GAGs: Function and Fixation in Bioprosthetic Heart Valves

描述(申请人提供)：生物人工心脏瓣膜(BHV)来源于戊二醛交联的猪主动脉瓣膜，每年用于数千例心脏瓣膜置换手术。这些装置在临床上常因变性和病理性钙化而失效。在无钙化的情况下，很少有人了解BHV的退行性失效。在之前的资助期间，我们发现瓣膜糖胺多聚糖(GAG)在组织固定和植入后丢失。GAG降解酶存在于瓣膜组织中或体内植入后渗入，是导致GAG退化的主要原因。BHV中GAG的丢失会导致组织的弯曲刚性降低、滞后消失和胶原蛋白结构恶化。保持加工组织中细胞外基质(ECM)的结构完整性对于持久的BHV至关重要。我们发现，单独的化学固定剂在预防BHV的GAG丢失方面只有部分有效。我们最近的结果表明，在常规的戊二醛(GLUT)交联之前，添加新霉素，一种GAG降解酶的抑制剂，结合碳二亚胺交联剂的化学固定，可以显著提高瓣膜GAG的稳定性。该项目的总体目标是将BHV的耐用性延长到20年以上。我们建议研究BHV在长达8亿次循环中的耐久性(25年的阀门功能寿命)。这种长期疲劳损伤研究在BHV领域是史无前例的。因此，我们将验证以下假设1)具有改进的细胞外基质稳定化和乙醇预处理以防止钙化的BHV将在体外长期弯曲疲劳期间和体内植入后抵抗退化。我们基于新霉素的新型交联方法将与临床上使用的乙醇抗钙化前处理相结合。A)GAG的稳定性将在体外储存和循环疲劳长达8亿次(超过25年的功能寿命)和b)在体内的大鼠皮下植入模型中进行测试。2)改善细胞外基质稳定性和乙醇预防钙化的BHV具有改善生物力学功能和增强长期耐久性的作用。我们将从与瓣膜功能相关的两种主要变形模式来研究天然GAG在保持GLUT-交联型猪主动脉瓣膜尖端生物力学性能中的作用：平面双轴拉伸和存在或不存在GAG时的弯曲。B)将研究牙槽骨在体外循环疲劳过程中的生物力学功能，最长可达8亿次循环(25年的功能寿命)。3)细胞外基质稳定性得到改善，乙醇处理防止钙化的BHV将被赋予更高的生物持久性。在绵羊二尖瓣置换术模型中，将比较GAG靶向化学/过剩/乙醇与临床使用的乙醇处理的过剩固定的BHV的退化和钙化。与公共卫生相关：约有17.5万名患者因瓣膜受损或功能障碍而需要心脏瓣膜置换。从化学固定的猪心脏瓣膜衍生的生物人工心脏瓣膜经常用于这一目的。这些瓣膜中的大多数在5-15年后由于退化和钙化而失效，需要再次更换。这项拨款提案正在研究新的化学固定剂，以延长这些瓣膜的功能寿命，从而使瓣膜的寿命超过患者。

DESCRIPTION (provided by applicant): Bioprosthetic heart valves (BHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are used annually in thousands of heart valve replacement surgeries. These devices often fail clinically due degeneration and pathologic calcification. Understanding degenerative failure of BHVs in the absence of calcification is rarely addressed. In the previous grant period, we showed that valvular glycosaminoglycans (GAGs) are lost during tissue fixation and after implantation. GAG-degrading enzymes either present in the valve tissue or infiltrated after in vivo implantation are a major cause of GAG degeneration. Loss of GAGs from BHVs leads to decreased tissue flexural rigidity, loss of hysteresis, and collagen structural deterioration. Maintaining the structural integrity of the extracellular matrix (ECM) in the processed tissues is essential for a durable BHV. We found that chemical fixatives alone are only partially effective in preventing GAG loss from BHVs. Our recent results show that addition of neomycin, an inhibitor of GAG-degrading enzymes, in combination with chemical GAG fixation by carbodiimide crosslinking prior to routine glutaraldehyde (GLUT) crosslinking leads to significantly better stabilization of valvular GAGs. The overall aim of this project is extend the durability of BHVs well beyond 20 years. We are proposing to study BHV durability for up to 800 million cycles (25 years of valve functional life). Such long-term fatigue damage study is unprecedented in the BHV field. Thus, we will test the following hypotheses.1) BHVs with improved extracellular matrix stabilization and ethanol pretreatment to prevent calcification will resist degeneration in vitro during extended flexural fatigue and after in vivo implantation. Our novel neomycin-based crosslinking procedure will be combined with clinically used ethanol anti-calcification pretreatment. a) GAG stability will be tested in vitro during storage and cyclic fatigue up to 800 million cycles (more than 25 years of functional life) and b) in vivo in a rat subdermal-implantation model. 2) BHVs with improved extracellular matrix stabilization and ethanol pretreatment for preventing calcification will have improved biomechanical function and enhanced long-term durability. The role of native GAGs in preserving the biomechanical performance of GLUT-crosslinked porcine aortic valve cusps will be studied in two major deformation modes associated with valve function: planar biaxial tension and flexure in presence or absence of GAGs. b) Cuspal biomechanical function during in vitro cyclic fatigue up to 800 million cycles (25 years of functional life) will be studied. 3) BHVs with improved extracellular matrix stabilization and ethanol pretreatment for preventing calcification will be endowed with improved biological durability. Degeneration and calcification of BHVs with GAG-targeted chemistry/GLUT/Ethanol will be compared with clinically used ethanol pretreated GLUT-fixed BHVs in a sheep mitral valve-replacement model. PUBLIC HEALTH RELEVANCE: About 175,000 patients need heart valve replacements due to damaged or dysfunctional valves. Bioprosthetic heart valves derived from chemically fixed pig heart valves are used frequently for this purpose. The majority of these valves fail after 5-15 years due to degeneration and calcification and need replacements again. This grant proposal is investigating new chemical fixatives that would improve functional life-time of these valves so that the valve could outlast the patient.