Tissue Engineered Aortic Heart Valves: Scaffolds and Stem Cells

描述(申请人提供)：全世界每年有近30万名患病的心脏瓣膜被替换，其中大多数使用的装置包括机械瓣膜、由非生物组织制成的装置或可存活的人类同种异体移植物。心脏瓣膜置换术的寿命限制在15-20年，主要原因是凝血风险、心内膜炎、变性、钙化以及无法生长和重塑。这项研究与公共健康高度相关，因为心脏瓣膜疾病是成人和儿童心血管疾病中非常重要的一章。我们的长期目标是开发活的组织工程瓣膜，这种瓣膜将持续一生，不容易出现并发症，具有生长和重塑的能力，从而最终影响数千名患者。我们的创新方案承认四个问题对于我们的方法是至关重要的：i)由部分稳定的胶原支架构建的结构，ii)由模仿天然心脏瓣膜组织结构的三层结构制成的解剖相似的3-D心脏瓣膜形状，iii)用于再繁殖和重塑的自体多能间充质干细胞，以及iv)诱导干细胞分化为能够维持基质动态平衡的瓣膜细胞的机械和生化线索。为了实现这些目标，我们建议开发部分稳定的胶原支架，这种支架在结构和功能上模仿主动脉瓣纤维层、室壁层和海绵层，将它们组装成自然心脏瓣膜形式的三层结构，并用人间充质干细胞填充它们。构建物将被安装在生物反应器中，以诱导干细胞分化为瓣膜间质细胞的类似物，并促进重塑。在具体目标1中，将从脱细胞的心包制备用作纤维层和室层的胶原层，并进行轻微的交联，以允许受控的生物降解。对于海绵层，高度多孔的胶原支架将从脱细胞的、弹性酶处理的动脉中制备，并富含瓣膜特异性的糖胺聚糖。然后将支架组装成组织学上类似的三层结构(纤维膜/海绵/室壁)，并通过在硅橡胶铸件上模压成形成类似天然主动脉根部的结构。工程化的主动脉根部将通过先进的机械分析进行表征，并在搏动瓣膜复制器中评估其功能。在具体目标2中，我们将制备人骨髓间充质干细胞。然后，干细胞将被种植到胶原蛋白凝胶上，并在Flexercell系统中受到可控的负荷状态。我们将评估表型变化和刺激干细胞分化为瓣膜间质细胞的能力。在具体目标3中，我们将海绵层包裹在三层支架内，用干细胞种植支架，并在生物反应器中进行体外循环构建。我们将在动态条件下评估不同时间点的细胞分化和基质重塑。

DESCRIPTION (provided by applicant): Worldwide, nearly 300,000 diseased heart valves are replaced annually, most of them with devices that include mechanical valves, devices made from non-living biological tissues or viable human allografts. Durability of heart valve replacements is limited to 15-20 years mostly due to coagulation risks, endocarditis, degeneration, calcification and failure to grow and remodel. This study is highly relevant to public health because heart valve disease is a very important chapter of cardiovascular diseases in adults and children. Our long-term objective is to develop living tissue-engineered valves that will last a life-time, will not be prone to complications, will have the ability to grow and remodel and thus ultimately impacting thousands of patients. Our innovative proposal acknowledges the vital importance of four issues that are unique to our approach: i) Constructs made from partially stabilized collagenous scaffolds, ii) Anatomically analogous 3-D heart valve shapes made form tri-layered structures that mimic the native heart valve histo-architecture, iii) Autologous multipotent mesenchymal stem cells for repopulation and remodeling and iv) Mechanical and biochemical cues to induce stem cell differentiation into valvular cells capable of maintaining matrix homeostasis. To accomplish these goals, we propose to develop partially stabilized collagen scaffolds that structurally and functionally mimic the aortic valve fibrosa, ventricularis and spongiosa layers, to assemble them into tri-layered constructs shaped in the form of natural heart valves and populate them with human mesenchymal stem cells. Constructs will be mounted in a bioreactor to induce differentiation of stem cells into analogues of valvular interstitial cells and promote remodeling. In Specific Aim 1, collagen layers to be used as fibrosa and ventricularis layers will be prepared from decellularized pericardium and lightly cross-linked to allow for controlled biodegradation. For the spongiosa layer, highly porous collagen scaffolds will be prepared from decellularized, elastase- treated arteries and enriched with valve-specific glycosaminoglycans. Scaffolds will be then assembled into histologically analogous tri-layered structures (fibrosa / spongiosa / ventricularis) and shaped into constructs resembling native aortic roots by molding on silicone rubber casts. Engineered aortic roots will be characterized by advanced mechanical analysis and their function evaluated in a pulsatile valve duplicator. In Specific Aim 2, we will prepare human mesenchymal stem cells. Stem cells will be then seeded onto collagen gels and subjected to controlled load regimes in a FlexerCell system. We will evaluate phenotypic changes and ability of stimulated stem cells to differentiate into valvular interstitial cells. In Specific Aim 3, we will encase spongiosa layer within the tri-layered scaffold, seed the scaffolds with stem cells, and subject constructs to in vitro cycling within a bioreactor. We will evaluate cell differentiation and matrix remodeling at various time-points in dynamic conditions.