Fluoride-Treated Bio-Resorbable Synthetic Hydroxyapatite Promotes Proliferation and Differentiation of Human Osteoblastic MG-63 Cells
When resorbable hydroxyapatite (HA) granules, which are used as a bone supplement material, were treated in neutral 4% sodium fluoride (NaF) solution, formation of a reactant resembling calcium fluoride was observed on the surface of the granules. Immediate and slow release of fluoride from fluoridated HA (HA+F) granules was observed after immersion in culture fluid, and the concentration increased over time to 1.25 ± 0.05 ppm F at 0.5 hours, 1.57 ± 0.12 ppm F at 24 hours, and 1.73 ± 0.15 ppm F at 48 hours. On invasion assay, migration of human osteoblast-like MG-63 cells exposed to the released fluoride was confirmed in comparison to the cells incubated with a nonfluoridated control sample (P < .01). In addition, fluoride added to the medium increased MG-63 cell proliferation in a manner dependent on fluoride concentrations up to 2.0 ppm (P < .05). At 5.0 ppm, however, fluoride significantly inhibited cell proliferation (P < .005). Activity of the osteogenic differentiation marker, alkaline phosphatase (ALP), also increased with fluoride after exposure for 1 week, increasing significantly at 1.0 ppm (P < .05). The promotion of MG-63 cell migration and proliferation, as well as increased ALP activity, suggested that fluoride released from the surface of resorbable HA granules, which were fluoridated by prior treatment with neutral 4% NaF solution, can provide a superb method to supply fluoride and promote osteogenic cell differentiation.
Fluoride released into culture medium from fluoridated hydroxyapatite granules. Release of fluoride from fluoridated hydroxyapatite granules was measured with a fluoride ion-selective electrode as described in Materials and Methods. Filled diamonds represent mean values with standard error (vertical bars).
MG-63 cell migration due to fluoride released from fluoridated hydroxyapatite granules. MG-63 cells were stained with hematoxylin-eosin after 24-hour culture in the presence of nonfluoridated hydroxyapatite granules (a) and fluoridated hydroxyapatite granules (b). Fluoride concentrations were less than 0.01 ppm F (a) and 1.51 ± 0.10 ppm F (b), respectively, after 24-hour incubation.
Figure 3. Cell migration in the presence of fluoridated or vehicle-treated fluoridated hydroxyapatite granules. Cell migration was assessed by invasion assay as described in Materials and Methods. Number of MG-63 cells, which migrated to the outer layer, was counted after the 24-hour incubation period with either fluoridated or mock-treated hydroxyapatite granules as in Figure 2. Vertical scale represents number in the field of view, 2.34 mm2. Fluoride concentrations were the same as in Figure 2. *P < .01. Figure 4. Effects of released fluoride on MG-63 osteoblastic cell proliferation. Cell proliferation was assessed by the MTS assay as described in Materials and Methods. Differences in MG-63 cells before and after 24-hour incubation with control hydroxyapatite medium were set at 100%. In culture medium with diluted, released fluoride, cell proliferation was significantly higher than in controls at 1.0 and 2.0 ppm F, and significantly lower than in controls at 5.0 ppm F. *P < .05. **P < .005. Figure 5. Effects of released fluoride on alkaline phosphatase (ALP) activity in MG-63 cells. ALP activity was measured after 1-week culture in the medium with diluted, low concentration of fluoride, 0.5, 1.0, and 2.0 ppm F. ALP activity was significantly higher with medium containing 1.0 ppm F than with control medium. *P < .05.
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