Extracellular Scaffold Elasticity and Binding Sites in Acinar Differentiation

描述（由申请人提供）：目前组织工程领域面临的最重大挑战之一是在工程组织中刺激和/或维持上皮细胞分化的能力。由于上皮细胞分泌功能对器官功能至关重要，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。人造功能分泌唾液的唾液腺结构将大大提高唾液腺功能减退患者的生活质量，但这种工程组织尚未产生，体外唾液腺泡分化仍然难以维持。细胞微环境在细胞分化中起着重要的作用，但关于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，组织工程中最有效的支架是从活组织中提取的脱细胞支架。由于组织工程的目标是能够合成比去细胞化的天然支架性能更好的支架，因此有必要了解天然细胞外基质的基本特征（化学、机械/弹性和拓扑特性）如何影响细胞分化。最近的研究已经确定了微环境弹性在决定间充质干细胞分化程度方面的重要性；然而，弹性在上皮组织分化调控中的意义尚未被研究。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，但化学信号与弹性之间的关系在很大程度上仍然未知。本项目的总体目标是确定底物弹性和细胞结合位点密度在调节下颌唾液腺（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，使用新型可调聚乙二醇水凝胶支架来解决这一问题。我们假设腺泡细胞分化需要一种具有最佳细胞结合位点的细胞外基质，这种基质在非典型底物刚性时被破坏。为了解决这一假设，我们提出在三个具体目标中使用可调聚乙二醇（PEG）基水凝胶：目标1。开发含有不同结合位点的不同弹性的聚乙二醇基水凝胶。目标2。确定弹性和细胞组织在使用水凝胶支架调节腺泡细胞分化中的作用。目标3。使用双层光刻技术创建用于原代细胞的微孔支架。缩写：AFM，原子力显微镜；Col IV，胶原IV型；ECM，细胞外基质；绿色荧光蛋白；IKVAV Isoleucine-Lysine-Valine-Alanine-Valine;聚乙二醇（PEG）；聚合酶链式反应；PEG-DMA PEG-dimethylacrylate;PEG-TMA PEG-trimethylacrylate;氧酰-2-甲基丙烯酸甲酯；波莫人,2 - ((prop-2-ynyloxy)甲基)环氧乙烷;SMG，下颌下唾液腺；上皮阻力，TER

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.