Regeneration of Multi-layered Vocal Fold Mucosa

描述(申请人提供)：声带(VF)粘膜是一个复杂的层状结构，由鳞状细胞上皮、基底膜和富含细胞外基质(ECM)的固有层组成。每一层粘膜都具有一套独特的功能，这些功能共同负责VF的免疫、运输和屏障能力，吸收相当大的冲击压力的能力，以及允许自我维持组织振荡以产生声音的良好粘弹性特性。由于创伤或疾病引起的室颤黏膜的破坏或破坏会导致严重的室颤振荡，通常会导致难治性发音困难。以前在VF组织修复和再生领域的工作主要集中在同质生物材料、单个细胞群体和单个或小群基因/蛋白质；然而，这些方法不足以试图重建这一动态生物和生物力学系统的内在复杂性。一种临床上有意义的方法来替代大的VF粘膜缺陷需要一个组织工程策略，该策略结合了分层结构、复杂的ECM网络和多种细胞类型。这项研究直接面对这些挑战，通过优化基于脱细胞VF ECM和VF成纤维细胞与角质形成细胞共培养的多层VF黏膜替代物。我们将使用组织工程、细胞和分子生物学、蛋白质组/糖凝胶和功能振动技术的新组合来进行这项研究；使我们能够直接评估细胞行为、整个ECM蛋白质和多糖网络的系统范围状态，以及这些工程结构与天然VF组织相比的振荡潜力。在受控的体外环境中，了解基质周转、细胞-细胞外基质相互作用和振动性能的动态变化，对于优化这一组织工程方法以用于未来的体内实验和最终的临床应用至关重要。除了实现这些治疗驱动的目标外，拟议的研究还将在蛋白质组和血糖水平上显著提高对天然VF黏膜的理解，并引入VF器官型共培养模型，该模型可广泛应用于VF上皮的形态发生和发育、生理、免疫和屏障功能的研究。

DESCRIPTION (provided by applicant): The vocal fold (VF) mucosa is a complex layered structure comprised of a squamous cell epithelium, basement membrane and lamina propria rich in extracellular matrix (ECM). Each mucosal layer holds a distinct set of functions that are together responsible for VF immune, transport and barrier capabilities, an ability to absorb considerable impact stress, and favorable viscoelastic properties allowing self-sustained tissue oscillation for voice production. Disruption or destruction of the VF mucosa due to trauma or disease has severe consequences for VF oscillation and generally results in a refractory dysphonia. Previous work in the area of VF tissue repair and regeneration has focused on homogenous biomaterials, individual cell populations, and individual or small groups of genes/proteins; however, these approaches are inadequate in attempting to reconstruct the inherent complexity of this dynamic biological and biomechanical system. A clinically meaningful approach to the replacement of large VF mucosal deficits requires a tissue engineering strategy that incorporates a layered structure, sophisticated ECM network and multiple cell types. The proposed research directly confronts these challenges by focusing on the optimization of a multi-layered VF mucosa substitute based on decellularized VF ECM and coculture of VF fibroblasts and keratinocytes. We will conduct this research using a novel combination of tissue engineering, cellular and molecular biology, proteomic/glycomic, and functional vibratory techniques; allowing us to directly evaluate cell behavior, the system-wide status of the entire ECM protein and glycan network, and the oscillatory potential of these engineered structures compared with native VF tissue. Understanding the dynamics of matrix turnover, cell- ECM interaction, and vibratory performance in a controlled in vitro environment is critical to optimizing this tissue engineering approach for future in vivo experiments and eventual clinical implementation. In addition to achieving these therapeutically driven goals, the proposed research will significantly improve understanding of the native VF mucosa at the proteomic and glycomic levels, and introduce a VF organotypic coculture model with wide-ranging applicability to the study of VF epithelial morphogenesis and development, physiologic, immunologic and barrier function.