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Supraoptimal intake of fluoride (F) induces structural defects in forming enamel, dentin and bone and increases the risk of bone fractures. In comparison to bone and dentin is formation of enamel most sensitive to low levels of F and the degree of enamel fluorosis depends on the mouse strain. What molecular mechanism is responsible for these differences in sensitivity is unclear. Maturation ameloblasts transport bicarbonates into enamel in exchange for Cl- to buffer protons released by forming apatites. We proposed that F-enhanced mineral deposition releases excess of protons that will affect mineralization in forming enamel. In this study we tested the hypothesis that increased sensitivity to F is associated with a reduced capacity of ameloblasts to buffer acids. Quantified electron probe microanalysis showed that enamel of F-sensitive C57Bl mice contained the same levels of Cl- as enamel of F-resistant FVB mice. Enamel of C57Bl mice was less mineral dense, contained less Ca but more Mg and K. Ameloblast modulation was much more impaired than in FVB mice. In enamel of FVB mice the levels of Mg correlated negative with Ca (r=-0.57, p=0.01) and with the Ca/P molar ratio (r=-0.32, p=0.53). In moderate and high acidic enamel the correlations between Mg and Ca/P ratio were strong (r=-0.75, p=0.08) to very strong negative (r=-0.98, p=0.0020), respectively. Correlations in enamel between F and Ca were (weak) negative but between F and Ca/P very high positive (r=+0.95, p=0.003) in high acidic enamel and less positive (r=0.45, p=0.27) in moderate acidic fluorotic enamel (r=0.45, p=0.27). Similar correlations between Mg and Ca/P or F and Ca/P were found in dentin and bone of fluorotic and Cftr null mice. These data are consistent with the concept that Mg delays but F increases maturation of crystals particularly when enamel is acidic. The sensitivity of forming enamel to F likely is due to the sensitivity of pH cycling to acidification of enamel associated with F-induced release of protons., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Supra-optimal intake of sodium fluoride (NaF) during early childhood results in formation of irreversible enamel defects. Monofluorophosphate (MFP) was considered as less toxic than NaF but equally cariostatic. We compared the potency of MFP and NaF to induce pre-eruptive sub-ameloblastic cysts and post-eruptive white spots and pits in developing hamster enamel. Hamster pups were injected subcutaneously with either NaF or MFP in equimolar doses of either 9 mg or 18 mg F/kg body weight. At 9 mg F/kg, MFP induced more but smaller sub-ameloblastic cysts with a collective cyst volume twice as large as that induced by NaF. Eight days after F injection, all F-injected groups had formed 4-6 white spots per molar, with an additional 2 pits per molar in the low MFP group. Twenty-eight days after injection, most white spots had turned into pits (5-6 per molar) and only the high MFP group still contained 2 white spots per molar. We conclude that parenterally applied MFP is more potent in inducing enamel defects than NaF. Most white spots formed turn into pits by functional use of the dentition. The higher potency of parenteral MFP may be associated with sustained elevated F levels in the enamel organ by enzymatic hydrolysis of MFP by alkaline phosphatase activity.

Enamel fluorosis is an irreversible structural enamel defect following exposure to supraoptimal levels of fluoride during amelogenesis. We hypothesized that fluorosis is associated with excess release of protons during formation of hypermineralized lines in the mineralizing enamel matrix. We tested this concept by analyzing fluorotic enamel defects in wild-type mice and mice deficient in anion exchanger-2a,b (Ae2a,b), a transmembrane protein in maturation ameloblasts that exchanges extracellular Cl(-) for bicarbonate. Defects were more pronounced in fluorotic Ae2a,b (-/-) mice than in fluorotic heterozygous or wild-type mice. Phenotypes included a hypermineralized surface, extensive subsurface hypomineralization, and multiple hypermineralized lines in deeper enamel. Mineral content decreased in all fluoride-exposed and Ae2a,b(-/-) mice and was strongly correlated with Cl(-). Exposure of enamel surfaces underlying maturation-stage ameloblasts to pH indicator dyes suggested the presence of diffusion barriers in fluorotic enamel. These results support the concept that fluoride stimulates hypermineralization at the mineralization front. This causes increased release of protons, which ameloblasts respond to by secreting more bicarbonates at the expense of Cl(-) levels in enamel. The fluoride-induced hypermineralized lines may form barriers that impede diffusion of proteins and mineral ions into the subsurface layers, thereby delaying biomineralization and causing retention of enamel matrix proteins.

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.

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.

Renal impairment in children is associated with tooth defects that include enamel pitting and hypoplasia. However, the specific effects of uremia on tooth formation are not known. In this study, we used rat mandibular incisors, which continuously erupt and contain all stages of tooth formation, to characterize the effects of uremia on tooth formation. We also tested the hypothesis that uremia aggravates the fluoride (F)-induced changes in developing teeth. Rats were subjected to a two-stage 5/6 nephrectomy or sham operation and then exposed to 0 (control) or 50 ppm NaF in drinking water for 14 days. The effects of these treatments on food intake, body growth rate, and biochemical serum parameters for renal function and calcium metabolism were monitored. Nephrectomy reduced food intake and weight gain. Intake of F by nephrectomized rats increased plasma F levels twofold and further decreased food intake and body weight gain. Uremia affected formation of dentin and enamel and was more extensive than the effect of F alone. Uremia also significantly increased predentin width and induced deposition of large amounts of osteodentin-like matrix-containing cells in the pulp chamber. In enamel formation, the cells most sensitive to uremia were the transitional-stage ameloblasts. These data demonstrate that intake of F by rats with reduced renal function impairs F clearance from the plasma and aggravates the already negative effects of uremia on incisor tooth development.

We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [3H]proline and uptake of 45Ca and acid-soluble 32PO4. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non-mineralizing) enamel matrix, increased matrix secretion but could not prevent F-induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front.

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F-) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5-20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of >or=5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F- is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.

Amelogenins are the major protein species synthesized by secretory ameloblasts and are believed to be involved in enamel mineralization. During enamel formation, amelogenins are progressively degraded into smaller fragments by protease activity. These amelogenin fragments are removed from the enamel extracellular space, thereby enabling full mineralization of the dental enamel. Enamel from fluorotic teeth is porous and contains more proteins and less mineral than sound enamel. In this study we examined the hypothesis that fluoride (F-) is capable of inhibiting the proteolysis of amelogenins in enamel being formed in organ culture. Hamster molar tooth germs in stages of secretory amelogenesis were pulse labeled in vitro with [3H]- or [14C] proline and subsequently pulse chased. The explants were exposed to F- at different days of chase (i.e., during secretory amelogenesis early after labeling, later after labeling or at stages just beyond secretory amelogenesis). Exposure of secretory stage explants to F- enhanced the release of radiolabeled fragments when F- was applied early after labeling but progressively less if applied later. In contrast, F- had no such effect in stages beyond secretion. The enhanced release of radiolabeled fragments in secretory stages was associated with a reduction of radioactivity in the soft tissue enamel organ indicating that fragmentation of enamel matrix proteins (mainly amelogenins) occurred intracellularly. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the fluorotic enamel contained less radiolabeled parent amelogenins (M(r) 28 kD and 26 kD) but more low-molecular-mass fragments than enamel from control explants. Our data indicate that F- promotes intracellular degradation of the newly synthesized parent amelogenins during secretory stage. Our in vitro data do not support the concept that F- impairs extracellular proteolysis of amelogenins, either in the secretory phase or in the stage just beyond the secretory phase.

The recovery of mineralization capacity of fluorotic enamel matrix was investigated in 3-day-old hamster first molar tooth germs already pre-exposed in organ culture to 10 parts/10(6) F- for 24 h during the secretory phase. The germs were then cultured for another 24 h in a fresh medium without F-. The unmineralized fluorotic enamel matrix secreted in vitro eventually mineralized in the absence of F- but the orientation of the crystals compared to those in the fluorotic enamel was disturbed, especially in the younger regions of the enamel nearest cervical-loop in which the underlaying fluorotic enamel was most hypermineralized; but least disturbed in the more mature parts of the enamel organ in which the fluorotic enamel was less hypermineralized. The subsequent culture in F(-)-free medium did not abolish or reduce the degree of hypermineralization induced by F- treatment during the initial 24 h of culture. It seems that in vitro the inhibitory effect of F- on enamel matrix mineralization during the secretory phase is completely reversible when the ion is removed from the matrix environment, i.e. F(-)-induced synthesis and secretion of defective enamel matrix is not the cause of the lack of matrix mineralization. The F(-)-induced hypermineralization seems to be irreversible.

Whether the interference by fluoride (F-) with secretory amelogenesis in vitro could be modulated by altering the levels of calcium (Ca) and inorganic phosphate (P) in the medium was investigated. Hamster first upper molar tooth germs in the secretory phase of amelogenesis were exposed to 10 microM-1.31 mM (0.2-25 parts/10(6)) of F- in vitro for 2 days in the presence of either low (1.2 mM), moderate (2.1 mM) or high (4.1 mM) levels of Ca, or moderate (1.6 mM) and high (3.6 mM) levels of P. The biosynthesis and secretion of enamel matrix proteins under each of the experimental conditions were examined by labelling with [3H]-proline during the last 24 h of culture, and mineralization by labelling with 45Ca and [32P]-orthophosphate. With moderate levels of Ca and P (control medium), F- increased the uptake of 45Ca and 32P in a dose-dependent manner; F- did not inhibit the synthesis of matrix proteins but to a moderate extent impaired their secretion. In explants grown in the presence of 52 microM of F- the superficial layers of enamel matrix deposited in vitro (fluorotic matrix) failed to mineralize. Increasing P levels in the medium had no clear histological effect, whereas lowering Ca levels sometimes seemed to aggravate the F- effect. Raising Ca levels improved the histological pattern: in spite of the presence of F-, high Ca levels allowed a limited mineralization of the superficial layer of fluorotic matrix along with a strong rise in mineralization of the deeper layers of pre-exposure enamel. High Ca levels also considerably reduced the cellular changes in secretory ameloblasts induced by 52 microM of F- and slightly counteracted the inhibition of matrix secretion, as measured biochemically. Some of the effects of F- on secretory amelogenesis in vitro can thus be reversed by raising Ca levels in the medium. Therefore, the effect of F- on secretory amelogenesis in vitro seems to be primarily interference with the enamel mineralization process per se and, secondarily, an impairment of matrix secretion.

The effects of fluoride (5, 10 and 20 parts/10(6) F-) were studied in vitro with light and electron microscopy in 5-day-old hamster maxillary second molar tooth germs explanted when most of the ameloblasts are in the secretory phase, and cultured for 24 h in the presence of F-. F- at all doses investigated induced hypermineralization of that enamel which had been secreted in vivo just prior to exposure to F-. The most intense hypermineralization was in the aprismatic enamel near the cervical loop region, where the in-vivo enamel layer was thinnest and gradually decreased (but was not abolished) with the increasing thickness of in-vivo formed enamel in the more mature parts of the enamel organ. The fluoride-induced hypermineralization in the aprismatic enamel layer did not stain at all with dilute toluidine blue solution and was therefore indistinguishable from the underlying dentine in light micrographs. The hypermineralization was due to growth in thickness of the enamel crystals, which in the aprismatic enamel layer resulted in a lateral fusion of all the enamel crystals. Thus fluoride administered during the secretory phase of enamel formation decontrols or even abolishes enamel crystal growth in length and promotes crystal growth in thickness so producing the hypermineralization of the pre-fluoride enamel. Enamel matrix secreted in the presence of fluoride did not mineralize.

The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed to fluoride (F-) in vitro was analyzed for its mineral content by means of the energy-dispersive x-ray microanalysis technique. The aim of this study was to obtain semi-quantitative data on the F(-)-induced hypermineralization patterns in the enamel and to confirm that the increase in electron density observed in micrographs of F(-)-treated enamel (Lyaruu et. al., 1986, 1987b) is indeed due to an increase in mineral content in the fluorotic enamel. The tooth germs were explanted during the early stages of secretory amelogenesis and initially cultured for 24 hr in the presence of 10 ppm F- in the culture medium. The germs were then cultured for another 24 hr without F-. In order to compare the ultrastructural results directly with the microprobe data, we used the same specimens for both investigations. The net calcium counts (measurement minus background counts) in the analyses were used as a measure of the mineral content in the enamel. The aprismatic pre-exposure enamel, deposited in vivo before the onset of culture, was the most hypermineralized region in the fluorotic enamel, i.e., it contained the highest amount of calcium measured. The degree of the F(-)-induced hypermineralization gradually decreased (but was not abolished) in the more mature regions of the enamel. The unmineralized enamel matrix secreted during the initial F- treatment in vitro mineralized during the subsequent culture without F-. The calcium content in this enamel layer was in the same order of magnitude as that recorded for the newly deposited enamel in control tooth germs cultured without F-.(ABSTRACT TRUNCATED AT 250 WORDS)

The prevalence of mottled enamel in the permanent dentition of children participating in a fluoride (F-) program at the dental school of the Vrije Universiteit (Amsterdam) was investigated in a study utilizing the Thylstrup-Fejerskov (TF) index. The randomly chosen children received a F- regime considered optimal by the Dutch Advisory Committee for Prevention of Oral and Dental Diseases. From the children examined (n = 83; 49 boys and 34 girls; mean age, 13 years and 5 months), 74% exhibited mottled enamel in a slight to moderate degree. More teeth were affected and the degree of mottling was higher when children started to use F- at an earlier age. Unintentional ingestion of toothpaste containing 0.15% F- during frequent toothbrushing in combination with the daily intake of F- tablets before the age of four may explain the high prevalence of mottled enamel. After these treatments, F- concentrations in plasma of young children can reach values which can directly affect the developing tooth germ.