Engineering Functional Salivary Glands Using Micropatterned Scaffolds

描述（由申请人提供）：数百万人患有口腔干燥症，或因缺乏唾液而导致的“口干”，导致多种症状导致生活质量下降，包括龋齿增加、口咽感染、吞咽困难（吞咽困难）和消化困难（粘膜炎）、味觉丧失和疼痛。由于目前对这些问题的治疗是不够的（1），有相当大的兴趣，创造一个人工唾液腺。体外维持唾液腺泡细胞的分化和功能是成功构建此类构建体的关键;然而，尚未实现这一突破。这主要是由于目前缺乏关于维持或诱导腺泡细胞分化所需的特异性细胞外信号的基础科学知识， 这在工程化功能性人工唾液腺的能力方面仍然是一个实质性的限制。需要体外测定系统来鉴定所需的细胞外信号。我们假设，通过3D支架中的微尺度图案化呈现给细胞的化学修饰的纳米纤维的组合将支持唾液腺泡细胞功能。本申请 提出了一个创新的，跨学科的战略，以建立一个高通量，非侵入性的测定系统，将用于感测唾液腺泡细胞分泌功能的活细胞生长 在支架材料上，这在以前是不可能的。一种基于MEMS的唾液传感器将被生产出来，用于精确测量在独特的支架组合上生长的细胞分泌的唾液分泌蛋白的浓度。一种新的多重免疫细胞化学方法将用于评估生物传感器测定后固定细胞的分化程度。使用创新的方法组合，纳米形貌，微图案化和化学信号将被独立调制，这样将确定支持腺泡细胞功能的参数的特定组合。将使用原代细胞和细胞系的组合评估支架维持腺泡分化或促进再分化的能力。我们将在四个具体的目标解决这个假设：目标1：开发一个高通量的MEMS探针阵列检测系统，以评估腺泡细胞的功能在体外。目的2：确定支持唾液腺泡细胞分化的理想的细胞构型。目的3：优化生物膜表面性质以增强涎腺腺泡细胞功能。目的4：确定一个最佳的微地形，以促进腺泡细胞功能。本申请中产生的体外唾液腺构建体将用于鉴定调节腺泡功能的途径，并且可以 用于药物筛选。由于这种应用而开发的原理将在未来应用于设计用于复制唾液腺和其他复杂分支器官的人工腺体的工程。

DESCRIPTION (provided by applicant): Millions of people suffer from xerostomia, or "dry mouth" resulting from lack of saliva, causing a decreased quality of life resulting from multiple symptoms, including increased dental caries, oropharyngeal infections, difficulties with swallowing (dysphagia) and digestion (mucositis), loss of taste, and pain. As current treatments for these problems are inadequate (1), there is considerable interest in creating an artificial salivary gland. Maintenance of salivary acinar cell differentiation and function in vitro is criticl to the successful engineering of such constructs; however, this breakthrough has not yet been achieved. This is primarily due to the current lack of basic scientific knowledge regarding the specific extracellular signals that are required to maintain or induce acinar cell differentiation, which remains a substantial limitation in the ability to engineer a functional artificial salivary gland. An in vitro assay system is needed to identify required extracellular signals. We hypothesize that the combination of chemically-modified nanofibers presented to cells via microscale patterning in a 3D scaffold will support salivary acinar cell function. This application proposes an innovative, interdisciplinary strategy to create a high-throughput, non-invasive assay system that will be used to sense salivary acinar cell secretory function in live cells grown on scaffold materials, which has not been previously possible. A MEMS-based saliva sensor will be produced to accurately measure the concentration of a salivary secretory protein secreted by cells grown on unique scaffold combinations. A novel multiplexed immunocytochemistry method will be used to assess the extent of differentiation in fixed cells following the biosensor assay. Using an innovative combination of methods, nanotopography, micropatterning, and chemical signaling will be independently modulated, such that specific combinations of parameters will be identified that support acinar cell function. Scaffolds will be assessed for their ability to maintin acinar differentiation or promote re-differentiation using a combination of primary cells and cell lines. We will address this hypothesis in four specific aims: Aim 1: Develop a high-throughput MEMS probe array assay system to evaluate acinar cell function in vitro. Aim 2: Identify the ideal nanofiber configuration to support salivary acinar cell differentiation. Aim 3: Optimize nanofiber surface properties to enhance salivary gland acinar cell function. Aim 4: Identify an optimal microtopography to promote acinar cell function. The in vitro salivary gland construct produced in this application will be useful to identify pathways regulating acinar function and can be applied for drug screening. The principles developed as a result of this application will be applied in the future towards engineering of artificial glands designed to replicate salivary gland and other complex branching organs.