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.

Bottom-up assembly of salivary gland microtissues for assessing myoepithelial cell function

“…[12] Our custom-designed HA hydrogels promoted spheroid assembly from dispersed hS/PCs. [14] We further discovered that hS/PCs cultured in HA gels decorated with bioactive peptides expressed a significantly higher levels of progenitor markers as compared to the unmodified HA gels. [12] The current study aimed to create physiologically relevant microtissues composed of myoepithelial cells and organized, acini-like spheroids to replicate the function of intact acini-hSMEC assemblies in tissue.…”

“…Custom-designed HA hydrogels have shown success in maintaining high viability of single hS/PCs and promoting the formation of spheroid structures in vitro . [13, 14] In this study, we utilized PEGDMI to crosslink HA-SH as rapid gelation is necessary to prevent the microtissue assemblies from settling. Our initial experiments showed that hSMECs remained on the outer surface of the hS/PC spheroids for up to 48 h in the HA gel without any biological signals.…”

“…[14] PEG (3,500 Da) bismaleimide (PEGDMI) was purchased from JenKem Technology USA (Plano, TX). Upon completion of the synthesis of HA-SH,[14, 21] the thoroughly dialyzed HA-SH solution was sterile filtered (0.22 µm) before lyophilization, and PEGDMI dissolved in PBS was sterile filtered before being used for cell culture purposes. The viscoelastic properties of HA/PEG gels were analyzed using a TA instruments DHR-3 rheometer with a 12 mm stainless steel geometry and 500 mm gap size at 37°C.…”

“…We have previously isolated and expanded hS/PCs,[11] confirmed their expression of stem/progenitor markers,[12] encapsulated them as dispersed single cells hyaluronic acid (HA)-based hydrogels[13], investigated the role of hydrogel properties on the formation of hS/PC spheroid,[14] and differentiated them to acinar cells using neurotransmitters. [12] Separately, consistent isolation, immunophenotypic characterization and functional analyses of hSMECs are essential for the generation of in vitro models with lobular cells.…”

Chemical synthesis of biomimetic hydrogels for tissue engineering

“…Cells in these structures formed cell-cell junctions, secreted basement membrane proteins and maintained key stem/progenitor markers. 132 Thus, incorporation of time-dependent, dynamic features into a covalently cross-linked HA network produces an adaptable hydrogel framework that promotes hS/PC assembly and supports early aspects of tissue morphogenesis.…”