International endodontic journal · 2015
Long-term exposure to pro-inflammatory cytokines inhibits the osteogenic/dentinogenic differentiation of stem cells from the apical papilla.
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Liu C, Xiong H, Chen K, Huang Y, Huang Y, Yin X
Аннотация
AIM: To investigate the in vitro effects of TNF-α and IL-1β on the osteogenic/dentinogenic differentiation of stem cells from the apical papilla (SCAP).
METHODOLOGY: SCAPs treated with 10 ng mL(-1) TNF-α and/or 5 ng mL(-1) IL-1β were defined as the treatment groups, with untreated cells defined as the control group. Cell proliferation was measured using the MTT assay and cell cycle analysis. Cells in all of the groups were cultured in the osteogenic/dentinogenic differentiation medium and processed for analysis at days 3, 7 and 14. The Alizarin Red S assay, alkaline phosphatase staining and real-time PCR were used to evaluate the differentiation capacity. Differences between the treatment groups and the control groups were analysed statistically using one-way anova and Dunnett's post-test.
RESULTS: Compared with the control group, the treated SCAPs were associated with a significantly greater proliferation activity at days 3 and 5 but reduced proliferation at day 9 (P < 0.05). Significantly lower calcium deposition and ALP activity were observed in the treatment groups at day 14 (P < 0.05), as well as reduced ALP, DSPP and DMP-1 expression levels (P < 0.05); however, treated SCAPs had significantly higher levels of DSPP and DMP-1 at day 7 (P < 0.05). In the control group, the ALP, DSPP and DMP-1 expression levels increased significantly from day 3 to day 14 (P < 0.05), but the treatment groups were not associated with increased expression from day 7 to day 14 (P > 0.05).
CONCLUSIONS: The pro-inflammatory cytokines TNF-α and IL-1β inhibited mineralization and osteogenic-/dentinogenic-related gene expression in SCAPs in vitro after long-term culturing. However, these cytokines induced cell proliferation and mineralization in short-term culture probably as a protective response. These findings help to elucidate the impact of inflammation on tooth development.
METHODOLOGY: SCAPs treated with 10 ng mL(-1) TNF-α and/or 5 ng mL(-1) IL-1β were defined as the treatment groups, with untreated cells defined as the control group. Cell proliferation was measured using the MTT assay and cell cycle analysis. Cells in all of the groups were cultured in the osteogenic/dentinogenic differentiation medium and processed for analysis at days 3, 7 and 14. The Alizarin Red S assay, alkaline phosphatase staining and real-time PCR were used to evaluate the differentiation capacity. Differences between the treatment groups and the control groups were analysed statistically using one-way anova and Dunnett's post-test.
RESULTS: Compared with the control group, the treated SCAPs were associated with a significantly greater proliferation activity at days 3 and 5 but reduced proliferation at day 9 (P < 0.05). Significantly lower calcium deposition and ALP activity were observed in the treatment groups at day 14 (P < 0.05), as well as reduced ALP, DSPP and DMP-1 expression levels (P < 0.05); however, treated SCAPs had significantly higher levels of DSPP and DMP-1 at day 7 (P < 0.05). In the control group, the ALP, DSPP and DMP-1 expression levels increased significantly from day 3 to day 14 (P < 0.05), but the treatment groups were not associated with increased expression from day 7 to day 14 (P > 0.05).
CONCLUSIONS: The pro-inflammatory cytokines TNF-α and IL-1β inhibited mineralization and osteogenic-/dentinogenic-related gene expression in SCAPs in vitro after long-term culturing. However, these cytokines induced cell proliferation and mineralization in short-term culture probably as a protective response. These findings help to elucidate the impact of inflammation on tooth development.