Fig. 4. Osteogenic differentiation effect of Chios gum mastic (CGM) in human dental pulp stem cells (hDPSCs).
(A) After treatment with 0, 5, and 10 μM of CGM in hDPSCs for 48 h, the expression of collagen type I was observed using confocal microscopy through immunofluorescence. Actin was stained with rhodamine phalloidin (red), collagen type I was stained with Alexa 488 (green), and nuclei were stained with DAPI (blue) (×400). (B) To determine the cell alkaline phosphatase (ALP) activity by CGM, hDPSCs were treated with 5 and 10 µM of CGM for 7 and 14 days. The ALP activity ratio was calculated in relation to the control group (for each 7 and 14 days), and the results were presented as a histogram. Results were expressed as mean ± SD and were derived from three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). (C, D) To measure ALP activity, we treated CGM under the same conditions as alizarin red staining and cultured the cells for 7 and 14 days. The color development observed after alizarin red staining was quantified based on the control group (for each 7 and 14 days) and expressed accordingly. Results were expressed as mean ± SD and were derived from three independent experiments (*p < 0.05, ***p < 0.001) (×40). (E) In order to confirm the effect of CGM on the odontogenic differentiation process at the mRNA level, hDPSCs were treated with 5 and 10 µM of CGM for 7 days, and the expression of bone and dentine-related factors (ALP, BSP, DMP-1, DSPP, OP, and RUNX2) in the cells was quantified by qPCR. The results were expressed as the mean ± SD. Data were derived from three independent experiments (**p < 0.01, ***p < 0.001).
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