IADR Abstract Archives
Histone-deacetylase-inhibition promotes pulpal repair mechanisms partly by modulating matrix-metalloproteinase-13 activity
Objectives: Application of the epigenetic modifiers, histone-deacetylase-inhibitors (HDACis), to dental pulp cell (DPC) cultures promotes mineralization and presents new opportunities for dental-tissue repair. Our previous transcriptomic analysis demonstrated that HDACi-induced DPC genes significantly modulated matrix metalloproteinase (MMP) and endochondral ossification pathways at relatively early time-points. To investigate the HDACi, suberoylanilide hydroxamic acid (SAHA) and a specific MMP-13 inhibitor, [pyrimidine-4,6-dicarboxylic acid, Bis-(4-Fluro-3-Methyl-Benzylamide)] in modulating regenerative processes in DPC cultures using mineralization, molecular, cellular and MMP assays.
Methods: MMP-13 inhibitor (MMP-13i) (0.5-10µM) was added to samples containing 50ng/ml of recombinant MMP-13 to confirm activity blocking and identify appropriate experimental parameters. Subsequently, HDACi (SAHA 1µM) and MMP-13i (1µM, 2µM), alone and in combination, were applied to rodent DPCs and stimulation of reparative processes was investigated by cell growth (24h, 5d) Alizarin red staining (14d, 21d) and qRT-PCR (24h, 5d). Furthermore, MMP-13 enzyme activity, protein expression (ELISA), wound healing and cell migration were assessed in a range of HDACi and MMP-13i supplemented cultures.
Results: The MMP-13i blocked enzyme activity in a dose-dependent manner, with significant decreases in activity >0.5µM MMP-13i. MMP-13i did not affect DPC growth, while 24h SAHA (1µM) significantly reduced cell numbers at 5d exposure. SAHA-supplementation significantly increased MMP-13 gene expression at relatively early time-points (24h, 5d), while protein expression as well as enzyme activity were modulated at later time-points (7d, 14d). Notably, MMP-13i application dose-dependently accelerated mineralization in both SAHA treated and non-treated cultures. MMP-13i supplementation also promoted the expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired.
Conclusions: Short-term low-dose SAHA-exposure promoted mineralization in DPCs, and modulated MMP expression and activity at relatively early time-points. MMP-13 inhibition further increased mineralization-associated events, but decreased cell migration indicating a potentially novel role for MMP-13 in pulpal repair processes.
Methods: MMP-13 inhibitor (MMP-13i) (0.5-10µM) was added to samples containing 50ng/ml of recombinant MMP-13 to confirm activity blocking and identify appropriate experimental parameters. Subsequently, HDACi (SAHA 1µM) and MMP-13i (1µM, 2µM), alone and in combination, were applied to rodent DPCs and stimulation of reparative processes was investigated by cell growth (24h, 5d) Alizarin red staining (14d, 21d) and qRT-PCR (24h, 5d). Furthermore, MMP-13 enzyme activity, protein expression (ELISA), wound healing and cell migration were assessed in a range of HDACi and MMP-13i supplemented cultures.
Results: The MMP-13i blocked enzyme activity in a dose-dependent manner, with significant decreases in activity >0.5µM MMP-13i. MMP-13i did not affect DPC growth, while 24h SAHA (1µM) significantly reduced cell numbers at 5d exposure. SAHA-supplementation significantly increased MMP-13 gene expression at relatively early time-points (24h, 5d), while protein expression as well as enzyme activity were modulated at later time-points (7d, 14d). Notably, MMP-13i application dose-dependently accelerated mineralization in both SAHA treated and non-treated cultures. MMP-13i supplementation also promoted the expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired.
Conclusions: Short-term low-dose SAHA-exposure promoted mineralization in DPCs, and modulated MMP expression and activity at relatively early time-points. MMP-13 inhibition further increased mineralization-associated events, but decreased cell migration indicating a potentially novel role for MMP-13 in pulpal repair processes.