Bilayered Vocal Fold Tissue Engineering

描述（由申请人提供）： 退伍军人遭受损失的喉的声带覆盖由于创伤或喉癌可能会遭受残疾的声音困难，治疗方案是有限的。振动替代组织将彻底改变喉部疾病的治疗。在这个建议中描述的工作的特点和优化组织工程声带覆盖（TE-VFC）。将从脂肪组织分离的成人干细胞在来源于血液产品冷沉淀物的纤维蛋白水凝胶内培养。结果是一个完全自体的三维组织替代品。这种方法的一个重大创新声带组织工程是同步复制的两个层（上皮和间充质）负责振动的天然声带覆盖。 该模型将被用来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，具有最接近地复制正常发育条件并产生双层上皮和间充质分化的“优化”情况。“对照”情况更典型的是标准组织培养条件，并产生无组织的细胞分化。 提出了三个研究方向。首先，使用免疫组织化学和原位杂交在TE-VFC和非上皮化对照中测定细胞外基质和基底膜沉积。通过跨上皮扩散测量上皮屏障功能。研究了两种情况下ECM重塑差异的机制，如基质金属蛋白酶分泌和前胶原mRNA。其次，间充质细胞表型在两种条件下通过蛋白质表达和收缩性来定义。研究了控制表型的潜在信号传导机制，包括EGF受体和TGF-β 1。最后，振动和发声进行评估，在这两种情况下，以及在去上皮TE-VFC，以确定上皮细胞在语音生产中的作用。将该结构连接到切除的喉的声带韧带上，模拟植入条件，用高速成像记录气流诱导的振动。 成功完成拟议的研究将通过比较上皮化和对照病例中的细胞表型、微观结构和功能，确定在发育中的组织结构中包括上皮是否有益。它还将确定上皮细胞影响相邻细胞表型和ECM重塑的机制，在受控系统中。这些发现与其他上皮化组织以及声带替换相关。最后，TE-VFC的这些体外研究将为进行动物和人体植入试验以治疗严重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.