Effects of fluoride on protein secretion and removal during enamel development in the rat

Enamel maturation consists of a loss of the early secreted matrix proteins and an increase in mineralization. This study investigated the changes in enamel proteins of the rat incisor, caused by the ingestion of fluoride at various stages of enamel formation. Rats were given 0, 10, 25, 50, or 100 ppm fluoride in drinking water for five weeks. Changes in the protein composition of the secretory, early-maturation, and late-maturation enamel were investigated by means of gel filtration chromatography and polyacrylamide gel electrophoresis. No differences were found between fluorosed and control enamel proteins in secretory enamel. In fluorosed early-maturation enamel, amelogenins were retained in larger quantities than in control enamel in animals ingesting 25 ppm fluoride or greater. At the late-maturation stage of enamel formation, only enamel from animals ingesting 100 ppm fluoride in drinking water contained more protein, when compared with control enamel. This study suggests that fluoride ingestion levels resulting in enamel fluorosis inhibit the mechanisms involved in the removal of proteins during enamel maturation.     

Changes in the fluoride-induced modulation of maturation stage ameloblasts of rats

The maturation stage of enamel development is characterized by a cyclic modulation of the ameloblasts between bands of smooth-ended cells and longer bands of ruffle-ended cells. There are cyclic patterns of calcein staining of and 45Ca uptake in the enamel associated with this cellular modulation. Rats were given 0, 75, 100, or 150 ppm fluoride in their drinking water. Fluoride disrupted the cyclic patterns of the maturation stage, resulting in fewer bands of smooth-ended ameloblasts, fewer calcein-stained stripes, and fewer cycles of 45Ca uptake. When animals were given water containing 0 ppm fluoride following ingestion of water containing 100 ppm fluoride, the pattern of calcein staining returned to that of the control enamel. The disruption of the cyclic patterns in the maturation stage and the increased protein content of maturation enamel seem to be among the early events in the development of fluorosis.     

Effect of low levels of fluoride in solution on enamel demineralization in vitro

The effect of low levels of fluoride in solution on in vitro enamel demineralization has been studied. Extracted human teeth were exposed to 0.1 M lactate solutions (at pH 4.3) which were partially saturated with respect to enamel mineral and which contained between 0.004 and 1 ppm fluoride. Enamel demineralization was monitored using SEM and polarized light microscopy. It was observed that, in the absence of fluoride, rapid enamel demineralization occurred, resulting in the formation of cavitations within 72 hours. Using the same demineralizing medium containing as little as 0.024 and 0.054 ppm fluoride, we observed a remarkable protection of the enamel surface. Subsurface enamel demineralization was, however, observed under these conditions as well as in a solution containing 0.154 ppm fluoride. When the demineralizing solution containing 1 ppm fluoride was used, no mineral loss was detected. The observed inhibition of enamel demineralization was also shown to be associated with a significant uptake of fluoride by the enamel mineral. These observations have been noted to correlate with an increase in solution supersaturation with respect to fluoridated apatitic species. The results obtained were found to be consistent with a proposed hypothesis that the net rate of enamel demineralization will be reduced in a demineralizing medium supersaturated with respect to less soluble fluoridated phases, due to the enhancement of the precipitation rate of fluoridated apatitic phases relative to the rate of dissolution of the original enamel surface.     

Effect of inhibition of net calcium uptake on net fluoride uptake in developing rat molars

Two methods were used to reduce net calcium uptake by the secretory stage enamel of developing rat molar explants. Neither method had a significant effect on fluoride uptake by the explants. These findings indicate that the mechanisms for uptake in the developing enamel are independent for calcium and fluoride.     

In-vitro study of cadmium-109 uptake by rat molar enamel during the secretory stage of formation

Rat molar explants, taken at an age when enamel formation was entirely in the secretory phase, were cultured for 4 h under conditions previously shown to support enamel-organ viability for 10–12h. The uptake of ¹⁰⁹Cd was compared in control explants and their contralateral pairs which had been either stripped of enamel organ, heat-treated, or metabolically inhibited. Evaluation of tracer uptake was by autoradiography, and by quantitatively measuring loss of ¹⁰⁹Cd from the medium, and uptake in the explants. The findings indicated that ¹⁰⁹Cd was readily absorbed by the forming enamel if the enamel organ was not present. Relative exclusion of the tracer from enamel during the secretory phase was dependent on the integrity and viability of the enamel organ. Binding of cadmium by plasma proteins was not required.     

The in vitro uptake of fluoride by secretory and maturation stage bovine enamel

The objectives of this study were to determine the specific surface area of secretory-stage and of maturation-stage enamel, to compare the fluoride uptake by isolated enamel at these two stages on a surface-area basis, and to examine the effect of the organic matrix on the fluoride uptake by whole enamel. Fetal bovine secretory and maturation stage enamel samples were collected, and a portion of the enamel at each developmental stage was treated with hydrazine for removal of the organic matrix. The specific surface areas of the enamel mineral, as determined by the multi-point BET method, were 59.3 m2/g in the secretory stage and 37.9 m2/g in the maturation stage. Whole and deproteinated enamel samples were equilibrated in buffered solutions containing 10(-5) to 10(-3) mol/L fluoride, and the uptake was measured with a fluoride specific electrode. The results indicate that the in vitro fluoride uptake was controlled solely by the surface area of the apatitic mineral and that the organic matrix did not contribute to the fluoride uptake.     

Effects of fluoride and/or trace elements on enamel solubility

Various studies have shown that the solubility of dental enamel can be reduced by exposing it to metal ions in the presence of fluoride. The purpose of this study was to determine the effects of various trace metal ions at two concentrations on dental enamel solubility and fluoride uptake. The solubilities of the enamel surfaces and their fluoride concentrations were first determined. Atypical teeth were discarded. Blocks of enamel were then divided into groups of five and solutions of salts of aluminium, strontium, titanium, molybdenum and vanadium followed by fluoride (4,000 ppm) were applied. Two successive layers were then etched off, using perchloric acid. Fluoride in the enamel was determined using an ion-specific electrode system. Calcium was determined by atomic absorption spectrophotometry. The values obtained were used to calculate the thicknesses of the layers removed. The results showed that combined application of aluminium and fluoride led to a marked reduction in solubility of enamel (p less than 0.001). Application of strontium (0.01 M) and fluoride also led to a significant reduction in enamel solubility (p less than 0.01). Pretreatment with titanium solution (both concentrations), aluminium or molybdenum solutions (higher concentrations) increased fluoride uptake in comparison with the control group. Statistically significant increase in fluoride uptake was seen only in those enamels treated with titanium solution at higher concentration when compared with those treated with fluoride alone.     

In vivo enamel fluoride uptake after use of fluoride products

PURPOSE: To evaluate fluoride uptake in enamel after use of products containing different fluoride components of distinct oral hygiene regimen in vivo. METHODS: 84 healthy subjects (24 females and 60 males aged 19-26 years, mean 22.2) recruited from the students attending the courses of the Medical Faculty of the University of Sassari, Italy, were randomly assigned to one of four treatments groups: A: treated with a dentifrice containing sodium monofluorophosphate (1250 ppm F-); B: treated with dentifrice with 1250 ppm F- from AmF; C: treated with an AmF toothpaste (1250 ppm F-) and a mouth rinse with 250 ppm F- from AmF; or D: treated with a NaMFP toothpaste (1250 ppm F-) plus 1250 ppm NaMFP varnish. Acid-etched enamel microbiopsies on the vestibular surface of the first maxillary premolar were collected at baseline (to), after 20 days of test product use (t1) and after 24 hours without treatment (t2). Fluoride concentration was measured using an ion-specific electrode. All measurements were made in triplicate and analyzed statistically using two-way factorial ANOVA for independent samples standard weighted-means analysis. RESULTS: The fluoride uptake in the enamel shows a considerable variation between the groups. Significant differences were found both within all products in terms of fluoride concentration in enamel for (P < 0.0001 for two-way ANOVA, and later Tukey HSD test). After 20 days of treatment, the group using AmF toothpaste plus AmF mouthrinse had a higher fluoride uptake than the groups using either of the other products (P < 0.05). In conclusion, all products lead to an appreciable amount of fluoride uptake in enamel; AmF products led to higher concentrations. The concentration of fluoride accumulated during treatment phase remained high after 24 hours following the cessation of use.     

Effect of calcium in model plaque on the anticaries activity of fluoride in vitro.

The uptake of calcium by a polysaccharide (agarose) gel used as a model for plaque from a two-step treatment (consisting of a calcium rinse followed by a fluoride treatment) and the effect of the deposited calcium in model plaque on caries lesion formation in enamel were determined. Calcium uptake was measured by treatment of the model plaques with [45Ca]-CaCl2 solutions with or without NaF. A two-step treatment consisting of calcium followed by fluoride produced a 100% increase in calcium content of model plaque, presumably due to the formation of CaF2, compared with a treatment with artificial saliva followed by calcium alone. The effects of these increased plaque minerals on caries lesion formation were studied by subjecting model-plaque-covered enamel blocks to a cyclic demineralization-remineralization treatment. Artificial-plaque-covered enamel blocks were treated daily with 180 ppm calcium for ten min, then 100 ppm fluoride for ten min, followed by demineralization for 16 h, and finally, remineralization for seven h and 40 min. After five days, the blocks were sectioned, and lesion formation was determined by microradiography-microdensitometry. Artificial plaque treated with a calcium rinse followed by a fluoride rinse reduced lesion size by 90%, compared with a 68% reduction by a fluoride rinse alone. When the experiment was repeated with a simulated pre-brush calcium rinse (180 ppm calcium) followed by a fluoride dentifrice suspension (110 ppm fluoride), lesion size was reduced by 46%, compared with a 32% reduction by the fluoride dentifrice suspension alone.     

Effect of calcium, given before or after a fluoride insult, on hamster secretory amelogenesis in vitro

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.     

Dental fluorosis developed in post-secretory enamel

The aim of this study was to test whether dental fluorosis can be produced by administration of chronic doses of fluoride during only the post-secretory stage of enamel mineralization. Eight control and eight experimental pigs matched by weight and litter were fed a low-fluoride diet (less than 0.05 mg F-/kg b.w. daily) from weaning to slaughter at 14 months. The test group received an oral dose of 2 mg F-/kg b.w. per day from 8 months of age. Lower fourth pre-molars were at the post-secretory stage at the start of fluoride administration (confirmed by tetracycline marker) and were just erupting at slaughter. All of the fourth pre-molar teeth from the test group developed diffuse enamel hypomineralization indistinguishable from human fluorosis. No such lesions were seen in any of the teeth from the control animals. It was concluded that enamel fluorosis may be caused by fluoride exposure in the maturation phase only. The pathogenic mechanism may be an effect either on the selective loss of protein or on the influx of mineral, both of which occur during the post-secretory or maturation stage of enamel formation.     

CaF2 in enamel biopsies 6 weeks and 18 months after fluoride treatment

Fluoride concentrations were studied in enamel biopsies from maxillary central incisors 6 weeks and 18 months after fluoride treatment. In the short-term study biopsies were obtained prior to and after treatment with acidic sodium or ammonium fluoride. The findings showed that large amounts of fluoride were deposited in enamel from NH4F treatment (mean concentration 84,723 ppm), indicating substantial CaF2 formation. NaF treatment resulted in mean fluoride concentrations of 7,818 ppm. In the 18-month study, biopsies from 58 placebo-treated teeth were analyzed for total fluoride (mean 1,733 ppm). Twenty-five additional biopsies from placebo-treated and 58 from NH4F-treated teeth were analyzed for KOH-soluble (CaF2) and KOH-nonsoluble (apatitic) fluoride. The mean values for total fluoride were 1,669 and 2,085 ppm in the placebo-treated and in the NH4F-treated groups, respectively. The corresponding mean values for KOH-nonsoluble fluoride were 1,467 and 1,731 ppm and for KOH-soluble fluoride 202 and 354 ppm, respectively. The increase in enamel fluoride after fluoride treatment was only marginally significant. Biopsies from the ammonium fluoride treated group were significantly more likely to have high (30 vs. 8%) and moderate (28 vs. 16%) CaF2 levels and less likely to have low levels than biopsies of placebo-treated teeth (chi-square = 8.0 with 2 d.f.; p = 0.018). It is concluded that very substantial amounts of CaF2 are present in enamel 6 weeks after treatment, and small amounts may persist in the surface enamel for as long as 18 months.     

The influence of plaque on reaction mechanism of MFP and NaF in vivo

A modified Intra-oral Cariogenicity Test was used to study the influence of plaque on the reaction mechanism of sodium monofluorophosphate (MFP) or sodium fluoride (NaF) in either sound or demineralized enamel in vivo. Volunteer students, wearing mouth appliances holding enamel blocks, rinsed their mouths with MFP or NaF solution (1,000 ppm F-) three times a day. The amount of loosely-bound and acquired fluoride was determined after an experimental period of five days in plaque-covered, demineralized (PCD); clean, demineralized (CD); plaque-covered, sound (PCS); and clean sound enamel (CS). While no measurable loosely-bound fluoride could be found after MFP treatment, NaF caused deposition of a significant amount of alkali-soluble fluoride in all experimental groups. After MFP rinses, fluoride concentration in the enamel was increased in the following order: CS, PCS, CD, and PCD. After NaF treatment, demineralized enamel exhibited a higher fluoride acquisition when compared with sound enamel. Plaque had a minor effect on F- acquisition. It is concluded that demineralization of enamel enhances F- uptake from both NaF and MFP solutions. In the presence of plaque, F- acquisition was additionally increased only after MFP rinses in vivo.     

Fluoride uptake and development of artificial erosions in bleached and fluoridated enamel in vitro

The aim of the present in vitro study was to evaluate the effect of carbamide peroxide (CP) bleaching on fluoride uptake in enamel. Additionally, the susceptibility for erosion in bleached and fluoridated enamel was tested. Each four enamel specimens were prepared from 44 bovine incisors. The four samples gained from each tooth were distributed among four groups (A-D) of 44 specimens each: A: (unbleached/unfluoridated) served for determination of baseline fluoride concentration; B: (unbleached/fluoridated) four times in 2000 ppm NaF solution (2 min); C: (bleached/unfluoridated) four times in 10% CP (8 h); D: (bleached/fluoridated) four times in 10% CP and fluoridation. In 22 specimens of each group both KOH-soluble and structurally bound fluoride were determined in the outermost 30 micro m of the enamel. In the remaining specimens erosions were induced by immersing the samples in 5 mL of 1% citric acid (20 min) and microhardness was evaluated before and after demineralization. Statistical analysis showed that KOH-soluble fluoride uptake was similar for group B and D specimens. Structurally bound fluoride uptake after fluoride application in unbleached samples was significantly higher than in bleached ones. Bleaching only resulted in a highly significant fluoride loss. In all samples erosion caused a significant microhardness loss. The study showed that pre-treatment of enamel with CP followed by fluoridation does not improve erosive resistance. Moreover the study reveals that CP treatment decreases concentration of structurally bound fluoride in enamel which could not be outweighed by fluoridation with 2000 ppm NaF. It is concluded that it is not feasible to improve fluoride uptake in enamel with a pre-treatment with 10% CP.     

不同浓度的含氟涂料对人牙釉质抗酸性的影响

目的：确定4种浓度的含氟涂料对人牙釉质抗酸性的影响。方法：采用22000ppm,10000ppm,5000ppm及2200ppm等4种不同浓度的含氟涂料处理离体人牙釉质后经脱矿实验，然后测量酸蚀液中钙的溶出量，并用激光荧光诊断仪对人工龋损进行定量诊断。结果：22000ppm组与10000ppm组的抗酸性无差别，而其余各组之间均有差别。结论：22000ppm及10000ppm的含氟涂料抑制牙釉质脱矿的效果比5000ppm及2200mmp的含氟涂料好，而将22000ppm的含氟涂料浓度降至10000ppm，并不影响它的防龋效果，因此，在临床应用时，可以考虑适应降低含氟涂氟浓度以提高安全性。     

Enamel fluoride uptake affected by site of application: comparing sodium and amine fluorides

In enamel fluoride uptake studies, the most frequently sampled site is the middle third of the buccal surface. Because different parts of the enamel surface vary in fluoride concentration, the present study investigated fluoride uptake at contrasting sites using two different topical agents. One was a neutral aqueous solution of sodium fluoride containing 2% w/w of fluoride, and the second was an aqueous solution of two amine fluorides containing 1% w/w of fluoride. The enamel of 10 pairs of clinically sound extracted human premolars was etched initially and after treatment with one of these agents on the cervical and middle thirds of the buccal surface and on the proximal surface, yielding the pre- and post-treatment fluoride concentrations of these sites at depths of 5 and 10 microns from the surface. Enamel treated with the amine F solution had significantly higher fluoride uptakes at all sites compared to the NaF-treated specimens. The differences in uptake from the two agents varied with site, being smallest for the buccal middle third enamel and greatest for the proximal enamel. It is suggested that these results relate to possible differences in enamel maturation or to the presence of initial proximal caries, and the greater affinity of amine fluoride for porous enamel. The findings emphasise the importance of obtaining site-specific data in the study of fluoride in enamel.     

Changes in dental fluorosis following an adjustment to the fluoride concentration of Hong Kong's water supplies

In June, 1978, the fluoride concentration in Hong Kong water supplies was reduced from 1.0 to 0.7 mg/L. The objectives of this study were (1) to determine whether, as a result of this minor adjustment, a consequent reduction in the prevalence and severity of dental fluorosis came about, and (2) to determine whether dental fluorosis develops during enamel secretion and primary mineralization or during the maturation stage of enamel development. Dental fluorosis was assessed by Dean's community fluorosis index (CFI) on upper central incisors in 2382 children aged from 7 (exposed to 0.7 mg/L only) to 13 years. The children were selected from four districts served with drinking water by four different water treatment stations. Differences in the distributions of dental fluorosis scores across ages were significant in all districts. The susceptibility to fluoride was assessed statistically through a series of analyses whereby the fluoride concentration in the drinking water (both coincident with enamel secretion and periods of enamel maturation) was correlated with CFI. It was concluded (1) that CFI values were reduced following a minor adjustment to the fluoride concentration in drinking water, (2) that dental fluorosis develops during the maturation stage of enamel development, (3) that the development of dental fluorosis may occur over a period of 16 to 24 months, commencing from 12 to 32 months following enamel secretion, and (4) that Dean's index is a suitable instrument for monitoring the effects on dental fluorosis of minor adjustments to the fluoride concentration in drinking water.     

Prenatal fluoride exposure: measurement of plasma levels and enamel uptake in the guinea pig

This study measured the effects of variations in the maternal fluoride intake on changes in maternal plasma, fetal plasma, and fetal enamel fluoride levels of near-term guinea pig pups. Pregnant sows were divided randomly into one of three groups whose drinking water was either de-ionized or contained 5 ppm or 20 ppm fluoride supplied ad libitum. At approximately 55 days' gestation, maternal and fetal plasma and fetal enamel samples were collected and assayed for fluoride. Results showed an increase in mean fluoride concentration for maternal plasma from the de-ionized to the 20-ppm group. Mean fetal plasma fluoride concentrations were lower at baseline and increased much less than did maternal plasma levels. Mean fetal enamel concentrations increased in a fashion similar to that of the maternal plasma levels and exhibited significant differences among all groups.     

In-vitro study of Zn uptake in developing rat enamel

Molar explants from 8-day-old rats were cultured for up to 12h. Addition of calf serum (10 per cent) to the defined medium (M199) markedly reduced ⁶⁵Zn uptake in enamel. Metabolic inhibition (DNP) or heat killing had no apparent effect on movement of tracer through the enamel organ to the enamel. Removal of the enamel organ prior to culture resulted in increased uptake of ⁶⁵Zn in the enamel.     

Mechanism and timing of fluoride effects on developing enamel

Fluoride appears to specifically interact with mineralizing tissues, causing an alteration of the mineralization process. In enamel, fluorosis results in a subsurface hypomineralization. This hypomineralized enamel appears to be directly related to a delay in the removal of amelogenins at the early-maturation stage of enamel formation. The specific cause for this delay is not known, although existing evidence points to reduced proteolytic activity of proteinases that hydrolyze amelogenin. This delay in hydrolysis of amelogenins could be due to a direct effect of fluoride on proteinase secretion or proteolytic activity, or to a reduced effectiveness of the proteinase due to other changes in the protein or mineral of the fluorosed enamel matrix. The formation of dental fluorosis is highly dependent on the dose, duration, and timing of fluoride exposure. The early-maturation stage of enamel formation appears to be particularly sensitive to the effects of fluoride on enamel formation. Although the risk of enamel fluorosis is minimal with exposure only during the secretory stage, this risk is greatest when exposure occurs in both secretory and maturation stages of enamel formation. The risk of fluorosis appears to be best related to the total cumulative fluoride exposure to the developing dentition.