Abstract
Research efforts have been made to develop human salivary gland (SG) secretory epithelia for transplantation in patients with SG hypofunction and dry mouth (xerostomia). However, the limited availability of human biopsies hinders the generation of sufficient cell numbers for epithelia formation and regeneration. Porcine SG have several similarities to their human counterparts, hence could replace human cells in SG modelling studies in vitro. Our study aims to establish porcine SG explant outgrowth models to generate functional secretory epithelia for regeneration purposes to rescue hyposalivation. Cells were isolated and expanded from porcine submandibular and parotid gland explants. Flow cytometry, immunocytochemistry, and gene arrays were performed to assess proliferation, standard mesenchymal stem cell, and putative SG epithelial stem/progenitor cell markers. Epithelial differentiation was induced and different SG-specific markers investigated. Functional assays upon neurostimulation determined α-amylase activity, trans-epithelial electrical resistance, and calcium influx. Primary cells exhibited SG epithelial progenitors and proliferation markers. After differentiation, SG markers were abundantly expressed resembling epithelial lineages (E-cadherin, Krt5, Krt14), and myoepithelial (α-smooth muscle actin) and neuronal (β3-tubulin, Chrm3) compartments. Differentiated cells from submandibular gland explant models displayed significantly greater proliferation, number of epithelial progenitors, amylase activity, and epithelial barrier function when compared to parotid gland models. Intracellular calcium was mobilized upon cholinergic and adrenergic neurostimulation. In summary, this study highlights new strategies to develop secretory epithelia from porcine SG explants, suitable for future proof-of-concept SG regeneration studies, as well as for testing novel muscarinic agonists and other biomolecules for dry mouth.
Highlights
Major salivary glands (SG) such as the submandibular and parotid glands are responsible for the bulk of resting and stimulated saliva secreted, respectively [1,2]
Primary cells from PG/SMG explant cultures exhibited a polygonal epithelial-like form and a spindle shape mesenchymal-like morphology (Figure 1C). These observations indicated these cultures are heterotypic and may possess the potential to recapitulate, in vitro, the epithelial secretory compartments found in the functional SG
The majority of primary cells at first subculture possessed a pro-mitotic proliferative activity shown after Ki67 immunostaining (Figure 1D, E)
Summary
Major salivary glands (SG) such as the submandibular and parotid glands are responsible for the bulk of resting and stimulated saliva secreted, respectively [1,2]. The irreversible damage to the innervated secretory epithelium of major SG prompts this hyposalivation phenomenon [5,6,7] These patients suffer from severe oral dryness [2,5,8], with oral tissue repercussions including infections, mucosal inflammation, pain, and tooth loss [9]. Current conventional therapies with saliva cholinergic agonists are not efficacious due to loss of secretory epithelia, in post-radiotherapy patients [4,7,10]. Such functional epithelia loss has led to tissue engineering strategies to model innervated SG epithelia in vitro for the development of future cell transplantation therapies into the injury site [11,12,13,14]
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