造织物外支架的牛颈静脉瓣膜返流为40mmHg压力下约8ml/min,压力达到60mmHg时,返流达到84ml/min,而单纯的牛颈静脉在20mmHg时返流量是0,在30,40,60mmHg压力下瓣膜返流量分别约32 ml/min,113 ml/min,207ml/min。两组血管统计学上有明显差异。
有外支架支持的复合体材料仅在60mmHg时出现1ml/min的渗漏。而单纯牛颈静脉管道在20,40,60mmHg压力下渗出量分别为1ml/min,5ml/min,13ml/min。与前组有显著统计学差异。
结论:通过结合人造纤维和生物工程血管的特点制备出来的纤维外支架可以起到增强牛颈静脉带瓣管道机械性能的作用。是解决生物工程血管机械性能不足问题的较理想方法。
第二章纤维织物外支架牛颈静脉带瓣管道重建犬右心室—肺动脉连接后组织学的变化及生物相容性研究
目的:
将牛颈静脉复合体用于重建犬右心室—肺动脉连接,并重点对植入后组织学(包括细胞和细胞外基质成分)的动态变化进行评价。
材料和方法:
本实验小组以前曾经有单纯用去细胞加光氧化交联处理的牛颈静脉重建右心室—肺动脉连接动物模型实验,作为对照。牛颈静脉外支架复合体建立犬重建右心室—肺动脉连接动物模型,分别在1个月、3个月、6个月后取出标本检测,观察其组织学的动态变化。分别于1月、3月和6月对犬处杀取出管道进行评价。评价指标包括(1)肉眼大体观察各血管和瓣膜形态、血栓和钙化情况等。(2)组织化学和免疫组织化学方法对植入后管道组织学进行动态评价。(3)采用胶原染色含量测定等方法对胶原代谢进行评价。
按生物材料处理方法不同将制备的牛颈静脉带瓣管道及浸提液分为5组,包括光氧化反应组、去细胞结合光氧化反应组、PC组、GA组。用细胞形态观察法观察细胞所受到的影响。
结果:
采用去细胞结合光氧化处理牛颈静脉带瓣管道重建犬肺动脉11例,2例手术中开放侧壁钳后室颤失血死亡,其余术后全部存活。术后1月、3月和术后6月各处杀3只获取组织标本;
大体观察:1月、3月、6月取出的牛颈静脉带瓣管道无动脉瘤样形成,质地柔软,所有牛颈静脉管壁内膜光滑,未见血栓及钙化形成,管腔通畅肺动脉端吻合口无增生狭窄,瓣膜得到了良好的保护。
光镜检测:包括(1)H&E染色;(2)标本的细胞评价;(3)胶原纤维的评价;(4)新生毛细血管等。胶原纤维在牛颈静脉-右心室两部分分界区域是连续的表明新生胶原纤维已经将吻合口连接起来。同时伴有大量的细胞浸润,包括成纤维母细胞、淋巴细胞等等。而大量的新生血管为这些生长替代建立了基础,纤维支架在这种良性的生长过程中看不出有不良的干扰作用。毛细血管的生成非常活跃,镜下可以看到大量的新生血管,尤其是吻合口周围以及纤维支架周围最明显,同时,与牛颈静脉相邻的心室和肺动脉也同时出现相应的改变,生成大量的毛细血管。毛细血管周围细胞浸润优势明显。胶原染色中,在开始是有明显分界的吻合口部位,心肌组织的胶原已经和生物血管胶原连接起来,分界不明显。这表明有新生胶原产生并将结构真正连接起来。彩色多谱勒心脏超声检查结果于术后1月、3月、6月分别进行心脏彩超检查,肺动脉直径与手术时变化不明显,瓣膜形态柔软,开放关闭正常,无异常返流。心脏功能指数正常。
去细胞结合光氧化反应复合体组血管片周边细胞生长基本正常,部分贴壁,细胞呈梭形,折光性好,细胞突伸展可,并见分裂细胞,与阴性对照相比,基本无区别;戊二醛组较阴性对照组细胞生长稍差,约50%贴壁,细胞呈梭形,折光性可;而阳性对照组细胞未见贴壁,仍呈悬浮状。
致癌实验未见肿瘤生长,结果为阴性。
结论:人造纤维织物牛颈静脉复合体有较好的组织反应性,生物安全性;纤维织物本身可以考虑利用可降解材料进一步降低异物反应。
Chapter One:The preparation of the complex of man made fibric stent and bovine jugular vein and the mechanic function testing
Objective:To increase the mechanic characteristic of bovine jugular veins conduit(BJVC) by using man made fiber stent outside,and to improve the tissue engineering method of BJVC.
Methods:Nylon and Dacron fibers were choosed to make the stent. To make a fibric stent:Firstly,a chain was sewn till become an enough length,and then connected the head and the tail together.Sew a certain mesh as wanted and then another until finish.
To make a bovine jugular vein conduit without any cells inside, including:1,drawing the materials;2,getting rid of the cells according to the three steps;3,the further dealing with BJVC by photooxidation.
Evaluation indexs:1,Observation in general in the color,flexibility, thickness and valve structure of BJVC.2,The observation in microscope.
Results:There was no extension of the BJVCs with fibric stent under pressure of 20mmHg.The extension was＜10%under pressure of 30mm and 40mmHg,untill 60mmHg,≈10%.While extension of the simple BJVCs under the pressure of 20mmHg reach 15%,and≈42%,53%,75% when 30,40,60mmHg.The difference of the two groups above has statistics significance.
The results of valve regurgitation volume sign:there was no regurgitation of the BJVCs with fibric stent under pressure of 20,30mmHg. The volume≈8ml/min when the pressure was 40mmHg,and≈84mmHg when the pressure was 60mmHg.While regurgitation volume of the simple BJVCs was 0 when 20mmHg,and≈32,113,207ml when the pressure was 30,40,60mmHg.The difference of the two groups above has statistics significance.
The results of leakage:there was no leakage of the BJVCs with fibric stent till pressure of 60mmHg,the volume was 1ml/min.While leakage volume of the simple BJVCs was 1,5,13ml/min when the pressure was 30, 40,60mmHg.The difference of the two groups above has statistics significance.
Conclusion:The BJVCs with fibric stent had the two advantages of BJVC and fibric stent so that it may be an ideal option of solving the mechanic insufficient of BJVC.
Chapter Two:Histological and biocompatibility evaluation of bovine jugular venous conduit complex treated with decellularization andphotooxidation technique
Objective:To investigate the histological change of the complex of fibric stent and BJVCs treated with decellularization and photooxidation technique in vivo and evaluate the possibility of which as a tissue engineering scaffold in clinical application.
Methods:BJVCs treated with decelluarization and photooxidation method were implanted in young dogs to reconstruct the connection of pulmonary arteries with right ventricles.The conduits were explanted and analyzed by gross examination,light microscopy in 1 month,6 months and 1 year after being implanted.Tissue collagen was assayed respectively to the explanted tissues.Neutral salt soluble fractions,acetic acid soluble fractions and pepsin-digested fractions of collagen were extracted and assayed for these samples.
Results:Of 11 dogs subjected implantation,2 died of ventricular fibrillation in operation and the other 9 ones survived.3 dogs sacrifaced in 1 month after operation,three in 3 month,3 dogs sacrifaced in 6 month.All the luminal wall were still soft and smooth without calcification, thrombosis and hemangioma.No graft stenosis was observed in all animals except that membrane-like intimal hyperplasia was found in the right-ventricle-side perianastomotic regions in one of 1-year dogs.The valves all functioned well and the thicknesses of which were not changed. Endothelium-like cells had migrated in the perianastomotic regions,and endothelium-like cells had also been found in valves and middle regions of BJVCs.Macrophages appeared in 1 month but disappeared in 6-month and 1-year dogs.
Carcinogenic test:To observe the canceration number of important organs in the whole body at 18 months after operation of implanting the valved bovine jugular vein conduit patches in the body of the SD mice. And there was no irritation reaction or tumor occurred in the test animals.
Conclusions:
The preliminary results in vivo showed decellularizaton and photooxidation treated BJVCs complex having growth potentiality and supporting endothelialization,with low-immunogenicity and low-calcification, which surpport the conduit as a tissue engineering scaffold for reconstructing the connection of pulmonary arteries with right ventricles.
目前,血管替代主要有三类材料。第一类是人造血管,制作材料以涤纶、聚四氟乙烯等应用较广,其优点主要是可选择性强,口径尺寸齐全,机械强度高可承受高血压。缺点是材料本身无法参与机体代谢,异物长期存在,腔内外细胞无法透过人造血管壁生长更替,内膜增厚明显,长期有使用寿命限制;本身是异物,易感染等等;第二类是自体血管,如大隐静脉、胸廓内动脉、挠动脉、胃网膜右动脉等,优点是组织相容性好,无排斥反应,缺点是受口径尺寸限制大,取材创伤大。中动脉以上口径没有可以用于替代的自体动脉血管来源。静脉替代动脉时无法长时间承受高压,发生曲张变形、瘤变,引起血流动力学改变,易导致血栓形成,或是增厚狭窄发生阻塞等。没有合适的肺动脉替代血管;第三类是生物工程血管,如人类脐静脉,异种材料如牛颈静脉带瓣管道以及以合成高分子材料骨架上种植内皮细胞体外培养血管等等。组织工程方法处理后的血管经过降低抗原性处理后排斥反应轻微,有天然胶原蛋白等结构作为基质,可以由机体代谢替换。所以组织工程血管理论上兼顾前两类血管的优点,如可选择口径尺寸限制少,可以参与机体代谢,有一定生长变化范围,可以早期内皮化等等。但是正因为可以参与组织代谢和降解,去抗原性处理本身也可导致管壁结构破坏,机械强度差或不稳定是其最大的问题。同时处理过程也较复杂等等。显然,第三类也就是生物工程血管是解决肺动脉替代瓶颈问题的合适材料,有比之其他两类代血管材料更加广阔的应用前景。
牛颈静脉带瓣管道是一种新型的肺动脉血管重建材料,主要应用于右室与肺动脉连接发育不良的复杂先天性心脏病的外科手术治疗。上世纪90年代国外开展牛颈静脉带瓣管道研究并应用于临床。牛颈静脉带瓣管道具有取材方便、类似人的肺动脉瓣结构的瓣膜,抗返流性能强,血管直径跨度范围大,更适合于临床婴幼儿病例。临床上已经有牛颈静脉带瓣管道产品,系采用常规生物组织交联剂戊二醛固定制备而成。
目前,肺动脉血管替代产品完全由国外公司垄断,价格高昂。单一血管材料价格相当于其他住院费用总和,病人负担沉重。临床应用迫切需要改良的、价格相对低廉的、有良好流体力学性能且组织相容性好的血管替代品种。国内吴忠仕等为了提高牛颈静脉带瓣管道的抗钙化性能,采用去细胞结合光氧化反应法制备的牛颈静脉带瓣管道已经进行了大量的实验研究并应用临床。
经过去细胞和光氧化处理的牛颈静脉带瓣管道在临床上取得了良好效果。但是,机械性能、防渗性能等等仍存在一些不足之处。比如在当肺动脉压力较高时,手术时发现牛颈静脉管道有一定的扩张变形,可能引起血流动力学的紊乱,有引发血栓形成、管道瘤样扩张、钙化等潜在可能,术后复查彩超常见到带瓣管道瓣膜返流的情况,以上原因多归于牛颈静脉壁本身以及处理过程导致的弹力蛋白和胶原蛋白不足。那么,如何在尽可能完全去除异体血管材料抗原性的同时保证其有足够的机械性能,或是使得静脉材料代替动脉时可以承受较高的血压,是扩大生物工程血管应用范围的一条正确思维路线。通过化学药品处理(交联)组织血管的机械强度足够,然而弹性不足,钙化严重、细胞毒性大等问题却成了最大制约。本课题试图通过纤维织物包裹、给予替代管道以支撑,防止其过度扩张,起到弥补弹力蛋白和胶原蛋白等结构力量不足的作用。这样,去细胞等酶解处理可以进一步降低移植物抗原性至适当程度,这样就减少了过度处理导致替代材料结构破坏、机械支持力不足的顾虑。
人造纤维支架一生物血管组合与同是人造纤维纺织而成的、目前广泛使用的”人造血管”不同之处在于,细胞可以自由地通过前者网眼的间隙内外双方向生长,代谢更替天然蛋白基质;而人造血管不能透过水分,也不能代谢,血管内外无法进行组织交换,内膜增生最终导致管腔狭窄闭塞。
本文将用来包裹组织工程血管的纤维织物称为“纤维织物外支架”,简称“外支架”。有外支架支持的组织工程血管称为“外支架生物血管复合体”,简称“复合体”。
第一章人造纤维织物支架与牛颈静脉带瓣管道复合体的制备和体外机械性能测试
目的:
利用人造纤维外支架提高牛颈静脉的机械性能,改进牛颈静脉的组织工程处理方法。
方法和材料:
人造纤维织物外支架的制备实验选择了尼龙和涤纶(聚酯)纤维作为外支架纤维材料。编链针编到所需长度以后,把编链最后一针和头端连接起来,然后按空隙大小的不同,编一定长度的编链网孔,如此反复直至编完。
细胞成分去除完全的牛颈静脉管道的制备包括:(1)牛颈静脉取材、(2)均按照三步法去细胞、(3)光氧化进一步处理。
评价指标:(1)大体观察牛颈静脉管壁的颜色,柔韧性,厚薄,及瓣膜结构的变化等。(2)显微镜观察新鲜、光氧化、去细胞和去细胞结合光氧化组管壁和瓣膜细胞排布、胶原和弹力膜等排布变化。结果:在20mmHg下带有人造织物外支架的牛颈静脉没有出现扩张。同样,在30mmHg和40mmHg压力下,复合体的膨胀形变仍不明显,均在10%以内。压力在60mmHg时,膨胀维持在10%左右。而单纯的牛颈静脉在20mmHg时膨胀率已经达到15%左右,在30,40,60mmHg压力下分别膨胀约42%,53%和75%。两组统计学上有明显差异。血管瓣膜返流量测定结果显示,在20mmHg,30mmHg压力下带有人造织物外支架的牛颈静脉瓣膜返流为40mmHg压力下约8ml/min,压力达到60mmHg时,返流达到84ml/min,而单纯的牛颈静脉在20mmHg时返流量是0,在30,40,60mmHg压力下瓣膜返流量分别约32 ml/min,113 ml/min,207ml/min。两组血管统计学上有明显差异。
有外支架支持的复合体材料仅在60mmHg时出现1ml/min的渗漏。而单纯牛颈静脉管道在20,40,60mmHg压力下渗出量分别为1ml/min,5ml/min,13ml/min。与前组有显著统计学差异。
结论:通过结合人造纤维和生物工程血管的特点制备出来的纤维外支架可以起到增强牛颈静脉带瓣管道机械性能的作用。是解决生物工程血管机械性能不足问题的较理想方法。
第二章纤维织物外支架牛颈静脉带瓣管道重建犬右心室—肺动脉连接后组织学的变化及生物相容性研究
目的:
将牛颈静脉复合体用于重建犬右心室—肺动脉连接,并重点对植入后组织学(包括细胞和细胞外基质成分)的动态变化进行评价。
材料和方法:
本实验小组以前曾经有单纯用去细胞加光氧化交联处理的牛颈静脉重建右心室—肺动脉连接动物模型实验,作为对照。牛颈静脉外支架复合体建立犬重建右心室—肺动脉连接动物模型,分别在1个月、3个月、6个月后取出标本检测,观察其组织学的动态变化。分别于1月、3月和6月对犬处杀取出管道进行评价。评价指标包括(1)肉眼大体观察各血管和瓣膜形态、血栓和钙化情况等。(2)组织化学和免疫组织化学方法对植入后管道组织学进行动态评价。(3)采用胶原染色含量测定等方法对胶原代谢进行评价。
按生物材料处理方法不同将制备的牛颈静脉带瓣管道及浸提液分为5组,包括光氧化反应组、去细胞结合光氧化反应组、PC组、GA组。用细胞形态观察法观察细胞所受到的影响。
结果:
采用去细胞结合光氧化处理牛颈静脉带瓣管道重建犬肺动脉11例,2例手术中开放侧壁钳后室颤失血死亡,其余术后全部存活。术后1月、3月和术后6月各处杀3只获取组织标本;
大体观察:1月、3月、6月取出的牛颈静脉带瓣管道无动脉瘤样形成,质地柔软,所有牛颈静脉管壁内膜光滑,未见血栓及钙化形成,管腔通畅肺动脉端吻合口无增生狭窄,瓣膜得到了良好的保护。
光镜检测:包括(1)H&E染色;(2)标本的细胞评价;(3)胶原纤维的评价;(4)新生毛细血管等。胶原纤维在牛颈静脉-右心室两部分分界区域是连续的表明新生胶原纤维已经将吻合口连接起来。同时伴有大量的细胞浸润,包括成纤维母细胞、淋巴细胞等等。而大量的新生血管为这些生长替代建立了基础,纤维支架在这种良性的生长过程中看不出有不良的干扰作用。毛细血管的生成非常活跃,镜下可以看到大量的新生血管,尤其是吻合口周围以及纤维支架周围最明显,同时,与牛颈静脉相邻的心室和肺动脉也同时出现相应的改变,生成大量的毛细血管。毛细血管周围细胞浸润优势明显。胶原染色中,在开始是有明显分界的吻合口部位,心肌组织的胶原已经和生物血管胶原连接起来,分界不明显。这表明有新生胶原产生并将结构真正连接起来。彩色多谱勒心脏超声检查结果于术后1月、3月、6月分别进行心脏彩超检查,肺动脉直径与手术时变化不明显,瓣膜形态柔软,开放关闭正常,无异常返流。心脏功能指数正常。
去细胞结合光氧化反应复合体组血管片周边细胞生长基本正常,部分贴壁,细胞呈梭形,折光性好,细胞突伸展可,并见分裂细胞,与阴性对照相比,基本无区别;戊二醛组较阴性对照组细胞生长稍差,约50%贴壁,细胞呈梭形,折光性可;而阳性对照组细胞未见贴壁,仍呈悬浮状。
致癌实验未见肿瘤生长,结果为阴性。
结论:人造纤维织物牛颈静脉复合体有较好的组织反应性,生物安全性;纤维织物本身可以考虑利用可降解材料进一步降低异物反应。
Chapter One:The preparation of the complex of man made fibric stent and bovine jugular vein and the mechanic function testing
Objective:To increase the mechanic characteristic of bovine jugular veins conduit(BJVC) by using man made fiber stent outside,and to improve the tissue engineering method of BJVC.
Methods:Nylon and Dacron fibers were choosed to make the stent. To make a fibric stent:Firstly,a chain was sewn till become an enough length,and then connected the head and the tail together.Sew a certain mesh as wanted and then another until finish.
To make a bovine jugular vein conduit without any cells inside, including:1,drawing the materials;2,getting rid of the cells according to the three steps;3,the further dealing with BJVC by photooxidation.
Evaluation indexs:1,Observation in general in the color,flexibility, thickness and valve structure of BJVC.2,The observation in microscope.
Results:There was no extension of the BJVCs with fibric stent under pressure of 20mmHg.The extension was＜10%under pressure of 30mm and 40mmHg,untill 60mmHg,≈10%.While extension of the simple BJVCs under the pressure of 20mmHg reach 15%,and≈42%,53%,75% when 30,40,60mmHg.The difference of the two groups above has statistics significance.
The results of valve regurgitation volume sign:there was no regurgitation of the BJVCs with fibric stent under pressure of 20,30mmHg. The volume≈8ml/min when the pressure was 40mmHg,and≈84mmHg when the pressure was 60mmHg.While regurgitation volume of the simple BJVCs was 0 when 20mmHg,and≈32,113,207ml when the pressure was 30,40,60mmHg.The difference of the two groups above has statistics significance.
The results of leakage:there was no leakage of the BJVCs with fibric stent till pressure of 60mmHg,the volume was 1ml/min.While leakage volume of the simple BJVCs was 1,5,13ml/min when the pressure was 30, 40,60mmHg.The difference of the two groups above has statistics significance.
Conclusion:The BJVCs with fibric stent had the two advantages of BJVC and fibric stent so that it may be an ideal option of solving the mechanic insufficient of BJVC.
Chapter Two:Histological and biocompatibility evaluation of bovine jugular venous conduit complex treated with decellularization andphotooxidation technique
Objective:To investigate the histological change of the complex of fibric stent and BJVCs treated with decellularization and photooxidation technique in vivo and evaluate the possibility of which as a tissue engineering scaffold in clinical application.
Methods:BJVCs treated with decelluarization and photooxidation method were implanted in young dogs to reconstruct the connection of pulmonary arteries with right ventricles.The conduits were explanted and analyzed by gross examination,light microscopy in 1 month,6 months and 1 year after being implanted.Tissue collagen was assayed respectively to the explanted tissues.Neutral salt soluble fractions,acetic acid soluble fractions and pepsin-digested fractions of collagen were extracted and assayed for these samples.
Results:Of 11 dogs subjected implantation,2 died of ventricular fibrillation in operation and the other 9 ones survived.3 dogs sacrifaced in 1 month after operation,three in 3 month,3 dogs sacrifaced in 6 month.All the luminal wall were still soft and smooth without calcification, thrombosis and hemangioma.No graft stenosis was observed in all animals except that membrane-like intimal hyperplasia was found in the right-ventricle-side perianastomotic regions in one of 1-year dogs.The valves all functioned well and the thicknesses of which were not changed. Endothelium-like cells had migrated in the perianastomotic regions,and endothelium-like cells had also been found in valves and middle regions of BJVCs.Macrophages appeared in 1 month but disappeared in 6-month and 1-year dogs.
Carcinogenic test:To observe the canceration number of important organs in the whole body at 18 months after operation of implanting the valved bovine jugular vein conduit patches in the body of the SD mice. And there was no irritation reaction or tumor occurred in the test animals.
Conclusions:
The preliminary results in vivo showed decellularizaton and photooxidation treated BJVCs complex having growth potentiality and supporting endothelialization,with low-immunogenicity and low-calcification, which surpport the conduit as a tissue engineering scaffold for reconstructing the connection of pulmonary arteries with right ventricles.
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