EXPERIMENTS FLUORIDE

ON LOCAL AND SYSTEMIC ACTION OF IN CARIES INHIBITION IN THE RAT R. H. LARSON, J. R. MELLBERG* and R. S!%+rNn@

National

Institute

*Colgate

of Dental Research. National Institutes of Health. Bethesda Md. 20014 and Research Center Piscataway. N.J. 08854. U.S.A.

Summary-Fluoride administered to rats by gastric intubation was associated with an increase in fluoride uptake by the enamel and caries inhibition, but to a lesser extent than in animals consuming the same amount of fluoride in the drinking water. These differences were more pronounced in the first and second molars which erupted within 2 or 3 days of the start of the experiment than on the third molars which had at least two weeks of pre-eruptive exposure to systemic fluoride.

METHODS

INTRODUCTION Much emphasis has been placed on the protective effect of fluoride in the enamel (Brudevold, McCann and Gran. 1965) and on variations in fluoride uptake of human enamel from different methods of fluoride administration (Brudevold, 1971). However, Englander and Mellberg (1976) found no association between fluoride concentration of enamel and caries in the human decidu& dentition. In two clinical studies (Mellberg and Nicholson, 1974a, b) fluoride uptake by enamel was higher when acidulated phosphate fluoride was applied than when neutral sodium fluoride was used, but rinses of the two substances produced similar caries inhibition. Larson rt al. (1976) found significant caries inhibition by fluoride in rats with no increase in the enamel fluoride. Fluoride is available for local and systemic effects when in drinking water (McClure. 1970) or tablets (DePaola and Lax, 1968), which are dissolved in the mouth and swished before swallowing. When fluoride is administered by other procedures, such as gels in special applicators (Englander. Keyes and Gestwicki. 1967), in mouthwashes (Horowitz. 1974) or in dentifrices (Heifetz and Horowitz, 1970). none is purposely swallowed; however, the amount of fluoride in solutions expectorated following oral treatment is significantly less than that in the original solution (Hellstrom, 1960; Ericsson and Forsman, 1969). Thus, only the systemic effect can be studied separately. and then only by delivering fluoride directly into the stomach with no direct local contact with the teeth. Bowen (1973) showed a significant increase in fluoride levels in dental plaque in monkeys given fluoride by gastric intubation, but he did not study the enamel uptake or caries inhibition. Our purpose was to evaluate both the systemic and direct local effects of fluoride by comparing both caries inhibition and enamel fluoride uptake in rats from fluoride delivered either by stomach intubation or by mouth.

Three groups of 21-22&y old Osborne-Mendel rats from the NIH colony were treated as follows for an g-week experimental period: (A) controls stomach-tubed 5 days per week with distilled water only, (B) stomach-tubed with distilled water and supplied with drinking water containing 5 parts/IO’ F 5 days per week, and (C) stomach-tubed 5 days per week with F solutions calculated tc provide the same amount of F as was consumed in the drinking water by animals in group B during the preceding 24 h. Stomach tubing was performed by the method of Crossland and Holloway (1971). Each group was composed of 24 animals fed caries test Diet 2000 (Keyes and Jordan. 1964). and infected with streptomycin-resistant Sfreptococcus mutans 67 I5 by placing 2 to 3 drops of an 18 h culture of the organism into the mouth of each animal 4 times during the first week of the experiment. Because caries-free teeth are needed for fluoride analysis of enamel. three additional groups of 10 animals each were maintained with the same variations in fluoride intake. but were not infected with Strep. mutans and were fed Diet 2114 (the same as Diet 2000 except that it contained 56 per cent corn starch and no sucrose). All animals were provided with diet and drinking water ad [ihiturn. The teeth were cleaned and scored for caries by the method of Keyes (1958) and individual teeth analyzed for fluoride as previously described (Mellberg and Larson, 1971; Nicholson, Taylor and Mellberg, 1974). The method of Scheffe (1953) was used for the statistical evaluation of group differences.

RESULTS Table I shows the levels of fluoride in the outer layer of enamel for each of the three mandibular molars in the three experimental groups. The level of fluoride in the control teeth was significantly (p < 0.01) less than for either of the fluoride-treated groups, and the animals drinking fluoridated water showed significantly higher levels of fluoride in the

-t Deceased. 437

R. H. Larson, J. R. Mellberg and R. Sennmg

438

Table 1. Mean and standard

error for fluoride (parts/lo’) type

Group

Number of animals

Control

8

F by stomach tube

6

F in drinking

8

water

in outer layer of enamel by treatment and tooth

1st molar

Type of tooth 2nd molar

3rd molar

89.0 f 8.5 271.7 + 43.5 607.8 f 73.5

14.5 * 4.0 291.2 * 38.9 694.2 k81.2

51.5 * 5.2 771.7 +71.5 1057.6 k 94.6

first and second molars than those receiving fluoride by intubation (p < 0.01). Fluoride uptake of individual molars was not significantly different (p < 0.01) between the three molars of the control animals, nor between the first and second molars of either groups of fluoride-treated animals. However, fluoride uptake in the third molars of the fluoride-treated animals was significantly (p < 0.01) higher than for the first and second molars, and was only slightly less for the intubated animals than for those drinking fluoridated water. Table 2 shows the mean number of carious enamel areas by tooth type and surface. A comparison of the total dentition shows that the animals drinking fluoridated water developed significantly (p < 0.01) fewer carious areas on both the smooth surfaces and in the sulci than either the intubated or control animals. The intubated animals developed significantly (p < 0.01) fewer carious areas than the controls on the smooth surfaces only. In the intubated animals there was significantly (p -=z0.01) greater caries inhibition on the third molars than on the first and second molars, whereas the animals drinking fluoridated water showed extremely low caries levels on all three molars. DISCUSSION The teeth of intubated animals had only systemic exposure to fluoride because the rat is unable to vomit (Hatcher and Weiss, 1923). The major source of oral fluoride in the intubated animals was probably in the saliva. Small amounts may have been present

in the gingival fluids and some exposure may have resulted from coprophagy, even though the animals were housed in screen-bottomed cages. Effects of fluoride exposure differed not only with respect to the route of administration but also for the first and second molars in contrast to the third. The first and second molars were at or near complete eruption at the start of the experiment (Larson and Fitzgerald, 1964). and fluoride exposure for these teeth was limited to the post-eruptive period. It was therefore not surprising that animals drinking fluoridated water, with consequent direct and systemic exposure, showed greater fluoride uptake and caries inhibition on the first and second molars than intubated animals. Unerupted teeth can be subjected to systemic fluoride only. Because the third molars of the rat do not erupt until 35 days of age or later, these teeth in both groups of fluoride treated animals were subjected to at least 14 days of pre-eruptive fluoride exposure. The finding that the third molars show the highest levels of fluoride uptake in both treated groups suggests that the two-week period prior to eruption is a highly critical period for fluoride uptake in enamel of rat molars. The slightly higher level of enamel fluoride in the third molars of animals drinking fluoridated water than in those of the intubated animals may be attributed to direct exposure of the teeth to fluoridated water immediately after eruption. Larson et al. (1976) found that the ability of rat molars to take up fluoride from dilute solutions decreases rapidly following eruption. The significantly higher level of F uptake by the third molars than the first and second molars in our experi-

Table 2. Treatment effect on mean and standard error for carious enamel areas on smooth and sulcal surfaces for the three molars separately and for the total dentition

Group

Number of animals

Control

24

F by stomach tube F in drinking water

24

22

1st molar Smooth Sulcal

Carious enamel areas 2nd molar 3rd molar Smooth Sulcal Smooth Sulcal

Total Smooth Sulcal

11.2 + 1.05 3.7 * 0.66

11.6 + 1.16 10.4 k 1.08

17.0 kO.99 4.8 +0.93

13.5 + 0.98 11.1 +0.76

7.0 +0.74 0.5 +0.17

2.3 -t_O.29 1.6 +0.32

30.2 *2.51 9.0 * 1.59

27.4 f2.16 23.1 + 1.89

0.4 10.26

5.1 + 0.92

0.2 +0.12

4.1 rfr0.59

0.0 0.0

0.8 +0.30

0.6 kO.37

10.6 * 1.53

Fluoride in caries inhibition

ment suggests that both pre- and post-eruptive exposure to fluoride is necessary for maximum uptake. Our study does not explain the mechanism by which fluoride inhibits caries but the different patterns of inhibition in the two fluoride-treated groups are of interest. Inhibition of sulcal caries in the animals drinking fluoridated water was apparently related to the frequent direct local contact between the sulci and the water; whereas intubated animals not having such contact showed essentially no caries inhibition in the sulci. That caries inhibition was directly related to enamel fluoride uptake provides no information as to the level of caries protection provided by enamel-bound fluoride. Intra-oral fluoride can inhibit caries in the rat without producing an increase in fluoride uptake (Larson et al.. 1976). Fluoride in the mouth would not only influence enamel uptake but also provide other local effects such as reduction in acid production by plaque bacteria (Edgar, Jenkins and Tatevossian. 1970) decrease in polysaccharide production (Bowen and Hewitt. 1974), reduction in release of calcium from exposed tooth surfaces (Birkeland. 1975) and possibly remineralization of early lesions. REFERENCES

Birkeland J. M. 1975. In vitro study on the mechanisms of action of fluoride in low concentrations. Curies Res. 9. ll(r118. Bowen W. H. 1973. The effect of single daily doses of fluoride on saliva, plaque and urine in monkeys (Mocaca fasciculnris). J. int. dent. Child. 4, 1l-14. Bowen W. H. and Hewitt M. J. 1974. Effect of fluoride on extracellular polysaccharide production by Strep@ coccus mutans. J. dent. Res. 53, 627-629. Brudevold F. 1971. Interaction of fluoride with human enamel. In: Chemistry and Physiology of Enamel. pp. 73-90. The University of-Michigan. _Brudevold F.. McCann H. Cr. and Gren P. 1965. Caries resistance as related to the chemistry of the enamel. In: Caries-Resistant Teeth, (Edited by Wolstenholme G. E. W.) pp. 121-148. Boston, Mass. Crossland L. M. and Holloway P. J. 1971. A technique for tube-feeding newborn rats and the effect of administration of various carbohydrate solutions on their subsequent caries susceptibility. Caries Res. 5. 144-l 50. DePaola P. F. and Lax M. 1968. The caries-inhibiting effect of acidulated phosphate-fluoride chewable tablets: A two-year double-blind study. J. Am. dent. Ass. 76. 554-557.

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Edgar W. M., Jenkins G. N. and Tatevossian A. 1970. The inhibitory action of fluoride on plaque bacteria. Br. dent. J. 128, 129-132. Englander H. R., Keyes P. H. and Gestwicki M. 1967. Clinical anticaries effect of repeated topical sodium fluoride applications by mouthpieces. J. Am. dent. Ass. 75, 638644.

Englander H. R. and Mellberg J. R. 1976. Failure to demonstrate an association between enamel fluoride concentration and dental caries in the deciduous dentition. J. dent. Res. 55, 707. Ericsson Y. and Forsman B. 1969. Fluoride retained from mouthrinses and dentifrices in preschool children. Caries Res. 3, 290-299.

Hatcher R. A. and Weiss S. 1923. Studies on vomiting. J. Pharmac.

exp. Ther.

22, 139-193.

Heifetz S. B. and Horowitz H. S. 1970. An appraisal of therapeutic dentifrices. J. Publ. Hlth Dent. 30, 2OG21 I. Hellstrom I. 1960. Fluorine retention following sodium fluoride mouthwashing. Acta odont. stand. 18, 263-278. Horowitz H. S. 1974. The prevention of dental caries by mouthrinsing with solutions of neutral sodium fluoride. Int.

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Keyes P. H. 1958. Dental caries in the molar teeth of rats. II. A method for diagnosing and scoring several types of lesions simultaneously. J. dent. Res. 37, 1088-1099. Keyes P. H. and Jordan H. V. 1964. Periodontal lesions in the Syrian hamster. III. Findings related to an infectious and transmissible component. Archs oral Biol. 9. 377xlO.

Larson R. H. and Fitzgerald R. J. 1964. Caries development in rats of different ages with controlled flora. Archs oral Biol. 9, 705-7 12. Larson R. H., Mellberg J. R., Englander H. R. and Senning R. 1976. Caries inhibition in the rat by water-borne and enamel bound fluoride. Caries Res. 10, 321-331. McClure F. J. 1970. Water Fluoridation: The Search and the Victory. National Institutes of Health, Dept. Health, Education and Welfare, Washington, D.C. Mellberg J. R. and Larson R. H. 1971. Fluoride concentrations in molars of rats fed a fluoridated diet. J. dent. Res. SO, 600-603. Mellberg J. R. and Nicholson C. R. 1974a. Weekly mouthrinsing with 3000 ppm of fluoride: The effect of concentration of fluoride in the enamel. J. Pub/. HIth Dent. 34, 2-7.

Mellberg J. R. and Nicholson C. R. 1974b. Fluoride uptake in uiuo by deciduous enamel of children from neutral fluoride and APF mouthrinses. Caries Res. 8, 148-154. Nicholson C. R., Taylor G. R. and Mellberg J. R. 1974. An automated device for removinp. enamel layers _ by_ dis-

solution. Caries Res. 8. 293-299. Scheffe H. 1953. The Analysis of Variance. Wiley, New York.

pp. 109-110.

Experiments on local and systemic action of fluoride in caries inhibition in the rat.

EXPERIMENTS FLUORIDE ON LOCAL AND SYSTEMIC ACTION OF IN CARIES INHIBITION IN THE RAT R. H. LARSON, J. R. MELLBERG* and R. S!%+rNn@ National Institu...
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