Incorporation of magnesium into rat dental enamel and its influence on crystallization

.4rchs oralEio/. Vol. 34,No. IO,pp. 767-771,1989 Printed in Great Britain. All rights reserved

0003-9969/89 .S3.00+0.00

Copyright 0 1989Pergamon Press plc

INCORPORATION OF MAGNESIUM INTO RAT DENTAL ENAMEL AND ITS INFLUENCE ON CRYSTALLIZATION P. SPENCER,* C. BARNES,J. MARTINI, R. GARCIA, C. ELLIOTT and R. DOREMUS Materials Engineering Department, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, U.S.A. (Accepted 2 May 1989) Summary-The

incorporation of magnesium into the enamel of immature rats was studied by chemical analysis and X-ray diffraction. Magnesium solutions were injected subcutaneously and later intra-orally into growing rats. Increased magnesium was detected in the enamel at the late developmental stage, and a delay in enamel mineralization resulted from increased magnesium uptake. Incisal enamel from the

Mg-injected rats had significant line-broadening on X-ray diffraction, characteristic of poorly crystallized enamel

INTRODUCTION The chemical composition and crystalline structure of dental enamel resemble those of the mineral hydroxylapatite. Relatively small differences in the dimensions of a unit cell, the degree of crystallinity, and the elemental structure have been linked to impurities in the lattice of enamel (Cutress, 1972; Featherstone et al., 1983; LeGeros, 1974; LeGeros, Bane1 and LeGeros, 1978; LeGeros, Miravite and Curzon, 1977). Although these impurities, which include Na, K, Mg, CO,, Sr and F, constitute only a fraction of the enamel, they are particularly effective at modifying or regulating its response to environmental agents (Curzon and Cracker, 1978; Featherstone and Nelson, 1980). As reported by Pate1 and Brown (1975), impurities in the lattice are largely responsible for the distinctly enhanced acid-solubility of human enamel compared to that of hydroxylapatite. Magnesium, one of the prominent inorganic contaminants in dental enamel (Asgar, 1956; Robinson, Weatherell and Hallsworth, 1981), is closely associated with acid-solubility (Feagin and Thiradilok, 1979; Pak and Diller, 1969) and thus, potentially, with tooth decay. When enamel is attacked by acid, as occurs during early dental caries, the first tissue removed is rich in magnesium (Besic et af., 1979; Cutress, 1978; Duncan and Featherstone, Hallsworth, Robinson and Weatherell, 1972; Zuoma et al., 1983). Animals on magnesium-supplemented diets frequently have increased levels of caries (McClure and McCann, 1960; Navia, 1970; Zuoma and Nuija, 1977). This increased acid-solubility, paralleled in hydroxylapatite in vitro, is perhaps caused by the adverse effect of magnesium on crystallization (Backra, Trauts and Simon, 1965; Eanes, Termine and Posner, 1967; LeGeros, 1984; Robinson, Hallsworth and Kirkham, 1984). *Address correspondence to: Dr Paulette Spencer, Department of Pediatric Dentistry, UMKC School of Dentistry, 650 East 25th Street, Kansas City, MO 65108, U.S.A.

In mature human enamel, the concentration of magnesium varies from 0.1% near the enamel surface to about 0.4% near the enamel-dentine junction (Robinson et al., 1981). Isolated deposits of very high concentrations of magnesium are associated with increased levels of protein in enamel (Landis and Navarro, 1983; Robinson et al., 1981). Whether the magnesium is an integral part of the protein or is loosely bound to it is uncertain. These magnesiumrich areas are, however, tooth sites that are recognized clinically as being particularly susceptible to caries. Little information is available on either the mechanism of uptake or the developmental stage during which magnesium is incorporated into enamel. In addition, efforts to isolate magnesium in the tooth and to study its effects have been frustrated by the low concentration of magnesium involved. Thus we have now sought to increase the concentration of magnesium in the tooth through a rigorous schedule of systemic supplementation, and to evaluate changes in the chemistry and crystalline structure of the enamel as a function of magnesium uptake. MATERIALS

Animals and schedule

AND METHODS

ofinjections

Immature Sprague-Dawley rats of either sex were provided with rat chow and distilled water ad libitum. Control and experimental groups were weighed every other day. Pups in the experimental groups received daily subcutaneous injections of MgS04 from day 1-18. These injections provided Mg at a concentration of 5 parts/lo6 of Mg per log of body weight. At day 19 until day 29, the experimental rats were injected intra-orally with MgSO, at a concentration of 12 parts/lo6 of Mg. The MgSO, solution was injected via a 27-gauge needle angled below the surface of the oral mucosa at the inferior border of the mandible and in line with the mesial margin of the central incisors. This technique deposited the magnesium solution close to the site of active mineralization of the mandibular incisors. 761

768

P.

SP+~NCER 621 oi

On day 30, the rats were killed by an overdose of nembutal. The jaws from each animal were immediately dissected and the molar teeth and incisors removed through careful dissection of the surrounding soft tissue and bone. Enamel from the incisors was isolated from the dentine and separated according to the stage of mineralization, using an ultra-thin diamond disc in a dental drill. The stages of mineralization in the rat incisor have been described as stage l-immature: stage 2-transitional; stage 3-maturing (Hiller, Robinson and Weatherell, 1975; Leblond and Warshawsky, 1979). These samples were immediately placed on dry ice in preparation for freeze-drying. Selected samples from the freeze-dried enamel were ground into fine particles in a percussion hammer mill (Spex Industries, Model 6700) bathed in liquid nitrogen.

Cu Kor radiation at 35 kV and 20 mA. Exposure times were 12--12.5 h. Samples of synthetic hydroxylapatite, prepared by MS Julie Martini in the Laboratory for Materials Engineering at Rensselaer Polytechnic Institute, were examined in the same manner as the enamel samples. RESULTS

Plasma spectrometry

These results are shown in Table I. The molar ratio of Ca/P was little different along the length of each tooth, either in controls (no injection of Mg) or in experimental samples (Mg-injected). The range for control samples was 1.42-1.50, with a mean of 1.48 i: 0.18 for all samples; the experimental samples had a range of 1.44-l .46, with a mean of 1.45 &-0.12. There was a significant difference (p < 0.001) in the molar ratio of Ca/P in segment III in controls (1 .SO) from that in experimental samples (1.44). The molar ratios of Mg/Ca and Mg/P in control samples showed little variation along the length of each tooth. The range for Mg/Ca (0.01-0.03) and the range for Mg/P (0.02-0.05) are similar to those reported for select density-fractions recovered from bovine teeth (Landis and Navarro, 1983). The experimental samples showed a significant increase in Mg from the immature enamel at the root apex to the more mature enamel at the tip. Molar ratios of Mg/Ca and Mg/P showed a three-fold increase between samples from segment I and those from segment III. At the root apex, the range for the molar ratio of Mg/Ca was 0.0 134.0 15, while towards the incisal tip this ratio was significantly increased (p < O.OOl), its range being 0.044-0.047. Similarly, the molar ratio of Mg/P from segment I ranged between 0.022 and 0.025, while the range at segment TITwas 0.066-0.076. There was no significant difference between segment I and segment II, however, in these ratios. Finally, the molar ratios of Mg/Ca and Mg/P from segment III of the experimental samples were significantly greater (p < 0.001) than the corresponding ratios in control samples.

Chemical anulysis by plasma spectrometry

Samples of freeze-dried and ground enamel representing each of the stages of mineralization were weighed in polyethylene vials and then dissolved overnight in 1.0 ml of 3 M HCl. The concentrations of magnesium, calcium and phosphorus in these solutions were determined by direct-current, plasmaemission spectrometry (Beckman Spectra Span). Concentrations of calcium and magnesium were also evaluated by atomic absorption spectrophotometry (Perkin-Elmer Model 2380). The synthetic apatites were similarly analysed. By plasma-emission mass spectroscopy, it was possible to measure Ca, P and Mg within each sample to an accuracy of 24%. Interference from the matrix was minimized by correlating samples with standards. The concentrations of Ca, P and Mg in dissolved hydroxylapatite was determined first, to give a standard calibration curve, and this standard was then compared with emissions for measured concentrations of Ca, P and Mg from enamel. Results for emissions from plasma were analysed statistically using Student’s t-test; the results were regarded as significant at p < 0.001. X-ray d#raction

Ground and freeze-dried senting the various stages incisors were examined by using a Dcbye-Scherrer

X -ra,v diffraction These results are shown in Table 2 and in Figs l-4. The control incisors had differences in their patterns of diffraction as a function of the ratio of Ca/P:

samples of enamel repreof mineralization of the powder X-ray diffraction camera and Ni-filtered

Table I. Chemical analysis by plasma-spectrometry of incisors of Mg-treated and control rats Segment I, molar ratios (n = 9)

Segment II,

Segment III,

molar ratios (n = IO)

molar ratios (n = 10)

Control

Me-treated

---.Control

i .45 0.007

1.46 0.02

1.45 0.01

1.44 0.02

1.51 0.03

1.44 0.05

0.014 0.002

0.012 0.001

0.017 0.001

0.016 0.003

0.028 0.001

0.049 0.009

0.020 0.001

0.023 0.001

0.026 0.003

0.027 0.004

0.045 0.005

0.07 1 0.009

Mg-treated

Control

Mg-treated

CajP

Mean SD MgiCa Mean SD &iP

Mean SD

Magnesium and crystallization of enamel

769

Table 2. Comparison of interplanar spacing of enamel of segments II and III in controls and in experimental samples (Exptal) with spacing in hydroxylapatite

segments

with lower from segment

Hydroxylapatite

II Controls

II Exptal

III Controls

III Exptal

8.17 5.26 3.88 3.44 3.17

8.248 5.288 3.910 3.440 3.175

8.259

8.237

8.162

3.891 3.439 3.168

3.927

3.897 3.444

3.08 2.814 2.778 2.720 2.631

3.094 2.828 2.770 2.721 2.634

3.087 2.826 2.780 2.727 2.634

3.072 2.822 2.774 2.724 2.627

2.528 2.262 2.148 2.065 2.000

2.539 2.270 2.156 2.059 1.998

2.269 2.155

2.538 2.268 2.159

2.273

1.943 1.890 1.841 1.806 1.780

1.948 1.89 1.844 1.798 1.785

I.949 1.897 1.841 1.811 1.791

I.947 1.897 1.842 1.815 1.786

1.946 1.893 1.842 1.811 I .790

1.754 1.722 1.503 1.474

1.757 1.719 1.505 1.479

1.760

1.755 1.717

1.756 I.719

Ca/P

ratios,

i.e.

immature

I, had fewer reflections and significant line-broadening in comparison to samples from mature enamel of segment III. The control samples produced diffraction patterns with peak positions characteristic of hydroxylapatite, and with only slight differences in d-spacing. Patterns of diffraction in enamel from Mg-injected rats showed line-broadening for each of the three segments. The weak and widened pattern for enamel collected from their segment III indicated significant interference by Mg in the process of mineralization. Both their segment II and III had fewer reflections and pronounced line-broadening in comparison to those produced by control samples. The results of diffraction analysis for samples from Mg-injected animals were characteristic of immature enamel. enamel

DISCUSSION

Bone is recognized to be the tissue of choice for measuring the body’s store of magnesium because it contains 65% of the magnesium in the body. Elevated levels of Mg are found in the mineral phase of bone as well as in dentine when dietary Mg is increased. Dentine, enamel and bone have similar inorganic compositions so in the active mineralizing phase of enamel in increased level of Mg should be registered in animals injected with Mg. We have used localized injections to deliver a high concentration of Mg to the site of active enamel mineralization. In contrast to previous studies done with dietary supplements of Mg (Eisenmann and Yaeger, 1969; Irving, 1944), these injections led to uptake of Mg. Hiller et al. (1975) found a decrease in Mg from immature to mature enamel in the rat incisor but we

1.717

3.239 2.829 2.783 2.733 2.637

found a sharp increase in Mg over this region, thus supporting the trends reported by Robinson et al. (1984). In bovine incisors, for example, Robinson noted an increase in magnesium at the border between transitional and maturing enamel and reported that the magnesium concentration rose from 0.25% in the younger tissue of stage I to 0.5% at the border between stages II and III. The molar ratio of Mg/P increased from 0.03 in stage I to 0.065 at the border between stages II and III. Our model provides a means of studying magnesium uptake in dental enamel according to the stage of mineralization. Localizing Mg in enamel is difficult because of its low concentration. In our model, Mg in the enamel approaches concentrations previously reported in dentine. The rise in Mg concentration and the rise in the molar ratio of Mg/P occur in an area similar to the border identified by Robinson et al. (1984). The high concentrations of Mg in this region and the low Ca/P ratio suggest a delay in enamel maturation as a result of enhanced Mg uptake. The results of our diffraction analysis indicate that the supplemental Mg interfered significantly with enamel mineralization. Patterns of diffraction from the experimental enamel were characteristic of immature enamel. LeGeros (1984) has reported limited incorporation of Mg into synthetic and biological apatites; she has further suggested a limited pattern of substitution of Mg for Ca in apatite. We have thus shown that localized supplemental Mg produces significant changes in enamel mineralization in the normal young rat. Enamel maturation was particularly inhibited in the transitional and mature stages. Magnesium uptake primarily occurred during the final stage of enamel development. There was a possible delay in maturation or a disordered

770

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SPENCER et al.

apatite that will not mature into healthy enamel. Therefore, Mg can directly influence enamel formation and is an important factor in enamel mineralization in vivo in this animal model. Acknowledgemenrs~We gratefully acknowledge the technical assistance of Mr Lloyd Colberg, MS Lisa Hurley and MS Andrea Kazmer. This work was supported in part by the National Institute of Dental Research, grant No. DE07054. REFERENCES

Asgar K. (1956) Chemical analysis of human teeth. J. dent. Res. 35, 742-748.

Backra B. N., Trauts 0. R. and Simon S. Z. (1965) Precipitation of calcium carbonates and phosphates. The effect of magnesium and fluoride ions on the spontaneous precipitation of calcium carbonates and phosphates. Archs oral Biol. 10, 731-~738. Besic F. Z., Knowles C. R., Ketter 0. and Wiemann M. R. (1979) Detailed electron probe microanalysis of three teeth sections with early enamel caries. J. dent. Res. 49, 111-118. Curzon M. E. G. and Cracker D. C. (1978) Relationships of trace elements in human tooth enamel to dental caries. Archs oral Biol. 23, 647-653.

Cutress T. W. (1972) The inorganic composition and solubility of dental enamel from several specific population groups. Archs oral Biol. 17, 93-109. Eanes E. E., Termine J. D. and Posner A. S. (1967) Amorphous calcium phosphate in skeletal tissues. C/in. Orthop. 53, 223-235.

Eisenmann D. R. and Yaeger J. A. (1969) Alterations in the formation of rat dentine and enamel induced by various ions. Archs oral Biol. 14, 1045-1064. Feagin F. F. and Thiradilok S. (1979) Effect of magnesium and fluoride on ion exchange and acid resistance of enamel. J. oral Path. 8, 23-27. Featherstone J. D. B. and Nelson D. G. A. (1980) The effect of fluoride, zinc, strontium, magnesium and iron on the crystal structural disorder in synthetic carbonated apatites. Aust. J. Chem. 33, 2363-2368. Featherstone J. D. B., Duncan J. F. and CutressT. W. (1978) Crystallographic changes in human tooth enamel during in vitro caries simulation. Archs oral Biol. 23, 405415. Featherstone J. D. B., Mayer I., Driessens F. C. M., Verbeeck R. M. H. and Heijhgers H. J. M. (1983) Synthetic apatites containing Na, Mg and CO, and their comparison with tooth enamel mineral. Calc. Tiss. Inf. 35, 169-171.

Hallsworth A. S., Robinson C. and Weatherell J. A. (1972) Mineral and magnesium distribution within the approxima1 carious lesion of dental enamel. Caries Res. 6, 15&168.

Hiller C. R., Robinson C. and Weatherell J. A. (1975) Variations in the composition of developing rat incisor enamel. Calc. Tiss. Res. 18, I-12. Irving J. T. (1944) Fluoride-like action of various substances on the teeth. Nature 154. 149-150. Landis W. J. and Navarro’ M. (1983) Correlated physicochemical and age changes in embryonic bovine enamel. Calc. Tbs. Ini. 35, 48-55. Leblond C. P. and Warshowsky H. (1979) Dynamics of enamel formation in the rat incisor tooth. J. dent. Res. 58(B). . ,. 950-975. LeGeros R. 2. (1974) The unit-cell dimension of human enamel apatite: effect of chloride incorporation. Archs oral Biol. 20, 63-7 1,

LeGeros R. Z. (1984) Incorporation of magnesium in synthetic and in biological apatites. In: Toofh Enamel IV (Edited by Fearnhead R. W. and Suga S.) pp. 32-36. Elsevier Science, New York. LeGeros R. Z., Miravite M. A. and Curzon M. E. J. (1977) The effect of some trace elements on the lattice parameters of human and synthetic apatites. Calc. Tiss. Res. Suppl. 22, 362-367. LeGeros R. Z., Bane1 G. and LeGeros R. (1978) Types of ‘H,O’ in human enamel and in precipitated apatites. Calc. Tiss. Res. 26, 11I-1 18. McClure F. J. and McCann H. G. (1960) Dental caries and composition of bones and teeth of white rats: effect of dietary mineral supplements. Archs oral Biol. 2, 151-161, Navia J. M. (1970) Effect of minerals on dental caries. In: Dietary Chemicals us Dental Caries (Edited by Gould R. F.) pp., 123-160. American Chemical Society, Washington. D.C. Pak C. Y. C. and Diller E. C. (1969) Ionic interaction with bone mineral. V. Effect of Mg+‘, citrate’-, F- and SOion the solubility dissolution and growth of bone mineral. Calc. Tiss. Res. 4, 69-77.

Pate1 P. R. and Brown W. E. (1975) Thermodynamic solubility product of human tooth enamel: powdered sample. J. dent. Rex 54, 728-736. Robinson C., Weatherell J. A. and Hallsworth A. S. (1981) Distribution of magnesium in mature human enamel. Curies Res. 15. l&77. Robinson C., Hallsworth A. S. and Kirkham J. (1984) Distribution and uptake of magnesium by developing deciduous bovine incisor enamel. Archs oral Biol. 29, 479482.

Zuoma H. and Nuija T. (1977) Caries reduction in rats by phosphate, magnesium and fluoride additions to diet with modifications of dental calculus and calcium of the kidneys and aorta. Caries Res. 11, lW108. Zuoma A. R.. Zuoma J., Raisanen J. and Hansen H. (1983) Effect of magnesium and fluoride on the fermentative dissolution of enamel by a Streptococcal layer as measured by micro-hardness tester and a proton probe microanalysis. Caries Res. 7, 43&438.

Plate 1 Fig. 1. X-ray diffraction film of segment II from control rats. Fig. 2. X-ray diffraction film of segment II from Mg-injected rats. Fig. 3. X-ray diffraction film of segment III from control rats. Fig. 4. X-ray diffraction film of segment III from Mg-injected rats.

Magnesium

and crystallization

Plate

I

of enamel

111