Calcif. Tiss. Res. 18, 91 100 (1975) 9 by Springer-Verlag 1975
Effect of Long-Term Administration of Fluoride on Physico-Chemical Properties of the Rat Incisor Enamel H. Shinoda Department of Pharmacology, Faculty of Dentistry, Tokyo Medical and Dental University Received June 13, accepted November 7, 1974 Rats were maintained on drinking water containing different amounts of fluoride (0, 9, 23 45, 68, and 113 ppm) for 70 days. Physieo-chemical properties of the incisor enamel were examined after fluoride administration, using contact microradiography, histoehemistry, and microhardness tests. The tooth enamel formed during high fluoride exposure showed marked hypocalcification. Much of organic substance in the enamel seemed to have been retained. In addition, the microhardness of enamel showed a marked decrease. These changes were most prominent in the outer region of enamel and were proportional to the concentration of fluoride administered. Such changes following fluoride administration indicated inhibition of enamel maturation, i.e., an inhibition of the progressive deposition of minerals and/or an inhibition of organic matrix withdrawal by ameloblasts. Enamel seemed more affected by fluoride than dentine. Key words: Fluoride - - Enamel - - Microradiography - - tIistochemistry - - Microharchless.
Introduction I n early studies on d e n t a l fluorosis, Ainsworth (1933) a n d R o h o l m (1937) observed t h a t m o t t l e d t e e t h in m a n were brittle, t h a t their crowns or incisal edges were apt to chip a n d t h a t fillings were n o t well retained. I n addition, it has been show~ e x p e r i m e n t a l l y t h a t the t e e t h formed d u r i n g high fluoride exposure were a b n o r m a l l y worn a n d inclined to fracture (Smith et al., 1932; Ishiki, 1957; Shupe a n d Alther, 1966). The reasons for such a decrease in the mechanical s t r e n g t h a n d resistance to wear are t h o u g h t to be closely related to d i s t u r b a n c e s of m i n e r a l i z a t i o n reported b y previous a u t h o r s (Darling a n d Brooks, 1959; N e w b r u n a n d Brudcvold, 1960; Otake, 1960; Gustafson, 1961; Yaeger, 1966; Kruger, 1970; Hasegawa, 1971). However, there seems to have been few systematic studies made on the physico-chemical n a t u r e of enamel in dental fluorosis. I n the present study, b y using contact microradiography, histochemical methods, arid microhardness test, changes in the incisor enamel were e x a m i n e d in rats which were given different doses of fluoride for 70 days.
Materials and Methods Sixty male rats of Wistar strain, 45 days old and with a mean body weight of 140 g, were divided into 6 groups of 10 animals. Groups of rats were maintained for 70 days on the drinking water ad libitum containing 0 (control group), 9, 23, 45, 68, and 113 ppm of fluorine respectively. These doses, decided according to the descriptions by Hodge and Smith (1965), For reprints: Dr. H. Shinoda Department of Pharmacology, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima 1-chome, Bunkyo-ku, Tokyo 113, Japan.
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and Ishiki (1957), were prepared by dissolving sodium fluoride (Wake Pure Chemical Industries Co., Ltd., Osaka) in deionized water. All the animals were fed on the standard laboratory diet (CE-2, CLEA Japan Inc., Tokyo) that contained 19 ppm of fluorine. During the experimental period, the room temperature was kept at 20-22 ~ and the relative humidity at 37-57 %. On the 71st day, the animals were killed by chloroform inhalation and then the maxillary and mandibular incisors were dissected free from surrounding bones and adhering soft tissues. For contact microradiography some of the right maxillary and mandibular incisors from each group were used. Each incisor was divided into three equal parts (apical, middle, and incisal). Only its incisal portion was used to examine the degree of calcification in the enamel. The incisal portion of the teeth was fixed in 10% neutral formalin, dehydrated, and embedded in Rigolac (Riken polyester resin, Shown Kobunshi Co., Ltd., Tokyo). The mid-sagittal ground sections, 30-35 ~zm thick, were prepared using a thin sectioning machine (Bronwill Co., Ltd., San Francisco, U.S.A.) followed by manual grinding. They were placed in close contact with Kodak 649-0 films (Eastman Kodak Co., Ltd., Rochester, U.S.A.) in a dark room and were mieroradiographed using a Softex CMR type apparatus (Softex Co., Ltd., Tokyo) at 7 kV and 3 mA for 10-20 min. Following X-ray exposure, they were mounted in Bioleit (Okenshoji Co., Ltd., Tokyo). Qualitative examinations of the microradiographs were made microscopically. For histochemical examinations, some of the right maxillary and mandibular incisors from each group were used. Only the incisal portion was fixed in 10% neutral formalin or 90% ethanol. Then, the mid-sagitta] ground sections of about 40 ~zm thick were also prepared. After dehydration, the enamel was tested for protein using the coupled tetrazonium method of Danielli (1947) and the dinitrofluorobenzene method of Burstone (1955). To measure the microhardness of the enamel, the left mandibular incisors from each group were used. They were stored in a refrigerator without fixation. Before measurement, transverse sections of about 1.5 mm thick were prepared from the midportion of the incisal part (Fig. 1) using a laboratory lathe. On these transverse sections, Knoop hardness of the enamel was examined from the labial surface towards the amelo-dentinal junction in three different points (Fig. 5a) using a mierohardness tester (Type MVK-C, Akashi Co., Ltd., Tokyo) with 100 g load applied for 30 sec. Similarly on the same section, the microhardness of dentine was examined from the amelo-dentinal junction towards the pulp in five different points (Fig. 5a). For the measurement of the microhardness of enamel in different regions of the tooth, the left maxillary incisors were cut mid-sagittally with a laboratory lathe. On the cut surface of the tooth, Knoop hardness of the enamel in different regions was examined longitudinally from the apical end towards the incisal edge at seven different points and at three additional points (Fig. 6a). The measurement was made approximately in the midportion of the whole thickness of the enamel in each point. Knoop hardness number (K.h.n.) at each measured point was obtained by the following equation, where L and d represent applied load (kg) and length of the major diagonal of indentation (mm) respectively: K.h.n. : 14.228 L/d 2 kg/mm 2 (Petty, 1971). Student's t-test was applied to examine the significance of difference between the means of measurements. Results
Contact Microradiography I n t h e c o n t r o l a n i m a l s , t h e o u t e r l a y e r of t h e e n a m e l s h o w e d h i g h e r r a d i o p a c i t y t h a n t h e m i d d l e a n d i n n e r layers. T h e r e was a t h i n l a y e r w i t h s l i g h t l y low r a d i o p a c i t y j u s t b e n e a t h t h e e n a m e l s u r f a c e b u t , in g e n e r a l , t h e r a d i o p a c i t y d e c r e a s e d t o w a r d s t h e a m e l o - d e n t i n a l j u n c t i o n (Fig. 2a). I n t h e e x p e r i m e n t a l groups, t h e r e w e r e v a r i o u s k i n d s of c h a n g e s in t h e l a y e r s of e n a m e l . T h e r a d i o l u c e n t s u b s u r f a c e a r e a s e e m e d t o e x p a n d t o w a r d s t h e a m e l o d e n t i n a l j u n c t i o n a n d t h e r a d i o l u c e n c y i n c r e a s e d as t h e f l u o r i n e c o n c e n t r a t i o n in t h e d r i n k i n g w a t e r b e c a m e h i g h e r , i.e., t h e s e r a d i o l u c e n t a r e a s w e r e l i m i t e d
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!
Fig. 1. Schematic representation of t~he longitudinal view of the rat mandibular incisor. Erupted part of the incisor, approximately one sixth of the whole length of the incisor from its ineisal edge as indicated by a solid line, was used for the mierohardness test
approximately to the outer layer in 9, 23, and 45 p p m groups (Fig. 2b~l), expanded to the middle layer in 68 p p m group (Fig. 2e) and to the inner layer in 113 p p m group (Fig. 2f). A thin surface layer with relatively high radiopaeity was observed in every group (Fig. 2a-i). Such radiotueent regions oecured periodically in animals given higher doses of fluoride (Fig. 3). I n addition, six to eight radiolucent layers running parallel to each other and slightly oblique to the enamel surface were observed. This change also was prominent in groups of animals given higher doses of fluoride, particularly in 113 p p m group (Figs. 2f and 3). Histochemical Examinations I n the mature enamel of control animals, the negative reactions obtained both with the coupled tetrazonium method and the dinitrofluorobenzene method (Fig. 4a), indicated t h a t no appreciable amounts of protein were present. The intensity of the reactiort increased as the dose of fluoride became higher. In 113 p p m group, the positive areas reached the inner layer of the enamel (Fig. 4b). Such positive areas also appeared periodically in the longitudinal sections. In addition, six to eight parallel layers as seen in the contact microradiographs also appeared in the enamel from 45, 68, and 113 p p m groups. The areas which exhibited the positive protein reactions seemed to correspond with the areas which showed increased radiolucency in the contact microradiographs. Microhardness Test
Figure 5b summarizes the results of microhardness test in the transverse sections of the mandibular incisors of rats given various doses of fluoride. The average value of Knoop hardness number in the outer enamel layer (El) in 9 p p m group already showed a significant decrease (p < 0.05) from t h a t of the control group. The hardness in the outer enamel layer decreased as the dose of fluoride became higher. The effect of fluoride on the microhardness of outer layer of the enamel was most prominent. I n 113 p p m group, the percentage reduction of hardness in the outer layer reached 79 % of the control value. The average values of mierohardness in both middle (E2) and inner (Ea) layers did not show significant changes between the control group and each of
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Fig. 2a--~
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Fig. 3. A microradiograph of the incisor enamel from 113 ppm group showing the periodical appearance of the radiolucent regions (E enamel; D dentine)
Fig. 4 a and b. Mid-sagittal sections of enamel in the incisal portion of r a t maxillary incisors tested by a coupled tetrazonium method (E enamel; D dentine). (a) 0 p p m group: negative reaction is observed. (b) 113 ppm group: the area exhibiting the positive reaction expands from subsurface towards the amelo-dentinal junction
Fig. 2 a - - f . Contact microradiographs of the mid-sagittal ground sections of enamel in rats given different doses of fluoride. The sections were prepared from the incisal portion of mandibular incisors. (E enamel; arrows = amelo-dentinal junction). (a) 0 ppm group: The outer layer showed a higher radiopacity. The radiolucency increased towards the amelodentinal junction. (b) 9 ppm group: The radioluceney of subsurface layer seemed to increase as compared with the control. (c)23 p p m group: Note the slightly increased radiolucency in the outer layer. (d) 45 p p m group : Several radiolueent layers running parallel to each other and slightly oblique to the enamel surface were observed. (e) 68 p p m group: The radioluceney was greater in the outer layer and expanded towards the amelo-dentinal junction. (f) 113 ppm group: The radiolucent region expanded more deeply towards the amelo-dentinal junction. The radiolucent layers running parallel to each other and slightly oblique to the enamel surface could be seen clearly
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Fig. 5. (a) Transverse section of the same portion of the incisor as shown by a solid line in Fig. i. E z - E a show the measured points of microhardness over the enamel and D1-D 5 show those over the dentine. (b) The microhardness of enamel and dentine in the transverse sections of the mandibular incisors of rats given different doses of fluoride for 70 days. The mean value of Knoop hardness number in each layer of enamel and dentine (see Fig. 5a) is plotted against the fluorine concentration in the drinking water. Number of animals in each group is shown in parentheses
9, 23, 45, a n d 68 p p m groups. H o w e v e r , in 113 p p m g r o u p , h a r d n e s s a n d E s s h o w e d m a r k e d d e c r e a s e s ( p < 0.01); t h e p e r c e n t a g e r e d u c t i o n s a n d 3 6 % of t h e c o n t r o l v a l u e s r e s p e c t i v e l y . T h e m i c r o h a r d n e s s of d e n t i n e in e a c h l a y e r (see F i g . 5 a , f r o m s h o w e d n o s i g n i f i c a n t c h a n g e s a f t e r a d m i n i s t r a t i o n of d i f f e r e n t doses in t h e p r e s e n t e x p e r i m e n t .
a t b o t h Ez b e i n g 52 % D 1 t o Ds) of f l u o r i d e
Fig. 6. (a) Schematic representation of the measured points of microhardness of enamel in different regions of the tooth. The left maxillary incisors were cut mid-sagittally. The whole length of incisor was divided arbitrarily into 8 equal parts. Then the seven (1 to 7) and three (1', 2', and 3 ]) additional points were examined. The measurement was made approximately in the midportion of the whole thickness of the enamel layer in each point. (b) The microhardness of enamel in different regions of the tooth (see Fig. 6a). Open circle represents the average value of Knoop hardnes number in each region. Each dot represents the value from each animal. Number of animals in each group is shown in parentheses
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Figure 6 b shows the results of microhardness test at different regions of enamel in each group of rats given different doses of fluoride. At points 1 and l' (see Fig. 6a), the enamel was soft and hardness could be measured only in a few samples in each group. From points 2 to 3', the increase in hardness was prominent in every group, suggesting the progressive deposition of minerals. From points 4 (or 3') to 7, no remarkable changes in the mierohardness were found in any groups. At points 3' and 6 in 68 p p m group, and at points 3', 4, 5, 6, and 7 in 113 p p m group, the average values were significantly low ( p ~ 0 . 0 1 ) as compared with those of the control group. Discussion
H u m a n mottled enamel has been examined by Darling and Brooks (1959), Newbrun and Brudevold (1960) and Gustafson (1961) using different histological techniques. Their findings could be summarized as follows. The outer area of the mottled enamel was diffusely hypocalcified. I n severe fluorosis, the hypocalcified area expanded towards the amelo-dentinal junction, but a thin, well-calcified layer always remained on the surface of enamel. The prisms in the affected enamel often had an irregular course. Layers of relatively well and poorly-calcified enamel could be seen running parallel to the accentuated incremental lines. The mieroradiograptfic findings in the present study of fluorosis in the rat incisors were essentially the same as their observations in human teeth. I t is supposed t h a t mineral deposition into the enamel was inhibited severely by the fluoride administration. Montelius et al. (1933) found no difference in the percentage of Ca and P (by weight) between normal and mottled human teeth, but this might have been due to the differences of mineral contents between normal and mottled enamel being small and, therefore, difficult to detect in whole enamel samples (Newbrun and Brudevold, 1960). The histochemical stains for protein showed a positive reaction in the hypocalcified region in the enamel from rats given high doses of fluoride, whereas almost no protein reaction was found in the control specimens. In the decalcified sections of human mottled teeth, the presence of thick organic matrix in the prism core and the interprismatic regions has been reported (Darling and Brooks, 1959). In the experimental fluorosis, acid-insoluble matrix remained even at the late stage of enamel formation in rats (Ishiki, 1957; Otake, 1960). An increased nitrogen content in the human mottled enamel has also been shown by Bowes and Murray (1936) and by Bhussry (1959). These finding agreed well with the present observations. An increase in the organic substances in the enamel formed during high fluoride exposure might be due to the entry of extraneous material into the hypocalcified regions (Bhussry, 1959). The maturation of enamel is characterized by the progressive deposition of mineral and the withdrawal of organic matrix and water (Burgess and Maclaren, 1965; Reith and Butcher, 1967). Thus, interference with maturation by fluoride m a y result not only in an increased organic content but also in the decreased mineral deposition (Fig. 2). The effect of fluoride on the microhardness of enamel have been examined by several investigators (Miyazaki et al., 1957; Herrmann, 1958; Nerbrun, 1960), but the detailed relationship between the dose of fluoride and the mierohardness
Effect of Fluoride on Physico-Chemical Properties of Enamel
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of e n a m e l a t different l a y e r s a n d a t v a r i o u s regions of t h e t o o t h has n o t been r e p o r t e d . I n t h e p r e s e n t e x p e r i m e n t , t h e h a r d n e s s of o u t e r e n a m e l l a y e r decreased as t h e dose of fluoride became higher, b u t t h e m i d d l e a n d i n n e r layers were n o t a p p r e c i a b l y affected b y t h e lower doses (see Fig. 5 b). These results closely r e l a t e d with t h e degree of calcification as shown b y t h e m i e r o r a d i o g r a p h i c e x a m i n a t i o n s (Figs. 2 a n d 5). I t is s u p p o s e d t h a t t h e o u t e r l a y e r is m o s t suseeptable to fluoride. A c c o r d i n g t o P i n d b o r g a n d W e i n m a n n (1959), t h e o u t e r l a y e r of e n a m e l m a t r i x is i o r m e d b y a m e l o b l a s t s a t t h e l a t e f o r m a t i v e stage. 0 t a k e (1960) has shown t h a t fluoride interferes m a i n l y w i t h a m e l o b l a s t s a t t h e late f o r m a t i v e a n d at t h e e a r l y m a t u r a t i v e stages. W a l t o n a n d E i s e n m a n n (1974) also h a v e shown electron microscopically t h a t cytologic features of a m e l o b l a s t s a t t h e f o r m a t i v e a n d t h e e a r l y m a t u r a t i v e stages were c o n s i s t e n t l y affected b y fluoride. I t is p r o b a b l e t h a t a m e l o b l a s t s a t various stages are m o r e or less sensitive to t h e fluoride exposure, b u t most sensitive a t t h e late f o r m a t i v e stage, a n d t h a t t h e i r s u b s e q u e n t functional d i f f e r e n t i a t i o n is interfered, so t h a t t h e y camlot achieve n o r m a l m i n e r a l d e p o s i t i o n a n d w i t h d r a w a l of organic m a t r i x in t h e i r s u b s e q u e n t stages. T h e r e h a v e been m a n y r e p o r t s t h a t dentinogenesis is also d i s t u r b e d b y t h e fluoride (Sehour a n d Smith, 1935; Yaeger, 1966). H o w e v e r , considering t h e results of p r e v i o u s r e p o r t s (Ishiki, 1957; Otake, 1960; Gustafson, 1961), amelogenesis seems to be more sensitive to fluoride t h a n dentinogenesis. I n t h e p r e s e n t microhardness test, each l a y e r of d e n t i n e showed no significant changes w i t h i n t h e doses of fluoride used in this e x p e r i m e n t . Consequently, t h e a b n o r m a l a b r a s i o n or f r a c t u r e of t h e t e e t h f o r m e d d u r i n g high fluoride e x p o s u r e , n a y m a i n l y be due to changes in t h e physieo-ehemieal p r o p e r t i e s of e n a m e l r a t h e r t h a n dentine.
Acknowledgements. The author is grateful to Professor H. Ogura for his continuous guidance and for many discussions, and to Professor M. Chiba, of Tsurumi University, for his valuable suggestions and kind help in preparing the manuscript. References Ainsworth, N. J.: Mottled teeth. Brit. dent. J. 55, 233-250 (1933) Bhussry, B. R. : ChemicM and physical studies of enamel from human teeth. IV. Density and nitrogen content of mottled enamel. J. dent. Res. 38, 369-373 (1959) Bowes, J. H., Murray, M. M. : A chemical study of "mottled teeth" from Maldon, Essex. Brit. dent. J. 60, 556 562 (1936) Burgess, R. C., Maclaren, C. M.: Proteins in developing bovine enamel. In: Tooth Enamel I (Stack, M. V., Fearnhead, 1% W., eds.), p. 74-8~. Bristol: John Wright &Sons 1965 Burstone, M. S. : An evaluation of histochemical methods for protein groups. J. Histochem. Cytochem. 8, 32-48 (1955) Danielli, J. F. : A study of techniques for the cytochemicM demonstration of nucleic acids and some components of proteins. Syrup. Soc. exp. Biol. l, 101-113 (1947) Darling, A. I., Brooks, A. W. : Some observations on the mottled enamel of fluorosis. J. dent. Res. 88, 1226-1227 (1959) Gustafson, A. G.: The histology of fluorosed teeth. Arch. oral Biol. 4, 67-69 (1961) Hasegawa, K. : Studies on the formative disturbances of the enamel in experimental chronic fluorosis. Jap. J. Oral Biol. 13, 239-254 (1971) Hodge, II. C., Smith, F. A. : Fluorine chemistry (J. 14. Simons, general ed.), vol. IV, p. 589-601. New York-London: Academic Press 1965
Herrmann, M.: Die Ver~nderung der H/~rte des Zahnes naeh toxiseben F]uorgaben (Tierexperimentelle Ergebnisse). Dtsch. zahn~trztl. Z. 13, 567-571 (1958) 7*
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Ishiki, T.: The histologic changes in the tooth and the salivary gland of the rat in the experimental fluorosis. J. Jap. Stomatol. Soc. 24, 1-18 (1957) Kruger, B. J. : The effect of different levels of fluoride on the ultrastructure of ameloblasts in the rats. Arch. oral Biol. 15, 109-114 (1970) Miyazaki, Y., Ishikawa, G., Kato, K. : The microhardness of the rat teeth following administration of fluoride. In: Medico-dental researches on fluorides (S. Utzino, ed.), p. 94-96. Tokyo: The Japan Society for the Promotion of Science 1957 Montelius, G., McIntosh, J. F., Ma, Y. C.: Chemical investigations of mottled enamel and brown stain. J. dent. Res. 13, 73-79 (1933) Newbrun, E. : Studies on the physical properties of fluorosed enamel-II. Microhardness. Arch. oral Biol. 2, 21-27 (1960) Ncwbrun, E., Brudevold, F.: Studies on the physical properties of fluorosed enamel-I. Microradiographic studies. Arch. oral Biol. 2, 15-20 (1960) Otake, T. : Pathohistological and histochemical studies on the formative disturbances of the enamel in experimental fluorosis. Odontology 48, 1-54 (1960) Petty, E. R. : Hardness testing. In: Measurement of mechanical properties (techniques of metals research, vol. V/II, R. F. Bunshar, ed.), p. 157-221. New York-London-SydneyToronto: Interscience Publishers 1971 Pindborg, J. J., Wcinmann, J. P.: Morphologic and functional correlations in the enamel organ of the rat incisor during amelogenesis. Acta anat. (Basel) 36, 367-381 (1959) Reith, E . J . , Butcher, E . O . : Microanatomy and histochemistry of amelogenesis. In: Structural and chemical organization of teeth (A. E. W. Miles, ed.), vol. I, p. 371-397. New York-London: Academic Press 1967 Roholm, K.: Fluorine Intoxication. A clinical-hygienic study, p. 33 and p. 218-219. Copenhagen-London: H. K. Lewis 1937 Schour, I., Smith, M. C.: Mottled teeth: An experimental and histologic analysis. J. Amer. dent. Ass. 22, 796-813 (1935) Shupe, J. L., Alther, E. W.: The effect of fluoride on livestock, with particular reference to cattle. In: Handbook of experimental pharmacology (Eichler, O., Farah, A., Herken, H., Welch, A. D., cds.), vol. XX/1, p. 307-354. Berlin-Heidelberg-New York: Springer 1966 Smith, M. C., Lantz, E., Smith, H. V. : The cause of mottled enamel. J. dent. Res. 12, 149-159 (1932) Walton, R. E., Eisenmann, D. 1%.: Ultrastructural examination of various stages of amelogenesis in the rat following parenteral fluoride administration. Arch. oral Biol. 19, 171-182 (1974) Yaeger, J. A. : The effect of high fluoride diet on developing enamel and dentine in the incisors of rats. Amer. J. Anat. 118, 665-684 (1966)
Effect of Long-Term Administration of Fluoride on Physico-Chemical Properties of the Rat Incisor Enamel H. Shinoda Department of Pharmacology, Faculty of Dentistry, Tokyo Medical and Dental University Received June 13, accepted November 7, 1974 Rats were maintained on drinking water containing different amounts of fluoride (0, 9, 23 45, 68, and 113 ppm) for 70 days. Physieo-chemical properties of the incisor enamel were examined after fluoride administration, using contact microradiography, histoehemistry, and microhardness tests. The tooth enamel formed during high fluoride exposure showed marked hypocalcification. Much of organic substance in the enamel seemed to have been retained. In addition, the microhardness of enamel showed a marked decrease. These changes were most prominent in the outer region of enamel and were proportional to the concentration of fluoride administered. Such changes following fluoride administration indicated inhibition of enamel maturation, i.e., an inhibition of the progressive deposition of minerals and/or an inhibition of organic matrix withdrawal by ameloblasts. Enamel seemed more affected by fluoride than dentine. Key words: Fluoride - - Enamel - - Microradiography - - tIistochemistry - - Microharchless.
Introduction I n early studies on d e n t a l fluorosis, Ainsworth (1933) a n d R o h o l m (1937) observed t h a t m o t t l e d t e e t h in m a n were brittle, t h a t their crowns or incisal edges were apt to chip a n d t h a t fillings were n o t well retained. I n addition, it has been show~ e x p e r i m e n t a l l y t h a t the t e e t h formed d u r i n g high fluoride exposure were a b n o r m a l l y worn a n d inclined to fracture (Smith et al., 1932; Ishiki, 1957; Shupe a n d Alther, 1966). The reasons for such a decrease in the mechanical s t r e n g t h a n d resistance to wear are t h o u g h t to be closely related to d i s t u r b a n c e s of m i n e r a l i z a t i o n reported b y previous a u t h o r s (Darling a n d Brooks, 1959; N e w b r u n a n d Brudcvold, 1960; Otake, 1960; Gustafson, 1961; Yaeger, 1966; Kruger, 1970; Hasegawa, 1971). However, there seems to have been few systematic studies made on the physico-chemical n a t u r e of enamel in dental fluorosis. I n the present study, b y using contact microradiography, histochemical methods, arid microhardness test, changes in the incisor enamel were e x a m i n e d in rats which were given different doses of fluoride for 70 days.
Materials and Methods Sixty male rats of Wistar strain, 45 days old and with a mean body weight of 140 g, were divided into 6 groups of 10 animals. Groups of rats were maintained for 70 days on the drinking water ad libitum containing 0 (control group), 9, 23, 45, 68, and 113 ppm of fluorine respectively. These doses, decided according to the descriptions by Hodge and Smith (1965), For reprints: Dr. H. Shinoda Department of Pharmacology, Faculty of Dentistry, Tokyo Medical and Dental University, Yushima 1-chome, Bunkyo-ku, Tokyo 113, Japan.
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and Ishiki (1957), were prepared by dissolving sodium fluoride (Wake Pure Chemical Industries Co., Ltd., Osaka) in deionized water. All the animals were fed on the standard laboratory diet (CE-2, CLEA Japan Inc., Tokyo) that contained 19 ppm of fluorine. During the experimental period, the room temperature was kept at 20-22 ~ and the relative humidity at 37-57 %. On the 71st day, the animals were killed by chloroform inhalation and then the maxillary and mandibular incisors were dissected free from surrounding bones and adhering soft tissues. For contact microradiography some of the right maxillary and mandibular incisors from each group were used. Each incisor was divided into three equal parts (apical, middle, and incisal). Only its incisal portion was used to examine the degree of calcification in the enamel. The incisal portion of the teeth was fixed in 10% neutral formalin, dehydrated, and embedded in Rigolac (Riken polyester resin, Shown Kobunshi Co., Ltd., Tokyo). The mid-sagittal ground sections, 30-35 ~zm thick, were prepared using a thin sectioning machine (Bronwill Co., Ltd., San Francisco, U.S.A.) followed by manual grinding. They were placed in close contact with Kodak 649-0 films (Eastman Kodak Co., Ltd., Rochester, U.S.A.) in a dark room and were mieroradiographed using a Softex CMR type apparatus (Softex Co., Ltd., Tokyo) at 7 kV and 3 mA for 10-20 min. Following X-ray exposure, they were mounted in Bioleit (Okenshoji Co., Ltd., Tokyo). Qualitative examinations of the microradiographs were made microscopically. For histochemical examinations, some of the right maxillary and mandibular incisors from each group were used. Only the incisal portion was fixed in 10% neutral formalin or 90% ethanol. Then, the mid-sagitta] ground sections of about 40 ~zm thick were also prepared. After dehydration, the enamel was tested for protein using the coupled tetrazonium method of Danielli (1947) and the dinitrofluorobenzene method of Burstone (1955). To measure the microhardness of the enamel, the left mandibular incisors from each group were used. They were stored in a refrigerator without fixation. Before measurement, transverse sections of about 1.5 mm thick were prepared from the midportion of the incisal part (Fig. 1) using a laboratory lathe. On these transverse sections, Knoop hardness of the enamel was examined from the labial surface towards the amelo-dentinal junction in three different points (Fig. 5a) using a mierohardness tester (Type MVK-C, Akashi Co., Ltd., Tokyo) with 100 g load applied for 30 sec. Similarly on the same section, the microhardness of dentine was examined from the amelo-dentinal junction towards the pulp in five different points (Fig. 5a). For the measurement of the microhardness of enamel in different regions of the tooth, the left maxillary incisors were cut mid-sagittally with a laboratory lathe. On the cut surface of the tooth, Knoop hardness of the enamel in different regions was examined longitudinally from the apical end towards the incisal edge at seven different points and at three additional points (Fig. 6a). The measurement was made approximately in the midportion of the whole thickness of the enamel in each point. Knoop hardness number (K.h.n.) at each measured point was obtained by the following equation, where L and d represent applied load (kg) and length of the major diagonal of indentation (mm) respectively: K.h.n. : 14.228 L/d 2 kg/mm 2 (Petty, 1971). Student's t-test was applied to examine the significance of difference between the means of measurements. Results
Contact Microradiography I n t h e c o n t r o l a n i m a l s , t h e o u t e r l a y e r of t h e e n a m e l s h o w e d h i g h e r r a d i o p a c i t y t h a n t h e m i d d l e a n d i n n e r layers. T h e r e was a t h i n l a y e r w i t h s l i g h t l y low r a d i o p a c i t y j u s t b e n e a t h t h e e n a m e l s u r f a c e b u t , in g e n e r a l , t h e r a d i o p a c i t y d e c r e a s e d t o w a r d s t h e a m e l o - d e n t i n a l j u n c t i o n (Fig. 2a). I n t h e e x p e r i m e n t a l groups, t h e r e w e r e v a r i o u s k i n d s of c h a n g e s in t h e l a y e r s of e n a m e l . T h e r a d i o l u c e n t s u b s u r f a c e a r e a s e e m e d t o e x p a n d t o w a r d s t h e a m e l o d e n t i n a l j u n c t i o n a n d t h e r a d i o l u c e n c y i n c r e a s e d as t h e f l u o r i n e c o n c e n t r a t i o n in t h e d r i n k i n g w a t e r b e c a m e h i g h e r , i.e., t h e s e r a d i o l u c e n t a r e a s w e r e l i m i t e d
Effect of Fluoride on Physico-Chemical Properties of Enamel
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!
Fig. 1. Schematic representation of t~he longitudinal view of the rat mandibular incisor. Erupted part of the incisor, approximately one sixth of the whole length of the incisor from its ineisal edge as indicated by a solid line, was used for the mierohardness test
approximately to the outer layer in 9, 23, and 45 p p m groups (Fig. 2b~l), expanded to the middle layer in 68 p p m group (Fig. 2e) and to the inner layer in 113 p p m group (Fig. 2f). A thin surface layer with relatively high radiopaeity was observed in every group (Fig. 2a-i). Such radiotueent regions oecured periodically in animals given higher doses of fluoride (Fig. 3). I n addition, six to eight radiolucent layers running parallel to each other and slightly oblique to the enamel surface were observed. This change also was prominent in groups of animals given higher doses of fluoride, particularly in 113 p p m group (Figs. 2f and 3). Histochemical Examinations I n the mature enamel of control animals, the negative reactions obtained both with the coupled tetrazonium method and the dinitrofluorobenzene method (Fig. 4a), indicated t h a t no appreciable amounts of protein were present. The intensity of the reactiort increased as the dose of fluoride became higher. In 113 p p m group, the positive areas reached the inner layer of the enamel (Fig. 4b). Such positive areas also appeared periodically in the longitudinal sections. In addition, six to eight parallel layers as seen in the contact microradiographs also appeared in the enamel from 45, 68, and 113 p p m groups. The areas which exhibited the positive protein reactions seemed to correspond with the areas which showed increased radiolucency in the contact microradiographs. Microhardness Test
Figure 5b summarizes the results of microhardness test in the transverse sections of the mandibular incisors of rats given various doses of fluoride. The average value of Knoop hardness number in the outer enamel layer (El) in 9 p p m group already showed a significant decrease (p < 0.05) from t h a t of the control group. The hardness in the outer enamel layer decreased as the dose of fluoride became higher. The effect of fluoride on the microhardness of outer layer of the enamel was most prominent. I n 113 p p m group, the percentage reduction of hardness in the outer layer reached 79 % of the control value. The average values of mierohardness in both middle (E2) and inner (Ea) layers did not show significant changes between the control group and each of
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Fig. 2a--~
Effect of Fluoride on Physico-Chemical Properties of Enamel
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Fig. 3. A microradiograph of the incisor enamel from 113 ppm group showing the periodical appearance of the radiolucent regions (E enamel; D dentine)
Fig. 4 a and b. Mid-sagittal sections of enamel in the incisal portion of r a t maxillary incisors tested by a coupled tetrazonium method (E enamel; D dentine). (a) 0 p p m group: negative reaction is observed. (b) 113 ppm group: the area exhibiting the positive reaction expands from subsurface towards the amelo-dentinal junction
Fig. 2 a - - f . Contact microradiographs of the mid-sagittal ground sections of enamel in rats given different doses of fluoride. The sections were prepared from the incisal portion of mandibular incisors. (E enamel; arrows = amelo-dentinal junction). (a) 0 ppm group: The outer layer showed a higher radiopacity. The radiolucency increased towards the amelodentinal junction. (b) 9 ppm group: The radioluceney of subsurface layer seemed to increase as compared with the control. (c)23 p p m group: Note the slightly increased radiolucency in the outer layer. (d) 45 p p m group : Several radiolueent layers running parallel to each other and slightly oblique to the enamel surface were observed. (e) 68 p p m group: The radioluceney was greater in the outer layer and expanded towards the amelo-dentinal junction. (f) 113 ppm group: The radiolucent region expanded more deeply towards the amelo-dentinal junction. The radiolucent layers running parallel to each other and slightly oblique to the enamel surface could be seen clearly
96
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Fig. 5. (a) Transverse section of the same portion of the incisor as shown by a solid line in Fig. i. E z - E a show the measured points of microhardness over the enamel and D1-D 5 show those over the dentine. (b) The microhardness of enamel and dentine in the transverse sections of the mandibular incisors of rats given different doses of fluoride for 70 days. The mean value of Knoop hardness number in each layer of enamel and dentine (see Fig. 5a) is plotted against the fluorine concentration in the drinking water. Number of animals in each group is shown in parentheses
9, 23, 45, a n d 68 p p m groups. H o w e v e r , in 113 p p m g r o u p , h a r d n e s s a n d E s s h o w e d m a r k e d d e c r e a s e s ( p < 0.01); t h e p e r c e n t a g e r e d u c t i o n s a n d 3 6 % of t h e c o n t r o l v a l u e s r e s p e c t i v e l y . T h e m i c r o h a r d n e s s of d e n t i n e in e a c h l a y e r (see F i g . 5 a , f r o m s h o w e d n o s i g n i f i c a n t c h a n g e s a f t e r a d m i n i s t r a t i o n of d i f f e r e n t doses in t h e p r e s e n t e x p e r i m e n t .
a t b o t h Ez b e i n g 52 % D 1 t o Ds) of f l u o r i d e
Fig. 6. (a) Schematic representation of the measured points of microhardness of enamel in different regions of the tooth. The left maxillary incisors were cut mid-sagittally. The whole length of incisor was divided arbitrarily into 8 equal parts. Then the seven (1 to 7) and three (1', 2', and 3 ]) additional points were examined. The measurement was made approximately in the midportion of the whole thickness of the enamel layer in each point. (b) The microhardness of enamel in different regions of the tooth (see Fig. 6a). Open circle represents the average value of Knoop hardnes number in each region. Each dot represents the value from each animal. Number of animals in each group is shown in parentheses
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98
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Figure 6 b shows the results of microhardness test at different regions of enamel in each group of rats given different doses of fluoride. At points 1 and l' (see Fig. 6a), the enamel was soft and hardness could be measured only in a few samples in each group. From points 2 to 3', the increase in hardness was prominent in every group, suggesting the progressive deposition of minerals. From points 4 (or 3') to 7, no remarkable changes in the mierohardness were found in any groups. At points 3' and 6 in 68 p p m group, and at points 3', 4, 5, 6, and 7 in 113 p p m group, the average values were significantly low ( p ~ 0 . 0 1 ) as compared with those of the control group. Discussion
H u m a n mottled enamel has been examined by Darling and Brooks (1959), Newbrun and Brudevold (1960) and Gustafson (1961) using different histological techniques. Their findings could be summarized as follows. The outer area of the mottled enamel was diffusely hypocalcified. I n severe fluorosis, the hypocalcified area expanded towards the amelo-dentinal junction, but a thin, well-calcified layer always remained on the surface of enamel. The prisms in the affected enamel often had an irregular course. Layers of relatively well and poorly-calcified enamel could be seen running parallel to the accentuated incremental lines. The mieroradiograptfic findings in the present study of fluorosis in the rat incisors were essentially the same as their observations in human teeth. I t is supposed t h a t mineral deposition into the enamel was inhibited severely by the fluoride administration. Montelius et al. (1933) found no difference in the percentage of Ca and P (by weight) between normal and mottled human teeth, but this might have been due to the differences of mineral contents between normal and mottled enamel being small and, therefore, difficult to detect in whole enamel samples (Newbrun and Brudevold, 1960). The histochemical stains for protein showed a positive reaction in the hypocalcified region in the enamel from rats given high doses of fluoride, whereas almost no protein reaction was found in the control specimens. In the decalcified sections of human mottled teeth, the presence of thick organic matrix in the prism core and the interprismatic regions has been reported (Darling and Brooks, 1959). In the experimental fluorosis, acid-insoluble matrix remained even at the late stage of enamel formation in rats (Ishiki, 1957; Otake, 1960). An increased nitrogen content in the human mottled enamel has also been shown by Bowes and Murray (1936) and by Bhussry (1959). These finding agreed well with the present observations. An increase in the organic substances in the enamel formed during high fluoride exposure might be due to the entry of extraneous material into the hypocalcified regions (Bhussry, 1959). The maturation of enamel is characterized by the progressive deposition of mineral and the withdrawal of organic matrix and water (Burgess and Maclaren, 1965; Reith and Butcher, 1967). Thus, interference with maturation by fluoride m a y result not only in an increased organic content but also in the decreased mineral deposition (Fig. 2). The effect of fluoride on the microhardness of enamel have been examined by several investigators (Miyazaki et al., 1957; Herrmann, 1958; Nerbrun, 1960), but the detailed relationship between the dose of fluoride and the mierohardness
Effect of Fluoride on Physico-Chemical Properties of Enamel
99
of e n a m e l a t different l a y e r s a n d a t v a r i o u s regions of t h e t o o t h has n o t been r e p o r t e d . I n t h e p r e s e n t e x p e r i m e n t , t h e h a r d n e s s of o u t e r e n a m e l l a y e r decreased as t h e dose of fluoride became higher, b u t t h e m i d d l e a n d i n n e r layers were n o t a p p r e c i a b l y affected b y t h e lower doses (see Fig. 5 b). These results closely r e l a t e d with t h e degree of calcification as shown b y t h e m i e r o r a d i o g r a p h i c e x a m i n a t i o n s (Figs. 2 a n d 5). I t is s u p p o s e d t h a t t h e o u t e r l a y e r is m o s t suseeptable to fluoride. A c c o r d i n g t o P i n d b o r g a n d W e i n m a n n (1959), t h e o u t e r l a y e r of e n a m e l m a t r i x is i o r m e d b y a m e l o b l a s t s a t t h e l a t e f o r m a t i v e stage. 0 t a k e (1960) has shown t h a t fluoride interferes m a i n l y w i t h a m e l o b l a s t s a t t h e late f o r m a t i v e a n d at t h e e a r l y m a t u r a t i v e stages. W a l t o n a n d E i s e n m a n n (1974) also h a v e shown electron microscopically t h a t cytologic features of a m e l o b l a s t s a t t h e f o r m a t i v e a n d t h e e a r l y m a t u r a t i v e stages were c o n s i s t e n t l y affected b y fluoride. I t is p r o b a b l e t h a t a m e l o b l a s t s a t various stages are m o r e or less sensitive to t h e fluoride exposure, b u t most sensitive a t t h e late f o r m a t i v e stage, a n d t h a t t h e i r s u b s e q u e n t functional d i f f e r e n t i a t i o n is interfered, so t h a t t h e y camlot achieve n o r m a l m i n e r a l d e p o s i t i o n a n d w i t h d r a w a l of organic m a t r i x in t h e i r s u b s e q u e n t stages. T h e r e h a v e been m a n y r e p o r t s t h a t dentinogenesis is also d i s t u r b e d b y t h e fluoride (Sehour a n d Smith, 1935; Yaeger, 1966). H o w e v e r , considering t h e results of p r e v i o u s r e p o r t s (Ishiki, 1957; Otake, 1960; Gustafson, 1961), amelogenesis seems to be more sensitive to fluoride t h a n dentinogenesis. I n t h e p r e s e n t microhardness test, each l a y e r of d e n t i n e showed no significant changes w i t h i n t h e doses of fluoride used in this e x p e r i m e n t . Consequently, t h e a b n o r m a l a b r a s i o n or f r a c t u r e of t h e t e e t h f o r m e d d u r i n g high fluoride e x p o s u r e , n a y m a i n l y be due to changes in t h e physieo-ehemieal p r o p e r t i e s of e n a m e l r a t h e r t h a n dentine.
Acknowledgements. The author is grateful to Professor H. Ogura for his continuous guidance and for many discussions, and to Professor M. Chiba, of Tsurumi University, for his valuable suggestions and kind help in preparing the manuscript. References Ainsworth, N. J.: Mottled teeth. Brit. dent. J. 55, 233-250 (1933) Bhussry, B. R. : ChemicM and physical studies of enamel from human teeth. IV. Density and nitrogen content of mottled enamel. J. dent. Res. 38, 369-373 (1959) Bowes, J. H., Murray, M. M. : A chemical study of "mottled teeth" from Maldon, Essex. Brit. dent. J. 60, 556 562 (1936) Burgess, R. C., Maclaren, C. M.: Proteins in developing bovine enamel. In: Tooth Enamel I (Stack, M. V., Fearnhead, 1% W., eds.), p. 74-8~. Bristol: John Wright &Sons 1965 Burstone, M. S. : An evaluation of histochemical methods for protein groups. J. Histochem. Cytochem. 8, 32-48 (1955) Danielli, J. F. : A study of techniques for the cytochemicM demonstration of nucleic acids and some components of proteins. Syrup. Soc. exp. Biol. l, 101-113 (1947) Darling, A. I., Brooks, A. W. : Some observations on the mottled enamel of fluorosis. J. dent. Res. 88, 1226-1227 (1959) Gustafson, A. G.: The histology of fluorosed teeth. Arch. oral Biol. 4, 67-69 (1961) Hasegawa, K. : Studies on the formative disturbances of the enamel in experimental chronic fluorosis. Jap. J. Oral Biol. 13, 239-254 (1971) Hodge, II. C., Smith, F. A. : Fluorine chemistry (J. 14. Simons, general ed.), vol. IV, p. 589-601. New York-London: Academic Press 1965
Herrmann, M.: Die Ver~nderung der H/~rte des Zahnes naeh toxiseben F]uorgaben (Tierexperimentelle Ergebnisse). Dtsch. zahn~trztl. Z. 13, 567-571 (1958) 7*
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Ishiki, T.: The histologic changes in the tooth and the salivary gland of the rat in the experimental fluorosis. J. Jap. Stomatol. Soc. 24, 1-18 (1957) Kruger, B. J. : The effect of different levels of fluoride on the ultrastructure of ameloblasts in the rats. Arch. oral Biol. 15, 109-114 (1970) Miyazaki, Y., Ishikawa, G., Kato, K. : The microhardness of the rat teeth following administration of fluoride. In: Medico-dental researches on fluorides (S. Utzino, ed.), p. 94-96. Tokyo: The Japan Society for the Promotion of Science 1957 Montelius, G., McIntosh, J. F., Ma, Y. C.: Chemical investigations of mottled enamel and brown stain. J. dent. Res. 13, 73-79 (1933) Newbrun, E. : Studies on the physical properties of fluorosed enamel-II. Microhardness. Arch. oral Biol. 2, 21-27 (1960) Ncwbrun, E., Brudevold, F.: Studies on the physical properties of fluorosed enamel-I. Microradiographic studies. Arch. oral Biol. 2, 15-20 (1960) Otake, T. : Pathohistological and histochemical studies on the formative disturbances of the enamel in experimental fluorosis. Odontology 48, 1-54 (1960) Petty, E. R. : Hardness testing. In: Measurement of mechanical properties (techniques of metals research, vol. V/II, R. F. Bunshar, ed.), p. 157-221. New York-London-SydneyToronto: Interscience Publishers 1971 Pindborg, J. J., Wcinmann, J. P.: Morphologic and functional correlations in the enamel organ of the rat incisor during amelogenesis. Acta anat. (Basel) 36, 367-381 (1959) Reith, E . J . , Butcher, E . O . : Microanatomy and histochemistry of amelogenesis. In: Structural and chemical organization of teeth (A. E. W. Miles, ed.), vol. I, p. 371-397. New York-London: Academic Press 1967 Roholm, K.: Fluorine Intoxication. A clinical-hygienic study, p. 33 and p. 218-219. Copenhagen-London: H. K. Lewis 1937 Schour, I., Smith, M. C.: Mottled teeth: An experimental and histologic analysis. J. Amer. dent. Ass. 22, 796-813 (1935) Shupe, J. L., Alther, E. W.: The effect of fluoride on livestock, with particular reference to cattle. In: Handbook of experimental pharmacology (Eichler, O., Farah, A., Herken, H., Welch, A. D., cds.), vol. XX/1, p. 307-354. Berlin-Heidelberg-New York: Springer 1966 Smith, M. C., Lantz, E., Smith, H. V. : The cause of mottled enamel. J. dent. Res. 12, 149-159 (1932) Walton, R. E., Eisenmann, D. 1%.: Ultrastructural examination of various stages of amelogenesis in the rat following parenteral fluoride administration. Arch. oral Biol. 19, 171-182 (1974) Yaeger, J. A. : The effect of high fluoride diet on developing enamel and dentine in the incisors of rats. Amer. J. Anat. 118, 665-684 (1966)
