INHIBITION OF HISTONE DEACETYLASES ENHANCES OSTEOGENICDIFFERENTIATION OF HUMAN PERIODONTAL LIGAMENT CELLS

Abstract

Objectives: When appropriately triggered, periodontal ligament (PDL) cells can differentiate into mineralized tissue forming cells, thus make it a good candidate for autologous bone graft. This study aimed to investigate the role of histone deacetylases (HDACs) in osteogenic differentiation of human PDL cells (hPDLs). The effect of HDAC inhibitor on an enhancement of bone regeneration by hPDL cells was also examined. Methods: Activity of HDACs was blocked in primary hPDLs using the inhibitor trichostatin A (TSA). Cell viability, gene expression, ALP activity and mineral deposition assays were used to assess osteoblast phenotypes. RUNX2, histone acetylation and HDACs expression were also observed by western blot analysis. In vitro 3D culture and mouse calvarial defect model were performed using co-polymer scaffold (PCL/PEG). Histomorphometric analysis, micro-CT scan were used to evaluate the in vivo effect of TSA on bone regeneration. The immunogenic activity of mice against allogenic hPDLs was verified by immunohistochemistry staining and ELISA. Results: During the osteogenic differentiation with TSA, osteoblast-related genes expression, ALP activity and bone nodule formation were accelerated. hPDLs highly expressed HDACs of class I (HDAC 1, 2, 3) and class II (HDAC 4, 6). During osteogenic differentiation, HDAC 3 expression gradually decreased. This effect was apparent in the presence of the inhibitor. The level of acetylated histone H3 increased during osteogenic differentiation while treatment with TSA induced histone H3 hyperacetylation and RUNX2 protein expression. TSA enhanced mineral deposition by hPDLs in in-vitro 3D culture model. In vivo bone regeneration potential of hPDLs in mouse calvarial defect model was significantly enhanced by TSA treatment. Micro-CT analysis demonstrated the significant increase of BV/TV in inhibitor treated groups at 4 and 8 weeks. Immunohistochemistry staining demonstrated human cells incorporate into newly form osseous tissues of mice calvarias. While ELISA of mice serum indicated no significant immune reactivity against xenogenic human cells. In conclusion: This study provided further insight into the roles of HDACs function in osteogenic differentiation of hPDLs. TSA enhanced both in vitro and in vivo bone regeneration potential of hPDLs making its more applicable for bone regeneration therapy.