Vergleichende in vivo Untersuchung verschiedener Konservierungsverfahren von allogenen Herzklappen

人类心脏瓣膜或同种异体心脏瓣膜是近乎完美的心脏瓣膜替代品。它们具有最佳的血液动力学特性，并能抵抗感染。尤其是在儿科患者中的缺点是可获得性有限、无法生长、退化和长期失败。后者的潜在罪魁祸首包括免疫反应和钙代谢增加。同种异体心脏瓣膜的第一次临床应用是作为同种异体心脏瓣膜，在抗生素孵育后移植，没有任何保存。自1968年以来，采用了标准的冷冻冷冻保存(SFC)方法，包括气相液氮保存。多光子成像分析(MIA)的应用使弹性蛋白和胶原的三维可视化可以通过诱导自体荧光来实现，而不需要在新鲜组织中固定、包埋和染色。MIA首次检测到SFC猪瓣膜弹性和胶原基质的部分破坏。由于胶原和弹性蛋白的总量保持不变，上述破坏的原因被推测为冰晶导致的细胞外基质(ECM)破坏。通过促进玻璃化，而不是在低温保存溶液玻璃化转变温度以下形成冰，可以避免低温保存期间发生的破坏性间质冰破坏。由于保存和加温是在不破坏任何组织的情况下完成的，玻璃化冷冻第一次能够完全保存心脏瓣膜中的细胞外基质。为了简化所需的基础设施，我们进一步开发了无冰冷冻保存(IFC)，它允许在80摄氏度的冰柜中存储高于溶液玻璃化转变温度的溶液。我们在最近的一项绵羊研究中应用了IFC，并能够证明维持ECM可以带来更好的瓣膜功能和减少退化。此外，IFC还具有抗炎作用，可阻止CD3阳性T细胞介导的反应。这种减弱的获得性免疫反应的确切机制尚不清楚。最近在猪组织与人类应答外周血白细胞组合中的研究表明，用IFC保存介质处理瓣膜组织会降低小叶的免疫原性，并诱导应答单核细胞向内皮样细胞分化。因此，在异种心脏瓣膜中应用IFC可能会克服器官匮乏的问题。通过省略受控速率冻结和液氮储存来简化，将提高成本效益，并允许在财政资源有限的第三世界和发展中国家应用。

Human heart valves or allografts represent nearly perfect heart valve substitutes. They have optimal hemodynamic characteristics and are resistant to infections. Disadvantages particularly in pediatric patients are limited availability, an inability to grow, degeneration and long-term failure. Potential culprits for the latter include immunological responses and an increased calcium metabolism. The first clinical use of allograft heart valves was as homovitals that were transplanted after antibiotic incubation without any preservation. Since 1968 standard frozen cryopreservation (SFC) has been employed including storage in vapour phase liquid nitrogen. Application of multiphoton imaging analysis (MIA) enables three dimensional visualization of elastin and collagen by induction of autofluorescence without fixation, embedding and staining in fresh tissue. MIA allowed for the first time detection of partial destruction of elastic and collagenous matrix in SFC porcine valves ex situ. As the overall amount of collagen and elastin remains unchanged the etiology of the above described destruction is postulated as freezing induced extracellular matrix (ECM) damage due to ice crystals. Disruptive interstitial ice damage that occurs during cryopreservation can be avoided by promoting vitrification, glass formation, instead of ice formation below the cryopreservation solutions glass transition temperature. As the preservation and warming is accomplished without any tissue destruction, vitrification enabled for the first time complete preservation of extracellular matrix in heart valves. In order to simplify the required infrastructure, we have further developed ice free cryopreservation (IFC), which permits storage above the solutions glass transition temperature in a 80C freezer. We applied IFC in a recent sheep study and were able to demonstrate that maintaining ECM results in better valve function and diminished degeneration. Furthermore, IFC is antiinflammatory preventing CD3 positive T cell mediated responses. The exact mechanism of this attenuated adaptive immune response remains unknown. Recent studies in a porcine tissue to human responder peripheral blood leukocyte combination have shown that treatment of valve tissue with IFC preservation medium reduces leaflet immunogenicity and induces responder monocyte differentiation towards endothelial like cells. Accordingly application of IFC for xenogeneic heart valves might overcome organ scarcity. Simplification by omitting controlled rate freezing and liquid nitrogen storage will improve cost effectiveness and allow application in third world and developing countries with limited financial resources.