Bilayered Vocal Fold Tissue Engineering

双层声带组织工程

• 批准号: 8659970
• 负责人: Jennifer L Long
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8659970
• 关键词: Acids; Adipose tissue; Animals; Autologous; Basement membrane; Breathing; Cell Differentiation process; Cells; Chemicals; Chronic; Cicatrix; Clinical; Collagen; Communication impairment; Deposition; Development; Diffusion; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Epithelium; Extracellular Matrix; Fatigue; Fibrin; Head and Neck Cancer; Human; Hydrogels; Image; Immunohistochemistry; In Situ Hybridization; In Vitro; Injury; Laryngeal Diseases; Larynx; Ligaments; Malignant neoplasm of larynx; Matrix Metalloproteinases; Measures; Membrane Protein Gene; Mesenchymal; Mesenchymal Differentiation; Messenger RNA; Modeling; Occupations; Operative Surgical Procedures; Outcome; Phenotype; Phonation; Physiology; Preparation; Production; Radiation therapy; Reflux; Research; Role; Signal Transduction; Simulate; Smoke; Social Interaction; Speed; Structure; System; Tissue Engineering; Tissues; Trauma; Veterans; Voice; Voice Quality; Work; arm; blood product; case control; clinical application; human adult stem cell; implantation; in vivo; innovation; pre-clinical; protein expression; public health relevance; research study; sound; stem cell differentiation; tissue culture; vibration; vocal cord

DESCRIPTION (provided by applicant): Veterans suffering loss of the larynx's vocal fold cover due to trauma or laryngeal cancer can suffer disabling voice difficulties, and treatment options are limited. A vibrating replacement tissue would revolutionize the treatment of laryngeal disorders. The work described in this proposal characterizes and optimizes a tissue-engineered vocal fold cover (TE-VFC). Adult human stem cells isolated from adipose tissue are cultured within fibrin hydrogel derived from the blood product cryoprecipitate. The result is a completely autologous three- dimensional tissue substitute. A significant innovation of this approach to vocal fold tissue engineering is th concomitant replication of the two layers (epithelial and mesenchymal) responsible for vibration in the native vocal fold cover. This model will be employed to systematically study fundamental issues of tissue engineering, stem cell differentiation, and vocal fold physiology. It is controllable, with an "optimized" case that most closely replicates normal developmental conditions and produces bilayered epithelial and mesenchymal differentiation. A "control" case is more typical of standard tissue culture conditions, and produces disorganized cell differentiation. Three research arms are proposed. First, extracellular matrix and basement membrane deposition are determined in the TE-VFC and the non- epithelialized control using immunohistochemistry and in situ hybridization. Epithelial barrier function is measured by trans-epithelial diffusion. Mechanisms for a difference in ECM remodeling between the two cases are investigated, such as matrix metalloproteinase secretion and pro-collagen mRNA. Second, mesenchymal cell phenotype is defined in the two conditions by protein expression and contractility. Potential signaling mechanisms controlling the phenotype are investigated, including EGF receptor and TGF- ¿1. Finally, vibration and phonation are assessed in both cases as well as in de-epithelialized TE-VFC to identify the role of epithelium in voice production. The constructs are attached to the vocal ligaments of excised larynges simulating implantation conditions, and airflow-induced vibration is recorded with high-speed imaging. Successful completion of the proposed research will determine whether including an epithelium in a developing tissue construct offers benefit, by comparing the cell phenotype, microstructure, and function in epithelialized and control cases. It will also identify mechanisms by which epithelial cells influence adjacent cell phenotype and ECM remodeling, in a controlled system. Findings will be relevant in other epithelialized tissues as well as this vocal fold replacement. Finally, these in vitro studies of the TE-VFC will provide necessary information to proceed with animal and human implantation trials to treat severe vocal fold scarring.

描述（由申请人提供）： 由于创伤或喉癌而导致的喉部声褶皱盖的退伍军人丧失了声音难度，并且治疗选择受到限制。振动替代组织将彻底改变喉部疾病的治疗。该提案中描述的工作表征并优化了组织工程的声带覆盖（TE-VFC）。从脂肪组织中分离出的成年人类干细胞在源自血液产物冷沉淀的纤维蛋白水凝胶中培养。结果是完全自体的三维组织替代品。这种对声带组织工程的方法的重大创新是，两层（上皮和间质）的复制是对天然声带盖中振动的负责。该模型将用于系统地研究组织工程，干细胞分化和声带生理学的基本问题。它是可控的，具有“优化”的情况，最紧密地复制正常的发育条件并产生双层上皮和间质分化。 “控制”病例更典型地是标准组织培养条件的典型，并产生混乱的细胞分化。提出了三个研究臂。首先，使用免疫组织化学和原位杂交在TE-VFC和非上皮化对照中确定细胞外基质和基底膜沉积。上皮屏障功能是通过跨上皮差异测量的。研究了两种情况之间ECM重塑差异的机制，例如基质金属蛋白酶分泌和促脂蛋白mRNA。其次，间充质细胞表型是通过蛋白质表达和收缩力在两种条件下定义的。研究了控制表型的潜在信号传导机制，包括EGF受体和TGF-�1。最后，在两种情况下以及在脱皮的TE-VFC中都评估了振动和发音，以识别上皮上皮在语音产生中的作用。这些构建体连接到模拟植入条件的切除的喉部的声带韧带，并通过高速成像记录气流诱导的振动。拟议研究的成功完成将确定通过比较细胞表型，微观结构和功能在上皮化和控制案例中的功能，是否在开发组织构建体中包括上皮。它还将确定上皮细胞在受控系统中影响相邻细胞表型和ECM重塑的机制。发现将与其他上皮组织以及这种声带更换有关。最后，这些对TE-VFC的体外研究将提供必要的信息，以进行动物和人植入试验，以治疗严重的人声折叠疤痕。