Tissue engineering a salivary biological system

这项工作的重要性在于每年有50万患有放疗后遗症的新头颈癌患者以及全球2.5亿干燥综合征患者。这些患者经历相当大的发病率和不适，例如口干、蛀牙、口腔感染和食物摄入减少。当唾液细胞被纤维化组织取代时，目前没有可用的适当治疗。目前在组织、细胞和分子水平上对人类唾液腺（SG）的了解来自动物模型、细胞系和患者活检，尽管它们很重要，但它们只能提供关于人类SG如何在健康和疾病期间实时发挥功能和修复的间接证据。构建SG组织的挑战是扩增分泌液体的细胞并为它们提供支撑支架。我们5年研究的主要目标是设计一个健康的生理唾液系统，以体外研究人类SG功能。我们提出三个具体目标。预期的结果是在选定的细胞组织水平上提供工程化人类唾液组织的离体模型，这也将有利于生理学、放射生物学、干细胞、药物筛选、细胞/组织工程和生物材料的研究。我们的长期目标是为严重口干患者设计/组装SG。 目的1：人类唾液类器官的扩增。我们最近表明，回收的组织支架可以用作“天然支架”。有趣的是，我们还发现，温和的酶消化导致3D唾液类器官保留其功能，增殖和基底膜。我们假设，通过在类器官周围保持最佳的3D构型和基质蛋白，腺泡细胞将有效地扩增。我们将用不同的酶类型、组合和浓度来测试这一假设，以优化细胞增殖和类器官大小，然后将它们接种在附着在显微镜载玻片上的天然支架上，或接种在人造3D凝胶中，用于延时研究。 目的2：离体SG组织模型。我们最近建立了一个人SG三维器官型切片培养模型（50-100 μ m厚）与一个完整的天然细胞外基质（ECM），保持腺泡功能14天。我们将使用该组织模型来测试移植的唾液细胞亚群是否可以整合/组装成完整的天然组织。共聚焦显微镜将跟踪细胞迁移和植入，而药物刺激试验将测量液体和蛋白质分泌。 目标3：生物打印3D唾液构建体。我们最近发现半透明的“蛋黄血浆”（EYP）作为一种廉价的凝胶，但有效地重现体内条件。我们将测试微调EYP物理特性（刚度，凝胶化）允许快速3D打印并选择性定位SG细胞的假设。延时显微镜将评估人SG细胞扩增。

The importance of this work is for the half-million of new head/neck cancer patients yearly with radiotherapy sequelae and also for 250 million Sjögren's syndrome patients worldwide. These patients experience considerable morbidity and discomfort such as dry mouth, tooth decay, oral infection and reduced food intake. There is currently no available adequate treatment when salivary cells are replaced by fibrotic tissue. Current knowledge on human salivary gland (SG) at the tissue-, cellular- and molecular-level comes from animal models, cell lines and patient biopsies, which despite their importance can only provide indirect evidence on how human SG can function and repair in real-time during health and disease. The challenge in building SG tissues is to expand fluid-secreting cells and to supply them with a supporting scaffold. The principal goal of our 5-year research is to engineer a healthy physiological salivary system to study human SG function ex vivo. We propose 3 specific aims. The expected outcome is to offer ex vivo models of engineered human salivary tissue at selected cellular organizational levels that will also benefit research in physiology, radiation biology, stem cell, drug screening, cell/ tissue engineering, and biomaterials. Our long-term goal is to engineer/ assemble a SG for patients with severe dry mouth. Aim 1: Expansion of human salivary organoids. We recently show that recycled tissue digests can be used as “native scaffolds”. Interestingly, we also uncover that gentler enzymatic digestion result in 3D-salivary organoids that preserve their function, proliferation and basement membrane. We hypothesize that, by keeping an optimal 3D-configuration and matrix protein around organoids, acinar cells will expand efficiently. We will test this hypothesis with different enzymatic types, combinations and concentrations to optimize cell proliferation and organoid size, and then seed them on native scaffolds attached to microscope slides, or in human-made 3D-gels, for time-lapse studies. Aim 2: Ex vivo SG tissue model. We recently establish a human SG 3D-organotypic slice culture model (50-100 um thick) with an intact native extracellular matrix (ECM) that maintains acini function for 14 days. We will use this tissue model to test if transplanted subpopulations of salivary cells can integrate/ assemble into an intact native tissue. Confocal microscopy will track cell migration and engraftment while drug-stimulated assays will measure fluid and protein secretion. Aim 3: Bioprinting a 3D salivary construct. We recently uncover translucent “egg yolk plasma” (EYP) as an inexpensive gel, yet effective in recapitulating in-vivo conditions. We will test the hypothesis that fine tuning EYP physical properties (stiffness, gelation) allows to 3D print rapidly and position selectively SG cells. Time-lapse microscopy will evaluate human SG cells expansion.