J Periodontal Implant Sci.  2013 Aug;43(4):168-176. 10.5051/jpis.2013.43.4.168.

The effects of dexamethasone on the apoptosis and osteogenic differentiation of human periodontal ligament cells

• Kim SM
• Kim YG
• Park JW
• Lee JM
• Suh JY

• KMID: 2027812
• DOI: http://doi.org/10.5051/jpis.2013.43.4.168

Abstract

PURPOSE
The purpose of the current study was to examine the effect of dexamethasone (Dex) at various concentrations on the apoptosis and mineralization of human periodontal ligament (hPDL) cells.
METHODS
hPDL cells were obtained from the mid-third of premolars extracted for orthodontic reasons, and a primary culture of hPDL cells was prepared using an explant technique. Groups of cells were divided according to the concentration of Dex (0, 1, 10, 100, and 1,000 nM). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed for evaluation of cellular viability, and alkaline phosphatase activity was examined for osteogenic differentiation of hPDL cells. Alizarin Red S staining was performed for observation of mineralization, and real-time polymerase chain reaction was performed for the evaluation of related genes.
RESULTS
Increasing the Dex concentration was found to reduce cellular viability, with an increase in alkaline phosphatase activity and mineralization. Within the range of Dex concentrations tested in this study, 100 nM of Dex was found to promote the most vigorous differentiation and mineralization of hPDL cells. Dex-induced osteogenic differentiation and mineralization was accompanied by an increase in the level of osteogenic and apoptosis-related genes and a reduction in the level of antiapoptotic genes. The decrease in hPDL cellular viability by glucocorticoid may be explained in part by the increased prevalence of cell apoptosis, as demonstrated by BAX expression and decreased expression of the antiapoptotic gene, Bcl-2.
CONCLUSIONS
An increase in hPDL cell differentiation rather than cellular viability at an early stage is likely to be a key factor in glucocorticoid induced mineralization. In addition, apoptosis might play an important role in Dex-induced tissue regeneration; however, further study is needed for investigation of the precise mechanism.

Keyword

Apoptosis; Cell differentiation; Cell survival; Dexamethasone; Periodontal ligament

MeSH Terms

Alkaline Phosphatase
Anthraquinones
Apoptosis
Bicuspid
Cell Differentiation
Cell Survival
Dexamethasone
Durapatite
Humans
Periodontal Ligament
Prevalence
Real-Time Polymerase Chain Reaction
Tetrazolium Salts
Thiazoles
Alkaline Phosphatase
Anthraquinones
Dexamethasone
Durapatite
Tetrazolium Salts
Thiazoles

Figure

• 

  Figure 1 Results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (optical density) of human periodontal ligament cells supplemented with different concentrations of dexamethasone (Dex) (0, 1, 10, 100, and 1,000 nM) on days 1, 4, and 7. Similar results were obtained in three separate experiments, and the data are presented as the mean±standard deviation. *Statistically significant difference, compared to the group without Dex treatment (P<0.05).

• 

  Figure 2 Alkaline phosphatase (ALP) activity of periodontal ligament cells supplemented with different concentrations of dexamethasone (Dex) (0, 1, 10, 100, and 1,000 nM) on days 5 and 10. Similar results were obtained in three separate experiments, and the data are presented as the mean±standard deviation. *Statistically significant difference, compared to the group without Dex treatment (P<0.05).

• 

  Figure 3 Macroscopic and microscopic features of mineralization in the control group (a) and the dexamethasone group with concentrations of of 1 nM (b), 10 nM (c), 100 nM (d), and 1,000 nM (e) after an osteogenic induction period of 7 (A), 14 (B), 21 (C), and 28 (D) days (Alizarin Red S staining, ×40).

• 

  Figure 4 Dose-dependent effects of dexamethasone (Dex) on the osteogenesis-related genes of human periodontal ligament cells cultured in osteogenic medium: (A) Cbfa-1, (B) OSX, (C) ALP, and (D) OCN. The cells were exposed to various concentrations (0, 1, 10, 100, and 1,000 nM) of Dex for seven days. Similar results were obtained in three separate experiments, and the data are presented as the mean±standard deviation. The graphs show the ratio of selected gene mRNAs to GAPDH mRNA from the real-time polymerase chain reaction results. *Statistically significant difference, compared to the group without Dex treatment (P<0.05).

• 

  Figure 5 Dose-dependent effects of dexamethasone (Dex) on the apoptosis-related genes of human periodontal ligament cells cultured in osteogenic medium: (A) BAX and (B) Bcl-2. The cells were exposed to various concentrations (0, 1, 10, 100, and 1,000 nM) of Dex for seven days. Similar results were obtained in three separate experiments, and the data are presented as the mean±standard deviation. The graphs show the ratio of selected gene mRNAs to GAPDH mRNA from the real-time polymerase chain reaction results. *Statistically significant difference, compared to the group without Dex treatment (P<0.05).

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