Vergleichende in vivo Untersuchung verschiedener Konservierungsverfahren von allogenen Herzklappen

人类心脏瓣膜或同种异体移植物代表了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。特别是在儿科患者中的缺点是有限的可用性，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瓣膜，在抗生素孵育后移植而不进行任何保存。自1968年以来，已采用标准冷冻低温保存（SFC），包括在气相液氮中储存。应用多光子成像分析（MIA）通过诱导自体荧光而无需在新鲜组织中固定、包埋和染色来实现弹性蛋白和胶原的三维可视化。MIA首次检测到SFC猪瓣膜中弹性和胶原基质的部分破坏。由于胶原蛋白和弹性蛋白的总量保持不变，上述破坏的病因被假定为由于冰晶引起的冷冻诱导的细胞外基质（ECM）损伤。在低温保存过程中发生的破坏性间隙冰损伤可以通过促进玻璃化、玻璃形成而不是在低温保存溶液玻璃化转变温度以下形成冰来避免。由于保存和加温是在没有任何组织破坏的情况下完成的，因此玻璃化首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 ℃的冰箱中储存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可改善瓣膜功能并减少退行性变。此外，IFC可预防CD3阳性T细胞介导的应答。这种减弱的适应性免疫应答的确切机制仍然未知。最近在猪组织与人反应者外周血白细胞组合中的研究表明，用IFC保存培养基处理瓣膜组织可降低瓣叶免疫原性，并诱导反应者单核细胞向内皮样细胞分化。因此，将IFC应用于异种心脏瓣膜可能会克服器官稀缺问题。通过省略控制速率冷冻和液氮储存的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.