Effects of inorganic phosphate on stem cells isolated from human exfoliated deciduous teeth

Aim: Calcium phosphate-based materials (CaP) are introduced as potential dental pulp capping materials for deciduous teeth. The present study investigated the influence of inorganic phosphate (Pi) on regulating stem cells isolated from human exfoliated deciduous teeth (SHED). Methodology: SHED cells were treated with 2.5 and 5 mM Pi and compared to controls receiving no additional Pi. Cell proliferation was examined using an MTT assay. Cell cycle progression and apoptosis were examined using flow cytometry analysis. Cell migration was evaluated using an in vitro scratch assay. Osteogenic and adipogenic differentiation were examined using alizarin red s and oil red o staining for mineral deposition and intracellular lipid accumulation, respectively. The mRNA expression profile was investigated using a high-throughput RNA sequencing technique. Results: SHED cells were isolated and validated via flow cytometry. Pi inhibited cell proliferation but did not affect the migratory ability of SHED. Pi increased the late apoptotic cell population while cell cycle progression was not altered. Pi upregulated osteo/odontoblastic gene expression and enhanced calcium deposition. Pi-induced mineralization in SHED was reversed by pretreatment of cells with foscarnet or p38 inhibitor. Pi treatment inhibited adipogenic differentiation as determined by decreased PPARg expression and reduced intracellular lipid accumulation. Interestingly, gene-expression profiling revealed that the upregulated genes were enriched in TFAP2 (AP2) family regulates transcription factor of cell cycle, TLR3-mediated TICAM1-dependent programmed cell death, and caspase activation via dependence receptors in the absence of ligand. On the contrary, the downregulated genes were enriched in MAPK (ERK) activation, muscarinic acetylcholine receptors, and synthesis of pyrophosphate in cytosol. Conclusion: Pi enhanced osteo/odontogenic differentiation but inhibited adipogenic differentiation in SHED.