Surface properties and ion release from fluoride-containing bioactive glasses promote osteoblast differentiation and mineralization in vitro

Abstract

Bioactive glasses (BG) are suitable for bone regeneration applications as they bond with bone and can be tailored to release therapeutic ions. Fluoride, which is widely recognized to prevent dental caries, is efficacious in promoting bone formation and preventing osteoporosis-related fractures when administered at appropriate doses. To take advantage of these properties, we created BG incorporating increasing levels of fluoride whilst holding their silicate structure constant, and tested their effects on human osteoblasts in vitro. Our results demonstrate that, whilst cell proliferation was highest on low-fluoride-containing BG, markers for differentiation and mineralization were highest on BG with the highest fluoride contents, a likely effect of a combination of surface effects and ion release. Furthermore, osteoblasts exposed to the dissolution products of fluoride-containing BG or early doses of sodium fluoride showed increased alkaline phosphatase activity, a marker for bone mineralization, suggesting that fluoride can direct osteoblast differentiation. Taken together, these results suggest that BG that can release therapeutic levels of fluoride may find use in a range of bone regeneration applications.

Figure 1

X-ray diffraction patterns between 18 and 60 °2θ collected from the surface of BG discs incubated in cell culture medium for a) 7, b) 14 and c) 28 days. Labels indicate apatite (Ap) and calcium carbonate (C).

Figure 2

Representative scanning electron microscopy images of cells adherent to BG discs containing a) 1.00, b) 9.28, and c) 17.76 mol% CaF₂ after 14 days in culture. Scale bars represent 50 μm, 30 μm, and 50 μm, respectively. d-l) Representative images of apatite formation on BG discs of selected compositions after 7, 14 and 28 days in culture. All other groups formed apatite on their surfaces similar to that noted in the 13.62 mol% CaF₂ group. Cracks in the underlying apatite layer resulted from sample dehydration. Scale bars represent 3 μm.

Figure 3

Cell viability staining of Saos-2 cells adherent to BG discs after 14 and 28 days in culture. Live cells appear green whilst dead cells are stained red. Scale bar = 500 μm.

Figure 4

a) Total number of Saos-2 adherent to BG discs after 7, 14 and 28 days in culture.* indicates significantly more cells in the indicated group compared to the 0 mol% CaF₂ group at the same time point. † significantly more cells than the 9.28 mol% CaF₂ group at the same time point. # significantly more cells than 13.62 mol% CaF₂ group at same time point. § significantly more cells than 17.76 mol% CaF₂ group at same time point. b) Total number of Saos-2 cells in cultures exposed to the dissolution products of BG discs for up to 28 days. * indicates significantly more cells in the indicated group compared to any other group at the same time point. † significantly fewer cells than any other group at the same time point. # significantly more cells than 13.62 mol% CaF₂ group at same time point. § significantly more cells than 17.76 mol% CaF₂ group at same time point.

Figure 5

a) Alkaline phosphatase activity of Saos-2 cells cultured on the surface of BG discs for 7, 14 and 28 days. * indicates significantly higher ALP activity per cell in the indicated group compared to Saos-2 on 0 mol% CaF₂ discs at the same time point. b) Alkaline phosphatase activity of Saos-2 cells exposed to the dissolution products of BG discs after 7, 14 and 28 days in culture. * significantly higher ALP activity compared to all other groups at the same time point. + significantly higher ALP activity than the 0 mol% CaF₂ group at same time point. ‡ significantly higher ALP activity than the 4.75 mol% CaF₂ group at same time point. † significantly higher ALP activity than 9.28 mol% CaF₂ group at same time point. # significantly higher ALP activity than 13.62 mol% CaF₂ group at the same time point.

Figure 6

Release of IL-6 from Saos-2 cultured with BG discs. * significantly higher levels of IL-6 were detected in the indicated group compared to all other groups at the same time point. & significantly higher levels compared to the 1.00 mol% CaF₂ group at the same time point; ‡ significant compared to 4.75 mol% CaF₂ group at the same time point; † significant compared to 9.28 mol% CaF₂ group at the same time point.

Figure 7

a) AlamarBlue^® activity after 7, 14 and 28 days in culture in Saos-2 treated with NaF for the indicated time periods. b) Alkaline phosphatase activity per mg protein after 7, 14 and 28 days in culture for Saos-2 treated with NaF for the indicated time periods. * significantly higher levels in the indicated group at the same time point compared to cultures treated for days 0-7. † significant compared to cultures treated for days 0-14. ‡ compared to cultures treated for days 7-14. # compared to cultures treated for days 0-28. § compared to cultures treated for days 14-28.