Bilayered Vocal Fold Tissue Engineering

描述(由申请人提供)： 因创伤或喉癌而失去喉部声带覆盖的退伍军人可能会患上致残嗓音困难，治疗选择有限。振动替代组织将彻底改变喉部疾病的治疗。本提案中描述的工作描述了组织工程声带覆盖(TE-VFC)的特征和优化。从脂肪组织中分离的成人干细胞在从血液产品冷沉淀物中提取的纤维蛋白水凝胶中培养。其结果是一种完全自体的三维组织替代品。这种声带组织工程方法的一个重要创新是复制了负责自然声带覆盖层振动的两层(上皮和间充质)。该模型将用于系统地研究组织工程学、干细胞分化和声带生理学的基本问题。它是可控的，具有最接近复制正常发育条件并产生双层上皮和间充质分化的“优化”病例。“对照”病例更典型的是标准的组织培养条件，并产生无序的细胞分化。提出了三个研究部门。首先，用免疫组织化学和原位杂交方法检测TE-VFC和非上皮化对照细胞外基质和基底膜的沉积情况。上皮屏障功能是通过跨上皮扩散来衡量的。研究了两种情况下ECM重塑不同的机制，如基质金属蛋白酶的分泌和前胶原基因的表达。第二，间充质细胞在这两种情况下的表型是通过蛋白表达和收缩能力来定义的。研究了控制表型的潜在信号机制，包括EGF受体和转化生长因子β1。最后，对这两种情况以及去上皮化的TE-VFC的振动和发声进行了评估，以确定上皮在发声中的作用。这些构造附着在切除的喉的声带韧带上，模拟植入条件，并用高速成像记录气流诱导的振动。这项拟议研究的成功完成将通过比较上皮化和对照病例中的细胞表型、微结构和功能，确定在发育中的组织结构中包括上皮细胞是否有好处。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。这一发现将适用于其他上皮化组织以及声带置换手术。最后，这些对TE-VFC的体外研究将为继续进行动物和人类植入试验治疗严重的声带瘢痕提供必要的信息。

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.