Head and neck cancer affects over 64,000 Americans every year. Most of these patients receive radiation therapy as part of their standard treatment, which leads to irradiation-induced xerostomia (dry mouth). Xerostomia drastically impairs these patients’ quality of life. Our lab proposes to develop a functional gland, suitable for re-implantation, from salivary-derived human stem/progenitor cells (hS/PCs) isolated from the patient before radiation therapy. Biocompatible hyaluronic acid (HA)-based hydrogels, functionalized with extracellular matrix (ECM)-derived peptides and seeded with hS/PCs and other support cells, will be used as a scaffold for bioengineering an autologous salivary gland replacement.

The first part of this work showed that hydrogel porosity and peptide content impacted hS/PC viability and 3D organization. Peptide-modified hydrogels maintained high hS/PC viability and yielded larger multicellular structures that better resembled a developing salivary gland. Use of an integrin ligand led to a higher number of multicellular structures and enhanced hS/PC proliferation. Specifically, migration-permissive (MP-HA) hydrogels led to the highest activation of integrin β1. In short, these experiments defined a hydrogel parameter space for hS/PC encapsulation and 3D culture that avoids confined, spheroidal multicellular assemblies in favor of asymmetric structures with early peripheral buds. Such features bring the models closer to the observed behavior of branching epithelial buds during salivary morphogenesis.

The second part of this work describes the isolation and characterization of human salivary-derived fibroblasts (hSFs), and their implementation in co-culture with hS/PCs. hSF-hS/PC 3D encapsulations were conducted either as fully mixed co-cultures, or as adjacent but discrete bilayers. Mixed co-cultures led to significantly higher overall cell viability and structure formation than discrete bilayer co-cultures and monocultures of either cell types. hSFs expressed basement membrane proteins in mixed co-cultures; basement membrane accumulated most in between single hSFs and multicellular hS/PC structures. Lastly, time-lapse imaging of mixed co-cultures illustrated that single cells and multicellular structures composed of either or both cell types were dynamically migrating and reorganizing in MP-HA over time. Thus, the use of MP-HA and a mesenchymal cell type enhanced overall cell viability, growth, and organization in a salivary gland regeneration model.