Extracellular Scaffold Elasticity and Binding Sites in Acinar Differentiation

描述(申请人提供)：组织工程领域目前面临的最大挑战之一是刺激和/或维持工程化组织中的上皮细胞分化的能力。由于上皮细胞的分泌功能对器官功能至关重要，因此了解调节和维持细胞分化的机制对于再生或工程功能组织至关重要。人工构建功能性唾液腺将极大地提高唾液功能低下患者的生活质量，但此类工程化组织尚未产生，唾液腺体外分化仍难以维持。细胞微环境在细胞分化中起着重要作用，但对调节细胞分化的微环境的具体特征知之甚少。工程支架往往不能模拟微环境，事实上，最有效的组织工程支架是从活组织中提取的脱细胞支架。由于组织工程学的目标是能够合成性能优于脱细胞天然支架的支架，因此有必要了解天然细胞外基质的基本特性(化学、机械/弹性和拓扑性质)如何影响细胞分化。最近的研究已经确认了微环境的弹性在决定间充质干细胞分化程度方面的重要性，然而，弹性在调节上皮组织分化中的意义还没有被研究。化学信号，包括生长调节因子和结合位点，已经得到了更广泛的研究，但化学信号和弹性之间的关系在很大程度上仍不清楚。本项目的总体目标是确定底物弹性和细胞结合部位密度在调节颌下腺(SMG)腺泡细胞分化中的作用。我们将使用细胞系和胚胎原代细胞来解决这一目标，使用新型可调的聚乙二醇水凝胶支架。我们假设腺泡细胞的分化需要一个顺应性的细胞外基质，该基质具有最佳的细胞结合部位，这种结合部位在非典型的底物刚性下被破坏。为了解决这一假设，我们建议在三个特定目标中使用可调的聚乙二醇基水凝胶：目的1.开发具有不同弹性的包含不同结合位置的聚乙二醇基水凝胶。目的2.利用水凝胶支架，确定弹性和细胞组织在调节腺泡细胞分化中的作用。目的3.使用双层光刻技术制作微孔支架，用于原代细胞。缩写：AFM，原子力显微镜；Col IV，IV型胶原蛋白；ECM，细胞外基质；GFP，绿色荧光蛋白；IKVAV，异亮氨酸-赖氨酸-丙氨酸-丙氨酸-Valine；聚乙二醇；聚合酶链式反应；聚乙二醇二甲酯；聚乙二醇三甲基丙烯酸酯；聚乙二醇三甲基丙烯酸酯；OMMA，甲基丙烯酸氧乙烷；POMO，2-(丙二酰氧基)甲基)环氧乙烷；SMG，颌下唾液腺；跨上皮阻力， 公共卫生相关性：该项目将确定细胞支架的必要弹性和细胞结合特性，以促进腺泡分化。这些结果将为未来在未来的提案中确定影响腺泡生成的其他因素提供一个框架。这些研究最终将使设计一种人工支架成为可能，以缓解干燥综合征患者的症状，以及其他导致唾液功能减退或口干的原因。

DESCRIPTION (provided by applicant): One of the most significant challenges currently facing the field of tissue engineering is the ability to stimulate and/or maintain epithelial cell differentiation in engineered tissues. Since epithelial cell secretory function is crucial to organ function, understanding the mechanisms regulating and maintaining cellular differentiation is critical to regenerating or engineering functional tissues. A man-made functional saliva-secreting salivary gland construct would greatly increase the quality of life for patients suffering from salivary hypofunction, but such engineered tissues have yet to be generated, and in vitro salivary acinar differentiation remains difficult to sustain. The cellular microenvironment plays a significant role in cell differentiation, and yet little is known regarding the specific characteristics of the microenvironment that regulate cell differentiation. Engineered scaffolds often fail to mimic the microenvironment and, in fact, the most effective scaffolds for tissue engineering are decellularized scaffolds derived from live tissue. Since the goal of tissue engineering is to be able to synthesize scaffolds that out-perform decellularized natural scaffolds, it is necessary to understand how the essential characteristics of the natural extracellular matrix (chemical, mechanical/elastic, and topological properties) affect cell differentiation. Recent studies have identified the importance of elasticity of the microenvironment in determining the extent of differentiation of mesenchymal stem cells; however, the significance of elasticity in regulation of epithelial tissue differentiation has not been investigated. Chemical signals, including growth regulatory factors and binding sites, have been much more extensively studied, but the relationship between chemical signals and elasticity remains largely unknown. The overall aim of this project is to define the function of substrate elasticity and cell binding site density in regulating submandibular salivary gland (SMG) acinar cell differentiation. We will use cell lines and embryonic primary cells to address this aim using novel tunable PEG hydrogel scaffolds. We hypothesize that acinar cell differentiation requires a compliant extracellular matrix having optimal cell binding sites which is disrupted at atypical substrate rigidities. To address this hypothesis, we propose to use tunable polyethylene-glycol (PEG)-based hydrogels in three specific aims: Aim 1. Develop PEG-based hydrogels of varied elasticity containing different levels of binding sites. Aim 2. Identify the contributions of elasticity and cell organization in modulating acinar cell differentiation using the hydrogel scaffolds. Aim 3. Use bilayer lithography to create microwell scaffolds for use with primary cells. Abbreviations: AFM, atomic force microscopy; Col IV, collagen type IV; ECM, extracellular matrix; GFP, green fluorescent protein; IKVAV, Isoleucine-Lysine-Valine-Alanine-Valine; PEG, poly(ethylene glycol); PCR, polymerase chain reaction; PEG-DMA, PEG-dimethylacrylate; PEG-TMA, PEG-trimethylacrylate; OMMA, oxiran-2-ylmethyl methacrylate; POMO, 2-((prop-2-ynyloxy)methyl)oxirane; SMG, submandibular salivary gland; transepithelial resistance, TER PUBLIC HEALTH RELEVANCE: This project will identify the necessary elastic and cell binding characteristics of a cellular scaffold to promote acinar differentiation. These results will provide a framework for future identification of other factors that influence acinar generation in a future proposal. These studies will eventually make possible the engineering of an artificial scaffold to alleviate symptoms in patients suffering from Sjogren's syndrome and other causes of salivary hypofunction, or dry mouth.