Liotta生物瓣膜对体内环境适应性的相关分析研究
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摘要
人工心脏瓣膜已经广泛应用于临床,取得了良好的效果。相对机械瓣膜而言,生物瓣膜不但具有优越的血流动力学性能、无需终身抗凝和抗感染强等优点,它还具有可以运用微创手术将其植入人体的潜力。经皮瓣膜置换术是近年来发展起来的一项微创瓣膜置换手术。随着经皮瓣膜置换术的不断发展,要求我们更多地去深入了解生物瓣膜对该技术的适应性,为临床研制出更为理想的生物瓣膜,为经皮瓣膜置换术的完善和发展奠定基础。因此,对常规手术和经皮瓣膜置换术植入的生物瓣膜临床使用后存在的问题进行研究分析具有非常重要的意义。
本论文对62个病人体内取出的LLP(Liotta Low Profile)猪主动脉瓣膜进行了形态学、组织学和矿物学等相关研究,从生物相容性、生物功能性和生物耐久性方面分析研究了Liotta生物瓣膜在人体环境中的适应性。这62个瓣膜中,有一部分是采用经皮瓣膜置换术植入病人体内的。我们重点关注的是瓣膜的纤维层、室面层以及松质层随着植入时间所发生的变化,以期了解瓣膜对血液流动的适应性、不同瓣膜化学处理方法的优缺点,从而为生产厂商优化生产加工和储存生物瓣膜提供理论依据。
这62个瓣膜失功能的原因有以下几方面:血流动力学、血栓形成和心内膜炎。其中使用寿命为短期(t≤1年)的瓣膜失功能原因为:血液渗透到瓣叶内部,纤维蛋白沉积,血栓形成;使用寿命为中期(1年﹤t≤5年)的瓣膜:内膜炎和血流动力学性能差;使用寿命为长期(t>5年)的瓣膜:钙化和撕裂所引起的血流动力学障碍。
研究目的:本课题通过对62个从人体内取出的LLP猪主动脉瓣膜进行形态学,组织学和矿物学等相关分析,从生物相容性、生物功能性和生物耐久性方面分析研究了Liotta生物瓣膜在人体环境中的适应性,作为第一代经皮瓣膜置换的生物瓣膜,它失功能的原因以及它是否适合用于经皮瓣膜置换术。
研究方法:采用62个从病人体内取出的LLP猪主动脉瓣膜。这些瓣膜植入人体的时间长短由几分钟到9年以上。其中10个瓣膜使用寿命为短期,19个瓣膜使用寿命为中期,另外33个瓣膜使用寿命为长期。通过形态学观察,分析瓣膜形态变化和失功能的相关性;通过组织学研究(包括光学显微镜,扫描电子显微镜和透射电镜),分析瓣膜两个表面和内部层的相关变化;通过矿物学分析研究(包括X射线衍射和核磁共振),检测瓣膜中钙盐的沉积以及测定钙化组分情况。
研究结果:
①使用寿命为短期的瓣膜:
1)形态学:这10个瓣膜均保有较为完好的血流动力学性能。其中撕裂和穿孔的瓣膜占20%。经皮瓣膜置换术植入的瓣膜中,有些瓣膜的瓣叶有轻微的损伤、撕裂或者瓣叶处于非自然状态。货架期较长的瓣膜植入病人体内后,在短时间内出现了严重的退化和钙化。
2)组织学:所有瓣膜表面上的内皮几乎完全脱落,暴露出下面的纤维结构。在纤维层表面可以看到有少量的内皮细胞分散存在。血小板常常存在于瓣叶表面纤维网状结构的凹陷处。瓣膜植入2个月后,血管翳慢慢地从缝合环的两面长出。在纤维层表面,宿主自身内皮细胞开始生长。大多数瓣膜的表面没有发现血栓形成,仅少数瓣膜的瓣叶内部或者缝合处存有血栓。随着植入时间的延续,瓣膜瓣叶表面的形态和结构持续发生着变化,血液中的蛋白沉积在瓣叶表面,形成一均匀的生物膜,厚约有10μm。蛋白生物膜的存在使瓣叶表面变得更光滑,在生物膜比较薄弱或者有凹陷的位置,发现了巨噬细胞的浸入。生物膜结构随瓣膜植入时间的延长由粒状变成纤维状。
3)矿物学:少数瓣膜出现较早瓣叶内的钙化或者瓣叶表面的钙化,且均为单瓣叶出现钙化现象。
②使用寿命为中期的瓣膜:
1)形态学:70%的瓣膜的生物功能性被减弱,主要原因是瓣叶的撕裂和钙化。瓣叶表面的钙化主要是由血栓和损伤所引起,瓣叶内的钙化主要发生在瓣叶压力集中的部位。少数瓣膜在瓣叶缝合处裂开。
2)组织学:血管翳沿着瓣叶表面发展的同时也向瓣叶内部发展,随着时间的延长,变得愈加严重,呈不连续断裂式发展,延续到瓣叶活动部位。纤维层表面的内皮层延伸到了瓣叶缝合处。在室面层上,无生物膜的区域有单核细胞和淋巴细胞附着。生物膜覆盖较好区域的生物膜中有少量的单核细胞和淋巴细胞存在。纤维层的结构保存较好。随着植入时间的延续,巨噬细胞、单核细胞和淋巴细胞由瓣叶表面开始向内部渗透,从而为瓣叶内部的钙化创造了条件。由于瓣叶表面结构的改变,瓣叶表面的钙化开始伴随着发展,纤维层表面较为明显。
3)矿物学:羟基磷灰石存在于所有的瓣膜内。
③使用寿命为长期的瓣膜:
1)形态学:极少数植入人体时间长的瓣膜有心内膜炎和血栓形成,但几乎所有的瓣膜都发生了撕裂和穿孔。约97%的瓣膜存在大量的钙化和多处撕裂,瓣膜的功能性严重减弱。由于钙化,瓣叶变硬;纤维层的瓣叶内的钙化转化为瓣叶表面的钙化沉淀物。纤维组织增生覆盖了瓣叶,因此严重影响瓣叶运动的灵活性。
2)组织学:部分瓣膜纤维层表面的内皮细胞层仍然存在,这些瓣膜的形态和瓣叶结构均保持完好。瓣叶表面的生物膜呈碎片状存在。细胞和血液碎片等渗透到了瓣叶内部松质层,瓣膜的结构受到严重的破坏,表现为细胞、纤维、细胞外基质和坏死组织混合在一起。瓣叶内的钙化越来越严重,已经穿破瓣叶组织成为瓣叶表面的钙化。
3)矿物学:所有的瓣膜均含有程度不同的钙沉淀物。
研究结论:
①生物相容性:LLP生物瓣膜具有较为良好的血流动力学特性和生物相容性,植入人体后极少产生由血流动力学不适或恶性组织反应而引发的血栓形成。但LLP生物瓣膜在以下几个方面还需要进一步改进:1)随植入人体时间的延续,瓣叶的表面变得粗糙。如能让瓣叶表面生长出一层内皮细胞,从而提高瓣叶表面的光滑度,将更为理想;2)应增强缝合环上的组织生长,从而减小缝合环与瓣叶之间的缝隙,以降低血液通过该缝隙的泄漏;3)应采取措施,不让血管翳延伸超过瓣叶。
②生物功能性:LLP生物瓣膜在正常的生理学条件下具有自如的开启、关闭性能,且在心房面没有发现任何的泄漏。然而,由病理学引起的生物瓣膜的退化会大大削弱瓣膜的生物功能性。虽然瓣叶的变硬没有严重地影响瓣膜的生物功能性,但是它会引起瓣叶的撕裂和破损,从而最终影响到瓣膜的生物功能性。因此,如何防止或延缓生物瓣膜的病理性退化是亟待解决的课题。
③生物耐久性:一个理想的生物瓣膜替代品的生物耐久性,应超过病人的寿命。但是由于病理的损害限制了生物瓣膜的发展,从而使其生物耐久性被大大地缩短了。Liotta生物瓣膜表面的内皮和基底膜在生产加工过程中受到了严重的破坏。纤维结构的暴露,加速了血液成分和细胞的渗透,从而为钙化的形成创造了条件,使瓣膜的生物耐久性被大大地缩短了。生物瓣膜组织表面内皮化是解决该问题的一个途径,然而体外内皮化方法受宿主内皮细胞来源、培养条件等因素的限制,体内内皮化受到生物瓣膜交联处理的细胞毒性作用等因素的影响,所以进展不大。本研究发现,瓣膜表面的蛋白生物膜不但可以提高瓣膜的生物相容性,同时可以作为一个保护屏障,防止血液成分和细胞渗入瓣叶。由此,我们建议可在瓣膜植入人体前,在生产加工过程中在瓣膜表面覆盖一层人工生物膜,作为屏障,阻止血液成分或组织液成分的渗入、钙盐沉积,预防瓣膜的变性和钙化,延长瓣膜的耐久性。
④货架期较长的瓣膜在较短的时间内便出现了严重的退化,因此建议植入人体的瓣膜货架期不应超过2年。
⑤Liotta生物瓣膜设计的构型独特,瓣架具有弹性,瓣架环为波浪形,支架的高度低,瓣叶的应力较小,且缝合环为瓣上型。这一设计的初衷是要使瓣膜更近于生理状态。然而,本研究发现,Liotta生物瓣膜并没有显示出明显的优势,有些瓣膜还发生了瓣叶缝合处的断裂。这说明,低剖面的设计影响了Liotta生物瓣膜的长期生物稳定性。
⑥通过对经皮瓣膜置换术植入体内的Liotta生物瓣膜形态学观察发现,瓣膜存在撕裂和损伤,部分瓣膜的瓣叶处于非自然状态,严重影响了瓣膜的生物功能性。这表明,目前的经皮瓣膜置换术影响生物瓣膜的生物功能性,手术技术有待进一步的提高,以降低手术过程中对瓣膜的损伤,否则,生物瓣膜是不适合用于经皮瓣膜置换术的。
For many years, artificial heart valves have been largely and successfully accepted for the replacement of pathological heart valves in humans. Bioprosthetic heart valves have good hemodynamics, and usually have good thrombo-resistance in comparison with mechanical heart valves. Moreover, they can be inserted in small diameter delivery conduits and implanted using percutaneous valve replacement techniques.
The development of percutaneous valves requires a particular understanding of the in vivo fate of bioprostheses as these valves are inserted through small diameter delivery conduits. In order to improve the bioprosthetic heart valves and develop suitable heart valves for the percutaneous valve replacement, it is very important to study systematically the bioprosthetic heart valves explanted from patients.
This study analysed sixty-two explanted Liotta Low Profile (LLP) porcine bioprostheses from patients by morphology, histology and mineralogy methods to highlight the issues related to their biocompatibility, biofunctionality and biodurability, through these aspects to know the adaptation of porcine valve in human environment. Among these 62 valves, some valves were implanted in patients by percutaneous valve replacement. A special attention was paid to ventricularis and to the fibrosa, as well as to the spongiosa. We tried to know their changes as time went on. The analysis of this series explanted devices that were obtained after a spectrum of duration provides abstract knowledge and experiment data for progress durability of bioprosthesis, also offers good suggestion of optimizations of the process and storage.
The etiology of the reoperations was as follows: hemodynamic, thrombosis and endocarditis . The primary failure modes leading to reoperation were as follows: in the short-term: blood infiltration, fibrin build-up, thrombosis; in the mid-term: endocarditis and hemodynamic insufficiency; and in the long-term: mineralization and tears, causing hemodynamic incompetence.
Objective: Sixty-two explanted Liotta porcine bioprostheses were revisited to highlight the issues related to their biocompatibility, biofunctionality and biodurability by morphology, histology and mineralogy, through these aspects to know their adaptation in human environment and to know the failure mechanisms as first percutaneous valve replacement and the adaptation for this technology.
Methods: Sixty-two LLP porcine valves from patients were harvested after a few hours to more than nine years of implantation: 10 short- term (i.e., t < 1 year) 19 mid-term (i.e., 1< t < 5 years) and 33 long-term (i.e., t > 5 years). Valves were examined macroscopically for gross morphology, and photographed with photomacroscopy equipment. The histological investigations involved light microscopy, scanning electron microscopy and transmission electrons microscopy. Mineralogy( involved X-ray and NMR) tests the mineral deposits of valves.
Results:
①Short term( t≤1 year):
1) Morphology: The 10 valves mostly preserved their hemodynamic biofunctionality. Tears and perforations were present on 20% valves. The valves, which were implanted by percutaneous valve replacement, were damaged, had some tears or were not in natural position. The valves were kept on the shelf for a long time, there were tissue degradation and calcification after short implantation.
2) Histology: Both surfaces of ventricularis and fibrosa were almost totally devoid of endothelium exposing fibrillous structures. A few scattered modified endothelial cells were still visible on the fibrosa. Platelets were frequently anchored in the invaginations of the surfaces entrapped in fibrin networks. After two months of implantation, a pannus developed slowly from the textile suturing ring on both sides with a fragile layer of endothelial like-cells on the fibrosa side that extended slowly. However, most of the surface was free from mural thrombi. In the very few valves, thrombi were anchored within the cusps or at the commissures. Progressively, the flow surface became covered with a proteins biofilm , about 10 um thick, both surfaces remained smooth. In the absence or weak of biofilm, small numbers of cells mainly macrophage as well as droplets were adhering to the flow surface of the ventricularis. The biofilm was becoming progressively more fibrillar as time went on.
3) Mineralogy: Few valves showed early endophitic or exophitic mineralization in one leaflet.
②Mid-term (1 < t≤5 years):
1) Morphology: The biofunctionality of the explanted devices at mid-term was seriously impaired in 70% bioprostheses due to tears and mineralizations. The exophitic mineralization was because of thrombi and damage, the endophitic mineralization was on stress area. Few valves were splited at the commissures.
2) Histology: The pannus developed on both surfaces, at the same time developing from outside to inside the leaflets. The pannus expanded more and more and developed irregularly until appositional area of leaflets. The endothelium covered fibrosa over the commissures. In the absence of biofilm, small numbers of cells mainly monocytes and lymphocytes as well as droplets were adhering to the flow surface of the ventricularis. Where the biofilm was on the flow surface and well developed, it incorporated number of monocytes and lymphocytes. The structure of fibrosa was generally well preserved. As time went, the macrophages, monocytes and lymphocytes were progressively penetrated from outside to inside the leaflets leading to the formation of void spaces and initiating some nidus of mineralization. The structure of the flow surfaces were changed to lead exophitic mineralization, specially on the fibrosa.
3) Mineralogy: The hydroxyapatite was present in all valves.
③Long-term implantation (t > 5 years)
1) Morphology: Endocarditis and thrombosis were much less frequent. Tears and perforations were present frequent on the long-term as almost all the devices were involved (97%). The biofunctionality of the explanted devices was seriously impaired. The leaflets became stiff due to the presence of calcification. On the fibrosa, endophitic mineralization became exophitic mineralization. Fibrinous vegetations covered the leaflets, the motility of the leaflets became progressively reduced.
2) Histology: The fragile layers of endothelial cells were observed on the fibrosa of few valves, so the structure of these valves were preserved better than others. The biofilm was frequently fragmented. The cells and blood debris penetrated within the spongiosa, the disorganization of the spongiosa was rapidly evidenced. The cells, the fibers and the amorphous extracellular matrix formed a homogeneous mass with tissue necrosis were progressing. The mineralization proved to be less and less endophitic: as they progressed, ruptures and crevices permitted the mineralization to become exophitic.
3)Mineralogy: The calcifications were present in all the valves more or less.
Conclusion:
①Biocompatibility: After the implantation of LLP, the capacity to become adapted to the blood and tissue environment without any thrombotic event related to flowing blood/valve leaflets interactions nor exacerbated tissue reaction at the site of anchorage of the bioprostheses. But we have to improve LLP from following aspects: 1) As the time goes after implantation, the flow surfaces become not smooth. If endothelial cells can develop on the flow surfaces, the surfaces of leaflets will become smooth and ideal. 2) To have more fibrous tissue for bioprostheses. The polyester of the sewing ring shall become encapsulated with fibrous tissue to prevent any endoleak. 3) Try to find a solution to prevent the pannus from extending over the leaflet.
②Biofunctionality: The valves shall be able to open and close under normal physiological conditions i.e. 130 mm Hg, 75 cycles/min ratio systole/diastole 45% without any leak detectable on the atrium side. The bioprosthesis must maintain its capacity over the years. Any pathological degeneration impairs the biofunctionality of the device. Stiffening of the leaflets can eventually be tolerated but holes and tears causing tears greatly handicap the biofunctionality. Anyway, preventing the pathological degeneration for bioprostheses is a big problem to solve.
③Biodurability: An ideal valve substitute shall have a biodurability overpassing the life expectancy of the patient. As the pathological damages to the bioprostheses develop, the biodurability is considerably reduced. During harvesting of LLP bioprosthesis in the process, the endothelium is destroyed as well as the basement membrane. The fibrillous structures are exposed, blood debris and blood cells can penetrate rapidly, so that leads to the formation of void spaces and initiating some nidus of mineralization, the biodurability is considerably reduced. The endothelial cells from host cover the flow surfaces of bioprostheses is a solution, but this way is limited by a lot of factors. From our study, we discovered that the biofilm can increase the biocompatibility of bioprosthesis, at the same time, it can be a protective barrier to prevent the penetrating of blood debris and blood cells. Therefore, we suggest that before implantation we cover the bioprostheses with an artificial biofilm, the artificial biofilm can be a protective barrier to prevent the penetrating of blood debris and blood cells and calcification to improve the biodurability.
④The valves, which kept on the shelf for a long time, were degraded in a short period after implantation. Therefore, we suggest that the shelf-life of bioprostheses should not exceed 2 years.
⑤Liotta bioprostheses have unique design and flexible stents, they are low profile valve, so shear strains reduce internal fibre strains and protect the leaflets against fatigue, Liotta valve is more like a natural heart valve. However, the Liotta bioprosthesis did not provide any clear cut advantage over standard porcine bioprosthesis and its long-term biostability appeared affected by its low profile design.
⑥We examined the morphology of the valves, which were implanted in the patients through percutaneous valve replacement, and discovered that these valves had tears and damage, some leaflets were not in the natural position. The biofunctionality was seriously impaired. Therefore, at the present the percutaneous valve replacement affects the biofunctionality of bioprostheses, it is very necessary to improve this technology and to decrease the damage at implantation. Otherwise, the bioprostheses do not adapt to percutaneous valve replacement.
本论文对62个病人体内取出的LLP(Liotta Low Profile)猪主动脉瓣膜进行了形态学、组织学和矿物学等相关研究,从生物相容性、生物功能性和生物耐久性方面分析研究了Liotta生物瓣膜在人体环境中的适应性。这62个瓣膜中,有一部分是采用经皮瓣膜置换术植入病人体内的。我们重点关注的是瓣膜的纤维层、室面层以及松质层随着植入时间所发生的变化,以期了解瓣膜对血液流动的适应性、不同瓣膜化学处理方法的优缺点,从而为生产厂商优化生产加工和储存生物瓣膜提供理论依据。
这62个瓣膜失功能的原因有以下几方面:血流动力学、血栓形成和心内膜炎。其中使用寿命为短期(t≤1年)的瓣膜失功能原因为:血液渗透到瓣叶内部,纤维蛋白沉积,血栓形成;使用寿命为中期(1年﹤t≤5年)的瓣膜:内膜炎和血流动力学性能差;使用寿命为长期(t>5年)的瓣膜:钙化和撕裂所引起的血流动力学障碍。
研究目的:本课题通过对62个从人体内取出的LLP猪主动脉瓣膜进行形态学,组织学和矿物学等相关分析,从生物相容性、生物功能性和生物耐久性方面分析研究了Liotta生物瓣膜在人体环境中的适应性,作为第一代经皮瓣膜置换的生物瓣膜,它失功能的原因以及它是否适合用于经皮瓣膜置换术。
研究方法:采用62个从病人体内取出的LLP猪主动脉瓣膜。这些瓣膜植入人体的时间长短由几分钟到9年以上。其中10个瓣膜使用寿命为短期,19个瓣膜使用寿命为中期,另外33个瓣膜使用寿命为长期。通过形态学观察,分析瓣膜形态变化和失功能的相关性;通过组织学研究(包括光学显微镜,扫描电子显微镜和透射电镜),分析瓣膜两个表面和内部层的相关变化;通过矿物学分析研究(包括X射线衍射和核磁共振),检测瓣膜中钙盐的沉积以及测定钙化组分情况。
研究结果:
①使用寿命为短期的瓣膜:
1)形态学:这10个瓣膜均保有较为完好的血流动力学性能。其中撕裂和穿孔的瓣膜占20%。经皮瓣膜置换术植入的瓣膜中,有些瓣膜的瓣叶有轻微的损伤、撕裂或者瓣叶处于非自然状态。货架期较长的瓣膜植入病人体内后,在短时间内出现了严重的退化和钙化。
2)组织学:所有瓣膜表面上的内皮几乎完全脱落,暴露出下面的纤维结构。在纤维层表面可以看到有少量的内皮细胞分散存在。血小板常常存在于瓣叶表面纤维网状结构的凹陷处。瓣膜植入2个月后,血管翳慢慢地从缝合环的两面长出。在纤维层表面,宿主自身内皮细胞开始生长。大多数瓣膜的表面没有发现血栓形成,仅少数瓣膜的瓣叶内部或者缝合处存有血栓。随着植入时间的延续,瓣膜瓣叶表面的形态和结构持续发生着变化,血液中的蛋白沉积在瓣叶表面,形成一均匀的生物膜,厚约有10μm。蛋白生物膜的存在使瓣叶表面变得更光滑,在生物膜比较薄弱或者有凹陷的位置,发现了巨噬细胞的浸入。生物膜结构随瓣膜植入时间的延长由粒状变成纤维状。
3)矿物学:少数瓣膜出现较早瓣叶内的钙化或者瓣叶表面的钙化,且均为单瓣叶出现钙化现象。
②使用寿命为中期的瓣膜:
1)形态学:70%的瓣膜的生物功能性被减弱,主要原因是瓣叶的撕裂和钙化。瓣叶表面的钙化主要是由血栓和损伤所引起,瓣叶内的钙化主要发生在瓣叶压力集中的部位。少数瓣膜在瓣叶缝合处裂开。
2)组织学:血管翳沿着瓣叶表面发展的同时也向瓣叶内部发展,随着时间的延长,变得愈加严重,呈不连续断裂式发展,延续到瓣叶活动部位。纤维层表面的内皮层延伸到了瓣叶缝合处。在室面层上,无生物膜的区域有单核细胞和淋巴细胞附着。生物膜覆盖较好区域的生物膜中有少量的单核细胞和淋巴细胞存在。纤维层的结构保存较好。随着植入时间的延续,巨噬细胞、单核细胞和淋巴细胞由瓣叶表面开始向内部渗透,从而为瓣叶内部的钙化创造了条件。由于瓣叶表面结构的改变,瓣叶表面的钙化开始伴随着发展,纤维层表面较为明显。
3)矿物学:羟基磷灰石存在于所有的瓣膜内。
③使用寿命为长期的瓣膜:
1)形态学:极少数植入人体时间长的瓣膜有心内膜炎和血栓形成,但几乎所有的瓣膜都发生了撕裂和穿孔。约97%的瓣膜存在大量的钙化和多处撕裂,瓣膜的功能性严重减弱。由于钙化,瓣叶变硬;纤维层的瓣叶内的钙化转化为瓣叶表面的钙化沉淀物。纤维组织增生覆盖了瓣叶,因此严重影响瓣叶运动的灵活性。
2)组织学:部分瓣膜纤维层表面的内皮细胞层仍然存在,这些瓣膜的形态和瓣叶结构均保持完好。瓣叶表面的生物膜呈碎片状存在。细胞和血液碎片等渗透到了瓣叶内部松质层,瓣膜的结构受到严重的破坏,表现为细胞、纤维、细胞外基质和坏死组织混合在一起。瓣叶内的钙化越来越严重,已经穿破瓣叶组织成为瓣叶表面的钙化。
3)矿物学:所有的瓣膜均含有程度不同的钙沉淀物。
研究结论:
①生物相容性:LLP生物瓣膜具有较为良好的血流动力学特性和生物相容性,植入人体后极少产生由血流动力学不适或恶性组织反应而引发的血栓形成。但LLP生物瓣膜在以下几个方面还需要进一步改进:1)随植入人体时间的延续,瓣叶的表面变得粗糙。如能让瓣叶表面生长出一层内皮细胞,从而提高瓣叶表面的光滑度,将更为理想;2)应增强缝合环上的组织生长,从而减小缝合环与瓣叶之间的缝隙,以降低血液通过该缝隙的泄漏;3)应采取措施,不让血管翳延伸超过瓣叶。
②生物功能性:LLP生物瓣膜在正常的生理学条件下具有自如的开启、关闭性能,且在心房面没有发现任何的泄漏。然而,由病理学引起的生物瓣膜的退化会大大削弱瓣膜的生物功能性。虽然瓣叶的变硬没有严重地影响瓣膜的生物功能性,但是它会引起瓣叶的撕裂和破损,从而最终影响到瓣膜的生物功能性。因此,如何防止或延缓生物瓣膜的病理性退化是亟待解决的课题。
③生物耐久性:一个理想的生物瓣膜替代品的生物耐久性,应超过病人的寿命。但是由于病理的损害限制了生物瓣膜的发展,从而使其生物耐久性被大大地缩短了。Liotta生物瓣膜表面的内皮和基底膜在生产加工过程中受到了严重的破坏。纤维结构的暴露,加速了血液成分和细胞的渗透,从而为钙化的形成创造了条件,使瓣膜的生物耐久性被大大地缩短了。生物瓣膜组织表面内皮化是解决该问题的一个途径,然而体外内皮化方法受宿主内皮细胞来源、培养条件等因素的限制,体内内皮化受到生物瓣膜交联处理的细胞毒性作用等因素的影响,所以进展不大。本研究发现,瓣膜表面的蛋白生物膜不但可以提高瓣膜的生物相容性,同时可以作为一个保护屏障,防止血液成分和细胞渗入瓣叶。由此,我们建议可在瓣膜植入人体前,在生产加工过程中在瓣膜表面覆盖一层人工生物膜,作为屏障,阻止血液成分或组织液成分的渗入、钙盐沉积,预防瓣膜的变性和钙化,延长瓣膜的耐久性。
④货架期较长的瓣膜在较短的时间内便出现了严重的退化,因此建议植入人体的瓣膜货架期不应超过2年。
⑤Liotta生物瓣膜设计的构型独特,瓣架具有弹性,瓣架环为波浪形,支架的高度低,瓣叶的应力较小,且缝合环为瓣上型。这一设计的初衷是要使瓣膜更近于生理状态。然而,本研究发现,Liotta生物瓣膜并没有显示出明显的优势,有些瓣膜还发生了瓣叶缝合处的断裂。这说明,低剖面的设计影响了Liotta生物瓣膜的长期生物稳定性。
⑥通过对经皮瓣膜置换术植入体内的Liotta生物瓣膜形态学观察发现,瓣膜存在撕裂和损伤,部分瓣膜的瓣叶处于非自然状态,严重影响了瓣膜的生物功能性。这表明,目前的经皮瓣膜置换术影响生物瓣膜的生物功能性,手术技术有待进一步的提高,以降低手术过程中对瓣膜的损伤,否则,生物瓣膜是不适合用于经皮瓣膜置换术的。
For many years, artificial heart valves have been largely and successfully accepted for the replacement of pathological heart valves in humans. Bioprosthetic heart valves have good hemodynamics, and usually have good thrombo-resistance in comparison with mechanical heart valves. Moreover, they can be inserted in small diameter delivery conduits and implanted using percutaneous valve replacement techniques.
The development of percutaneous valves requires a particular understanding of the in vivo fate of bioprostheses as these valves are inserted through small diameter delivery conduits. In order to improve the bioprosthetic heart valves and develop suitable heart valves for the percutaneous valve replacement, it is very important to study systematically the bioprosthetic heart valves explanted from patients.
This study analysed sixty-two explanted Liotta Low Profile (LLP) porcine bioprostheses from patients by morphology, histology and mineralogy methods to highlight the issues related to their biocompatibility, biofunctionality and biodurability, through these aspects to know the adaptation of porcine valve in human environment. Among these 62 valves, some valves were implanted in patients by percutaneous valve replacement. A special attention was paid to ventricularis and to the fibrosa, as well as to the spongiosa. We tried to know their changes as time went on. The analysis of this series explanted devices that were obtained after a spectrum of duration provides abstract knowledge and experiment data for progress durability of bioprosthesis, also offers good suggestion of optimizations of the process and storage.
The etiology of the reoperations was as follows: hemodynamic, thrombosis and endocarditis . The primary failure modes leading to reoperation were as follows: in the short-term: blood infiltration, fibrin build-up, thrombosis; in the mid-term: endocarditis and hemodynamic insufficiency; and in the long-term: mineralization and tears, causing hemodynamic incompetence.
Objective: Sixty-two explanted Liotta porcine bioprostheses were revisited to highlight the issues related to their biocompatibility, biofunctionality and biodurability by morphology, histology and mineralogy, through these aspects to know their adaptation in human environment and to know the failure mechanisms as first percutaneous valve replacement and the adaptation for this technology.
Methods: Sixty-two LLP porcine valves from patients were harvested after a few hours to more than nine years of implantation: 10 short- term (i.e., t < 1 year) 19 mid-term (i.e., 1< t < 5 years) and 33 long-term (i.e., t > 5 years). Valves were examined macroscopically for gross morphology, and photographed with photomacroscopy equipment. The histological investigations involved light microscopy, scanning electron microscopy and transmission electrons microscopy. Mineralogy( involved X-ray and NMR) tests the mineral deposits of valves.
Results:
①Short term( t≤1 year):
1) Morphology: The 10 valves mostly preserved their hemodynamic biofunctionality. Tears and perforations were present on 20% valves. The valves, which were implanted by percutaneous valve replacement, were damaged, had some tears or were not in natural position. The valves were kept on the shelf for a long time, there were tissue degradation and calcification after short implantation.
2) Histology: Both surfaces of ventricularis and fibrosa were almost totally devoid of endothelium exposing fibrillous structures. A few scattered modified endothelial cells were still visible on the fibrosa. Platelets were frequently anchored in the invaginations of the surfaces entrapped in fibrin networks. After two months of implantation, a pannus developed slowly from the textile suturing ring on both sides with a fragile layer of endothelial like-cells on the fibrosa side that extended slowly. However, most of the surface was free from mural thrombi. In the very few valves, thrombi were anchored within the cusps or at the commissures. Progressively, the flow surface became covered with a proteins biofilm , about 10 um thick, both surfaces remained smooth. In the absence or weak of biofilm, small numbers of cells mainly macrophage as well as droplets were adhering to the flow surface of the ventricularis. The biofilm was becoming progressively more fibrillar as time went on.
3) Mineralogy: Few valves showed early endophitic or exophitic mineralization in one leaflet.
②Mid-term (1 < t≤5 years):
1) Morphology: The biofunctionality of the explanted devices at mid-term was seriously impaired in 70% bioprostheses due to tears and mineralizations. The exophitic mineralization was because of thrombi and damage, the endophitic mineralization was on stress area. Few valves were splited at the commissures.
2) Histology: The pannus developed on both surfaces, at the same time developing from outside to inside the leaflets. The pannus expanded more and more and developed irregularly until appositional area of leaflets. The endothelium covered fibrosa over the commissures. In the absence of biofilm, small numbers of cells mainly monocytes and lymphocytes as well as droplets were adhering to the flow surface of the ventricularis. Where the biofilm was on the flow surface and well developed, it incorporated number of monocytes and lymphocytes. The structure of fibrosa was generally well preserved. As time went, the macrophages, monocytes and lymphocytes were progressively penetrated from outside to inside the leaflets leading to the formation of void spaces and initiating some nidus of mineralization. The structure of the flow surfaces were changed to lead exophitic mineralization, specially on the fibrosa.
3) Mineralogy: The hydroxyapatite was present in all valves.
③Long-term implantation (t > 5 years)
1) Morphology: Endocarditis and thrombosis were much less frequent. Tears and perforations were present frequent on the long-term as almost all the devices were involved (97%). The biofunctionality of the explanted devices was seriously impaired. The leaflets became stiff due to the presence of calcification. On the fibrosa, endophitic mineralization became exophitic mineralization. Fibrinous vegetations covered the leaflets, the motility of the leaflets became progressively reduced.
2) Histology: The fragile layers of endothelial cells were observed on the fibrosa of few valves, so the structure of these valves were preserved better than others. The biofilm was frequently fragmented. The cells and blood debris penetrated within the spongiosa, the disorganization of the spongiosa was rapidly evidenced. The cells, the fibers and the amorphous extracellular matrix formed a homogeneous mass with tissue necrosis were progressing. The mineralization proved to be less and less endophitic: as they progressed, ruptures and crevices permitted the mineralization to become exophitic.
3)Mineralogy: The calcifications were present in all the valves more or less.
Conclusion:
①Biocompatibility: After the implantation of LLP, the capacity to become adapted to the blood and tissue environment without any thrombotic event related to flowing blood/valve leaflets interactions nor exacerbated tissue reaction at the site of anchorage of the bioprostheses. But we have to improve LLP from following aspects: 1) As the time goes after implantation, the flow surfaces become not smooth. If endothelial cells can develop on the flow surfaces, the surfaces of leaflets will become smooth and ideal. 2) To have more fibrous tissue for bioprostheses. The polyester of the sewing ring shall become encapsulated with fibrous tissue to prevent any endoleak. 3) Try to find a solution to prevent the pannus from extending over the leaflet.
②Biofunctionality: The valves shall be able to open and close under normal physiological conditions i.e. 130 mm Hg, 75 cycles/min ratio systole/diastole 45% without any leak detectable on the atrium side. The bioprosthesis must maintain its capacity over the years. Any pathological degeneration impairs the biofunctionality of the device. Stiffening of the leaflets can eventually be tolerated but holes and tears causing tears greatly handicap the biofunctionality. Anyway, preventing the pathological degeneration for bioprostheses is a big problem to solve.
③Biodurability: An ideal valve substitute shall have a biodurability overpassing the life expectancy of the patient. As the pathological damages to the bioprostheses develop, the biodurability is considerably reduced. During harvesting of LLP bioprosthesis in the process, the endothelium is destroyed as well as the basement membrane. The fibrillous structures are exposed, blood debris and blood cells can penetrate rapidly, so that leads to the formation of void spaces and initiating some nidus of mineralization, the biodurability is considerably reduced. The endothelial cells from host cover the flow surfaces of bioprostheses is a solution, but this way is limited by a lot of factors. From our study, we discovered that the biofilm can increase the biocompatibility of bioprosthesis, at the same time, it can be a protective barrier to prevent the penetrating of blood debris and blood cells. Therefore, we suggest that before implantation we cover the bioprostheses with an artificial biofilm, the artificial biofilm can be a protective barrier to prevent the penetrating of blood debris and blood cells and calcification to improve the biodurability.
④The valves, which kept on the shelf for a long time, were degraded in a short period after implantation. Therefore, we suggest that the shelf-life of bioprostheses should not exceed 2 years.
⑤Liotta bioprostheses have unique design and flexible stents, they are low profile valve, so shear strains reduce internal fibre strains and protect the leaflets against fatigue, Liotta valve is more like a natural heart valve. However, the Liotta bioprosthesis did not provide any clear cut advantage over standard porcine bioprosthesis and its long-term biostability appeared affected by its low profile design.
⑥We examined the morphology of the valves, which were implanted in the patients through percutaneous valve replacement, and discovered that these valves had tears and damage, some leaflets were not in the natural position. The biofunctionality was seriously impaired. Therefore, at the present the percutaneous valve replacement affects the biofunctionality of bioprostheses, it is very necessary to improve this technology and to decrease the damage at implantation. Otherwise, the bioprostheses do not adapt to percutaneous valve replacement.
引文
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