The Impact of Fluoride on Ameloblasts and the Mechanisms of Enamel Fluorosis

Intake of excess amounts of fluoride during tooth development cause enamel fluorosis, a developmental disturbance that makes enamel more porous. In mild fluorosis, there are white opaque striations across the enamel surface, whereas in more severe cases, the porous regions increase in size, with enamel pitting, and secondary discoloration of the enamel surface. The effects of fluoride on enamel formation suggest that fluoride affects the enamel-forming cells, the ameloblasts. Studies investigating the effects of fluoride on ameloblasts and the mechanisms of fluorosis are based on in vitro cultures as well as animal models. The use of these model systems requires a biologically relevant fluoride dose, and must be carefully interpreted in relation to human tooth formation. Based on these studies, we propose that fluoride can directly affect the ameloblasts, particularly at high fluoride levels, while at lower fluoride levels, the ameloblasts may respond to local effects of fluoride on the mineralizing matrix. A new working model is presented, focused on the assumption that fluoride increases the rate of mineral formation, resulting in a greater release of protons into the forming enamel matrix

Enamel pits in hamster molars, formed by a single high fluoride dose, are associated with a perturbation of transitional stage ameloblasts

Excessive intake of fluoride (F) by young children results in formation of enamel subsurface porosities and pits, called enamel fluorosis. In this study, we used a single high dose of fluoride administered to hamster pups, to determine the stage of ameloblasts most affected by fluoride, and whether pit formation was related to F-related sub-ameloblastic cyst formation. Hamster pups received a single subcutaneous injection of either 20 mg or 40 mg NaF/kg body weights, were sacrificed 24 h later, and the number of cysts formed in the first molars counted. Other pups were sacrificed 8 days after F-injection when the first molars had just erupted, to score for enamel defects. All F-injected pups formed enamel defects in the upper half of the cusps in a dose-dependent way. After injection of 20 mg NaF/kg an average of 2.2 white spots per molar was found but no pits. At 40 mg NaF/kg, almost 4.5 spots per molar were counted as well as 2 pits per molar. The defects in erupted enamel were located in the upper half of the cusps, sites where cysts had formed at the transition stage of ameloblast differentiation. These results suggest that transitional ameloblasts, located between secretory and maturation stage ameloblasts, are most sensitive to the effects of a single high dose of fluoride. Fluoride- induced cysts formed earlier at the pre-secretory stage were not correlated to either white spots or enamel pits, suggesting that damaged ameloblasts overlying a fluoride induced cyst regenerate and continue to form enamel

Fate of fluoride-induced subameloblastic cysts in developing hamster molar tooth germs

White opacities and pits are developmental defects in enamel caused by high intake of fluoride (F) during amelogenesis. We tested the hypothesis that these enamel pits develop at locations where F induces the formation of sub-ameloblastic cysts. We followed the fate of these cysts during molar development over time. Mandibles from hamster pups injected with 20 mg NaF/kg at postnatal day 4 were excised from 1 h after injection till shortly after tooth eruption, 8 days later. Tissues were histologically processed and cysts located and measured. Cysts were formed at early secretory stage and transitional stage of amelogenesis and detected as early 1 h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16 h post-injection. The size of the cysts was about the same, i.e., 0.46 ± 0.29 × 106 μm3 at 2 h and 0.50 ± 0.35 × 107 μm3 at 16 h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16 h post-injection. With time new ameloblasts repopulated and covered the enamel surface in the cystic area. Three days after injection all cysts had disappeared and the integrity of the ameloblastic layer restored. After eruption, white opaque areas with intact enamel surface were found occlusally at similar anatomical locations as late secretory stage cysts were seen pre-eruptively. We conclude that at this moderate F dose, the opaque sub-surface defects with intact surface enamel (white spots) are the consequence of the fluoride-induced cystic lesions formed earlier under the late secretory-transitional stage ameloblasts

Ameloblast modulation and transport of Cl-, Na+, and K+ during amelogenesis

Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na+K+-dependent calcium transporter NCKX4 and the Na+-dependent HPO42- (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl− was strongly reduced; 2) K+ and Na+ accumulated (Na+ not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na+K+-dependent calcium transporter (likely NCKX4) and a Na+-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca2+, Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization

Horizontal Transmission of Mutans Streptococci in Children

It has not been established whether transmission of mutans streptococci occurs between unrelated children older than 4 years of age. The aim of the study was to investigate the possible transmission of mutans streptococci genotypes from child to child in kindergarten. We studied 96 children (ages 5-6 yrs) in three San Francisco Bay Area public schools. Mutans streptococci colonies from each child were isolated from selective culture on Mitis Salivarius Sucrose Bacitracin agar. We used arbitrary primed polymerase chain reactions to determine the mutans streptococci genotypes. Two children (not siblings) in each of the three schools (6%) shared an identical amplitype of S. mutans, unique to each pair. The 19 S. sobrinus amplitypes were found in 12 children, and all were unique to each child. The presence of matching genotypes of S. mutans demonstrates horizontal transmission of this species between unrelated children aged 5-6 years

NBCe1 in Mouse and Human Ameloblasts may be Indirectly Regulated by Fluoride

Enamel biomineralization results in a release of protons into the enamel matrix, causing an acidification of the local microenvironment. This acidification, which may be enhanced by more rapid mineral deposition in the presence of fluoride, must be neutralized by the overlying ameloblasts. The electrogenic sodium bicarbonate co-transporter NBCe1 has been localized in mouse ameloblasts, and has been proposed to have a role in matrix pH regulation. In this study, transcript analysis by PCR showed NBCe1-A present in human ameloblasts, whereas mouse ameloblasts expressed NBCe1-B. In situ hybridization and qPCR in mouse and fetal human incisors showed that NBCe1 mRNA was up-regulated as ameloblasts differentiated. Ingestion of 50 ppm fluoride resulted in an up-regulation of NBCe1 mRNA in maturation-stage mouse ameloblasts in vivo, as compared with controls. NBCe1 expression was up-regulated by low pH, but not by fluoride, in human ameloblast-lineage cells in vitro. The up-regulation of NBCe1 in vivo as enamel maturation and mineralization progressed provides evidence that NBCe1 participates in pH modulation during enamel formation. Up-regulation of NBCe1 in fluorosed maturation ameloblasts in vivo, with no effect of fluoride in vitro, supports the hypothesis that fluoride-enhanced mineral deposition results in acidification of the mineralizing enamel matrix