Effects of EZH2 inhibitor, trichostatin A, and 5-azacytidine combinatorial treatment on osteogenic differentiation of dental pulp stem cells

Epigenetic mechanisms play regulatory roles in dental pulp stem cell (DPSC) differentiation. The molecules that modulate these mechanisms can be used to enhance DPSC differentiation in experimental studies and clinical applications. We investigated the combined effects of an epigenetic modulator enhancer of zeste homologue 2 inhibitor (EZH2i), trichostatin A (TSA), and 5-azacytidine (5-AZA) on the osteogenic differentiation of DPSCs. To assess osteogenic differentiation, we measured alkaline phosphatase activity, calcium deposition, and expression of osteogenic differentiation marker genes (RUNX2, BMP2, and ALPL) after 7 or 21 days of combinatorial drug treatment in normal cell culture medium or osteo-inductive medium (OIM). No synergistic effects were observed for any possible combination of EZH2i, TSA, or 5-AZA. However, the effects of these drugs and their combinations depend on the time and culture conditions. We confirmed that EZH2i and TSA have positive effects on the osteogenic differentiation of DPSCs. EZH2i activates the expression of key regulatory genes (RUNX2, BMP2, and ALPL) directly, whereas TSA interacts with signalling pathways induced by supplements in OIM to activate these genes. •There is no synergism between EZH2i, TSA and 5-AZA, when DPSC cells are treated with all possible drug combinations.•EZH2i affects osteogen regulatory genes (RUNX2, BMP-2, ALPL) on a direct manner.•TSA affects RUNX2, BMP-2, and ALPL indirectly via genes of signaling pathways involved in osteogen differentiation.