Bottom-Up Assembly of Functional Salivary Gland Tissues

项目摘要 尽管治疗策略取得了进步，口干症(或口干)仍然是一个永久性和破坏性的副作用。 放射治疗头颈部癌症的效果，降低了约5万名癌症患者的生活质量 我们的目标是开发组织工程学方法来恢复唾液功能。我们已经隔离了 放射治疗前患者的唾液腺干细胞/祖细胞(HS/PC)。我们已经创造了 可调节的水凝胶基质，维持祖细胞状态，诱导谱系特异性分化并促进 从分散的HS/PC发展成有组织的多细胞球体。另外，我们已经设计了 唾液腺微组织在HS/PC来源的腺泡样核之间显示协调的钙激活 以及周围的肌上皮细胞。然而，功能性腺体具有广泛的分枝，两极化的腺泡， 而且互联管道还没有实现。在这里，我们提出一种自下而上的方法来建立 功能性唾液腺使用定义的形状、几何形状和组成的多细胞组件。我们会 合成能够概括基底膜和间质基质关键特性的水凝胶支架 在发育中的器官中。我们将重建血管、神经和间充质成分 在体外培养组织形态发生和在体内维持组织动态平衡的工程环境。在……里面 目的1，我们将利用四嗪连接，S和黄杨之间的生物正交高效环加成反应。 四嗪和张力烯烃，用于建立细胞指导基质。我们将调整我们现有的 方法制备含有隔离乙酰胆碱类似物卡巴胆碱(CCH)的微凝胶。在目标2中，我们将 利用非粘附性水凝胶微孔制备由HS/PC组成的多细胞上皮组件 和CCH仓库。生成的微组织将被包裹在具有生物活性的合成基底膜中。 刺激前胸腺祖细胞表型发育的多肽。我们将产生内皮细胞 由人唾液腺内皮细胞(HSECs)为核心和人的外壳组成的显微组织 间充质干细胞。我们将在人工合成的细胞中共培养上皮和内皮微组织 具有定义的细胞引导提示的细胞外基质，以帮助建立分层整合的 组织组装。在目标3中，具有整合的微血管和结合神经营养的工程化腺体 将在切除的无瘤大鼠腮腺床内植入神经突蛋白因子。酶促释放 神经突触素将促进种植体的神经支配。组织超微结构，生物标志物表达，腺体形态， 生物整合和功能将在不同的结构配置下进行评估。我们将审问一下 工程化的微环境刺激分化，触发极化，促进分支。整体而言 假设HS/PC与hSECs/hMSCs在3D合成基质中共培养，显示生化， 从本地器官中识别出的几何和机械线索将组装成具有功能的唾液组织。 我们的研究将有助于确定治疗口干症的生物工程方法。

Project Summary Despite advances in treatment strategies, xerostomia (or dry mouth) remains a permanent and devastating side effect of radiotherapy for head and neck cancers, reducing the quality of life for ~50,000 cancer patients each year in the U.S. We aim to develop tissue-engineering approaches to restore salivary function. We have isolated human salivary gland stem/progenitor cells (hS/PCs) from patients prior to radiotherapy. We have created tunable hydrogel matrices that maintain the progenitor status, induce lineage-specific differentiation and promote the development of organized multicellular spheroids from dispersed hS/PCs. Separately, we have engineered salivary gland microtissues that exhibit coordinated calcium activation between hS/PC-derived acini-like core and the surrounding myoepithelial cells. However, a functional gland with extensive branching, polarized acini, and interconnected ducts has not yet been realized. Here, we propose a bottom-up approach to establish functional salivary glands using multicellular assemblies of defined shape, geometry and composition. We will synthesize hydrogel scaffolds that recapitulate key features of the basement membrane and the interstitial matrix in the developing organ. We will reconstitute the vascular, neural and mesenchymal components in the engineered environment to foster tissue morphogenesis in vitro and to maintain tissue homeostasis in vivo. In Aim 1, we will exploit tetrazine ligation, the bioorthogonal and highly efficient cycloaddition reaction between s- tetrazine and strained alkenes, for the establishment of cell-instructive matrices. We will adapt our established methods to generate microgels containing sequestered acetylcholine analog, carbachol (CCh). In Aim 2, we will employ non-adhesive hydrogel microwells to produce multicellular epithelial assemblies consisting of hS/PCs and CCh depots. The resultant microtissue will be encased in a synthetic basement membrane with bioactive peptides to stimulate the development of proacrinar progenitor phenotype. We will generate endothelial microtissues consisting of a core of human salivary gland endothelial cells (hSECs) and a shell of human mesenchymal stem cells (hMSCs). We will co-culture the epithelial and endothelial microtissues in a synthetic extracellular matrix with defined cell-guidance cues to aid in the establishment of a hierarchically integrated tissue assembly. In Aim 3, the engineered gland with integrated microvasculature and conjugated neurotrophic factor, neurturin, will be implanted in the resected parotid bed of athymic rats. Enzymatically triggered release of neurturin will promote implant innervation. Tissue ultrastructure, biomarker expression, gland morphology, biointegration and function will be assessed under various construct configurations. We will interrogate how the engineered microenvironments stimulate differentiation, trigger polarization and promote branching. The overall hypothesis is that hS/PCs co-cultured with hSECs/hMSCs in 3D synthetic matrices displaying biochemical, geometrical and mechanical cues identified from the native organs will assemble into functional salivary tissues. Our investigations will help define bioengineering approaches toward the management of xerostomia.