A Hydrogel-Based Cellular Model of the Human Vocal Fold

 描述（由申请人提供）：当声带被来自气管的气流驱动成波浪状运动时产生声音，将空气动力学能量和气流转换成声音形式的声能。这种巨大的机械多功能性的关键在于组织的独特结构和组成。每个声带由一个柔软的结缔组织振动层组成，称为固有层（LP），夹在肌肉和上皮层之间。LP的结构和力学从肌肉到上皮逐渐变化。许多环境、机械和病理因素会损害这一脆弱的组织，导致影响数百万美国人的各种声音障碍。目前声带疾病的治疗选择是有限的，新的程序的发展一直缓慢，由于组织的不可及性，易受损伤，以及动物模型从人体组织的解剖差异。该项目旨在设计一个可靠的，生理相关的体外组织模型，可用于研究声带发育，健康和疾病，更重要的是，促进新治疗方案的开发和测试。所提出的工作的中心假设是，显示具有组织样各向异性的分层和梯度结构的拟声带合成细胞外基质（sECM）将为驻留细胞提供建立适当组织结构的指导线索。来自sECM的初始模板效应将通过应用生理相关的振动刺激来进一步加强，最终产生可行的和功能性的声带组织模型。在目标1中，我们将使用模块化构建块并在明确定义的界面上采用快速生物正交反应来创建sECM。所得的sECM将由底部纤维层、基底膜样顶层和具有交联密度和生化信号梯度的中间凝胶层组成。在目标2中，我们将产生和表征干细胞衍生的声带上皮细胞。分化的上皮细胞将在由原代人声带成纤维细胞（VFF）填充的sECM上生长。将确定培养条件以促进工程化LP的上皮形成。在目标3中，我们将制造自振荡组织构建体，其由支撑在无细胞合成水凝胶上的负载VFF的sECM组成，其几何形状和力学反映了声带肌肉的几何形状和力学。将在标准细胞培养条件下维持的构建体定期转移至振荡生物反应器或机械刺激。在工程化的声音折叠模拟微环境下，VFF将主动重塑合成环境，分泌天然基质组分，并与系留的上皮细胞通讯以建立有粘性和功能性的组织。总的来说，拟组织合成基质、多能干细胞和振动培养装置的组合为可靠和可行的声带组织模型的工程化提供了令人兴奋的机会。

 DESCRIPTION (provided by applicant): 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. The key to this great mechanical versatility lie in the unique structure and composition of the tissue. Each vocal fold consists of a pliable vibratory layer of connective tissue, known as the lamina propria (LP), sandwiched between a muscle and an epithelial layer. The structure and mechanics of the LP change gradually from the muscle to the epithelium. Numerous environmental, mechanical and pathological factors can damage this delicate tissue, resulting in a wide spectrum of voice disorders that affect millions o Americans. Current treatment options for vocal fold disorders are limited, and the development of new procedures has been slow owing to the inaccessibility of the tissue, its susceptibility to damage, and the anatomical differences of animal models from the human tissue. 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. The central hypothesis of the proposed work is that vocal fold-mimetic synthetic extracellular matrices (sECMs) displaying a layered and gradient structure with tissue- like anisotropy will provide the resident cells with guidance cues for the establishment of appropriate tissue structures. The initial template effects from the sECMs will be further reinforced by the application of physiologically relevant vibratory stimulations, ultimately producing a viable and functional vocal fold tissue model. In Aim 1, we will create sECMs using modular building blocks and employing a rapid bioorthogonal reaction at well-defined interfaces. The resultant sECM will consist of a bottom fibrous layer, a basement membrane-like top layer and a middle gel layer with a gradient of crosslinking density and biochemical signals. In Aim 2, we will produce and characterize stem cell-derived vocal fold epithelial cells. The differentiated epithelial cells will be grown on sECMs populated by primary human vocal fold fibroblasts (VFFs). Culture conditions will be identified to foster the epithelialization of the engineered LP. In Aim 3, we will fabricate a self- oscillating tissue construct, consisting of the VFF-laden sECM supported on a cell-free synthetic hydrogel with geometry and mechanics reflecting that of the vocal fold muscle. The construct, maintained under standard cell culture conditions, will be regularly transferred to an oscillatory bioreactor or mechanical stimulations. Under the engineered, vocal fold-mimetic microenvironment, VFFs will actively remodel the synthetic environment, secrete native matrix components, and communicate with the tethered epithelial cells to establish a cohesive and functional tissue. Overall, the combination of tissue-mimetic synthetic matrix, pluripotent stem cells and a vibratory culture device offers an exciting opportunity for the engineering of reliable and viable vocal fold tissue models.