Effect of temperature and fluoride on ameloblasts in vitro

Abstract

Several external agents may alter the delicate balance between stimulatory factors, enamel proteins and cells during tooth development and enamel mineralization. Fluoride is well known as a specific and effective caries prophylactic agent, excess fluoride may, however, promote disturbances in enamel development and mineralization of teeth known as dental fluorosis. A systemic condition during tooth development, such as high fever, may also affect the mineralization process and produce a pattern of enamel defects in the dentition. We have examined the effect of fluoride and heat, alone or in combination, on protein and gene expression in murine ameloblasts. Murine ameloblasts (LS-8) has been exposed to heat (40C for 24 h) and various concentrations of sodium fluoride (40 μg/ml and 200 μg/ml), alone or in combination, for 1, 3 and 7 days. Cell culture medium and cells have been harvested at each time point. mRNA was isolated using Dynal beads (Dynal, Norway), and mRNA expression of various genes were quantified by real-time PCR. Secretion of cytokines and interleukins to the medium has been measured using 20-plex kit (BioSource) and Luminex technology. We observed no significant toxic effect of fluoride or heat on the cells, measured as lactate dehydrogenase activity (LDH) in the cell culture medium. Alkaline phospatase (ALP) activity in the culture medium was not affected by the treatments, neither was osteocalcin mRNA expression. Amelogenin (AMEL) mRNA expression and the expression of matrix metalloperptidase 20 (MMP-20) were significantly reduced with the highest concentration of fluoride. There was an acute increase in the expression of enamelin by both 40 μg/ml and 200 μg/ml fluoride after 1 day, whereas fluoride in combination with heat had no effect. After 7 days of exposure to the highest concentration of fluoride the enamelin mRNA expression was significantly reduced compared to control. The secretion of vascular endothelial growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and interferon inducible protein 10 (IP-10) to the medium was reduced by 80% of control with fluoride. The observed effects were time-and dose dependent. The reduction in amelogenin, MMP-20 and enamelin expression, all proteins involved in the structural organization of apatite crystals during enamel mineralization, as well as a reduction in vascular signalling factors with high dosages of fluoride may be mechanisms involved in the development of enamel defects.