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.

Project Narrative This project seeks to develop an alternative treatment method for xerostomia or dry mouth, a devastating side effect of radiation therapy that affects 68-85% of head and neck cancer patients. We will develop a bottom-up approach to recreate a functional salivary gland using patient-derived cells with regenerative potential and artificial extracellular matrices that recapitulate the native microenvironments of the developing organ. The goal is to produce an engineered construct with branched architecture consisting of secretory acini and connected ducts.