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， 将空气动力学能量和气流转换成声音形式的声能。每个声带由 结缔组织的柔韧振动层，称为固有层（LP），夹在肌肉之间， 和复层鳞状上皮（EP）。许多环境、机械和病理因素可以 损伤这一脆弱的组织，导致声带疤痕，影响数百万美国人， 治疗方案。虽然对声带瘢痕形成的病理生理学有普遍的共识， 控制持续纤维化的分子和细胞机制仍然知之甚少。其他研究 纤维化疾病表明成纤维细胞、上皮细胞和间质基质是纤维化的积极参与者， 纤维化该项目旨在设计一种可靠的，生理相关的体外组织模型， 用于研究声带发育、健康和疾病，更重要的是， 开发和测试新的治疗方案。我们打算开发一种微工程器官芯片， 将上皮细胞和间充质细胞整合在具有内置气流的组织模拟结构中， 发声使用微流体模型，我们将研究上皮损伤如何引发纤维化， 纤维化细胞外基质（ECM）维持纤维化，以及肌成纤维细胞增殖和基质沉积 继续不减。最后，我们将使用已显示有效性的抗纤维化生长因子校准我们的模型 在治疗声带疤痕，并测试一个有前途的药理学抑制剂，以前没有测试 在声带疤痕的情况下。高效的生物正交四嗪连接将用于建立 初始LP基质周围的健康成纤维细胞，并引入组成和机械改变， 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，通过采用发展模式，并通过系统的操作， 工程微环境。压阻应变传感器嵌入在微流体的侧壁中 通道将用于原位监测组织硬度和EP渗透性。微工程组织模型 将根据细胞表型、微观结构、机械性能和生理功能来表征。 为了比较的目的，一个独立的，人类大小的声带模型将开发和特点 采用在喉科学领域建立的方法。本项目产生的数据应 对声带瘢痕形成相关基础研究产生重大影响，并提供关键信息， 治疗决策在不久的将来。

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.