Bioengineering and Cell-derived Strategies for Salivary Gland Regeneration

Sammanfattning: Xerostomia (dry mouth symptoms) is a group of incurable debilitating conditions of salivary glands caused by aging, radiation/chemical exposure, or aberrant inflammation in the salivary glands. During this PhD thesis, we aimed to evaluate whether cell-derived strategies (e.g., extracellular vesicles, EVs) could be a potential new therapy to ameliorate salivary gland injury and restore function after radiotherapy or in autoimmune diseases. In addition, we aimed to develop new imaging techniques for both 2D and 3D analysis of larger samples which allows for quantification of disease and regenerative features. Firstly, we constructed an in vivo murine model of 25 Gy irradiation-induced salivary gland damage to evaluate the potential of human dental pulp stem cell (hDPSCs)-derived EVs. EVs were injected 3x weekly via tail vein, beginning immediately after irradiation. Salivary gland function was evaluated 18 days after irradiation using salivary gland flow rate (SFR), gene expression (by qRT-PCR) and histopathology. Next, we tested different methods to generate PCSS using a vibratome and evaluated the slices in terms of viability (by WST-1), gene expression (by qRT-PCR), secreted α-amylase activity (by α-amylase assay kit) and histological/light sheet fluorescence microscopy (LSFM) three-dimensional imaging. Following irradiation, SFR decreased while senescence-associated β-galactosidase-positive cells (via immunofluorescences) and senescence-related genes and secretory-phenotypes (e.g., p21 and MMP3 in qRT-PCR) increased. SFR was unchanged following EVs treatment, but senescence-associated genes and secretory-phenotypes decreased. We also demonstrated that in an animal model of Sjögren’s syndrome, which exhibit dry mouth symptoms, that hDPSCs-EVs could inhibit the acquisition of the senescent phenotype in salivary gland epithelial cells (SGECs) and alleviate the loss of glandular function. EVs were also found to perform these effects through an underlying immunomodulatory mechanism. For PCSS, we developed protocols to produce viable slices of controled thicknesses which retained the ability to secrete functional α-amylase for at least two days in ex vivo culture. Phenotypic salivary gland cell epithelial markers (e.g., Keratin 5 and Aquaporin 5) increased over time in PCSS (by qRT-PCR), indicating the retention of cells that are necessary for salivary glands’ function. We developed workflows to perform LSFM 3D visualization in whole salivary glands as well as the PCSS model. In conclusion, hDPSCs-EVs reduced senescence of salivary gland epithelial cells in both murine irradiation and Sjögren’s syndrome models and may become a promising future for xerostomia patients. For the murine PCSS, we successfully established an executable operating procedure at the methodological level to reliably generate viable and functional murine PCSS and developed new state-of-the-art analytical methods (such as LFSM 3D imaging and qRT-PCR) to increase the diversity of objective tools to evaluate PCSS. Therefore, this work laid the foundation for the future application of other therapies (such as irradiation therapy or EVs therapy) to the PCSS model. Those future applications could include drug screening or mechanism of injury study. At the same time, we developed a sustainable histology process to reduce xylene utilization in histological processing for salivary gland tissue processing. Therefore, this work has developed a set of in vitro and in vivo experiments with state-of-the-art methods to better understand disease mechanisms and to evaluate new therapies for salivary glands.