­­­TEGDMA Cytotoxicity on DPSCs is Mediated Through Wnt/beta-catenin Signaling

Method: DPSCs cultures were established from extracted third molars of healthy donors 15-25 years-old. Cultures were characterized for stem cell markers using flow cytometry. Cells were exposed to TEGDMA (0.5-2mM) with or without the presence of the Wnt activator lithium chloride (LiCl) at non-toxic concentrations (LiCl:1-10mM). TEGDMA cytotoxicity with/without LiCL was evaluated by the MTT assay. Expression of key molecules of the Wnt/beta-catenin pathway, including LEF-1, GSK-3b and beta-catenin was assessed by Real-time PCR. Western Blotting was used to evaluate cytosolic accumulation of beta-catenin and translocation into nucleus in TEGDMA- and/or LiCl-treated cultures. 

Result: DPSCs cultures were strongly positive (>95% of the population) for CD90, CD81, CD49f, Nestin, Nanog and in partly for CD105 (75%), CD146 (57%), STRO-1 (4.3%), CD24 (4.8%), Oct3/4 (25%), CD271 (2.5%). CD45, CD34, SSEA-3 and TRA-1-60 were not expressed (<1%). Our results showed that LiCl is a weak inducer of the Wnt/beta-catenin signaling. TEGDMA showed a concentration-dependent cytotoxicity which was not influenced by the presence of LiCl (1-10mM). Exposure of DPSCs to TEGDMA in combination with LiCl (2.5-10mM) caused upregulation of the expression of LEF-1 (up to 9 times) and GSK3b (3.5 times), whereas beta-catenin mRNA expression remained unaltered. Western Blotting revealed, however, accumulation of cytosolic beta-catenin and translocation into nucleus both in TEGDMA (1, 2mM) and TEGDMA/LiCl co-treated cultures, indicative of activation of the Wnt canonical signaling.

Conclusion: These findings suggest that DPSCs repair responses to monomers, like TEGDMA, leached from resin-based materials, are mediated through activation of the canonical Wnt/beta-catenin signaling. These data give evidence on the important role of Wnt signaling in pulp repair responses to restorative materials.