Inducing epithelial morphogenesis in human salivary acinar-like cells cultured in 3D hyaluronic acid, laminin-modified hydrogels

Duncan, Randall L.Witt, Robert L.Radiation therapy used to treat head and neck cancers results in salivary gland hypofunction and xerostomia. Salivary gland dysfunction is concurrent with a steady decline in oral health. The creation of a bioengineered salivary gland would provide a potential long term treatment option for those suffering from xerostomia, or dry mouth. The goal of this project was to create an extracellular matrix (ECM): hyaluronic acid (HA)-based hydrogel culture system to promote the survival, growth, and morphogenesis of human salivary acinar-like cells (hSACs) into higher-ordered, branched structures. Ultimately, we aim to create hSAC structures capable of secreting fluid and salivary-specific proteins in a vectorial fashion to be used for engineering a fully functional artificial salivary gland to be implanted into patients suffering from xerostomia. Human salivary tissue was obtained from patients undergoing head and neck surgery under the approval of the Institutional Review Board (IRB) at Christiana Care Health Systems (CCHS) and the University of Delaware (UD). hSACs were identified in tissue explant culture. Gene expression and protein level analysis showed that hSACs express a variety of stem/progenitor cell markers in both two- and three-dimensional culture systems. Here we report the effects of fibroblast growth factors, FGF7 and FGF10 on human salivary gland acinar-like cells (hSACs) grown in three-dimensional hyaluronic acid:laminin hydrogels. hSAC spheroids encapsulated in our culture system self-assemble into spheroid structures after seven days. Stimulation with FGFR2b ligands, FGF7 or FGF10 showed sustained hSAC proliferation and specific modes of morphogenesis. FGF7 treatment promoted the formation of epithelial cleft-like and lobule-like structures, whereas the addition of FGF10 to hSAC structures induced duct-like elongations. We found that hSACs treated with FGF7 and FGF10 increase fibronectin protein levels and deposition within the hydrogel network; however, FGF7 but not FGF10 increased protein levels of fibronectin-binding ?5-integrin. FGF10 treatment followed by FGF7 addition resulted in more complex morphogenesis than either ligand alone. Interestingly, simultaneous stimulation with FGF7 and FGF10 had a minimal effect on overall hSAC morphogenesis. Sustained culture of hSAC structures treated with FGF10-heparin followed by FGF7 began to form lumens. To assess the capability of hSAC differentiation, acinar cell biomarker α-amylase protein expression was investigated. We observed amylase staining in hSAC structures undergoing morphogenesis. Interestingly, hSACs treated with EGFR ligand, HB-EGF, following sequential addition of FGF10 and FGF7 express ductal marker, cytokeratin 19 (CK19). In summary, FGFR2b ligands FGF7 and FGF10 are capable of inducing morphogenesis of primary salivary gland cells into structures reminiscent of native salivary gland architectures, which can be used to restore glandular function in tissue engineering applications.University of Delaware, Department of Biological SciencesM.S