A Hydrogel-Based Cellular Model of the Human Vocal Fold

项目摘要 当声带被来自气管的气流驱动成波浪状运动时产生声音， 将空气动力能和气流转化为声音形式的声能。每个声带由 夹在肌肉之间的一层柔软的结缔组织振动层，称为固有层(LP) 和复层鳞状上皮(EP)。许多环境、机械和病理因素都可以 破坏这种脆弱的组织，导致声带疤痕，影响数百万患有有限疾病的美国人 治疗方案。虽然关于声带疤痕的病理生理学有一个普遍的共识，但 控制持续性纤维化的分子和细胞机制仍然知之甚少。关于其他方面的研究 纤维化疾病提示成纤维细胞、上皮细胞和间质基质在 纤维化。该项目旨在设计一种可靠的、与生理相关的体外组织模型，该模型可以 用于研究声带的发育、健康和疾病，更重要的是，有助于 开发和测试新的治疗方案。我们计划开发一种微工程器官芯片， 将上皮细胞和间充质细胞以组织模拟的形式与内置气流整合在一起，以刺激 发声。利用微流体模型，我们将研究上皮损伤如何引发纤维化，如何 纤维性细胞外基质(ECM)维持纤维化以及肌成纤维细胞的增殖和基质沉积 继续有增无减。最后，我们将用一种已经显示出疗效的抗纤维化生长因子来校准我们的模型。 治疗声带疤痕，并测试一种以前没有测试过的有前景的药物抑制剂 在声带疤痕的背景下。将使用高效的生物正交川芎嗪结扎法建立 围绕健康成纤维细胞的初始LP基质，并引入成分和机械改变， 促进成纤维细胞活化。多能和多能干细胞将被引导分化为声带- 像上皮细胞和成纤维细胞一样，通过采用发育范式和通过系统地操纵 工程化的微环境。嵌入微流控芯片侧壁的压阻式应变传感器 通道将用于现场监测组织硬度和EP通透性。微工程化组织模型 将从细胞表型、微观结构、机械性能和生理功能方面进行表征。 为了比较的目的，将开发一个独立的、真人大小的声带模型并对其进行表征 采用在喉科领域确立的方法。从该项目生成的数据应该 显著影响与声带疤痕相关的基础研究，并提供关键信息 在不久的将来做出治疗决策。

Project Summary Voice is produced when the vocal folds are driven into a wave-like motion by the airstream from the trachea, converting aerodynamic energy and airflow into acoustic energy in the form of sound. Each vocal fold consists of a pliable vibratory layer of connective tissue, known as the lamina propria (LP), sandwiched between a muscle and a stratified squamous epithelium (EP). Numerous environmental, mechanical and pathological factors can damage this delicate tissue, resulting in vocal fold scarring that affects millions of Americans with limited treatment options. Although there is a general consensus on the pathophysiology of vocal fold scarring, the molecular and cellular mechanisms that control unremitting fibrosis remain poorly understood. Studies on other fibrotic diseases suggest that fibroblasts, epithelial cells and the interstitial matrix are active players in fibrogenesis. This project aims to engineer a reliable, physiologically relevant in vitro tissue model that can be used to investigate vocal fold development, health, and disease, and more importantly, to facilitate the development and testing of new treatment options. We propose to develop a microengineered organ chip that integrates the epithelial and mesenchymal cells in a tissue-mimetic configuration with built-in airflow to stimulate phonation. Using the microfluidic model, we will investigate how damage to the epithelium initiates fibrosis, how the fibrotic extracellular matrix (ECM) sustains fibrosis and how myofibroblast proliferation and matrix deposition continue unabated. Finally, we will calibrate our model with an antifibrotic growth factor that has shown efficacy in treating vocal fold scarring, and test a promising pharmacological inhibitor that has not been previously tested in the context of vocal fold scarring. Highly efficient bioorthogonal tetrazine ligation will be used to establish the initial LP matrix surrounding healthy fibroblasts and to introduce compositional and mechanical alterations that promote fibroblast activation. Pluripotent and multipotent stem cells will be guided to differentiate into vocal fold- like epithelial cells and fibroblasts by adopting a development paradigm and through systematic manipulation of the engineered microenvironment. Piezoresistive strain sensors embedded in the sidewalls of the microfluidic channels will be used to monitor tissue stiffness and EP permeability in situ. The microengineered tissue model will be characterized in terms of cell phenotype, microstructure, mechanical properties and physiological function. For comparison purposes, a stand-alone, human-sized vocal fold model will be developed and characterized employing methodologies established in the laryngology field. Data generated from this project should significantly impact fundamental research related to vocal fold scarring and provide critical information on therapeutic decision-making in the near future.