The Selective Histone Deacetylase Inhibitor MI192 Enhances the Osteogenic Differentiation Efficacy of Human Dental Pulp Stromal Cells

Abstract

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells' epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time-dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G₂/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.

Keywords: HDAC inhibitor; MI192; bone tissue engineering; epigenetics; histone deacetylase; human dental pulp stromal cells.

Figure 1

Experimental outline investigating the effects of altering hDPSCs epigenetic functionality with MI192 on their osteogenic capacity.

Figure 2

Effects of MI192 on hDPSCs morphology and viability. (A) hDPSCs morphology. Scale bar = 100 μm. (B) Metabolic activity of hDPSCs, quantified with the AlamarBlue assay. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05.

Figure 3

The influence of MI192 on hDPSCs epigenetic functionality. HDAC activity levels (A) immediately post MI192 treatment and (B) 1 week post MI192 treatment. H3K9 histone acetylation levels (C) immediately post MI192 treatment and (D) 1 week post MI192 treatment. Data are expressed as mean ± SD (n = 3). * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.

Figure 4

Effects of MI192 treatment on hDPSCs cell cycle progression. Data are expressed as mean ± SD (n = 3). The significance levels shown are the test group compared to the basal control for that time point. * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.

Figure 5

Effect of MI192 pre-treatment on hDPSCs ALPSA. Cells were pre-treated with/without MI192 for 48 h before culture in osteogenic conditions for 14 days. Data are expressed as mean ± SD (n = 3). ** p ≤ 0.01.

Figure 6

The effects of MI192 pre-treatment (2 μM MI192 for 48 h) on hDPSCs osteogenic differentiation and mineralisation. Osteoblast-related (A) gene expression, (B) protein production, (C,D) calcium deposition (scale bar = 100 µm)) (day 28) and (E) mineral nodule formation (day 28) (scale bar = 50 µm). Data are expressed as mean ± SD (n = 3). ** p ≤ 0.01 and *** p ≤ 0.001.