Biological mechanisms of dental fluorosis relevant to the use of fluoride supplements

Fluorosis occurs when fluoride interacts with mineralizing tissues, causing alterations in the mineralization process. In dental enamel, fluorosis causes subsurface hypomineralizations or porosity, which extend toward the dentinal-enamel junction as severity increases. This subsurface porosity is most likely caused by a delay in the hydrolysis and removal of enamel proteins, particularly amelogenins, as the enamel matures. This delay could be due to the direct effect of fluoride on the ameloblasts or to an interaction of fluoride with the proteins or proteinases in the mineralizing matrix. The specific mechanisms by which fluoride causes the changes leading to enamel fluorosis are not well defined; though the early-maturation stage of enamel formation appears to be particularly sensitive to fluoride exposure. The development of fluorosis is highly dependent on the dose, duration, and timing of fluoride exposure. The risk of enamel fluorosis is lowest when exposure takes place only during the secretory stage, but highest when exposure occurs in both secretory and maturation stages. The incidence of dental fluorosis is best correlated with the total cumulative fluoride exposure to the developing dentition. Fluoride supplements can contribute to the total fluoride exposure of children, and if the total fluoride exposure to the developing teeth is excessive, fluorosis will result.     

Biological mechanisms of fluorosis and level and timing of systemic exposure to fluoride with respect to fluorosis.

Enamel fluorosis can occur following either an acute or chronic exposure to fluoride during tooth formation. Fluorosed enamel is characterized by a retention of amelogenins in the early-maturation stage, and by the formation of a more porous enamel with a subsurface hypomineralization. The mechanisms by which fluoride affects enamel development include specific effects on both the ameloblasts and on the developing enamel matrix. Maturation-stage ameloblast modulation is more rapid in fluorosed enamel as compared with control enamel, and proteolytic activity in fluorosed early-maturation enamel is reduced as compared with controls. Secretory enamel appears to be more susceptible to the effects of fluoride following acute fluoride exposure, such as may occur with the use of fluoride supplements. However, both human and animal studies show that the transition/early-maturation stage of enamel formation is most susceptible to the effects of chronic fluoride ingestion at above-optimal levels of fluoride in drinking water.     

Enamel proteases in secretory and maturation enamel of rats ingesting 0 and 100 PPM fluoride in drinking water

Dental enamel formed during ingestion of high levels of fluoride in drinking water has an increased organic content in the maturation stage, which may be due to a delay in the breakdown of amelogenins during the early-maturation stage of enamel formation. This delay in the breakdown of amelogenins in fluorosed enamel suggests an effect of fluoride on enamel proteases which hydrolyze the early secreted enamel proteins. In this study, we compared the proteases present in fluorosed and control secretory-stage and maturation-stage enamel. Enamel was demineralized and separated in SDS gels containing 0.1% gelatin. After incubation in 100 mmol/L Tris-HCl, pH 8, with 10 mmol/L CaCl2, the gels were stained with Coomassie Blue, and proteases were seen as clear zones of degraded gelatin. Similar bands of proteolytic activity were seen in fluorosed and in control enamel. In the maturation stage, more proteases were present than in the secretory stage of enamel formation. Less digestion of gelatin substrate occurred in several proteases found in the fluorosed maturation-stage enamel as compared with the control maturation-stage enamel. This suggests that the amount of protease secreted or the activity of the proteases may be altered in fluorosed maturation-stage enamel.     

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.     

Fluoride down-regulates the expression of matrix metalloproteinase-20 in human fetal tooth ameloblast-lineage cells in vitro

Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10  µ M fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10  µ M NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10  µ M NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel.     

Effects of fluoride on rat dental enamel matrix proteinases

Enamel fluorosis is characterised by increased porosity and a delay in the removal of enamel matrix proteins as the enamel matures. Amelogenin is the primary matrix protein in secretory-stage dental enamel. As enamel matures, amelogenins are hydrolysed by a number of enamel proteinases, including matrix metalloproteinase-20 (MMP-20 or enamelysin) and serine proteinase. Here, the effect of ingested fluoride on the relative activity of proteinases in the enamel matrix and the specific effect of fluoride on MMP-20 activity were examined. Proteinase activity relative to total enamel matrix protein was measured by fluorescence assay of enamel matrix dissected from rats given 0, 50, or 100 parts per 10(6) fluoride in their drinking water. To determine the specific effect of fluoride on the activity of MMP-20, the hydrolysis of a full-length recombinant human amelogenin by recombinant MMP-20 (rMMP-20) in the presence of 0, 2, 5, 10 or 100 microM fluoride was compared by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). In addition, a fluorescent peptide assay was developed to quantify enzyme activity against the tyrosine-rich amelogenin peptide cleavage site. In the late maturation stage, total proteinase activity per unit protein was lower in the fluoride-exposed rats than in the control rats. This in vivo finding indicates that fluoride ingestion can alter the relative amount of active proteinase in mature enamel. Hydrolysis of amelogenin at neutral pH by rMMP-20 was reduced in the presence of 100 microM F. In the peptide assay, rMMP-20 activity was significantly reduced by concentrations of fluoride as low as 2 microM at pH 6, with no significant effect at pH 7.2. These in vitro assays show that micromolar concentrations of fluoride can alter metalloproteinase activity, particularly when the pH is reduced to 6.0. These studies suggest that the effects of fluoride on enamel matrix proteinase secretion or activity could be involved in the aetiology of fluorosis in enamel and other mineralising tissues.     

Possible function of matrix proteins in fluoride incorporation into enamel mineral during porcine amelogenesis

The present study was undertaken to elucidate the mechanism of fluoride incorporation into secretory enamel mineral, with porcine enamel used as a model. Although the fluoride content in the enamel varied greatly among the animals, we observed that the fluoride-to-calcium ratio in the enamel tissue was maximal at the beginning of the secretory stage; the F/Ca ratio decreased (and leveled off) with the advancement of mineralization. In vitro work showed that some of the fluoride in the secretory enamel tissue was removed with the extraction of organic matter, mostly amelogenins. Furthermore, coating hydroxyapatite crystals with enamel matrix proteins resulted in a retardation of fluoride incorporation into the crystals when exposed to fluoride solutions, as a result of an inhibition of apatite reprecipitation. We also confirmed that the growth kinetics of fluoridated apatite onto HA seeds decreased with increasing coverage of the seed surface with the enamel proteins. All the results of the present study strongly suggest that the fluoride incorporation into enamel mineral during the secretory stage may be regulated by the kinetics of mineralization, which is highly dependent on the driving force for precipitation and the presence of proteinaceous inhibitors, mainly amelogenins.     

Cadherin-related neuronal receptors in incisor development

Cadherins are cell adhesion molecules that are critical for tissue development. In this report, we identified members of the cadherin family cadherin-related neuronal receptors (CNRs) 1 and 5 expressed in rat incisors by the differential display method. Quantitative RT-PCR revealed that CNR1 mRNA is expressed in the secretory stage but reduced in the early-maturation stage, while CNR5 mRNA is expressed in both these stages. In situ hybridization showed that strong expression of CNR1 is strong in the secretory stage, but reduced in the early phase and diminished in the late phase of the early-maturation stage. CNR5 mRNA is expressed almost at the same levels in the secretory and in the early phase of the early-maturation stages but is absent in the late phase of the early-maturation stage. Both CNR1 and 5 mRNA are continuously expressed in odontoblasts. Immunohistology showed that CNR proteins are expressed in the secretory and early-maturation stages of ameloblasts, but no protein expression at the late-maturation stage was observed. CNR proteins were continuously expressed in odontoblasts. We found that recombinant CNR1 binds dental epithelial and mesenchymal cells through N-terminal domain EC1 in vitro. These results suggest that CNR1 and CNR5 may play an important role in enamel and dentin formation, probably through cell-cell and/or cell-matrix interactions.     

Clinical and structural features and possible pathogenic mechanisms of dental fluorosis

ABSTRACT— The aim of the present review has been to give a survey of our present knowledge concerning dental fluorosis and to discuss possible pathogenic mechanisms of this disease. Based on a discussion of the principles behind the Community Index of dental fluorosis it is stressed that absence of harmful community effects should not be confused with a complete absence of fluoride-induced biological changes. The index values apply to communities and they do not equivalate individual diagnosis. Structurally, an increase in fluoride intake results in an increase in degree and extent of porosity of the enamel. Further, dentin changes may be observed. It is stressed that the clinical and structural features should be kept in mind when possible pathogenic mechanisms of dental fluorosis are discussed. It is obvious that the enamel changes described may not necessarily be a result of a fluoride damage of secretory ameloblast as commonly assumed. Thus, structural changes similar to those of enamel fluorosis may result from an impairment of the process of enamel maturation. This can either be due to a fluoride-induced change in composition or rheologic features of enamel matrix, or be a result of a disturbance of the cellular processes during enamel maturation. In addition, it is suggested that fluoride may act directly on the poorly understood processes of mineralization in vivo. Finally, results indicate that fluoride interferes with calcium homeostasis, and it cannot be entirely excluded that dental fluorosis may be a result of a more generalized effect of fluoride. It is concluded that much more basic research on fluoride metabolism and formation of mineralized tissues is needed to better understand the effect of fluoride on dental hard tissues.     

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.     

Effect of prenatal and postnatal fluoride on the human deciduous dentition. A literature review

Fluoride passes from the mother to fetal teeth. Much of the fluoride is taken up in secretory enamel, probably by the forming mineral apatite crystals. Some is retained with residual proteins. The low concentration of fluoride in the inner enamel is incorporated mainly during the secretory stage, while the enhanced concentration in the surface enamel is produced during the much longer maturation stage. Mature, hard enamel is generally absent during fetal life. The clinical question is whether prenatal fluoride imparts an additional benefit to the universally accepted effect of postnatal fluoride. In general, surface enamel fluoride levels of deciduous teeth increase with increasing pre- and postnatal fluoride administration. A consistent level of caries protection has been reported with pre- and postnatal administration of fluoride unrelated to the acquisition of fluoride in the surface enamel. Many children develop enamel opacities in their deciduous dentition related by various factors to enamel mineralization disturbances in drinking water areas even low in fluoride. Accumulation of fluoride due to an increased fluoride intake is a feature of fluorosed enamel in the deciduous as well as permanent dentition. The resulting mature fluorosed enamel retains a relatively high proportion of immature matrix proteins onto the crystal surface. The degree of fluorosis of the deciduous dentition is less compared with that of the permanent dentition, due probably to a partial protection afforded by the maternal loss of fluoride, formerly known as the "placental barrier".     

Risk of enamel fluorosis in nonfluoridated and optimally fluoridated populations: considerations for the dental professional

BACKGROUND: Few studies have evaluated the impact of specific fluoride sources on the prevalence of enamel fluorosis in the population. The author conducted research to determine attributable risk percent estimates for mild-to-moderate enamel fluorosis in two populations of middle-school-aged children. METHODS: The author recruited two groups of children 10 to 14 years of age. One group of 429 had grown up in nonfluoridated communities; the other group of 234 had grown up in optimally fluoridated communities. Trained examiners measured enamel fluorosis using the Fluorosis Risk Index and measured early childhood fluoride exposure using a questionnaire completed by the parent. The author then calculated attributable risk percent estimates, or the proportion of cases of mild-to-moderate enamel fluorosis associated with exposure to specific early fluoride sources, based on logistic regression models. RESULTS: In the nonfluoridated study sample, sixty-five percent of the enamel fluorosis cases were attributed to fluoride supplementation under the pre-1994 protocol. An additional 34 percent were explained by the children having brushed more than once per day during the first two years of life. In the optimally fluoridated study sample, 68 percent of the enamel fluorosis cases were explained by the children using more than a pea-sized amount of toothpaste during the first year of life, 13 percent by having been inappropriately given a fluoride supplement, and 9 percent by the use of infant formula in the form of a powdered concentrate. CONCLUSIONS: Enamel fluorosis in the nonfluoridated study sample was attributed to fluoride supplementation under the pre-1994 protocol and early toothbrushing behaviors. Enamel fluorosis in the optimally fluoridated study sample was attributed to early toothbrushing behaviors, inappropriate fluoride supplementation and the use of infant formula in the form of a powdered concentrate. CLINICAL IMPLICATIONS: By advising parents about the best early use of fluoride agents, health professionals play an important role in reducing the prevalence of clinically noticeable enamel fluorosis.     

Fluoride content of infant formulae in Australia

The prevalence of dental fluorosis in Australia and the United States of America has increased in both optimally fluoridated and non-fluoridated areas. This has been attributed to an increase in the fluoride level of food and beverages through processing with fluoridated water, inadvertent ingestion of fluoride toothpaste, and the inappropriate use of dietary supplements. A major source of fluoride in infancy is considered to be infant formula which has been implicated as a risk factor for fluorosis in a number of studies. In this study the fluoride content of the infant formulae commonly used in Australia was determined. The acid diffusible fluoride of each powdered formula was isolated by microdiffusion and measured using a fluoride ion-specific electrode. The fluoride content of milk-based formulae ranged from 0.23 to 3.71 μg F/g and for soy-based formulae from 1.08 to 2.86 μg F/g. When reconstituted, according to the manufacturer's directions, with water not containing fluoride, the formulae ranged in fluoride content from 0.031 to 0.532 ppm, with the average fluoride content 0.240 ppm. Using average infant body masses and suggested volumes of formula consumption for infants 1–12 months of age, possible fluoride ingestion per kg body mass was estimated. None of the formulae, if reconstituted using water containing up to 0.1 ppm F, should provide a daily fluoride intake above the suggested threshold for fluorosis of 0.1 mg F/kg body mass. However, W reconstituted with water containing 1.0 ppm F they should all provide a daily fluoride intake of above the suggested threshold for fluorosis with intakes up to 2–3 times the recommended upper ‘optimal’ limit of 0.07 mg/kg body mass. Under these conditions the water used to reconstitute the formulae would provide 65–97 per cent of the fluoride ingested. These figures are likely to be overestimates due to the intake of nutrients from other sources reducing formulae consumption and also due to the lower bioavallability of fluoride from milk-based formulae. Further, it is generally believed that the maturation stage of enamel formation is the critical period for fluorosis development by chronic, above-threshold fluoride exposure. The maturation stage for the anterior permanent teeth, however, is after the first twelve months of life where fluoride intake from infant formula consumption per kg body mass is highest. The level of fluoride in the commonly used Australian formulae would suggest that infant formula consumption alone is unlikely to be a risk factor for dental fluorosis in a non-fluoridated community, but could make a major contribution to an infant's daily fluoride intake. However, prolonged consumption (beyond 12 months of age) of infant formula reconstituted with optimally-fluoridated water could result in excessive amounts of fluoride being ingested during enamel development of the anterior permanent teeth and therefore may be a risk factor for fluorosis of these teeth.     

Fluoride and enamel defects

The concentration of fluoride in drinking water is the major determinant of the prevalence and severity of dental fluorosis in a community. Fluorosis is more prevalent and discernible in permanent teeth than in primary teeth; the intensity can range from barely perceptible, whitish striations in enamel to confluent pitting and dark staining. The traditional belief is that fluorosis is produced only during the secretory stages of ameloblastic activity. Some recent reports suggest that the maturation stages of enamel development are as important as or even more important than the secretory stages as the time when fluorosis can be produced. The question of timing remains unresolved. Many questions also remain about general and individual physiologic variations in relation to susceptibility to dental fluorosis. Good criteria for differential diagnosis exist to distinguish dental fluorosis from non-fluoride enamel opacities. An increasing number of reports indicates that the prevalence of fluorosis may be increasing among children in fluoridated and non-fluoridated communities. Reasons for the increases may relate to misuse of dietary fluoride supplements, ingestion of fluoride toothpastes, or increasing amounts of fluoride in foods or the atmosphere. The intensity of the increased fluorosis is in the milder categories and is not generally unsightly. It should be recognized that a small amount of fluorosis may be an alternative to a greater prevalence of dental caries, a disease that may produce cosmetic problems and sequelae worse than those produced by fluorosis.     

Enamel mineralization disturbances in 12-year-old children with known early exposure to fluorides

Abstract Enamel changes and caries experience were studied in 134 12-year-old children with a known early exposure to fluoride tablets and/or fluoride containing toothpaste. The influence of birth weight and breast-feeding period was also analyzed. A clinical intact enamel was found in 32% of the children. Enamel fluorosis was found in 45% and localized opacities or hypoplasias in 40% Children who had consumed fluoride tablets for a period of at least 12 months from the age of 6 months ran a 5.4 times greater risk of developing enamel fluorosis than children with no such consumption. No such risk could be shown in children who at 6 or 12 months of age started to use fluoride toothpaste. There was no statistically significant reduction in the prevalence of smooth surface caries or fillings in children with an exposure to fluoride tablets.     

Fluorosis: is it really a problem?

BACKGROUND: Scientists have noted an association between mottled enamel and fluoride exposure since the early 1900s. By the mid-1900s, they also recognized that fluoride intake was related to lower caries incidence. To harness the protective effect of fluoride while limiting the occurrence of fluorosis, dental researchers have recommended that the fluoride level in chinking water be 1 part per million or less. OVERVIEW: Despite the recognition that fluoride levels in water can be controlled to offer caries protection with minimal risk of fluorosis, the cosmetic defect continues to appear. However, although the word "fluorosis" conjures up images of brown stained and pitted enamel, such severe cases rarely are seen in the United States. Children in this country are exposed to fluoride from numerous sources and the appearance of mild fluorosis is not unusual. CONCLUSIONS AND PRACTICE IMPLICATIONS: In most cases, fluorosis is a minor cosmetic defect that should not be cause for alarm. Dentists should educate their patients about the optimal range of fluoride intake for caries protection, sources of fluoride and the possibility of fluorosis.     

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.     

Fluorosis: diagnosis, risk assessment and epidemiology

Fluorosis is the most widespread side-effect of fluoride use and appears as discrete white spots on the enamel up to severe enamel dysplasia. There are different techniques for scoring fluorosis in epidemiological surveys. In the literature there is no uniform way of selection of teeth and data processing. Fluorosis risk is determined by environmental factors such as water and food fluoride content as well as individual factors such as use (or misuse) of fluoride supplements and fluoridated oral hygiene products. In a group of Flemish schoolchildren, fluorosis prevalence is low and mainly related to use of fluoride supplements and toothpaste in childhood.     

Fluoride metabolism and fluorosis

OBJECTIVES: This paper is primarily concerned with the only proven risk associated with water fluoridation: enamel fluorosis. Its purpose is to review current methods of measuring enamel fluorosis, its aetiology and metabolism. A further objective is to identify risk factors to reduce the prevalence of enamel fluorosis and employ methods to manage such risk factors. DATA: The prevalence of enamel fluorosis is increasing in Ireland and internationally. A critical period has been identified at which teeth are most at risk of developing enamel fluorosis: 15-24 months of age for males and 21-30 months of age for females. The data included took these two factors into account. SOURCE: A thorough narrative review of published literature was conducted to identify studies concerning the aetiology and metabolism of enamel fluorosis. Risk factors for fluorosis were identified from these studies. STUDY SELECTION: As it is the pre-eruptive phase of enamel development which represents the greatest risk to developing enamel fluorosis, studies examining sources of fluoride ingestion for young children were selected. These included studies on ingestion of fluoride toothpaste by young children, fluoride supplementation and infant formula reconstituted with fluoridated water. CONCLUSIONS: There is evidence that the age at which tooth brushing with fluoride toothpastes is commenced and the amount of fluoride placed on the brush are important risk factors in the incidence of dental fluorosis. It is recommended that brushing should not commence until the age of 2 and that a pea-sized amount (0.25 g) of toothpaste should be placed on the brush.     

Fluoride incorporation into apatite crystals delays amelogenin hydrolysis

Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1,000, or 4,000 ppm F(-). After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4), bound protein was characterized by reverse-phase high-performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100-CAP was significantly reduced in a dose-dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100-CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100-CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride-containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.