Hydrogel Network Topology Impacts Salivary Cluster Morphology in Tissue-Engineered Matrices

Objectives: Tissue engineering approaches toward salivary gland regeneration offer a potential therapeutic option for head-and-neck cancer patients with radiation-induced xerostomia. In these workflows, in vitro culture of cells within a supportive matrix is a common preparatory step, to develop organized cell structures that mimic the native tissue’s structure and/or function. We have previously described the 3D culture of primary human salivary stem/progenitor cells (hS/PCs) as spheroids within hyaluronate (HA)-based hydrogels, and their progressive deposition of peripheral basement membrane. We hypothesized that changes in hydrogel composition and porosity would impact hS/PC growth dynamics and maturation.
Methods: hS/PCs were isolated from human parotid gland biopsies, and encapsulated within HA matrices at ~3E6 cells/mL. The composition of each matrix was varied to include either poly(ethylene glycol) (PEG)-functional integrin-binding sites (RGD) or matrix metalloproteinase (MMP)-labile sites, or their scrambled non-functional equivalents. Cell-laden gels were cultured over 25 days, and assessed for viability, morphology, proliferation, extracellular matrix production patterns, and extent of activated integrin localization across each population of hS/PC clusters, via fluorescent staining and confocal microscopy.
Results: hS/PCs maintained high viability in all matrices. In unmodified HA hydrogels, hS/PCs consistently grew as spherical multicellular structures, surrounded by a distinct peripheral ring of laminin and both interior and exterior fibronectin. Conversely, HA matrices modified with PEG-functional peptides promoted formation of larger multicellular structures, with wrinkled peripheries, and more punctate laminin and fibronectin between cell extrusions. RGD-functional matrices additionally induced a larger number of hS/PC structures, and higher activated integrin expression.
Conclusions: 3D culture of epithelial cells commonly results in spheroidal structures that do not progress toward a desired anisotropic, branched morphology that is characteristic of mature glands. The present work identifies matrix modifications in porosity and biofunctional peptide incorporation that are an early step toward deriving these more complex, phenotypic morphologies, and are desirable for salivary gland regeneration.