Regeneration of Multi-layered Vocal Fold Mucosa

描述（由申请方提供）：声带（VF）粘膜是一种复杂的分层结构，由鳞状上皮细胞、基底膜和富含细胞外基质（ECM）的固有层组成。每个粘膜层都具有一组不同的功能，这些功能共同负责VF免疫、运输和屏障能力，吸收相当大的冲击应力的能力，以及允许自我维持组织振荡以产生声音的有利粘弹性。由于创伤或疾病引起的VF粘膜的破坏或破坏对VF振荡具有严重后果，并且通常导致难治性发声困难。VF组织修复和再生领域的先前工作集中在同质生物材料、个体细胞群和个体或小群体的基因/蛋白质;然而，这些方法不足以试图重建这种动态生物学和生物力学系统的固有复杂性。一个临床上有意义的方法来取代大VF粘膜缺陷需要一个组织工程策略，结合分层结构，复杂的ECM网络和多种细胞类型。所提出的研究直接面对这些挑战，专注于优化的多层VF粘膜替代物的基础上脱细胞VF ECM和VF成纤维细胞和角质形成细胞的共培养。我们将使用组织工程，细胞和分子生物学，蛋白质组学/糖组学和功能振动技术的新组合进行这项研究;使我们能够直接评估细胞行为，整个ECM蛋白和聚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。了解在受控的体外环境中基质周转、细胞- ECM相互作用和振动性能的动力学对于优化这种组织工程方法以用于未来的体内实验和最终的临床实施是至关重要的。除了实现这些治疗驱动的目标，拟议的研究将显着提高理解的天然VF粘膜在蛋白质组学和糖组学水平，并介绍了VF器官型共培养模型具有广泛的适用性VF上皮形态发生和发展的研究，生理，免疫和屏障功能。

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.