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）： 由于外伤或喉癌而失去喉部声带覆盖的退伍军人可能会遭受发声困难，而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描述并优化了组织。 -工程声带盖（TE-VFC）。从脂肪组织中分离出的成人干细胞在源自血液产品冷沉淀物的纤维蛋白水凝胶中进行培养。这种声带组织工程方法的一个重大创新是同时复制负责天然声带覆盖层振动的两层（上皮层和间质层）。组织工程、干细胞分化和折叠发声生理学的基本问题是可控的，具有最接近地复制正常发育条件并产生双层上皮和间质分化的“优化”情况。 “对照”情况是标准组织培养条件下更典型的情况，并提出了三个研究方向：首先，使用免疫组织化学在 TE-VFC 和非上皮化对照中测定细胞外基质和基底膜沉积。原位杂交通过跨上皮扩散测量上皮屏障功能，研究了两种情况之间 ECM 重塑的差异机制，例如基质金属蛋白酶分泌和其次，间充质细胞表型是通过蛋白质表达和收缩性来定义的，研究了控制表型的潜在信号机制，包括 EGF 受体和 TGF-β。 1. 最后，对两种情况以及去上皮 TE-VFC 中的振动和发声进行评估，以确定上皮在发声中的作用。这些结构附着在模拟植入条件和气流的切除喉的声带韧带上。通过高速成像记录诱发的振动，成功完成拟议的研究将通过比较细胞表型、微观结构、确定在发育的组织结构中包含上皮是否有益。它还将在受控系统中确定上皮细胞影响邻近细胞表型和 ECM 重塑的机制，这些发现将与其他上皮组织以及声带替换相关。 TE-VFC 的体外研究将为动物和人体植入试验提供必要的信息，以治疗严重的声带疤痕。