title: Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells.
abstract: BACKGROUND
  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.
authors.affiliation: Division of Restorative Dentistry and Periodontology, Dublin Dental University Hospital, Trinity College Dublin, Dublin, Ireland. Hal.Duncan@dental.tcd.ie
authors.forename: Henry F
authors.initials: HF
authors.lastname: Duncan
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Smith
authors.forename: Garry J P
authors.initials: GJ
authors.lastname: Fleming
authors.forename: Paul R
authors.initials: PR
authors.lastname: Cooper
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002113:Calcification, Physiologic / Q000187:drug effects; D053148:Caspase 3 / Q000187:drug effects; D002452:Cell Count; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000187:drug effects; D003782:Dental Pulp / Q000166:cytology / Q000187:drug effects*; D003810:Dentinogenesis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D016326:Extracellular Matrix Proteins / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D006877:Hydroxamic Acids / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D012038:Regeneration / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects; D014635:Valproic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000187:drug effects
chemical_list: C494773:Bak1 protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; C012589:trichostatin A; D014635:Valproic Acid; D053148:Caspase 3