Bottom-Up Assembly of Functional Salivary Gland Tissues

项目摘要 尽管治疗策略取得了进展，但口干症（或口干）仍然是一种永久性和破坏性的疾病 放射治疗对头颈部癌症的影响，降低了约50，000名癌症患者的生活质量 我们的目标是开发组织工程方法来恢复唾液功能。我们已经分离出 人唾液腺干/祖细胞（hS/PC）。我们创造了 可调节的水凝胶基质，其维持祖细胞状态，诱导谱系特异性分化并促进 从分散的hS/PC发育成有组织的多细胞球体。另外，我们设计了 在hS/PC衍生的腺泡样核心之间表现出协调的钙激活的唾液腺微组织 以及周围的肌上皮细胞然而，一个有广泛分支的功能性腺体，极化腺泡， 并且互连的管道还没有实现。在这里，我们提出了一个自下而上的方法来建立 使用具有确定形状、几何形状和组成的多细胞组装体的功能性唾液腺。我们将 合成概括基底膜和间质基质关键特征的水凝胶支架 发育中的器官。我们将重组血管，神经和间质成分， 在一些实施方案中，本发明提供了一种工程化环境，以在体外促进组织形态发生并在体内维持组织稳态。在 目的1，我们将开发四嗪连接，生物正交和高效的s- 四嗪和应变烯烃，用于建立细胞指导矩阵。我们将调整我们现有的 制备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 使用非粘附性水凝胶微孔产生由hS/PC组成的多细胞上皮组装体 和CCh仓库。所产生的微组织将被包裹在具有生物活性的合成基底膜中， 肽以刺激前分泌腺祖细胞表型的发育。我们将在血管内 由人唾液腺内皮细胞（hSEC）的核心和人唾液腺内皮细胞（hSEC）的外壳组成的微组织 间充质干细胞（hMSCs）。我们将在一个人工合成的 细胞外基质与确定的细胞指导线索，以帮助建立一个分层整合的 组织组装。在目标3中，将具有整合的微血管和结合的神经营养素的工程化腺体 将因子Neurturin植入切除的无胸腺大鼠腮腺床中。酶促触发释放 Neurturin将促进植入物神经支配。组织超微结构，生物标志物表达，腺体形态， 将在各种结构配置下评估生物整合和功能。我们将审问 工程微环境刺激分化，触发极化和促进分支。整体 假设在3D合成基质中与hSEC/hMSC共培养的hS/PC显示生物化学， 从天然器官识别的几何和机械线索将组装成功能性唾液组织。 我们的调查将有助于确定生物工程方法对口干症的管理。

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.