Tissue Engineered Aortic Heart Valves: Scaffolds and Stem Cells

描述（由申请人提供）：在全球范围内，每年有近300，000例患病心脏瓣膜被置换，其中大多数被置换的器械包括机械瓣膜、由非活体生物组织或活体人体同种异体移植物制成的器械。心脏瓣膜置换术的耐用性仅限于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。这项研究与公共卫生高度相关，因为心脏瓣膜疾病是成人和儿童心血管疾病的一个非常重要的章节。我们的长期目标是开发活的组织工程瓣膜，这些瓣膜将持续一生，不易发生并发症，具有生长和重塑的能力，从而最终影响成千上万的患者。我们的创新提案承认我们的方法所特有的四个问题的至关重要性：i）由部分稳定的胶原支架制成的构造，ii）由模拟天然心脏瓣膜组织结构的三层结构制成的解剖学上类似的3-D心脏瓣膜形状，iii）用于再增殖和重塑的自体多能间充质干细胞，和iv）诱导干细胞分化为能够维持基质稳态的瓣膜细胞的机械和生物化学线索。为了实现这些目标，我们建议开发部分稳定的胶原蛋白支架，其在结构和功能上模仿主动脉瓣纤维层、心室层和海绵层，将它们组装成以天然心脏瓣膜形式成形的三层结构，并用人类间充质干细胞填充它们。将构建体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，将从脱细胞心包制备用作纤维层和心室层的胶原蛋白层，并轻度交联以允许受控生物降解。对于海绵层，高度多孔的胶原蛋白支架将由脱细胞的弹性蛋白酶处理的动脉制备，并富含瓣膜特异性糖胺聚糖。然后将支架组装成组织学上类似的三层结构（纤维/海绵/心室），并通过在硅橡胶铸件上模塑成型为类似天然主动脉根的结构。工程主动脉根部将通过先进的机械分析进行表征，并在脉动瓣膜复制器中评价其功能。在具体目标2中，我们将制备人间充质干细胞。然后将干细胞接种到胶原凝胶上，并在FlexerCell系统中接受受控的负载方案。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。在特定目标3中，我们将在三层支架内包裹海绵层，用干细胞接种支架，并使构建体在生物反应器内进行体外循环。我们将评估动态条件下不同时间点的细胞分化和基质重塑。 公共卫生相关性：心脏瓣膜是心脏腔室内的瓣状组织，在心动周期的每一秒都打开和关闭，以允许血液流过心脏。人工心脏瓣膜通常通过手术进行更换，但可用的人工装置并不理想，并且在植入后15-20年内失效，主要是因为它们由非生物材料制成。每年有30多万患者需要新的和改进的设备。我们正在开发由多层组织支架组成的活材料，我们将患者的细胞添加到其中，并将整个设备塑造成天然心脏瓣膜的形式。这种组织工程设备具有适应和改造患者的潜力，因此将通过治疗成人和儿童的心血管疾病产生全球影响。

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. PUBLIC HEALTH RELEVANCE: Heart valves are flap-like tissues inside the heart chambers that open and close every second of the cardiac cycle to allow blood to flow through the heart. Diseased heart valves are routinely replaced by surgery, but available artificial devices are less than optimal and fail within 15-20 years after implantation, mostly because they are made of non-living materials. New and improved devices are needed for more than 300,000 patients every year. We are developing living materials comprised of layers of tissue scaffolds to which we add the own patients' cells and shape the entire device in the form of a natural heart valve. This tissue engineered device has the potential to adapt and remodel with the patient, and thus will have a global impact by treating cardiovascular diseases in adults and children.