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.