Community Dent Oral Epidemiol 1999; 27: 41–7 Copyright C Munksgaard 1999Printed in Denmark . All rights reserved

ISSN 0301-5661

P. K. DenBestenDivision of Pediatric Dentistry, DepartmentBiological mechanisms of dentalof Growth and Development, University ofCalifornia at San Francisco, San Francisco,California, USAfluorosis relevant to the use of

fluoride supplementsDenBesten PK: Biological mechanisms of dental fluorosis relevant to the use offluoride supplements. Community Dent Oral Epidemiol 1999; 27: 41–7. C Munks-gaard, 1999

Abstract – Fluorosis occurs when fluoride interacts with mineralizing tissues,causing alterations in the mineralization process. In dental enamel, fluorosis causessubsurface hypomineralizations or porosity, which extend toward the dentinal-enamel junction as severity increases. This subsurface porosity is most likelycaused by a delay in the hydrolysis and removal of enamel proteins, particularlyamelogenins, as the enamel matures. This delay could be due to the direct effectof fluoride on the ameloblasts or to an interaction of fluoride with the proteinsor proteinases in the mineralizing matrix. The specific mechanisms by which fluo-ride causes the changes leading to enamel fluorosis are not well defined; though Key words: enamel; fluoride; fluorosis,

review; supplementsthe early-maturation stage of enamel formation appears to be particularly sensitiveto fluoride exposure. The development of fluorosis is highly dependent on the Division of Pediatric Dentistry, Department

of Growth and Development, Box 0640,dose, duration, and timing of fluoride exposure. The risk of enamel fluorosis isUniversity of California at San Francisco, Sanlowest when exposure takes place only during the secretory stage, but highestFrancisco, CA, 94019, USA

when exposure occurs in both secretory and maturation stages. The incidence of Tel: π1 415 502 6383dental fluorosis is best correlated with the total cumulative fluoride exposure to Fax: π1 415 476 1499

E-mail: pkdb/itsa.ucsf.eduthe developing dentition. Fluoride supplements can contribute to the total fluorideexposure of children, and if the total fluoride exposure to the developing teeth is Accepted without peer review 9 Novemberexcessive, fluorosis will result. 1998

Exposure of the developing tooth organ to exces-sive amounts of fluoride can result in a mineraliza-tion defect of the enamel that is referred to as fluo-rosis. Fluorotic enamel has an altered structure andappearance that becomes more severe as theamount and duration of fluoride ingestion increase(1–3). The clinical appearance of dental fluorosis ischaracterized by bilateral opaque white areas in theenamel. With increasing levels of fluoride inges-tion, the enamel becomes striated, mottled, and/orpitted. In severe fluorosis, the opaque areas maybecome stained yellow to dark brown (4–7).

Histopathologically, nonpitted fluorotic enamelshows a subsurface porosity below a well-mineral-ized surface zone (Fig. 1) (8–11). This subsurfaceporosity is what produces the whiter, more opaqueappearance of the enamel. With increasingly severefluorosis, the porosity extends toward the dentinal-

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enamel junction, and the enamel surface can breakdown after eruption, resulting in pitting of theenamel (7). Fejerskov and co-workers (1, 12, 13)have shown that the severity of dental fluorosis isdirectly related to the amount of fluoride in theenamel, and the subsurface porosity.

Models for studying fluorosis

Several studies have been done to determine themechanisms by which fluoride interacts with thedeveloping enamel to result in fluorosis. The mod-els used for study include tooth organ culture withboth short-term and long-term fluoride exposure(14–16), animal studies following single injectionsof fluoride (17–21), multiple injections of fluoride(22), and chronic ingestion of fluoride in food orwater. Human studies include epidemiologic anal-

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Fig. 1. Polarized light micrograph of fluorosed enamel inwater. The well-mineralized (negatively birefringent) surfacelayer is indicated by the shorter arrows, and the hypomin-eralized (positively birefringent) subsurface layer, character-istic of fluorosed enamel, is indicated by the longer arrows.

yses to determine the critical time at which fluorideaffects the developing tooth to result in enamelfluorosis. The results of these studies suggest anumber of possible mechanisms, including a sys-temic effect of fluoride on calcium homeostasis; al-tered protein secretion; impaired matrix biosynthe-sis; direct effects on extracellular proteins and pro-teinases; and specific effects on cell metabolismand function.

Normal enamel development

It is necessary to understand the mechanisms con-trolling normal enamel formation to determinehow fluoride affects enamel development. Dentalenamel is formed by the ameloblasts, which arecells that differentiate from the dental lamina. Theameloblasts differentiate into polarized cells andbegin to secrete an extracellular matrix. The mor-phologic features of the ameloblasts and the ap-pearance of the extracellular matrix which theyproduce have been used to define the stages ofenamel development (23). Although various classi-fications for stages of enamel formation exist, thestages are most often identified as presecretory, se-cretory, transition, and maturation.

During the presecretory stage, the differentiatingameloblasts acquire their phenotype and prepareto secrete the organic matrix of enamel. In the se-cretory stage, the secreted protein consists primari-ly of amelogenins, which are hydrophobic proteinsthat are later removed from the maturing enamelmatrix. Other proteins, such as tuftelin, enamelin,ameloblastins, and metalloproteinases, are secreted

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in lesser quantities. As the secretory stage contin-ues, these matrix proteins begin to be hydrolyzed.The cells then shorten into transition-stage amelo-blasts and begin to secrete proteinases, which begina rapid degradation of amelogenins.

The early-maturation stage follows, with ahighly porous matrix overlaid with maturation-stage ameloblasts. The maturation stage is charac-terized by modulating cycles of ruffle-borderedand smooth-bordered ameloblasts. These cells re-move the rest of the amelogenin from the maturingenamel matrix and direct the final mineralizationof the enamel. At the end of maturation, the cellslose their polarity and become the reduced enamelepithelium, which is lost from the surface of thetooth when the tooth erupts into the oral cavity.

Effects of fluoride on enameldevelopment

Effects on cell functionAt high levels of fluoride, a major effect appears tobe the reduced secretion of enamel proteins (16, 24,25). However, this effect of fluoride on secretory-stage enamel appears to be mostly reversible (25–27). The primary effects of fluoride occur duringthe early maturation stage, when fluoride canrapidly accumulate (25–28).

Chronic exposure of animals to high levels offluoride in their food or water causes multiple ef-fects on amelogenesis, including the induction ofabnormal modulation cycles within maturation-stage ameloblasts. The number of cycles decreasesin a dose-dependent manner with increasing levelsof fluoride exposure (27, 28) Alterations in themodulation of ameloblasts would affect the pro-cessing of the matrix proteins and subsequent min-eralization of the mature enamel.

Effects on matrix mineralizationThe beginning of enamel maturation is defined bya secondary influx of mineral ions and is charac-terized by a white opaque zone which was de-scribed in rat incisors by Hiller et al. (29) and latershown to be present in most species (30). The whiteopaque zone and the preceding transitional enamelare also characterized by a selective uptake offluoride (31), possibly because of the high degreeof hydration at this stage (32, 33) which would al-low free access to the tissue (34). The increased up-take of fluoride at this stage may partly explain thesusceptibility of early-maturation enamel to the ef-fects of fluoride. The relatively high concentrations

Fluoride mechanisms relevant to supplement usage

of fluoride that are found in the enamel matrix atthe transition/early-maturation stage of enamelformation could reduce the availability of ioniccalcium, resulting in reduced proteolytic activity atthis critical stage (35, 36).

A study by Bronckers and co-workers (16) of theeffects of fluoride on the mineralization of hamstermolars in tooth organ culture showed that fluoridein the medium irreversibly affected the existingmineralizing matrix by producing a rapid deposi-tion of crystals and disrupting their growth.Furthermore, fluoride in the medium interferedwith the deposition of crystals in the new matrix.However, when fluoride was removed from themedium, the newly formed matrix recovered andmineralized normally. These findings suggest thatfluoride may interfere with nucleating sites in thematrix, perhaps by labile binding of the fluoride tothe nucleating sites (35).

After exposure to fluoride, the nature of the min-eral component of enamel is altered. Fluorosedenamel has an increased concentration of magne-sium (30) and in bone mineral, fluoride increasesmanganese and decreases concentrations of car-bonate, citrate (37), and zinc (38). These changes inthe mineral chemistry could affect mineral-matrixinteractions and enzyme activity. For example, ithas been suggested that enamel proteins producedin the presence of fluoride may be more tightlybound to fluorapatite, thereby making them lessaccessible to degradation by enamel proteinases(39).

Studies using histochemical staining (40, 41),scanning electron microscopy (8, 9, 42, 43), andquantitative measurement of nitrogen (44) and car-bon (45, 46) have shown an increase in the amountof organic material in fluorosed enamel as a resultof fluoride ingestion. Studies on the effects of fluo-ride on developing enamel in rats have comparedthe protein composition of enamel in rats ingestinghigh levels of fluoride in drinking water with thatin normal rats. In the fluorosed enamel, amelogen-in proteins were retained longer in the maturationstage as compared to control enamel (47–49) (Fig.2).

The mechanism responsible for the delay in hy-drolysis and removal of amelogenin has not yetbeen determined. However, fluoride may alter thequantity or activity of extracellular proteinasesneeded to degrade enamel proteins during thematuration stage of amelogenesis (47, 50, 51).Although proteinases are present in the enamel inboth the secretory and the maturation stages, the

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Fig. 2. Diagram of ameloblasts showing the cell morphologyat different developmental stages. The relative amount ofprotein in the enamel matrix corresponding to the ameloblastmorphology, is shown in the graph below. The protein con-tent of fluorosed enamel is higher at the transition and early-maturation stages of formation, as compared to normalenamel. RA, ruffle-ended ameloblasts; SA, smooth-endedameloblasts.

proteinases most active in the hydrolysis of amelo-genin are the serine proteinases, which are presentat the maturation stage (52–55). There is some evi-dence to suggest that the proteolytic activity of ser-ine proteinases is reduced in fluorosed enamel,compared to normal enamel (51). The delayed re-moval of amelogenin may be an important mecha-nism in the development of fluorosis by delayingthe growth of enamel crystals, so that when thetooth erupts, the enamel remains incompletelymineralized.

Metabolic factors influencing enamelfluorosis

Metabolic factors that affect the plasma levels offluoride and hence the severity of enamel fluorosisinclude body weight, rate of skeletal growth, andperiods of bone remodeling. Fluoride is rapidly ab-sorbed from the plasma by the bones of the devel-oping skeleton (56), owing to the large surface areaof loosely organized crystallites in the developingskeleton (57).

Rapid growth, resulting in increased absorptionof fluoride into the bone, may have several effects.Removal of fluoride from the blood stream mayinitially reduce the degree of enamel fluorosis byreducing the amount of fluoride available to thedeveloping enamel. However, if fluoride does ac-cumulate in the bone, a relatively large reservoir of

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fluoride may be present in the bones of the devel-oping skeleton and may be released locally (58, 59).Angmar-Mansson and co-workers (17, 22) showedthat in rats given a single injection of fluoride, thesubsequently erupting enamel continued to showfluorosis after the serum fluoride levels hadreached baseline levels. This result indicates a con-tinuous local release of fluoride around the devel-oping ameloblasts, presumably from the surround-ing bone. Although the specific fluorotic lesionmay occur during the early-maturation stage oftooth enamel formation, the accumulation of fluo-ride earlier in tooth development may contributeto the level of exposure when enamel fluorosis be-gins.

The metabolic uptake of fluoride is also affectedby other factors, such as nutrition, altitude, and re-nal activity (3, 57). Calcium has been shown to in-hibit fluoride absorption (60), and certain diets,such as those high in protein, lower the gastric pHand result in increased fluoride resorption andhigher levels of fluoride in plasma (57, 61, 62).Fluoride supplements would be unlikely to affectsystemic calcium homeostasis, unless an underly-ing metabolic problem exacerbated the effects offluoride. Because fluoride is largely excretedthrough the renal tubules, any disturbances of re-nal function, such as renal insufficiency, can en-hance fluoride retention and result in fluorosis.

Exposure to fluoride – timing,duration and dose

The severity of fluorosis is related to the timing,duration, and dose of exposure to fluoride (1, 63).In both animal studies (64, 65) and human studies(66), fluoride has been shown to affect the matura-tion stage of enamel formation without previousexposure of enamel to fluoride in the secretorystage. In animal studies as well, fluoride exposureduring the secretory stage alone did not affectameloblast modulation in the maturation stage(27).

Epidemiologic studies support experimentalfindings that the transition and early-maturationstages of enamel formation are highly susceptibleto the effects of fluoride (36, 59, 67–70). The import-ance of timing in the development of enamel fluo-rosis was shown in a study by Ishii & Suckling (71).Investigators recorded the degree of fluorosis inchildren who were initially drinking 7.8 mg/Lfluoride in drinking water, which was subse-quently changed to 0.2 mg/L fluoride. They found

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moderate to severe fluorosis of the upper leftcentral incisor in children aged 35 through 42months at the changeover, when the tooth wouldhave been in the maturation stage of formation. Incontrast, children aged 11 to 33 months at thechangeover (and thus mostly in the secretory stage)had very mild, questionable, or no fluorosis. Simi-larly, in a study by Pendrys & Katz (72) using theFluorosis Risk Index, fluoride supplements usedonly during the first year of life (secretory stage)held little risk of fluorosis.

These studies have led to the recommendationthat fluoride supplementation should begin afterthis critical period for enamel formation. Althoughthe secretory stage of enamel formation appears tobe affected only by high levels of fluoride, expo-sure to fluoride during this stage clearly increasesthe risk of fluorosis. Although these studies sug-gest that the maturation stage is most sensitive tothe effects of fluoride, the duration of exposure tofluoride before the early-maturation stage does af-fect the severity of enamel fluorosis (59, 71, 73).

The level of fluorosis in a population is relatedto the level of exposure to ingested fluoride for thatpopulation (2, 74). Some investigators have soughtto determine a threshold fluoride level belowwhich enamel fluorosis would not occur. Angmar-Mansson and co-workers (75) postulated that tem-porary peak values rather than elevated fastingvalues are responsible for the occurrence of enamelfluorosis and that the peak values must approachabout 10 mmol to alter enamel formation by theameloblasts. However, Myers (76) noted that thedistinct pattern of increasing prevalence of fluoro-sis with increasing levels of fluoride in the water isnot compatible with the concept of a thresholdlevel for the action of fluoride ion on the enamelorgan (76). The effects of fluoride ingestion appearto be cumulative during tooth formation (1, 2, 27,36, 69, 77).

Summary and conclusions

Excessive ingestion of fluoride results in the de-layed maturation of dental enamel, and subse-quently more porous surface enamel. It is notknown whether fluoride can accumulate in thecells and what local fluoride concentrations areavailable to developing ameloblasts. However, it isapparent that the dose and the duration of fluorideexposure are critical factors in the formation ofenamel fluorosis (1, 4, 5).

The risk factors can be summarized as follows:

Fluoride mechanisms relevant to supplement usage

– Lowest risk occurs with fluoride exposure onlyduring the secretory stage (∞15 months of age).

– Highest risk occurs with fluoride exposure dur-ing both secretion and maturation stages.

– Risk increases with increasing fluoride dose (i.e.,ingestion of fluoride from multiple sources, in-cluding fluoride supplements).The effects of fluoride are cumulative rather than

requiring a specific threshold dose. Therefore, thelonger the exposure to fluoride supplements, thegreater the risk of enamel fluorosis. Recommenda-tions for optimal doses of fluoride supplementsshould take into account the total ingestion offluoride from all sources.

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