Release of bio-active dentine extracellular matrix components by histone deacetylase inhibitors (HDACi)

Abstract

Aim: To characterize dentine matrix component (DMC) release and smear layer removal by histone deacetylase inhibitors (HDACis). Methodology: DMCs were extracted from powdered human dentine over 14 days using three HDACis, valproic acid (VPA), trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) and compared with a control extractant, 10% (w/v) EDTA. Protein compositions of the resultant extracts were analysed by 1D-polyacrylamide gel electrophoresis (1D-PAGE), TGF-β-1 and MMP-9 ELISAs and a high-throughput growth factor antibody array. Dentine discs with a standardized smear layer were prepared from human molars and treated with EDTA (17% w/v), polyacrylic acid (PA) (20% v/v) and the experimental HDACis prior to analysis by scanning electron microscopy. Parametric ELISA data were analysed using one-way anova and Tukey's post hoc test, whilst nonparametric smear layer data were analysed by Kruskal–Wallis test and Mann–Whitney U-test (P < 0.05). Results: HDACis did not remove smear layer in the presence or absence of PA pre-treatment (P ≥ 0.478). 1D-PAGE analysis demonstrated different protein profiles for EDTA and HDACi extracts. All HDACi solutions released TGF-β-1 although less effectively than EDTA (P < 0.001), whilst MMP-9 was extracted in significantly higher concentration by EDTA and VPA compared with TSA (P < 0.012). Antibody array analysis demonstrated the ability of HDACis to extract a complex cocktail of established/novel growth factors from dentine, albeit significantly less efficiently than EDTA for certain cytokines (TGF-β-1, PDGF-AA, VEGF-A) and significantly more effectively for others (GDF-15, IGF-1, EGRF-1, NGFR, BDNF, SCF-R). Conclusions: HDACi release a range of bioactive DMCs that could promote dentine repair processes in vivo; however, they are ineffective at removing smear layer.