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Micromolar Fluoride Alters Ameloblast Lineage Cells in vitro

Department of Orofacial Sciences, University of California at San Francisco, 513 Parnassus Ave. S-704, San Francisco, CA 94143-0422, USA

View larger version (24K): [in this window] [in a new window]   Figure 1. Characterization of ameloblast-lineage cells by immunohistochemistry and real-time PCR. (A) DSP immunostaining showed strong staining by most ameloblast-lineage cells. Select cells stained positive for KLK-4 (B) and TNF-(C). Controls showed only nuclear staining (D). Scale bar: 20 µm. (E) Quantitative PCR showed mRNA expression of TNF, DSPP, and KLK4 in pooled ameloblast-lineage cells exposed to either 0 or 10 µM F (RQ = relative quantity). Average (SD) values of RQ of the gene tested: (KLK4) RQctrl = 1.21 ± 0.37, RQF = 0.90 ± 0.31; (TNF) RQctrl = 0.81 ± 0.51, RQF = 1.06 ± 0.71; (DSPP) RQctrl = 0.87 ± 0.63, RQF = 0.43 ± 0.14 (n = 3). (F) DSPP was up-regulated in the youngest (group 3) cells derived from second molars exposed to F. Cells from incisors (group 1) or canines and first molars (group 2) were not affected. (G) TNF was up-regulated in the youngest F-exposed cells.

View larger version (17K): [in this window] [in a new window]   Figure 2. Osteogenesis-related genes expressed in relatively high levels by ameloblast-lineage cells derived from pooled teeth in the cap and bell stages. There were no differences detected between 10-µM-NaF-treated groups and the control cells. Average (SD) values of normalized signal strength of the gene tested: (Control) ITGA3, 0.42 (0.16); COL17A1, 0.29 (0.20); TNF, 0.32 (0.14); EGFR, 0.61 (0.25); FN1, 1.03 (0.39); GAPDH 1 (0); ITGB1, 0.39 (0.19); RUNX2, 0.71 (0.29); VEGF, 0.18 (0.05); (10-µM-NaF-treated groups) ITGA3, 0.22 (0.04); COL17A1, 0.18 (0.09); TNF, 0.22 (0.08); EGFR, 0.47 (0.06); FN1, 0.83 (0.28); GAPDH 1 (0); ITGB1, 0.40 (0.20); RUNX2, 0.74 (0.10); VEGF, 0.24 (0.22) (n = 3).

View larger version (12K): [in this window] [in a new window]   Figure 3. Cell proliferation rates were measured by immunofluorescent staining of Brdu incorporation, quantitated as relative light units per sec (rlu/s). Fluoride concentrations less than 256 µM promoted ameloblast-lineage cell proliferation, with the peak effect at 16 µM, while the higher levels significantly inhibited cell proliferation relative to control cells. Average (SD) values of groups: 0 µm, 71.9 (19.5); 4 µm, 126.7 (62.5); 16 µm, 164.6 (44.7); 64 µm, 134.3 (48.2); 256 µm, 113.4 (38.9); 1024 µm, 20.5 (12.7); 4096 µm, 14.4 (4.8) (n = 6).

View larger version (28K): [in this window] [in a new window]   Figure 4. Ameloblast-lineage cells exposed to fluoride. (A–C) Flow cytometry of ameloblast-lineage passage 2 cells isolated from 21-week-old fetal tissue exposed to: (A) 0 µM F, (B) 10 µM F, or (C) 20 µM F. PI, propidium iodide; Hoechst, Hoechst 33342. The cell group labeled R4 refers to an early-apoptotic population (PI- and increased Ho342 fluorescence), R5 is the late-apoptotic population (intermediate intensity of PI and decreased Ho342 fluorescence), and R2 is the viable cell population. (D) The apoptotic index shows that cells exposed to fluoride had significantly increased apoptosis. The average (SD) apoptotic index of each group: (21W) control, 6.87 (1.44); 10 µm NaF, 11.39 (2.70); 20 µm NaF, 13.84 (1.22); (22W) control, 4.98 (0.73); 10 µm NaF, 9.92 (0.99); 20 µm NaF, 11.43 (1.40) (n = 3). (E) TUNEL staining showed positive clusters of apoptotic cells in green. Scale bar = 20 µm.

Journal of Dental Research, Vol. 86, No. 4, 336-340 (2007)
DOI: 10.1177/154405910708600407


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