Calcif. Tiss. Res. 18, 1--12 (1975) 9 by Springer-Verlag 1975
Original Papers Variations in the Composition of Developing Rat Incisor Enamel C h r i s t o p h e r R. Hiller, Colin Robinson, a n d J o h n A. W e a t h e r e l l Department of Oral Biology, School of Dentistry, University of Leeds Received August 7, accepted October 14, 1974 The developing enamel of rat incisors was dissected into a series of samples extending from the newly-formed partially-mineralised matrix to the mature enamel. Chemical analysis showed that, on a dry weight basis, the tissue achieved the composition of mature enamel well before the completion of mineral uptake. The enamel at this stage was porous and relatively soft. As more mineral was acquired, its hardness increased. Throughout the developing region, the Ca: P ratio remained fairly constant, but the CO2: P and Mg: P ratios both decreased due, apparently, to dilution by an influx of relatively CO2- and Mg-free mineral. Key words: Rat incisor - - Enamel - - Composition - - Mineralisation. Introduction
Histological studies h a v e shown t h a t e n a m e l develops in t w o stages : a p a r t i a l l y mineralised m a t r i x is first f o r m e d a n d t h e n c o n v e r t e d i n t o h a r d , m i n e r a l i s e d e n a m e l (Marsland, 1951, 1952; Crabb, 1959; Allan, 1967). Chemical a n a l y s i s has i n d i c a t e d t h a t t h e m i n e r a l i s a t i o n process involves a loss of p r o t e i n a n d w a t e r in a d d i t i o n to t h e u p t a k e of m i n e r a l (Deakins, 1942; Gloek et al., 1942; W e i n m a n n et al., 1942). T h e q u a n t i t a t i v e d e t a i l s of this process a n d t h e n a t u r e of t h e first m i n e r a l p h a s e laid d o w n are, however, largely u n k n o w n . A n a t t e m p t has b e e n m a d e t o o b t a i n q u a n t i t a t i v e i n f o r m a t i o n a b o u t t h e p a t t e r n of m i n e r a l d e p o s i t i o n in e n a m e l a n d t o d e t e r m i n e t h e v a r i a t i o n s in m i n e r a l c o m p o s i t i o n which occur d u r i n g d e v e l o p m e n t . T h e d e v e l o p i n g e n a m e l of t h e c o n t i n u o u s l y - g r o w i n g r a t incisor was dissected into a series of contiguous pieces of equal l e n g t h e x t e n d i n g from t h e growing r o o t a p e x of t h e t o o t h t o t h e m a t u r e region of enamel. B y a n a l y s i n g t h e s e pieces, q u a n t i t a t i v e d a t a has been o b t a i n e d a b o u t v a r i a t i o n s in t h e m i n e r a l t h r o u g h o u t t h e d e v e l o p i n g region of enamel. Materials and Methods
Preparation o/ Teeth. Young male Wistar rats (200-250 g) were killed with chloroform. The mandibles were removed and the lower incisors dissected out, care being taken to avoid damaging the soft developing enamel. Any adhering blood and soft tissue was carefully wiped from the enamel surface with paper tissue. On dissection of the tooth from the mandible, part of the enamel quickly dried to form an opaque white region, about 7 mm long, which contrasted sharply with the translucent enamel towards the root apex. The boundary between the translucent and the opaque enamel ('opaque boundary') was well-defined and seemed to correspond in position to the V-shaped boundary previously observed by Hiller (1971) in ground sections. In the 35 rats examined, it was
For reprints: Dr. J. A. Weatherell Department of Oral Biology, School of Dentistry, Blundell Street, Leeds, LS 1 3EU, England. 1 Calcif. Tiss. Res,, Vol. 18
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situated a t a consistent distance (6.5• mm) from the growing root apex, measuring along the mid-line of the labial surface. I n paired t e e t h from the same r a t its distance from the root apex was less variable (6.5• mm). Dissection o/ Enamel Pieces /rom Whole Teeth. B y cutting t h r o u g h t h e enamel to the underlying dentine, pieces of enamel could be separated from the tooth. About 25 contiguous samples of equal length (0.5 mm) were t h u s obtained in a sequence extending from near the root apex to the maturing enamel. The chemical changes associated with enamel development could then be assessed a t regular intervals during each developmental stage, 0.5 m m representing a b o u t 24 h growth in t o o t h length. I t was also possible in this way to determine changes in t h e total a m o u n t of CO 2, Mg, etc., occurring along the tooth. These samples encompassed a spectrum of development from the young, partially-mineralised enamel near the root apex to the older, highly-mineralised a n d almost m a t u r e enamel roughly halfway along the tooth (Fig. 1). During the dissection, the exact position of the opaque boundary was recorded. This provided a convenient reference point a n d enabled the results obtained from different teeth to be compared. Dissection o/Enamel Particles/rom Embedded Tooth Sections. To relate the results of chemical analysis to enamel volume, it was necessary to use embedded material so t h a t the volumes of samples could be determined. The t e e t h were embedded as described b y Hallsworth et al. (1972) Freshly-dissected t e e t h were snap-frozen in Freon (--30~ transferred to liquid nitrogen (--196 ~ a n d freeze-dried. The teeth were t h e n immersed in methaerylate monomer a t --48 ~ in vacuo, the v a c u u m released and the t e e t h allowed to imbibe the methaerylate a t 24 ~ for 24 h. Polymerisation was t h e n carried out a t 37 ~ for 48 h a n d the embedded t e e t h ground to produce longitudinal sections a b o u t 250 ~ m thick. Using a fine-pointed scalpel, the enamel was dissected into a series of 0.5 mm-long particles extending from the apex to the m a t u r e enamel. Determination o/the Volumes o/ Embedded Enamel Particles. The densities of methaerylateembedded particles were measured in a density gradient column (Weidmann et al., 1967), the methaerylate preventing the enamel imbibing the density-gradient fluid. After weighing each particle, its volume was calculated from the weight a n d density; reproducibility of the density measurement was < • 1.0 % S t a n d a r d Deviation. I t should be pointed out t h a t the enamel was embedded in methacrylate solely to prevent imbibition b y the density gradient fluid. The densities obtained were not therefore true tissue densities a n d were used only to calculate the external volumes of the particles. I n view of the shrinkage which occurs in methacrylate as it polymerises, tests were carried out to check t h a t the freeze-drying a n d embedding h a d not artifaetually altered the enamel volume. Teeth were removed from freshly killed rats a n d some of the enamel was dissected from one side of the tooth under saline. The tooth was then viewed from the side in order to examine and photograph the remaining enamel in vertical section a t different stages of development. I t was then freeze-dried and embedded in methaerylate as described above. Photographs of the tooth (a) in saline, (b) after freeze-drying a n d (e) when embedded in methaerylate, revealed no change in enamel thickness. Preparation o/ Enamel Samples /or Analysis. The enamel samples were weighed on a Cahn Gram electrobalance (4-0.2 ~g). The pieces dissected from the whole t o o t h weighed 20-100 ~tg; the smaller particles of enamel, dissected from the embedded sections, weighed only 5-50 ~g. All samples, except those required for carbonate determination, were ashed for 24 h a t 575 ~ to remove t h e organic m a t r i x and, in the case of the embedded material, the methacrylate polymer. The ashed enamel was t h e n reweighed. Determination o/Calcium. Ashed enamel samples were dissolved in 0.025 M perehlorie acid to give concentrations of a b o u t 8 ~g ash/ml. The calcium concentration of these solutions (about 3 ~g Ca/ml) was determined spectrophotometrically (Robinson a n d Weatherell, 1968). The reproducibility of the determination was 4-1% S . D . Determination o/Magnesium. Ashed enamel samples were dissolved in 0.1 N HC1 to give concentrations varying between 2 ~g ash/ml (samples near the root apex) a n d 20 ~g ash/ml {mature enamel). The magnesium concentrations of these solutions, which varied between 40-90 ng Mg/ml, were measured using a Pye Unicam SP90 Atomic Absorption Speetrophotometer. The effect of variations in sample concentration a n d P concentration on the determination of Mg was examined. Determinations carried out on pooled m a t u r e h u m a n enamel a t
Variations in the Composition of I)eveloping Rat Incisor Enamel
Root apex
3
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Meture enamel
Opaque boundary Fig. 1. Diagram of a rat mandibular incisor showing the area of enamel sampled for analysis and the position of the opaque boundary, which was used as a reference point
concentrations from 5 to 30 ~zg enamel/ml showed that the values obtained for Mg:P ratio (0.028 -4-7 % S.I).) were unaffected by sample concentration. Other tests showed that variations in the Mg: P ratio from 0.01-co also had no significant effect on the Mg determination ( • 5 %
s.i)). The reproducibility of the determination was ! 5 % S. I). at 30 ng Mg/ml and • 3 % S.D. at 90 ng Mg/ml. Determination o/ Carbonate. Unashed enamel samples were weighed and dissolved in perchloric acid on a haemocytometer slide. The carbonate content of the samples was determined by measuring the volume of CO2 gas liberated as described by Weatherell and Robinson (1968). The reproducibility of the determination was • 7 % S. I). Determination o/ Phosphorus. Phosphorus was determined by the method of Chen et al. (1956). Following the determination of calcium, magnesium and carbonate, aliquots of the enamel solutions were mixed with an equal volume of a solution consisting of 4 vol 0.625% ammonium molybdate in 1.5 N H2SOa and 1 vol 10% ascorbic acid. After incubating at40 ~for 1.5 h, the absorbance of the solutions was measured at 820 nm. The reproducibility of the determination was • 1% S. I).
Results Variation in Mineral Content Relative to Weight o/Enamel. T h e variations in Ca a n d P c o n c e n t r a t i o n per d r y weight of e n a m e l were measured i n teeth from 13 animals. Similar p a t t e r n s of calcium a n d phosphorus d i s t r i b u t i o n were f o u n d i n every t o o t h (Figs. 2 a a n d 2 b). Calcium c o n c e n t r a t i o n increased from a b o u t 15% n e a r t h e growing apex of the t o o t h to a b o u t 17 % a t a p o i n t 3 m m before t h e opaque b o u n d a r y . I t t h e n rose more r a p i d l y to reach a b o u t 26 % a t the opaque b o u n d a r y itself and, a t a p o i n t 3 - 4 m m b e y o n d it, reached a m a x i m u m v a l u e of a b o u t 37 % calcium. Phosphorus c o n c e n t r a t i o n followed a similar p a t t e r n , increasing from a b o u t 7 % n e a r t h e growing apex of the t o o t h to a b o u t 8 % a t a p o i n t 3 m m before t h e opaque b o u n d a r y . I t t h e n rose more r a p i d l y to reach a b o u t 13 % P a t the opaque b o u n d a r y itself and, a t a p o i n t 3 - 4 m m b e y o n d it, reached a m a x i m u m value of a b o u t 17.5% phosphorus. A l t h o u g h Ca a n d P concentrations a t t a i n e d their m a x i m u m values per weight of enamel (37% Ca a n d 17.5% P) 3 - 4 m m b e y o n d t h e opaque b o u n d a r y , the enamel a t this p o i n t was still relatively soft a n d several more samples could be cut with a scalpel before its hardness approached t h a t of m a t u r e enamel. 1.
C.R. Hiller st al.
4
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20 Phosphorus
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Fig. 2 a and b. Variation in percent Ca and P per dry weight along the developing enamel of teeth from 13 rats, showing means and standard deviations. The opaque boundary is indicated by the hatched line
Relative to Volume o/ Enamel. The variations in Ca and P concentration per unit volume of enamel (w/v) were measured in teeth from 6 animals. Calcium distribution was measured in 5 of these and phosphorus distribution measured in all 6 teeth (Fig. 3). Similar patterns of distribution were found in every tooth. Although the opaque boundary was clearly visible in air-dried teeth, it was not easily detected in freeze-dried teeth. I t was not, therefore, possible to mark the position of the opaque boundary in the freeze-dried teeth used for volume determination. Measurements carried out on air-dried pairs of incisors from 10 rats
Variations in the Composition of Developing Rat Incisor Enamel
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Fig. 3. Variation in Ca and P per volume of enamel along the developing region of teeth from 6 rats established, however, t h a t the position of the opaque boundary in adjacent teeth differed only b y ~ 0.25 ram. To determine the position of the opaque boundary in a freeze-dried tooth, the distance of the boundary from the root apex was therefore determined in an air-dried tooth and its position assumed to be the same in the adjacent (freeze-dried) tooth from the same animal. Figure 3 shows that, from the root apex to a point 2 m m before the opaque boundary, calcium concentration per unit volume of enamel appeared to rise only slightly, if at all, most of the values lying between 0.12 and 0.22 g Ca/cm s. I t then increased rather steeply to reach about 0.3 g Ca/cm 8 at the opaque boundary itself and, at a point about 7 m m beyond it, reached a m a x i m u m value of 0.9-1.0 g
Ca/cm 8. Phosphorus concentration followed a similar pattern, rising only slightly between the growing apex of the tooth and a point 2 m m before the opaque
6
C.R. Hiller et al.
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Fig. 4. Comparison of weight-related (solid lines) and volume-related (dotted lines) calcium concentration along the developing enamel. Three pairs of adjacent incisors were analysed. In one tooth from each pair, calcium was related to weight of enamel and, in the other, to volume of enamel. Pairs of teeth are identified by a common symbol. The graphs have been normalised; each result is plotted as a percentage of the total increase along the tooth
boundary, most of the values lying between 0.06 and 0.11 g P/cm 8. I t then increased to about 0.15 g P/era 8 at the opaque boundary itself, reaching a value of 0.45-0.5 g P/cm 3 at a point about 7 mm beyond it. Measurements could not be made beyond this point, since the enamel fragmented during dissection. These results indicated that the distribution pattern of Ca and P determined on a weight basis was different from that determined on a volume basis. I t was not possible to make a direct comparison between two sets of data from the same tooth, since the dry weight of embedded enamel could not be determined and the volume of the air-dried material was not known. To obtain as close a comparison as possible, pairs of adjacent incisors from three rats were analysed. I n one tooth from each pair calcium was related to enamel weight and in the other, adjacent, tooth the enamel volume. The results are presented in Fig. 4 where, to bring both sets of data to the same scale, the results of calcium per weight and calcium per volume of enamel have been expressed as a percentage of the total increase along the tooth. On a weight basis, about 65% of the increase in Ca concentration occurred before, and about 35 % after, the opaque boundary. In contrast, on a volume basis, only about 20% of the increase in calcium concentration occurred before, and about 80% after, the opaque boundary. Also, whereas calcium per
Variations in the Composition of Developing Rat Incisor Enamel
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Fig. 5. Variation in Ca:P molar ratio along the developing enamel of teeth from 21 rats, showing means and standard deviations
weight enamel reached levels characteristic of mature enamel at a point 3 m m beyond the opaque boundary, calcium per volume did not reach mature levels until 7 m m after this boundary.
Variation in Composition o/the Mineral Calcium: Phosphorus Ratio. No consistent change in Ca :P ratio along the enamel was found, although larger variations tended to occur in the younger enamel near the apex of the tooth (Fig. 5). Some variation occurred between teeth from different animals, mean values varying from 1.53 4-0.19 S.D. to 1.74 4-0.12 S.D. The mean molar calcium:phosphorus ratio of all the (385) samples of developing enamel analysed from 21 teeth was 1.60 4- 0.12 S.D. Carbonate. I n the 10 teeth examined, the total amount of CO 2 present in the 0.5 mmlong samples of enamel increased from about 0.2 ~g C02 at the root apex to about 1.0 ~g C02 in the maturing tissue. The percentage carbonate in enamel (w/w) also tended to increase slightly between the youngest tissue (about 1.4% COs) and the mature enamel (about 2.0 % CO2). Three sets of results are shown in Fig. 6. Relating these same results to the phosphorus content of the enamel, however, the CO~:P molar ratio invariably fell from about 0.16 in the youngest region to about 0.07 in the mature enamel (Fig. 7). Magnesium. I n the 6 teeth examined, the total amount of Mg present in the 0.5 mmlong samples of enamel varied from about 250-500 ng Mg, but showed no overall change between the root apex and the maturing tissue. The percentage magnesium in enamel (w/w) decreased steeply, howevel, between about 1.3% Mg in the youngest tissue to about 0.6% at about 2 m m from the root apex. After this point, the concentration of Mg was variable but seemed to decrease further to about 0.4 % Mg in the mature enamel (Fig. 8). The Mg: P molar ratio followed a somewhat similar pattern (Fig. 9), decreasing from about 0.3 in the youngest tissue to 0.1 at a point 2 m m from the root apex. After this point, M g : P ratio decreased further to reach about 0.02 in the mature enamel.
8
C.R. Hiller et al.
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Fig. 6. Variation in carbonate concentration in enamel between the root apex and mature tissue of teeth from 3 rats, expressed as percent COS per dry weight of enamel
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Fig. 7. Variation in CO~:P (molar) ratio between the root apex and mature enamel (data from the 3 teeth shown in Fig. 6)
Discussion T h e p a t t e r n of Ca a n d P d i s t r i b u t i o n t h r o u g h o u t t h e d e v e l o p i n g e n a m e l was consistent in all t e e t h , a l t h o u g h t h e r e were obvious differences b e t w e e n d a t a r e l a t e d t o d r y weight of e n a m e l a n d t h a t r e l a t e d to e n a m e l volume. On a d r y weight basis, Ca a n d P c o n c e n t r a t i o n s increased slowly from t h e r o o t a p e x u n t i l
Variations in the Composition of Developing Rat Incisor Enamel
.
9
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Fig. 8. Variation in magnesium concentration in enamel between the root apex and mature tissue of teeth from 6 rats
about 2 m m before the opaque boundary whereas, on a volume basis, they remained relatively constant. Both sets of data then rose steeply but, although the dry weight-based reached a m a x i m u m about 3 m m after the opaque boundary, the volume-based data continued to rise until, at a point 7 m m after the boundary, the enamel became too hard to cut. I n Fig. 4 this point has been taken to represent full maturation, although the possibility of some further maturation cannot be ruled out. The difference between the two sets of data in Fig. 4 was presumably due to the fact that, whereas volume-based measurements related mineral content to the total volume occupied b y mineral, matrix and water, dry weight-based measurements related mineral content only to the total weight of mineral and matrix, taking no account of any water which had evaporated prior to analysis. The difference between these two sets of data therefore reflected porosity in the tissue. The increase along the developing region in Ca or P per dry weight of enamel would be due, in part, to the established removal of organic matrix from the tissue and evaporation of the water which had replaced it. This would not affect the values of Ca and P per volume of enamel which, provided the overall volume of the tissue remained constant, would increase only if the total amount of Ca and P
110
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.
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Fig. 9. Variation in Mg:P (molar) ratio between the root apex and mature enamel (data from the 6 teeth shown in Fig. 8)
in the tissue had increased. Not surprisingly, in view of the loss of matrix, the volume-based values rose less rapidly than the weight-based values. The two curves still differed, however, even when Ca and P per weight had reached its m a x i m u m value, i.e. when most of the matrix had been withdrawn. At this point the enamel remained soft enough to cut and its dramatic change in appearance on drying suggested that, here, the tissue had contained a considerable amount of water. The subsequent increase in Ca and P per volume of enamel, accompanied b y the increasing hardness of the tissue, probably reflected the replacement of water as further mineral was acquired. This view is supported by the demonstration t h a t 3~P-labelled phosphate, taken up in vivo b y opaque (maturing) enamel was relatively difficult to remove b y back-exchange with 'cold' phosphate (Robinson et al., 1974), suggesting t h a t phosphate, and therefore presumably mineral, is built into this region of the tissue. An alternative possibility, proposed b y Starkey (1971), t h a t the maturation process involves a contraction of enamel with a consequent increase in mineral density, was also considered. To check this point, an incisor tooth was freeze-dried and embedded in methacrylate, this procedure having been shown to produce no alteration in tissue volume (see page 4). The embedded tooth was then ground
Variations in the Composition of Developing Rat Incisor Enamel
11
transversely in stages from the root apex through the developing enamel to the mature pigmented region. From photo-mierographs taken at intervals throughout the grinding process, measurements showed t h a t the enamel reached its m a x i m u m thickness near the opaque boundary. After this, although enamel thickness fluctuated (-4-5%), no systematic reduction indicative of tissue contraction was observed. Examination of the average composition of the mineral fraction along the developing enamel revealed no consistent variation in C a : P ratio. This conflicted with our earlier preliminary report t h a t the Ca :P ratio of rat incisor enamel decreased gradually as the enamel minerallsed (Hiller et al., 1971). Subsequent investigations showed t h a t in the previous analysis on unashed samples, matrix had interfered with the phosphorus determinations. The observed decrease in C02: P and Mg: P ratios along the developing enamel region is of some interest, since both CO~ and Mg have been associated with poorly-crystallised apatites (Trautz, 1960; LeGeros et al., 1972). I t seems possible, therefore, t h a t the decrease in CO~:P and M g : P ratios, from relatively high values near the root apex to relatively low values in mature enamel, might explain why the crystallinity of the mineral gradually increases as the tissue matures (I~ylen et al., 1963). The reason for this decrease in CO~:P and M g : P ratios is not fully established but, since there seemed to be no fall in the total carbonate or magnesium content of the tissue, the high ratios near the root apex are probably diluted b y relatively CO~- and Mg-free mineral acquired as the enamel matures. I n view of the effect of C02 and Mg on the properties of crystallltes, it is worthwhile to consider the implications of these findings in the resistance of enamel to caries. The first stage of caries occurs in the interprismatie regions of enamel (Darling, 1961) and seems to involve a preferential removal of CO S- and Mg-rieh material (Coolidge and Jaeobs, 1957; Hallsworth et al., 1972; Hallsworth et al., 1973). I t is therefore perhaps in the interprismatic enamel t h a t least dilution of the original CO~ and Mg concentrations occurs.
Acknowledgement. The authors wish to acknowledge the financial support of the Medical Research Council of Great Britain (Grant No: 971/447/B). References Allan, J . H . : Maturation of enamel. In: Structural and chemical organization of teeth, A. E. W. Miles, ed., p. 467-494. London-New York: Academic Press 1967 Chen, P. S., Toribara, T. Y., Warner, H. : Mierodetermination of phosphorus. Analyt. Chem. 28, 1756-1758 (1956) Coolidge, T. B., Jaeobs, M. H. : Enamel carbonate in caries. J. dent. Res. 86, 765-768 (1957) Crabb, H. S. M.: The pattern of mineralization in human dental enamel. Proc. roy. Soc. Mcd. 52, 118-122 (1959) Darling, A. I.: The selective attack of caries on the dental enamel. Ann. roy. Coll. Surg. 29, 354-369 (1961) Deakins, M. : Changes in the ash, water and organic content of pig enamel during calcification. J. dent. Res. 21, 429-435 (1942) Glock, G. E., Mellanby, H., Mellanby, M., Murray, M. M., Thewlis, J. : A study of the development of dental enamel in dogs. J. dent. Res. 21, 183-199 (1942) Hallsworth, A. S., Robinson, C., Weatherell, J.A.: Mineral and magnesium distribution within the approximal carious lesion of dental enamel. Caries Res. 6, 156-168 (1972)
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Hallsworth, A. S., Weatherell, J. A., Robinson, C. : Loss of carbonate during the first stages of enamel caries. Caries Res. 7, 345-348 (1973) Hiller, C. R. : Studies on the developing enamel of the rat incisor. Ph.D. Thesis, Leeds (1971) Hiller, C. R., Weatherell, J. A., Hallsworth, A. S. : Changes in the Ca]P ratio of developing enamel. J. dent. Res. 50, 694 (1971) LeGeros, R. Z., Shirra, W. P., LeGeros, J. P. : Amorphous calcium phosphates. J. dent. Res. 51, (Special Issue), 122 (1972) Marsland, E. A. : A histological investigation of amelogenesis in rats. I. Matrix formation. Brit. dent. J. 91, 251-261 (1951) Marsland, E. A. : A histological investigation of amelogenesis in rats. II. Maturation. Brit. dent. J. 92, 109-119 (1952) Nylen, M. U., Eanes, E. D., Omnell, K. A. : Crystal growth in rat enamel. J. Cell Biol. 18, 109-123 (1963) Robinson, C., Hiller, C. R., Weatherell, J. A.: Uptake of 82P-labelled phosphate into developing rat incisor enamel. Calcif. Tiss. Res. 15, 143-152 (1974) Robinson, C., Weatherell, J . A . : The micro-determination of calcium in mammalian hard tissues. Analyst 93, 722-728 (1968) Starkey, W. E. : Dimensional changes associated with enamel maturation in rabbits. Arch. oral Biol. 16, 479-493 (1971) Trautz, O.R.: Crystallographic studies of calcium carbonate phosphate. Ann. N.Y. Aead. Sci. 85, 145-160 (1960) Weatherell, J. A., Robinson, C. : Micro-determination of carbonate in dental enamel. Analyst 93, 244-248 (1968) Weidmann, S. M., Weatherell, J. A., Hamm, Stella M. : Variations of enamel density in sections of human teeth. Arch. oral Biol. 12, 85-97 (1967) Wehunann, J . P . , Wessinger, G.D., Reed, G.: Correlation of chemical and histological investigations on developing enamel. J. dent. Res. 21, 171-182 (1942)
Original Papers Variations in the Composition of Developing Rat Incisor Enamel C h r i s t o p h e r R. Hiller, Colin Robinson, a n d J o h n A. W e a t h e r e l l Department of Oral Biology, School of Dentistry, University of Leeds Received August 7, accepted October 14, 1974 The developing enamel of rat incisors was dissected into a series of samples extending from the newly-formed partially-mineralised matrix to the mature enamel. Chemical analysis showed that, on a dry weight basis, the tissue achieved the composition of mature enamel well before the completion of mineral uptake. The enamel at this stage was porous and relatively soft. As more mineral was acquired, its hardness increased. Throughout the developing region, the Ca: P ratio remained fairly constant, but the CO2: P and Mg: P ratios both decreased due, apparently, to dilution by an influx of relatively CO2- and Mg-free mineral. Key words: Rat incisor - - Enamel - - Composition - - Mineralisation. Introduction
Histological studies h a v e shown t h a t e n a m e l develops in t w o stages : a p a r t i a l l y mineralised m a t r i x is first f o r m e d a n d t h e n c o n v e r t e d i n t o h a r d , m i n e r a l i s e d e n a m e l (Marsland, 1951, 1952; Crabb, 1959; Allan, 1967). Chemical a n a l y s i s has i n d i c a t e d t h a t t h e m i n e r a l i s a t i o n process involves a loss of p r o t e i n a n d w a t e r in a d d i t i o n to t h e u p t a k e of m i n e r a l (Deakins, 1942; Gloek et al., 1942; W e i n m a n n et al., 1942). T h e q u a n t i t a t i v e d e t a i l s of this process a n d t h e n a t u r e of t h e first m i n e r a l p h a s e laid d o w n are, however, largely u n k n o w n . A n a t t e m p t has b e e n m a d e t o o b t a i n q u a n t i t a t i v e i n f o r m a t i o n a b o u t t h e p a t t e r n of m i n e r a l d e p o s i t i o n in e n a m e l a n d t o d e t e r m i n e t h e v a r i a t i o n s in m i n e r a l c o m p o s i t i o n which occur d u r i n g d e v e l o p m e n t . T h e d e v e l o p i n g e n a m e l of t h e c o n t i n u o u s l y - g r o w i n g r a t incisor was dissected into a series of contiguous pieces of equal l e n g t h e x t e n d i n g from t h e growing r o o t a p e x of t h e t o o t h t o t h e m a t u r e region of enamel. B y a n a l y s i n g t h e s e pieces, q u a n t i t a t i v e d a t a has been o b t a i n e d a b o u t v a r i a t i o n s in t h e m i n e r a l t h r o u g h o u t t h e d e v e l o p i n g region of enamel. Materials and Methods
Preparation o/ Teeth. Young male Wistar rats (200-250 g) were killed with chloroform. The mandibles were removed and the lower incisors dissected out, care being taken to avoid damaging the soft developing enamel. Any adhering blood and soft tissue was carefully wiped from the enamel surface with paper tissue. On dissection of the tooth from the mandible, part of the enamel quickly dried to form an opaque white region, about 7 mm long, which contrasted sharply with the translucent enamel towards the root apex. The boundary between the translucent and the opaque enamel ('opaque boundary') was well-defined and seemed to correspond in position to the V-shaped boundary previously observed by Hiller (1971) in ground sections. In the 35 rats examined, it was
For reprints: Dr. J. A. Weatherell Department of Oral Biology, School of Dentistry, Blundell Street, Leeds, LS 1 3EU, England. 1 Calcif. Tiss. Res,, Vol. 18
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situated a t a consistent distance (6.5• mm) from the growing root apex, measuring along the mid-line of the labial surface. I n paired t e e t h from the same r a t its distance from the root apex was less variable (6.5• mm). Dissection o/ Enamel Pieces /rom Whole Teeth. B y cutting t h r o u g h t h e enamel to the underlying dentine, pieces of enamel could be separated from the tooth. About 25 contiguous samples of equal length (0.5 mm) were t h u s obtained in a sequence extending from near the root apex to the maturing enamel. The chemical changes associated with enamel development could then be assessed a t regular intervals during each developmental stage, 0.5 m m representing a b o u t 24 h growth in t o o t h length. I t was also possible in this way to determine changes in t h e total a m o u n t of CO 2, Mg, etc., occurring along the tooth. These samples encompassed a spectrum of development from the young, partially-mineralised enamel near the root apex to the older, highly-mineralised a n d almost m a t u r e enamel roughly halfway along the tooth (Fig. 1). During the dissection, the exact position of the opaque boundary was recorded. This provided a convenient reference point a n d enabled the results obtained from different teeth to be compared. Dissection o/Enamel Particles/rom Embedded Tooth Sections. To relate the results of chemical analysis to enamel volume, it was necessary to use embedded material so t h a t the volumes of samples could be determined. The t e e t h were embedded as described b y Hallsworth et al. (1972) Freshly-dissected t e e t h were snap-frozen in Freon (--30~ transferred to liquid nitrogen (--196 ~ a n d freeze-dried. The teeth were t h e n immersed in methaerylate monomer a t --48 ~ in vacuo, the v a c u u m released and the t e e t h allowed to imbibe the methaerylate a t 24 ~ for 24 h. Polymerisation was t h e n carried out a t 37 ~ for 48 h a n d the embedded t e e t h ground to produce longitudinal sections a b o u t 250 ~ m thick. Using a fine-pointed scalpel, the enamel was dissected into a series of 0.5 mm-long particles extending from the apex to the m a t u r e enamel. Determination o/the Volumes o/ Embedded Enamel Particles. The densities of methaerylateembedded particles were measured in a density gradient column (Weidmann et al., 1967), the methaerylate preventing the enamel imbibing the density-gradient fluid. After weighing each particle, its volume was calculated from the weight a n d density; reproducibility of the density measurement was < • 1.0 % S t a n d a r d Deviation. I t should be pointed out t h a t the enamel was embedded in methacrylate solely to prevent imbibition b y the density gradient fluid. The densities obtained were not therefore true tissue densities a n d were used only to calculate the external volumes of the particles. I n view of the shrinkage which occurs in methacrylate as it polymerises, tests were carried out to check t h a t the freeze-drying a n d embedding h a d not artifaetually altered the enamel volume. Teeth were removed from freshly killed rats a n d some of the enamel was dissected from one side of the tooth under saline. The tooth was then viewed from the side in order to examine and photograph the remaining enamel in vertical section a t different stages of development. I t was then freeze-dried and embedded in methaerylate as described above. Photographs of the tooth (a) in saline, (b) after freeze-drying a n d (e) when embedded in methaerylate, revealed no change in enamel thickness. Preparation o/ Enamel Samples /or Analysis. The enamel samples were weighed on a Cahn Gram electrobalance (4-0.2 ~g). The pieces dissected from the whole t o o t h weighed 20-100 ~tg; the smaller particles of enamel, dissected from the embedded sections, weighed only 5-50 ~g. All samples, except those required for carbonate determination, were ashed for 24 h a t 575 ~ to remove t h e organic m a t r i x and, in the case of the embedded material, the methacrylate polymer. The ashed enamel was t h e n reweighed. Determination o/Calcium. Ashed enamel samples were dissolved in 0.025 M perehlorie acid to give concentrations of a b o u t 8 ~g ash/ml. The calcium concentration of these solutions (about 3 ~g Ca/ml) was determined spectrophotometrically (Robinson a n d Weatherell, 1968). The reproducibility of the determination was 4-1% S . D . Determination o/Magnesium. Ashed enamel samples were dissolved in 0.1 N HC1 to give concentrations varying between 2 ~g ash/ml (samples near the root apex) a n d 20 ~g ash/ml {mature enamel). The magnesium concentrations of these solutions, which varied between 40-90 ng Mg/ml, were measured using a Pye Unicam SP90 Atomic Absorption Speetrophotometer. The effect of variations in sample concentration a n d P concentration on the determination of Mg was examined. Determinations carried out on pooled m a t u r e h u m a n enamel a t
Variations in the Composition of I)eveloping Rat Incisor Enamel
Root apex
3
Tip
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Wh~e22e~qU e
Meture enamel
Opaque boundary Fig. 1. Diagram of a rat mandibular incisor showing the area of enamel sampled for analysis and the position of the opaque boundary, which was used as a reference point
concentrations from 5 to 30 ~zg enamel/ml showed that the values obtained for Mg:P ratio (0.028 -4-7 % S.I).) were unaffected by sample concentration. Other tests showed that variations in the Mg: P ratio from 0.01-co also had no significant effect on the Mg determination ( • 5 %
s.i)). The reproducibility of the determination was ! 5 % S. I). at 30 ng Mg/ml and • 3 % S.D. at 90 ng Mg/ml. Determination o/ Carbonate. Unashed enamel samples were weighed and dissolved in perchloric acid on a haemocytometer slide. The carbonate content of the samples was determined by measuring the volume of CO2 gas liberated as described by Weatherell and Robinson (1968). The reproducibility of the determination was • 7 % S. I). Determination o/ Phosphorus. Phosphorus was determined by the method of Chen et al. (1956). Following the determination of calcium, magnesium and carbonate, aliquots of the enamel solutions were mixed with an equal volume of a solution consisting of 4 vol 0.625% ammonium molybdate in 1.5 N H2SOa and 1 vol 10% ascorbic acid. After incubating at40 ~for 1.5 h, the absorbance of the solutions was measured at 820 nm. The reproducibility of the determination was • 1% S. I).
Results Variation in Mineral Content Relative to Weight o/Enamel. T h e variations in Ca a n d P c o n c e n t r a t i o n per d r y weight of e n a m e l were measured i n teeth from 13 animals. Similar p a t t e r n s of calcium a n d phosphorus d i s t r i b u t i o n were f o u n d i n every t o o t h (Figs. 2 a a n d 2 b). Calcium c o n c e n t r a t i o n increased from a b o u t 15% n e a r t h e growing apex of the t o o t h to a b o u t 17 % a t a p o i n t 3 m m before t h e opaque b o u n d a r y . I t t h e n rose more r a p i d l y to reach a b o u t 26 % a t the opaque b o u n d a r y itself and, a t a p o i n t 3 - 4 m m b e y o n d it, reached a m a x i m u m v a l u e of a b o u t 37 % calcium. Phosphorus c o n c e n t r a t i o n followed a similar p a t t e r n , increasing from a b o u t 7 % n e a r t h e growing apex of the t o o t h to a b o u t 8 % a t a p o i n t 3 m m before t h e opaque b o u n d a r y . I t t h e n rose more r a p i d l y to reach a b o u t 13 % P a t the opaque b o u n d a r y itself and, a t a p o i n t 3 - 4 m m b e y o n d it, reached a m a x i m u m value of a b o u t 17.5% phosphorus. A l t h o u g h Ca a n d P concentrations a t t a i n e d their m a x i m u m values per weight of enamel (37% Ca a n d 17.5% P) 3 - 4 m m b e y o n d t h e opaque b o u n d a r y , the enamel a t this p o i n t was still relatively soft a n d several more samples could be cut with a scalpel before its hardness approached t h a t of m a t u r e enamel. 1.
C.R. Hiller st al.
4
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Calcium
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20 Phosphorus
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Fig. 2 a and b. Variation in percent Ca and P per dry weight along the developing enamel of teeth from 13 rats, showing means and standard deviations. The opaque boundary is indicated by the hatched line
Relative to Volume o/ Enamel. The variations in Ca and P concentration per unit volume of enamel (w/v) were measured in teeth from 6 animals. Calcium distribution was measured in 5 of these and phosphorus distribution measured in all 6 teeth (Fig. 3). Similar patterns of distribution were found in every tooth. Although the opaque boundary was clearly visible in air-dried teeth, it was not easily detected in freeze-dried teeth. I t was not, therefore, possible to mark the position of the opaque boundary in the freeze-dried teeth used for volume determination. Measurements carried out on air-dried pairs of incisors from 10 rats
Variations in the Composition of Developing Rat Incisor Enamel
5
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Fig. 3. Variation in Ca and P per volume of enamel along the developing region of teeth from 6 rats established, however, t h a t the position of the opaque boundary in adjacent teeth differed only b y ~ 0.25 ram. To determine the position of the opaque boundary in a freeze-dried tooth, the distance of the boundary from the root apex was therefore determined in an air-dried tooth and its position assumed to be the same in the adjacent (freeze-dried) tooth from the same animal. Figure 3 shows that, from the root apex to a point 2 m m before the opaque boundary, calcium concentration per unit volume of enamel appeared to rise only slightly, if at all, most of the values lying between 0.12 and 0.22 g Ca/cm s. I t then increased rather steeply to reach about 0.3 g Ca/cm 8 at the opaque boundary itself and, at a point about 7 m m beyond it, reached a m a x i m u m value of 0.9-1.0 g
Ca/cm 8. Phosphorus concentration followed a similar pattern, rising only slightly between the growing apex of the tooth and a point 2 m m before the opaque
6
C.R. Hiller et al.
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Fig. 4. Comparison of weight-related (solid lines) and volume-related (dotted lines) calcium concentration along the developing enamel. Three pairs of adjacent incisors were analysed. In one tooth from each pair, calcium was related to weight of enamel and, in the other, to volume of enamel. Pairs of teeth are identified by a common symbol. The graphs have been normalised; each result is plotted as a percentage of the total increase along the tooth
boundary, most of the values lying between 0.06 and 0.11 g P/cm 8. I t then increased to about 0.15 g P/era 8 at the opaque boundary itself, reaching a value of 0.45-0.5 g P/cm 3 at a point about 7 mm beyond it. Measurements could not be made beyond this point, since the enamel fragmented during dissection. These results indicated that the distribution pattern of Ca and P determined on a weight basis was different from that determined on a volume basis. I t was not possible to make a direct comparison between two sets of data from the same tooth, since the dry weight of embedded enamel could not be determined and the volume of the air-dried material was not known. To obtain as close a comparison as possible, pairs of adjacent incisors from three rats were analysed. I n one tooth from each pair calcium was related to enamel weight and in the other, adjacent, tooth the enamel volume. The results are presented in Fig. 4 where, to bring both sets of data to the same scale, the results of calcium per weight and calcium per volume of enamel have been expressed as a percentage of the total increase along the tooth. On a weight basis, about 65% of the increase in Ca concentration occurred before, and about 35 % after, the opaque boundary. In contrast, on a volume basis, only about 20% of the increase in calcium concentration occurred before, and about 80% after, the opaque boundary. Also, whereas calcium per
Variations in the Composition of Developing Rat Incisor Enamel
7
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E
1"8f I
IIIII II
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1.6
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Fig. 5. Variation in Ca:P molar ratio along the developing enamel of teeth from 21 rats, showing means and standard deviations
weight enamel reached levels characteristic of mature enamel at a point 3 m m beyond the opaque boundary, calcium per volume did not reach mature levels until 7 m m after this boundary.
Variation in Composition o/the Mineral Calcium: Phosphorus Ratio. No consistent change in Ca :P ratio along the enamel was found, although larger variations tended to occur in the younger enamel near the apex of the tooth (Fig. 5). Some variation occurred between teeth from different animals, mean values varying from 1.53 4-0.19 S.D. to 1.74 4-0.12 S.D. The mean molar calcium:phosphorus ratio of all the (385) samples of developing enamel analysed from 21 teeth was 1.60 4- 0.12 S.D. Carbonate. I n the 10 teeth examined, the total amount of CO 2 present in the 0.5 mmlong samples of enamel increased from about 0.2 ~g C02 at the root apex to about 1.0 ~g C02 in the maturing tissue. The percentage carbonate in enamel (w/w) also tended to increase slightly between the youngest tissue (about 1.4% COs) and the mature enamel (about 2.0 % CO2). Three sets of results are shown in Fig. 6. Relating these same results to the phosphorus content of the enamel, however, the CO~:P molar ratio invariably fell from about 0.16 in the youngest region to about 0.07 in the mature enamel (Fig. 7). Magnesium. I n the 6 teeth examined, the total amount of Mg present in the 0.5 mmlong samples of enamel varied from about 250-500 ng Mg, but showed no overall change between the root apex and the maturing tissue. The percentage magnesium in enamel (w/w) decreased steeply, howevel, between about 1.3% Mg in the youngest tissue to about 0.6% at about 2 m m from the root apex. After this point, the concentration of Mg was variable but seemed to decrease further to about 0.4 % Mg in the mature enamel (Fig. 8). The Mg: P molar ratio followed a somewhat similar pattern (Fig. 9), decreasing from about 0.3 in the youngest tissue to 0.1 at a point 2 m m from the root apex. After this point, M g : P ratio decreased further to reach about 0.02 in the mature enamel.
8
C.R. Hiller et al.
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Fig. 6. Variation in carbonate concentration in enamel between the root apex and mature tissue of teeth from 3 rats, expressed as percent COS per dry weight of enamel
0.25
0.20 "E E 0.15 O_ cq o CO
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.
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Fig. 7. Variation in CO~:P (molar) ratio between the root apex and mature enamel (data from the 3 teeth shown in Fig. 6)
Discussion T h e p a t t e r n of Ca a n d P d i s t r i b u t i o n t h r o u g h o u t t h e d e v e l o p i n g e n a m e l was consistent in all t e e t h , a l t h o u g h t h e r e were obvious differences b e t w e e n d a t a r e l a t e d t o d r y weight of e n a m e l a n d t h a t r e l a t e d to e n a m e l volume. On a d r y weight basis, Ca a n d P c o n c e n t r a t i o n s increased slowly from t h e r o o t a p e x u n t i l
Variations in the Composition of Developing Rat Incisor Enamel
.
9
!
0
I i --i I I I mrn
Fig. 8. Variation in magnesium concentration in enamel between the root apex and mature tissue of teeth from 6 rats
about 2 m m before the opaque boundary whereas, on a volume basis, they remained relatively constant. Both sets of data then rose steeply but, although the dry weight-based reached a m a x i m u m about 3 m m after the opaque boundary, the volume-based data continued to rise until, at a point 7 m m after the boundary, the enamel became too hard to cut. I n Fig. 4 this point has been taken to represent full maturation, although the possibility of some further maturation cannot be ruled out. The difference between the two sets of data in Fig. 4 was presumably due to the fact that, whereas volume-based measurements related mineral content to the total volume occupied b y mineral, matrix and water, dry weight-based measurements related mineral content only to the total weight of mineral and matrix, taking no account of any water which had evaporated prior to analysis. The difference between these two sets of data therefore reflected porosity in the tissue. The increase along the developing region in Ca or P per dry weight of enamel would be due, in part, to the established removal of organic matrix from the tissue and evaporation of the water which had replaced it. This would not affect the values of Ca and P per volume of enamel which, provided the overall volume of the tissue remained constant, would increase only if the total amount of Ca and P
110
C. R . H i l l e r et
al.
0.3
-~ 0.2 (X_
0.1 ~
0!
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.
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Fig. 9. Variation in Mg:P (molar) ratio between the root apex and mature enamel (data from the 6 teeth shown in Fig. 8)
in the tissue had increased. Not surprisingly, in view of the loss of matrix, the volume-based values rose less rapidly than the weight-based values. The two curves still differed, however, even when Ca and P per weight had reached its m a x i m u m value, i.e. when most of the matrix had been withdrawn. At this point the enamel remained soft enough to cut and its dramatic change in appearance on drying suggested that, here, the tissue had contained a considerable amount of water. The subsequent increase in Ca and P per volume of enamel, accompanied b y the increasing hardness of the tissue, probably reflected the replacement of water as further mineral was acquired. This view is supported by the demonstration t h a t 3~P-labelled phosphate, taken up in vivo b y opaque (maturing) enamel was relatively difficult to remove b y back-exchange with 'cold' phosphate (Robinson et al., 1974), suggesting t h a t phosphate, and therefore presumably mineral, is built into this region of the tissue. An alternative possibility, proposed b y Starkey (1971), t h a t the maturation process involves a contraction of enamel with a consequent increase in mineral density, was also considered. To check this point, an incisor tooth was freeze-dried and embedded in methacrylate, this procedure having been shown to produce no alteration in tissue volume (see page 4). The embedded tooth was then ground
Variations in the Composition of Developing Rat Incisor Enamel
11
transversely in stages from the root apex through the developing enamel to the mature pigmented region. From photo-mierographs taken at intervals throughout the grinding process, measurements showed t h a t the enamel reached its m a x i m u m thickness near the opaque boundary. After this, although enamel thickness fluctuated (-4-5%), no systematic reduction indicative of tissue contraction was observed. Examination of the average composition of the mineral fraction along the developing enamel revealed no consistent variation in C a : P ratio. This conflicted with our earlier preliminary report t h a t the Ca :P ratio of rat incisor enamel decreased gradually as the enamel minerallsed (Hiller et al., 1971). Subsequent investigations showed t h a t in the previous analysis on unashed samples, matrix had interfered with the phosphorus determinations. The observed decrease in C02: P and Mg: P ratios along the developing enamel region is of some interest, since both CO~ and Mg have been associated with poorly-crystallised apatites (Trautz, 1960; LeGeros et al., 1972). I t seems possible, therefore, t h a t the decrease in CO~:P and M g : P ratios, from relatively high values near the root apex to relatively low values in mature enamel, might explain why the crystallinity of the mineral gradually increases as the tissue matures (I~ylen et al., 1963). The reason for this decrease in CO~:P and M g : P ratios is not fully established but, since there seemed to be no fall in the total carbonate or magnesium content of the tissue, the high ratios near the root apex are probably diluted b y relatively CO~- and Mg-free mineral acquired as the enamel matures. I n view of the effect of C02 and Mg on the properties of crystallltes, it is worthwhile to consider the implications of these findings in the resistance of enamel to caries. The first stage of caries occurs in the interprismatie regions of enamel (Darling, 1961) and seems to involve a preferential removal of CO S- and Mg-rieh material (Coolidge and Jaeobs, 1957; Hallsworth et al., 1972; Hallsworth et al., 1973). I t is therefore perhaps in the interprismatic enamel t h a t least dilution of the original CO~ and Mg concentrations occurs.
Acknowledgement. The authors wish to acknowledge the financial support of the Medical Research Council of Great Britain (Grant No: 971/447/B). References Allan, J . H . : Maturation of enamel. In: Structural and chemical organization of teeth, A. E. W. Miles, ed., p. 467-494. London-New York: Academic Press 1967 Chen, P. S., Toribara, T. Y., Warner, H. : Mierodetermination of phosphorus. Analyt. Chem. 28, 1756-1758 (1956) Coolidge, T. B., Jaeobs, M. H. : Enamel carbonate in caries. J. dent. Res. 86, 765-768 (1957) Crabb, H. S. M.: The pattern of mineralization in human dental enamel. Proc. roy. Soc. Mcd. 52, 118-122 (1959) Darling, A. I.: The selective attack of caries on the dental enamel. Ann. roy. Coll. Surg. 29, 354-369 (1961) Deakins, M. : Changes in the ash, water and organic content of pig enamel during calcification. J. dent. Res. 21, 429-435 (1942) Glock, G. E., Mellanby, H., Mellanby, M., Murray, M. M., Thewlis, J. : A study of the development of dental enamel in dogs. J. dent. Res. 21, 183-199 (1942) Hallsworth, A. S., Robinson, C., Weatherell, J.A.: Mineral and magnesium distribution within the approximal carious lesion of dental enamel. Caries Res. 6, 156-168 (1972)
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Hallsworth, A. S., Weatherell, J. A., Robinson, C. : Loss of carbonate during the first stages of enamel caries. Caries Res. 7, 345-348 (1973) Hiller, C. R. : Studies on the developing enamel of the rat incisor. Ph.D. Thesis, Leeds (1971) Hiller, C. R., Weatherell, J. A., Hallsworth, A. S. : Changes in the Ca]P ratio of developing enamel. J. dent. Res. 50, 694 (1971) LeGeros, R. Z., Shirra, W. P., LeGeros, J. P. : Amorphous calcium phosphates. J. dent. Res. 51, (Special Issue), 122 (1972) Marsland, E. A. : A histological investigation of amelogenesis in rats. I. Matrix formation. Brit. dent. J. 91, 251-261 (1951) Marsland, E. A. : A histological investigation of amelogenesis in rats. II. Maturation. Brit. dent. J. 92, 109-119 (1952) Nylen, M. U., Eanes, E. D., Omnell, K. A. : Crystal growth in rat enamel. J. Cell Biol. 18, 109-123 (1963) Robinson, C., Hiller, C. R., Weatherell, J. A.: Uptake of 82P-labelled phosphate into developing rat incisor enamel. Calcif. Tiss. Res. 15, 143-152 (1974) Robinson, C., Weatherell, J . A . : The micro-determination of calcium in mammalian hard tissues. Analyst 93, 722-728 (1968) Starkey, W. E. : Dimensional changes associated with enamel maturation in rabbits. Arch. oral Biol. 16, 479-493 (1971) Trautz, O.R.: Crystallographic studies of calcium carbonate phosphate. Ann. N.Y. Aead. Sci. 85, 145-160 (1960) Weatherell, J. A., Robinson, C. : Micro-determination of carbonate in dental enamel. Analyst 93, 244-248 (1968) Weidmann, S. M., Weatherell, J. A., Hamm, Stella M. : Variations of enamel density in sections of human teeth. Arch. oral Biol. 12, 85-97 (1967) Wehunann, J . P . , Wessinger, G.D., Reed, G.: Correlation of chemical and histological investigations on developing enamel. J. dent. Res. 21, 171-182 (1942)
