Effects of p38 mitogen-activated protein kinase on osteogenic differentiation of human periodontal ligament stem cells in inflammatory microenvironment.

OBJECTIVE

To investigate the expression of mitogen-activated protein kinase (MAPK) in the chronic periodontitis tissue-derived in the periodontal ligament stem cells (PDLSCs) and explore its effect on the osteogenic differentiation of human PDLSCs in inflammatory microenvironment.

METHODS

PDLSCs were obtained from human healthy individuals (H-PDLSCs) and patients with periodontitis (P-PDLSCs). The tumor necrosis factor (TNF)-Α and interleukin (IL)-1Β secretion and mRNA expression levels of H-PDLSCs and P-PDLSCs were detected using enzyme-linked immunosorbent assay and real-time quantitative PCR. Immunofluorescence staining was used for determining the protein levels of p38 in PDLSCs. The levels of p38 and p-p38 following culture in osteogenic medium for 7 d of H-PDLSCs and P-PDLSCs were detected using Western blotting. After the PDLSCs were stimulated with SB-203580,the p38 MAPK specific inhibitor, for 30 min and then in osteogenic induction process for 7 days,the expression levels of the osteogenic gene Runx2 and alkaline phosphatase (ALP) were determined by real-time quantitative PCR, and bone formation ability of PDLSCs was tested by alizarin red (AR) staining.

RESULTS

The secretions of TNF-Α and IL-1Β were significantly higher in P-PDLSCs compared with H-PDLSCs (68.80 ± 6.70 vs. 34.10 ± 3.07,P=0.001;57.10 ± 4.23 vs. 26.90 ± 2.58,P=0.000). The same trend was seen in the gene expression levels of both TNF-Α and IL-1Β in PDLSCs (PTNF-Α=0.011,P IL-1Β=0.009). p38 was more strongly induced in P-PDLSCs cells than in H-PDLSCs.The basal level of p38 in H-PDLSCs was lower than that in P-PDLSCs cells cultured in the basic medium. However,the level of p-p38 was increased in H-PDLSCs than in P-PDLSCs under osteogenic condition. Treatment of PDLSCs with SB-203580 and then cultures under osteogenic differentiation lead to significantly decreased expressions of Runx2 and ALP in both H-PDLSCs and P-PDLSCs (H-PDLSCs:P(Runx2)=0.044, PALP=0.036;P-PDLSCs:P(Runx2)=0.017, PALP=0.004).

CONCLUSIONS

p38 MAPK is involved in the inflammatory response of PDLSCs in the chronic inflammatory microenvironment. The inhibition of p38 by SB-203580 also remarkably suppresses the osteogenic differentiation of PDLSCs in a chronic inflammatory microenvironment.