Relevant immunomodulatory effects have been proposed following allogeneic cell-based therapy with human periodontal ligament stem cells (hPDLSCs). This study aimed to examine the influence of shear stress on the immunosuppressive capacity of hPDLSCs. Cells were subjected to shear stress at different magnitudes (0.5, 5 and 10 dyn/cm${}^2$). The expression of immunosuppressive markers was evaluated in shear stress-induced hPDLSCs using qRT-PCR, western blot, enzyme activity and enzyme-linked immunosorbent assays. The effects of a shear stress-derived condition medium (SS-CM) on T cell proliferation were examined using a resazurin assay. Treg differentiation was investigated using qRT-PCR and flow cytometry analysis. Our results revealed that shear stress increased mRNA expression of IDO and COX2 but not TGF-β1 and IFN-γ. IDO activity, kynurenine and active TGF-$\beta$1 increased in SS-CM when compared to the non-shear stress-derived conditioned medium (CTL-CM). The amount of kynurenine in SS-CM was reduced in the presence of cycloheximide and ERK inhibitor. Subsequently, T cell proliferation decreased in SS-CM compared to CTL-CM. Treg differentiation was promoted in SS-CM, indicated by FOXP3, IL-10 expression and CD4${}^{+}$CD25${}^{hi}$CD127${}^{lo/-}$ subpopulation. In conclusion, shear stress promotes kynurenine production through ERK signalling in hPDLSC, leading to the inhibition of T cell proliferation and the promotion of Treg cell differentiation.

Intermittent compressive force influences human periodontal ligament (PDL) cell behavior that facilitates periodontal tissue regeneration. In response to mechanical stimuli, Yes-associated protein (YAP) has been recognized as a mechanosensitive transcriptional activator that regulates cell proliferation and cell fate decisions. This study aimed to investigate whether compressive forces influence cell proliferation and cell fate decisions of human PDL cells via YAP signaling. YAP expression was silenced by shRNA. The effect of YAP on cell proliferation, adipogenesis and osteogenesis of PDL cells under ICF loading were determined. Adipogenic differentiation bias upon ICF loading was confirmed by fourier-transform infrared spectroscopy (FTIR). The results revealed that ICF-induced YAP promotes osteogenesis, but it inhibits adipogenesis in PDL cells. Depletion of YAP results in PDL cells that are irresponsive to ICF and, therefore, the failure of the PDL cells to undergo osteogenic differentiation. This was shown by a significant reduction in calcium deposited in the CF-derived osteoblasts of the YAP-knockdown (YAP-KD) PDL cells. As to control treatment, reduction of YAP promoted adipogenesis, whereas ICF-induced YAP inhibited this mechanism. However, the adipocyte differentiation in YAP-KD cells was not affected upon ICF treatment as the YAP-KD cells still exhibited a better adipogenic differentiation that was unrelated to the ICF. This study demonstrated that, in response to ICF treatment, YAP could be a crucial mechanosensitive transcriptional activator for the regulation of PDL cell behavior through a mechanobiological process. Our results may provide the possibility of facilitating PDL tissue regeneration by manipulation of the Hippo-YAP signaling pathway.

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.

Objectives: The aim of this study was to investigate the effect of mechanical force on possible dynamic changes of the matrix proteins deposition in the PDL upon in vitro mechanical and in vivo occlusal forces in a rat model with hypofunctional conditions.Materials and methods: Intermittent compressive force (ICF) and shear force (SF) were applied to human periodontal ligament stem cells (PDLSCs). Protein expression of collagen I and POSTN was analyzed by western blot technique. To establish an in vivo model, rat maxillary molars were extracted to facilitate hypofunction of the periodontal ligament (PDL) tissue of the opposing mandibular molar. The mandibles were collected after 4-, 8-, and 12-weeks post-extraction and used for micro-CT and immunohistochemical analysis.Results: ICF and SF increased the synthesis of POSTN by human PDLSCs. Histological changes in the hypofunctional teeth revealed a narrowing of the PDL space, along with a decreased amount of collagen I, POSTN, and laminin in perivascular structures compared to the functional contralateral molars.Conclusion: Our results revealed that loss of occlusal force disrupts deposition of some major matrix proteins in the PDL, underscoring the relevance of mechanical forces in maintaining periodontal tissue homeostasis by modulating ECM composition.