title: Tuning Hydrogel Properties to Promote the Assembly of Salivary Gland Spheroids in 3D.
abstract: Current treatments for chronic xerostomia, or "dry mouth", do not offer long-term therapeutic benefits for head and neck cancer survivors previously treated with curative radiation. Towards the goal of creating tissue-engineered constructs for the restoration of salivary gland functions, we developed new hyaluronic acid (HA)-based hydrogels using thiolated HA (HA-SH) and acrylated HA (HA-AES) with a significant molecular weight mismatch. Four hydrogel formulations with varying HA concentration, (1-2.4 wt%) and thiol/acrylate ratios (2/1 to 36/1) and elastic moduli (G': 35 to 1897 Pa, 2 h post-mixing) were investigated. In our system, thiol/acrylate reaction was initiated rapidly upon mixing of HA-SH/HA-AES to establish thioether crosslinks with neighboring ester groups, and spontaneous sulfhydryl oxidation occurred slowly over several days to install a secondary network. The concurrent reactions cooperatively create a cell-permissive network to allow for cell expansion and aggregation. Multicellular spheroids formed readily from a robust ductal epithelial cell line (Madin-Darby Canine Kidney, MDCK cells) in all hydrogel formulations investigated. Primary salivary human stem/progenitor cells (hS/PCs), on the other hand, are sensitive to the synthetic extracellular environment, and organized acini-like structures with an average diameter of 50 µm were obtained only in gels with G' ≤ 216 Pa and a thiol/acrylate ratio ≥18. The spheroid size and size distribution were dependent on the HA content in the hydrogel. Cells in hS/PC spheroids formed tight junctions (occludin), remained viable and proliferative, secreted structural proteins (collagen IV and laminin) found in the basement membrane and maintained key stem/progenitor markers. We conclude that incorporation of time-dependent, dynamic features into a covalently crosslinked HA network produces an adaptable hydrogel framework that promotes hS/PC assembly and supports early aspects of salivary morphogenesis, key to reconstitution of a fully functional implantable salivary gland.
authors.affiliation: Department of Materials Science and Engineering, University of Delaware, 210 South College Ave., Newark, DE 19716, USA.
authors.forename: Tugba
authors.initials: T
authors.lastname: Ozdemir
authors.affiliation: Department of Materials Science and Engineering, University of Delaware, 210 South College Ave., Newark, DE 19716, USA.
authors.forename: Eric W
authors.initials: EW
authors.lastname: Fowler
authors.affiliation: Department of Materials Science and Engineering, University of Delaware, 210 South College Ave., Newark, DE 19716, USA.
authors.forename: Shuang
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of BioSciences, Rice University, 6100 Main St., Houston, TX 77005, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Harrington
authors.affiliation: Department of Biological Sciences, University of Delaware, 210 South College Ave., Newark, DE, 19716, USA; Helen F. Graham Cancer Center and Research Institute, Christiana Care Health Systems, 4701 Ogletown Stanton Rd., Newark, DE, 19713, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Witt
authors.affiliation: Department of BioSciences, Rice University, 6100 Main St., Houston, TX 77005, USA; Department of Biological Sciences, University of Delaware, 210 South College Ave., Newark, DE, 19716, USA; Department of Bioengineering, Rice University, 6100 Main St., Houston, TX 77005, USA.
authors.forename: Mary C
authors.initials: MC
authors.lastname: Farach-Carson
authors.affiliation: Department of Biological Sciences, University of Delaware, 210 South College Ave., Newark, DE, 19716, USA; Helen F. Graham Cancer Center and Research Institute, Christiana Care Health Systems, 4701 Ogletown Stanton Rd., Newark, DE, 19713, USA; Department of Biomedical Engineering, University of Delaware, 210 South College Ave., Newark, DE 19716, USA.
authors.forename: Swati
authors.initials: S
authors.lastname: Pradhan-Bhatt
authors.affiliation: Department of Materials Science and Engineering, University of Delaware, 210 South College Ave., Newark, DE 19716, USA; Department of Biological Sciences, University of Delaware, 210 South College Ave., Newark, DE, 19716, USA; Department of Biomedical Engineering, University of Delaware, 210 South College Ave., Newark, DE 19716, USA.
authors.forename: Xinqiao
authors.initials: X
authors.lastname: Jia
keywords: Elastic Modulus; Hyaluronic Acid-Based Hydrogels; Permissive Network; Salivary Human Stem/Progenitor Cells; Thiol/Acrylate Ratio; Xerostomia