Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells

Abstract

Introduction: Histone deacetylase inhibitors (HDACis) alter the homeostatic balance between 2 groups of cellular enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs), increasing transcription and influencing cell behavior. This study investigated the potential of 2 HDACis, valproic acid (VPA) and trichostatin A (TSA), to promote reparative processes in pulp cells as assayed by viability, cell cycle, and mineralization analyses.

Methods: VPA (0.125-5 mmol/L) and TSA (12.5-400 nmol/L) were applied to a pulp-derived cell population and compared with unsupplemented controls. Cell proliferation and viability were evaluated by trypan blue staining and cell counting, whereas cell cycle and apoptosis were analyzed by immunocytochemical staining with antibodies for p53, phosphorylated p53, Bcl-2 homologous antagonist/killer (BAK), caspase-3 and p21(WAF1/CIP), and DNA staining with Hoechst 33342. For mineralization analysis, cultures were stained with Alizarin red and quantified spectrophotometrically. Relative gene expression levels of mineralization associated markers were analyzed using reverse-transcriptase polymerase chain reaction. One-way analysis of variance and Tukey post hoc tests were applied to the data (P < .05).

Results: VPA and TSA reduced cell proliferation dose dependently with no significant effect on cell viability except at 400 nmol/L TSA. The transcription factor p21(WAF1/CIP) was significantly increased at the highest concentration of TSA but not VPA. Significant increases (P < .05) in the apoptosis marker protein active caspase-3 and cell cycle alterations were only evident at the maximum concentrations of TSA/VPA, whereas HDACi-induced mineralization per cell was stimulated dose dependently with a significant increase in the expression of the dentinogenic-associated transcript, dentine matrix protein-1.

Conclusions: These results indicate that HDACis are capable of epigenetically modulating pulp cell behavior, signifying their therapeutic potential for augmenting biomaterials, and stimulating regenerative responses in the damaged pulp.