Carbonate Content in Developing Human and Bovine Enamel M. SYDNEY-ZAX" 2, I. MAYER3, and D. DEUTSCH1"4 'Dental Research Unit, Hebrew University-Hadassah Faculty of Dental Medicine, Ein Karem, Jerusalem, Israel; and 3Department of Inorganic and Analytical Chemistry, Hebrew University, Givat Ram, Jerusalem, Israel

The present study describes the detailed changes in carbonate distribution throughout the different stages of development in human and bovine enamel, from early formation to maturation. Twenty-two human maxillary and mandibular deciduous anterior teeth and 46 bovine teeth were studied. The major mineral ions, calcium and phosphorus, were also analyzed to facilitate comparison of carbonate content with progressing mineralization. The results showed that as enamel matured and mineral concentration increased, carbonate concentration decreased. The observed decrease in percent carbonate per weight enamel mineral may be due to dilution by an influx of relatively carbonate-free mineral, and could, in part, explain the observed increase in crystallinity of enamel mineral as the tissue matures. J Dent Res 70(5):913-916, May, 1991

Introduction. Histological, microradiographic, and biochemical studies have shown that enamel development in the human tooth (Eastoe and Camilleri, 1971; Deutsch et al., 1984; Deutsch and Shapira, 1987; Deutsch, 1989) occurs, as in animal teeth (Deakins, 1942; Hiller et al., 1975; Deutsch et al., 1979; LeBlond and Warshawsky, 1979), in at least three distinct stages: forming, maturing, and mature. In the forming stage, the mineral ions (calcium and phosphorus) are present at relatively low concentrations in an organic matrix that is rich in proteins and water. During the maturation phase, most of the qgt'anic matrix disappears from the tissue, which thus becomes porous and hydrated. This porosity is subsequently occluded by further uptake of mineral ions until the tissue finally becomes non-porous, hard, mature enamel (Deakins, 1942; Hiller et al., 1975; Deutsch and Shapira, 1987). Mature enamel contains approximately 96% mineral by tissue weight (85% by volume); the remainder is composed of organic material and water. The principal inorganic constituent of enamel is hydroxyapatite, Ca10(PO4)6(OH)2, which tends to incorporate a large number of trace elements (Weatherell and Robinson, 1973; LeGeros, 1981). One constituent that is incorporated into developing enamel is carbonate. The incorporation of carbonate is known to affect both the physical structure as well as the chemical stability of dental enamel. As early as 1949, Hardwick coined carbonate as the Achilles' heel of dental enamel, because enamel rich in carbonate was found to be particularly vulnerable to acid attack. More recent studies by Hallsworth et al. (1972) have shown that enamel rich in carbonate and magnesium preferReceived for publicaton March 28, 1990 Accepted for publication December 18, 1990 2Present address: Department of Anatomy, Cornell University, New York State College of Veterinary Medicine, Ithaca, New York 14853-

entially dissolves in the early caries lesion. A high carbonateto-phosphate ratio has also been associated with teeth particularly susceptible to acid attack (Sobel, 1962). Structurally, carbonate may replace either the phosphate or the hydroxyl group in the hydroxyapatite lattice (LeGeros, 1981). In A-type substitution, carbonate replaces hydroxyl and causes expansion of the a-axis and contraction of the c-axis; in Btype substitution, carbonate replaces phosphate and causes contraction in the a-axis and expansion in the c-axis. While most of the carbonate in enamel is believed to be of the B type, Elliott et al. (1985) have reported that 11 ± 1% is of the A type. In addition, some of the carbonate may be intercrystalline. Most recently, Beshah et al. (1990) identified a third site for carbonate in enamel mineral and postulated that the location may be on the surfaces of the crystals or in perturbed areas inside the crystals. Carbonated hydroxyapatite is more acid-labile than either calcium hydroxyapatite or fluorapatite (LeGeros, 1981; LeGeros and Tung, 1983). Carbonate is abundant in the body, and its presence in the mineral phase may depend on the metabolic activity of the cells near the forming crystallites (Weatherell and Robinson, 1973). In fact, the decrease in carbonate content toward the enamel surface may reflect the decreased metabolic activity of the secretary ameloblasts as they approach the enamel surface (Weatherell et al., 1974). Despite the vast information on the effect of carbonate on mature enamel and synthetic calcium hydroxyapatite (Hallsworth et al., 1973; LeGeros, 1981), only limited information is available concerning the concentration and distribution of carbonate during enamel development (Hiller et al., 1975; Landis and Navarro, 1983) with no previous study of carbonate content in developing human enamel. In view of carbonate's profound effects on both the physical structure and chemical stability of dental enamel, the present study was undertaken to determine the distribution and concentration of carbonate throughout the different stages of enamel development (forming, maturing, and mature) in a series of human and bovine teeth.

Materials and methods. Since human and bovine teeth are of limited growth, no single tooth contains the full spectrum of mineralization. The stage of development depends on tooth age and tooth type (Robinson et al., 1978; Deutsch et al., 1984). To characterize the distribution and concentration of carbonate in developing human enamel throughout all the stages of development, a series of 22 teeth representing stages from early formation to maturation was selected. The 46 bovine teeth included forming and maturing enamel and, in addition, mature unerupted and erupted enamel. The teeth were dissected from the jaws under a stereo dissecting microscope. Tissue paper was used to clean the teeth carefully of adhering soft tissues and blood. Special care was taken to avoid any damage to the developing enamel.

The teeth were then air-dried and stored. The different stages of enamel development were determined by inspection of the teeth under ultraviolet light (RobDownloaded from jdr.sagepub.com at UNIV OF MICHIGAN on June 24, 2015 For personal use only. No other uses without permission. 913

6401 4To whom reprint requests should be addresssed Supported by NIH Research Grant RO1 DE05780

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Fig. 1-Changes in carbonate distribution and concentration in labial and lingual enamel through the different stages of development (forming, maturing, and mature) in a series of human teeth of different ages. Sampling distance from the cervical margin of each tooth is indicated (mm). Reference points that enabled the results from different teeth to be compared were either the border between forming and maturing enamel or the border between maturing and mature enamel in the respective teeth.

inson et al., 1978; Deutsch and Shapira, 1987). Following identification of the various areas of development in the teeth studied, the borders between the various stages were scored with a scalpel. In the study of bovine mature enamel, the border between erupted and unerupted enamel was delineated at the time of tooth dissection from fresh bovine mandibles. Similarly to previous studies on human teeth (Deutsch and Gedalia, 1980; Deutsch and Pe'er, 1982; Deutsch et al., 1984), enamel was dissected into a contiguous series of enamel particles along the tooth from the oldest enamel at the incisal edge to the youngest enamel at the cervical margin, in the plane of the tooth's long central axis. For human teeth, both labial and lingual enamel samples were dissected in this manner. The enamel particles were then weighed in a Cahn 25 electrobalance. Enamel particles weighed between 0.2 and 2.0 mg, and each was analyzed individually. Determination of carbonate content was performed by the infrared method of Featherstone et al. (1984). In this method, the ratio of the extinction coefficient of the IR carbonate band at about 1415 cm-' to the extinction coefficient of the phosphate band at about 575 cm-' is linearly related to the carbonate content of the carbonated apatite. This relationship was determined by use of specially prepared carbonated apatite samples of known carbonate content, and whose carbonate content was independently determined directly by gas chromatography (Nelson and Featherstone, 1982). This method facilitates carbonate estimation to a better than 10% accuracy in the range of 1-12% wt/wt. The calibration curve used to determine carbonate content in the enamel particles studied was that of Featherstone et al. (1984). Synthetic carbonated apatite standards prepared in our laboratory that contained 1.2, 3.0, and 11.2% carbonate, respectively (Mayer et al., 1985), were used for verification of the accuracy of the carbonate determinations. Each enamel particle was combined with 120 mg of spectroscopic grade KBr (Sigma Chemical Co., St. Louis, MO), which serves as an inert matrix in infrared determinations. The samples were each ground in an agate mortar and pestle and then pressed under vacuum in a Perkin-Elmer die, under a 9000-kg load, into a 13-mm-diameter pellet. Infrared spectra

were obtained from 200-4000 cm-' on an infrared spectrophotometer (Model 597, Perkin-Elmer, Norwalk, CT). The concentration and distribution of calcium and phosphorus were also determined throughout the different stages of development to facilitate direct comparison of carbonate content with mineral content. For removal of the organic matrix, the enamel samples were ashed in platinum boats at 575TC for 24 h. The calcium content of the ash was determined by atomic absorption spectrophotometiy (Perkin-Elmer Model 403 atomic absorption spectrophotometer) (accuracy + 5%), and phosphorus content was analyzed according to the method of Chen et al. (1956) (accuracy + 1%). Calcium and phosphorus contents were expressed on a tissue-weight basis. Statistical analysis of the data was carried out by the F test for inequality of variance and Student's t test.

Results. The average carbonate content in the labial-forming human enamel was 5.18 ± 0.39% per weight enamel mineral (n = 13). In the maturing enamel, carbonate content fell to 3.80 ± 0.91% per weight enamel mineral (n = 12). This difference between forming and maturing enamel was statistically significant at the 99.5% confidence interval. Finally, in the mature enamel, carbonate content fell even further, to 3.04 + 0.39% per weight enamel mineral (n = 10). The decrease in carbonate content in mature, as compared with maturing, enamel was statistically significant at the 99.0% confidence interval. In the lingual-forming human enamel, carbonate content averaged 5.37 + 0.34% per weight enamel mineral (n = 13). Maturing lingual enamel contained 3.93 + 0.75% carbonate per weight enamel mineral (n = 11), significantly less than that in the forming enamel at the 99.5% confidence interval. Mature lingual enamel contained 3.46 ± 0.56% carbonateper weight enamel mineral (n = 9), significantly less than that in the maturing enamel at the 99.0% confidence interval. For the developing bovine teeth, forming enamel averaged 5.46 ± 0.62% carbonate per weight enamel mineral (n = 35), while the maturing averaged 3.78 + 0.60% carbonateper weight enamel mineral (n = 16). This decrease in carbonate

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CARBONATE CONTENT IN DEVELOPING ENAMEL

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Fig. 2-Changes in carbonate, calcium, and phosphorus concentrations, and Ca/P ratio of human enamel through the different stages of development (forming, maturing, and mature). Sampling distance from the cervical margin of each tooth is indicated (mm). Reference points that enabled the results from different teeth to be compared were either the border between forming and maturing enamel or the border between maturing and mature enamel ,in the respective teeth. content from forming to maturing stages was significant at the

99.5% confidence interval. In another set of studies, carbonate concentration was determined in 11 bovine teeth which contained mature unerupted and erupted enamel. The carbonate content in the erupted bovine enamel (4.06 0.24%, n = 11) was slightly lower than that of the unerupted mature enamel (4.27 + 0.54%, n = 8), but the difference was not statistically significant. Fig. 1 illustrates the distribution and concentration of carbonate on a mineral-weight basis across the different stages of development in human enamel, in a series of 22 teeth, covering the spectrum of enamel development from early formation to maturation. The results for both labial and lingual enamel are shown. The highest carbonate content was present in the forming enamel. The concentration then began to decrease markedly at the late formation/early maturation stage to reach lowest values in the mature enamel. Fig. 2 compares the carbonate concentration and distribution throughout development in human labial enamel with the cal-

cium and phosphorus distributions. Ca/P ratio (wt/wt) is also shown. As the enamel matured and mineral content increased, as expressed by increasing calcium and phosphorus concentrations, carbonate content decreased. Ca/P ratio remained fairly constant throughout development, averaging 2.05 ± 0.09 wt/ wt.

Fig. 3 depicts the changes in carbonate concentration across the different zones of development (forming, maturing, and mature) in both labial and lingual enamel in a series of four developing human teeth of different ages, demonstrating the overall decrease in carbonate content as enamel matured.

Discussion. The present study describes the detailed changes in carbonate concentration in developing human and bovine enamel through the different stages of development from formation to maturation. The mineral content, as expressed by calcium and phosphorus concentrations on a mineral-weight basis, has also

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SYDNEY-ZAX et al.

Finally, this decrease in carbonate concentration as mineralization of enamel increases is consistent with the increased crystallinity observed as enamel matures (Nylen et al., 1963; Landis and Navarro, 1983).

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been analyzed to facilitate direct comparison of carbonate content with progressing mineralization. The results show that the carbonate concentration, which was highest in the forming enamel, began to decrease at the beginning of the maturing stage (a stage where the mineral concentration of developing enamel increases abruptly), to reach lowest levels in the mature tissue. Since carbonate concentration was measured in this study on a mineral-weight basis, the observed decrease in carbonate concentration with increasing mineralization of enamel in the human tooth may reflect dilution of carbonate-rich mineral with carbonate-free mineral. A similar finding was reported by Hiller et al. (1975) in the continuously growing rat incisor. Hiller reported that, on a mineral-weight basis, CO2 concentration decreased as the enamel mineralized, but on a tissue-weight basis, CO2 concentration remained relatively constant across forming, maturing, and mature enamel. The results for the carbonate concentration on a mineral-weight basis in the mature enamel agree well with those obtained previously in mature human enamel (Weatherell and Robinson, 1968; Weatherell et al., 1968, 1974). In view of the effect of carbonate on the stability of enamel mineral, it is important to consider the implications of these findings in the resistance of enamel to dental caries. Darling (1961) reported that the first stage of caries occurs in the interprismatic regions of enamel, and it seems to involve a preferential dissolution of carbonate-rich mineral (Coolidge and Jacobs, 1957; Hallsworth et al., 1972, 1973). Hiller et al. (1975) have suggested that it may be in the interprismatic enamel that the least dilution of the original carbonate concentration occurs.

REFERENCES BESHAH, K.; REY, C.; GLIMCHER, M.J.; SCHIMIZU, M.; and GRIFFIN, R.G. (1990): Solid State Carbon-13 and Proton NMR Studies of Carbonate Containing Calcium Phosphates and Enamel, J Solid State Chemistry 84:71-81. CHEN, P.S.; TORIBARA, T.Y.; and WARNER, H. (1956): Microdetermination of Phosphorus, Anal Chem 28:1756-1758. COOLIDGE, T.B. and JACOBS, M.H. (1957): Enamel Carbonate in Caries, JDent Res 36:765-768. DARLING, A.I. (1961): The Selective Attack of Caries on Dental Enamel, Ann R Coll Surg 29:354-369. DEAKINS, M. (1942): Changes in the Ash, Water, and Organic Content of Pig Enamel During Calcification, J Dent Res 21:429-435. DEUTSCH, D. (1989): Structure and Function of Enamel Gene Products, Anat Rec 224:189-210. DEUTSCH, D.; EL-ATTAR, I.; ROBINSON, C.; and WEATHERELL, J. (1979): Rate and Timing of Enamel Development in the Deciduous Bovine Incisor, Arch Oral Biol 24:407-413. DEUTSCH, D. and GEDALIA, 1. (1980): Chemically Distinct Stages in Developing Fetal Human Enamel, Arch Oral Biol 25:635-639. DEUTSCH, D. and PE'ER, E. (1982): Development of Enamel in Human Fetal Teeth, J Dent Res 61(Sp Iss):1543-1551. DEUTSCH, D. and SHAPIRA, L. (1987): Changes in Mineral Distribution and Concentration During Enamel Development in the Deciduous Human Maxillary and Mandibular Teeth, Growth 51:334-341. DEUTSCH, D.; SHAPIRA, L.; ALAYOFF, A.; LEVIEL, D.; YOELI, Z.; and ARAD, A. (1984): Protein and Mineral Changes During Prenatal and Postnatal Development and Mineralization of Human Deciduous Enamel. In: Tooth Enamel IV, R.W. Fearnhead and S. Suga, Eds., Amsterdam: Elsevier Publishing Co., pp. 234-239. EASTOE, J.E. and CAMILLERI, G.E. (1971): Pattern of Protein Distribution in Enamel During Maturation. In: Tooth Enamel II, R.W. Feamhead and M.V. Stack, Eds., Bristol: John Wright, pp. 119-123. ELLIOTT, J.C.; HOLCOMB, D.W.; and YOUNG, R.A. (1985): Infrared Determination of the Degree of Substitution of Hydroxyl by Carbonate Ions in Human Dental Enamel, Calcif Tissue Int 37:372-375. FEATHERSTONE, J.D.B.; PEARSON, S,; and LEGEROS, R.Z. (1984): An Infrared Method for Quantification of Carbonate in Carbonated Apatites, Caries Res 18:63-66. HALLSWORTH, A.S.; ROBINSON, C.; and WEATHERELL, J.A. (1972): Mineral and Magnesium Distribution Within the Approximal Carious Lesion, Caries Res 6:156-168. HALLSWORTH, A.S.; WEATHERELL, J.A.; and ROBINSON, C. (1973): Loss of Carbonate During the First Stages of Enamel Caries, Caries Res 7:343-348. HARDWICK, J.L. (1949): Enamel Caries: A Chemico-Physical Hypothesis. Phase One-Chemical, Br Dent J 87:137-142. HILLER, C.R.; ROBINSON, C.; and WEATHERELL, J.A. (1975): Variation in the Composition of Developing Rat Incisor Enamel, Calcif Tissue Res 18:1-12. LANDIS, W.J. and NAVARRO, M. (1983): Correlated Physicochemical and Age Changes in Embryonic Bovine Enamel, Calcif Tissue Int 35:48-55. LEBLOND, C.P. and WARSHAWSKY, H. (1979): Dynamics of Enamel Formation in the Rat Incisor Tooth, J Dent Res 58(B):950-975. LEGEROS, R.Z. (1981): Apatites in Biological Systems, Frog Crystal Growth Charact 4:1-45. LEGEROS, R.Z. and TUNG, M.S. (1983): Chemical Stability of Carbonate- and Fluoride-containing Apatites, Caries Res 17:419-429. MAYER, I.; FEATHERSTONE, J.D.B.; NAGLER, R.; NOEJOVICH, M.; DEUTSCH, D.; and GEDALIA, I. (1985): The Thermal Decomposition of Mgcontaining Carbonate Apatites, J Solid State Chem 56:230-235. NELSON, D.G.A. and FEATHERSTONE, J.D.B. (1982): Preparation, Analysis, and Characterization of Carbonated Apatites, Calcif Tissue Int 34:S69-S81. NYLEN, M.; EANES, E.D.; and OMNELL, K.A. (1963): Crystal Growth in Rat Enamel, J Cell Biol 18:109-123. ROBINSON, C.; FUCHS, P.; DEUTSCH, D.; and WEATHERELL, J.A. (1978): Four Chemically Distinct Stages in Developing Enamel from Bovine Incisor Teeth, Caries Res 12:1-11. SOBEL, A.E. (1962): Dietary Phosphate and Caries Susceptibility, Dent Prog 2:4852. WEATHERELL, J.A. and ROBINSON, C. (1968): Micro Determination of Carbonate in Dental Enamel, Analyst 93:244-248. WEATHERELL, J.A. and ROBINSON, C. (1973): The Inorganic Composition of Teeth. In: Biological Mineralization, I. Zipkin, Ed., New York: John Wiley and Sons, Inc., pp. 43-74. WEATHERELL, J.A.; ROBINSON, C.; and HALLSWORTH, A.S. (1974): Variations in the Chemical Composition of Human Enamel, J Dent Res 53:180192. WEATHERELL, J.A.; ROBINSON, C.; and HILLER, C.R. (1968): Distribution of Carbonate in Thin Sections of Dental Enamel, Caries Res 2:1-9.

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