Tissue Engineered Aortic Heart Valves: Scaffolds and Stem Cells

描述（由申请人提供）：在世界范围内，每年更换近30万个病变心脏瓣膜，其中大多数设备包括机械瓣膜，由非活体生物组织制成的设备或有活力的人类同种异体移植物。心脏瓣膜置换术的持续时间限制在15-20年，主要是由于凝血风险、心内膜炎、变性、钙化和不能生长和重塑。这项研究与公共卫生高度相关，因为心脏瓣膜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，它将持续一生，不会容易出现并发症，具有生长和重塑的能力，从而最终影响成千上万的患者。我们的创新建议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结构，ii)由模拟天然心脏瓣膜组织结构的三层结构制成的解剖上类似的三维心脏瓣膜形状，iii)用于再生和重塑的自体多能间充质干细胞，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，在结构和功能上模拟主动脉瓣纤维层、脑室层和海绵层，将它们组装成天然心脏瓣膜形状的三层结构，并用人间充质干细胞填充。构建物将安装在生物反应器中，诱导干细胞分化为瓣膜间质细胞的类似物，并促进重塑。在Specific Aim 1中，将从脱细胞心包制备用作纤维层和脑室层的胶原层，并轻度交联以允许可控的生物降解。对于海绵状层，高多孔性胶原蛋白支架将由脱细胞、弹性酶处理的动脉制备，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/海绵状/脑室），并在硅橡胶模具上成型，形成类似天然主动脉根部的结构。工程主动脉根部将以先进的力学分析为特征，并在脉冲瓣膜复制器中评估其功能。在特异性目标2中，我们将制备人间充质干细胞。干细胞将被植入胶原蛋白凝胶中，并在FlexerCell系统中接受可控负载。我们将评估受刺激的干细胞分化为瓣膜间质细胞的表型变化和能力。在特异性目标3中，我们将在三层支架内包裹海绵层，在支架上植入干细胞，并在生物反应器内进行体外循环。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. 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.