Bottom-Up Assembly of Functional Salivary Gland Tissues

项目概要 尽管治疗策略取得了进步，口干症（或口干症）仍然是一个永久性的、具有破坏性的问题 放射治疗对头颈癌的影响，降低了约 50,000 名癌症患者的生活质量 在美国，我们的目标是开发组织工程方法来恢复唾液功能。我们已经隔离了 放疗前取自患者的人类唾液腺干/祖细胞 (hS/PC)。我们已经创建了 可调节的水凝胶基质可维持祖细胞状态，诱导谱系特异性分化并促进 从分散的 hS/PC 发展有组织的多细胞球体。我们分别设计了 唾液腺微组织在 hS/PC 衍生的腺泡样核心之间表现出协调的钙激活 以及周围的肌上皮细胞。然而，具有广泛分支、极化腺泡的功能性腺体， 和互连管道尚未实现。在这里，我们提出一种自下而上的方法来建立 使用具有确定形状、几何形状和成分的多细胞组件来实现功能性唾液腺。我们会 合成水凝胶支架，概括基底膜和间质基质的关键特征 在发育中的器官中。我们将重建血管、神经和间质成分 工程化环境可促进体外组织形态发生并维持体内组织稳态。在 目标 1，我们将利用四嗪连接，即 s- 之间的生物正交高效环加成反应 四嗪和应变烯烃，用于建立细胞指导基质。我们将调整我们既定的 产生含有螯合乙酰胆碱类似物卡巴胆碱（CCh）的微凝胶的方法。在目标 2 中，我们将 采用非粘附性水凝胶微孔生产由 hS/PC 组成的多细胞上皮组件 和 CCh 仓库。由此产生的微组织将被包裹在具有生物活性的合成基底膜中 刺激前腺祖表型发育的肽。我们将产生内皮细胞 由人唾液腺内皮细胞 (hSEC) 核心和人唾液腺内皮细胞外壳组成的微组织 间充质干细胞（hMSC）。我们将在合成的培养皿中共培养上皮和内皮微组织 具有明确的细胞引导线索的细胞外基质，有助于建立分层整合的细胞外基质 组织组装。在目标 3 中，工程化腺体具有集成的微脉管系统和结合的神经营养物质 神经营养因子将被植入无胸腺大鼠切除的腮腺床中。酶促释放 neurturin 将促进种植体神经支配。组织超微结构、生物标志物表达、腺体形态、 将在各种构建配置下评估生物整合和功能。我们将询问如何 工程微环境刺激分化、引发极化并促进分支。整体 假设 hS/PC 与 hSEC/hMSC 在 3D 合成基质中共培养，显示生化、 从天然器官中识别出的几何和机械线索将组装成功能性唾液组织。 我们的研究将有助于定义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.