Iop Laura, Bonetti Antonella, Naso Filippo, Rizzo Stefania, Cagnin Stefano, Bianco Roberto, Dal Lin Carlo, Martini Paolo, Poser Helen, Franci Paolo, Lanfranchi Gerolamo, Busetto Roberto, Spina Michel, Basso Cristina, Marchini Maurizio, Gandaglia Alessandro, Ortolani Fulvia, Gerosa Gino
Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua, Italy.
Department of Experimental and Clinical Medicine, University of Udine, Udine, Italy.
PLoS One. 2014 Jun 18;9(6):e99593. doi: 10.1371/journal.pone.0099593. eCollection 2014.
Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.
组织工程心脏瓣膜被提议作为具有更长耐久性和生长潜力的新型可行替代品。然而，在植入前它们需要在生物反应器中进行广泛的体外细胞预处理。在此，在大型动物模型中研究了未经预处理的脱细胞心脏瓣膜在体内自发自我再生的倾向。对脱细胞猪主动脉瓣进行评估，用于越南猪（n = 11）的右心室流出道（RVOT）重建，随访6个月（n = 5）和15个月（n = 6）。重新定位的天然瓣膜（每次n = 2）被视为对照。通过组织学、免疫组织化学、电子显微镜和基因表达研究取出瓣膜的组织和细胞成分。组织学、免疫组织化学、超微结构、代谢和转录组学分析显示，大多数替代品在15个月的植入期内持续表现出体内足够的血流动力学性能和体外渐进性再填充，没有钙化、纤维化和/或血栓形成的迹象。定植细胞表现出类似天然的表型，并积极合成新的细胞外基质成分，如胶原蛋白和弹性纤维。在再填充的瓣膜中，特别是在再生动脉壁的中层和外膜中发现了新的成熟血管，即毛细血管和血管滋养管。这些发现与血管基因转录上调相关。在组织学和超微结构水平上观察到了神经再生的特征。具有修复性M2表型的巨噬细胞群体在再填充的瓣膜中高度富集。事实上，免疫组织化学和转录组学模式未显示任何不良/免疫反应方面。在分化的细胞成分中，鉴定出几种细胞表达典型的胚胎、造血、神经和间充质谱系的干细胞标志物，其数量明显多于对照瓣膜，且具有特定的拓扑分布。在临床前模型中实现最长随访后，未经预处理的脱细胞同种异体瓣膜为体内细胞归巢和组织重塑提供了合适的微环境。这些有前景的瓣膜替代品采用简单、省时且经济高效的程序制造，有望成为一种有效的替代方案，特别是对于儿科患者。
