Earliest enamel deposits of the rat incisor examined by electron microscopy electron diffraction and electron probe microanalysis.код для вставкиСкачать
THE ANATOMICAL RECORD 220:233-238 (1988) Earliest Enamel Deposits of the Rat Incisor Examined by Electron Microscopy, Electron Diffraction, and Electron Probe Microanalysis WILLIAM J. LANDIS, GRACE Y. BURKE, JULIA R. NEURINGER, MARY C. PAINE, ANTONIO NANCI, PAUL BAI, AND HERSHEY WARSHAWSKY Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedic Surgery, Harvard Medical School at the Children’s Hospital, Boston, Massachusetts 02115 (w.J. L., G.Y B . , J. R.N., M. C.P); Departement de Stomatologie, FacultC de MCdecine Dentaire, Universitt de MontrCal, Montreal, QuCbec, Canada H3C 3T9 (A.N.); Department of Anatomy, McGill University, Montreal, Quebec, Canada H3A 2B2 (PB., H. W ) ABSTRACT In order to describe initial events in enamel mineralization and to help characterize inorganic-organic interactions in this tissue, the earliest rod and interrod enamel in mandibular incisors from normal young adult (100 gm) rats, perfused with 100% ethylene glycol, has been studied by transmission electron microscopy, selected area electron diffraction, and high-spatial-resolution electron probe microanalysis. Diffraction and probe data were correlated precisely from the same extracellular regions of the tissue. Sites were examined progressively as a function of location a) from the most recently deposited enamel adjacent to ameloblasts toward the dentin-enamel junction and b) from the apical portion of the tooth longitudinally toward its incisal end. Electron diffraction patterns consistent with that of a poorly crystalline hydroxyapatite were generated at all locations. Diffraction characteristics changed only slightly toward that of more crystalline hydroxyapatite at different locations. Earliest apical enamel generated molar Ca/P ratios in a range of 0.99-1.46 (average 1.24 0.15). Molar Ca/P ratios of the first enamel interrod elements increased from 1.24 a t ameloblast-enamel boundaries to 1.40 at the dentin-enamel junction, small changes corresponding to those observed in electron diffraction characteristics. - In the biomineralization of the variety of species comprising both invertebrates and vertebrates, a general concept of mechanism holds that the deposition of a mineral phase is ultimately controlled by a specific organic matrix serving as the framework for crystal nucleation and growth. Supporting this idea in a circumstantial way are observations in bone (Glimcher and Krane, 19681, dentin (Katchburian, 19731, and calcifying tendon (Landis, 1985, 1986) that hydroxyapatite crystals are highly oriented such that their crystallographic c-axes lie parallel to the long axes of collagen fibrils in which they are found. Furthermore, the crystals are deposited initially in the collagen hole zones (Hodge and Petruska, 1963). In the mollusk, there is additional evidence indicating a critical interaction between the organic matrix and composite inorganic crystals of aragonite (Weiner et al., 1983; Weiner and Traub, 1984). While such circumstantial data provide certain evidence for a n intimate inorganic-organic relationship in biological mineralization, a n explicit description would be more compelling. For this purpose, both the mineral and the matrix in a given species must be definitively known from a molecular perspective. In no case has this yet been accomplished, but, as a step toward this goal, this article examines the earliest deposits of a mineral phase in enamel from the continuously erupting incisors 0 1988 ALAN R.LISS, INC. - of young adult rats. This model maintains biologically a relatively homogeneous population of its mineral phase particles (Leblond and Warshawsky, 1979), and the preparative methods employed adequately preserve the chemical and physical integrity of the inorganic matrices (Landis and Glimcher, 1978; Landis et al., 1977). In this instance the initial deposits of rod and interrod enamel from animals perfused with anhydrous ethylene glycol have been systematically examined by transmission electron microscopy, selected area electron diffraction, and electron probe microanalysis to localize and identify the extracellular mineral phase and to determine changes in its chemical and physical nature with tissue age and maturation. The data help characterize the extracellular matrices of dental tissues and some of the basic events of enamel mineralization as a first approach to describing molecular interactions between inorganic and organic tissue components. MATERIALS AND METHODS Normal male Sherman rats, approximately 100 gm in weight, were treated according to the general procedure of Bishop and Warshawsky (1982). Animals were anesthetized with sodium pentabarbitone by intraperitoneal Received April 8, 1987; accepted July 23, 1987. 234 W.J. LANDIS ET AL. injection and then perfused through the left ventricle of the heart with lactated Ringer’s solution for 1-2 min and with 100% ethylene glycol for a n additional 10-15 min. Incisors were dissected from mandibular bone and the apical third of each was sliced with a clean razorblade transversely to the long axis of the tooth. Specimens (<1 mm3) were then placed in 100% ethylene glycol in scintillation vials, and the anhydrous treat- ment of Landis et al. (1977) was continued through ?ample embedment in Epon. Silver sections (-800 A ) of tissue, containing initial and inner enamel secretion regions as determined from 1-pm-thick sections stained with toluidine blue, were obtained with diamond knives, floated on 100% ethylene glycol, and collected on 75mesh carbon-reinforced Parlodion-coated copper grids (Landis et al., 1977). Grids were dried with filter paper 4 ? h 40 C. 60 9&3 100 120 1 4 0 160 160 200 228 2 4 0 CHRNNEL N O - 235 ELECTRON OPTICS OF RAT INCISOR ENAMEL Fig. 7. Selected area electron diffraction (80 keV) of the same region of initial enamel in the zone of secretion from which probe data were obtained (Figs. 2 and 5). The pattern has a small number of poorly resolved, low-intensity reflections. The major reflection appearing is a relatively wide, bright ring at d = 2.78 A (the unresolved triplet d = 2.81, 2.78, and 2.72 A corresponding to hkl = 211 + 112 + 300, respectively). This and a very weak reflection at d = 3.44 A (hkl = 002, not labeled) are consistent with that of a poorly crystalline hy- droxyapatite (see Fig. 12 of Landis and Glimcher, 1978). Magnification of transmission image was x 15,000. Fig. 8. Selected area electron diffraction of the region of inner enamel corresponding to Figures 3 and 6. The pattern is slightly different from that shown in Figure 7 but only in that the weak reflections a t d = 3.44 A and 2.78 A are somewhat better resolved. Magnification of transmission image was ~15,000. and then placed under vacuum for several hours to remove residual glycol. Sections were left completely unstained for electron optical examination. Methods for electron microscopy, electron diffraction, and high-spatial-resolution (electron beam diameter < l o 0 A ) electron probe X-ray microanalysis were followed as described in detail elsewhere (Landis and , . Glimcher, 1982). As before (Landis and Glimcher, 1978, Fig. 2. Enlargement of Figure 1showing the first observable initial a JEoL loo' Or EM 300 was for enamel deposits (E), evident as small irregularly shaped masses of 1982)3 thin. ribbon-like crvstals (inset) adiacent to a laver of dentin (D). The m~CrOscOPYand diffraction, and a modified JEoL JSM proximal portions 6f secretory amefoblasts form 6lunt and wide Tomes' 50A opeiated in scanning transmission mode for probe processes WP) without interdigitations with enamel. Dentin is consid- microanalysis. Areas having a 4-pm diameter were deerably more electron-dense than enamel in such unstained sections. fined at the specimen for selected area electron diffrac~7,000;inset, ~35,000. tion. Evaporated aluminum and thallous chloride were Fig. 3. Enlargement of Figure 1 illustrating the first interrod ele- used as electron diffraction calibration standards. Dements (IR)and short, interdigitating portions of Tomes' processes bP). Interrod growth regions (IGR) exist at the tips of the interrod elements. spite maintenance of high vacuum in the electron probe, While the first row of rod profiles is not yet elaborated at this location, the regions of enamel investigated were susceptible to a few of the earliest rods (R) are being formed in the spaces occupied some contamination during data acquisition; X-ray by the processes. D, dentin. x 3,000. counts were accumulated over 360-sec integrated detectFig. 4. Energy-dispersive X-ray spectrum generated from a region of ing time periods with a static spot probe. Quantitative immature enamel (Point 1 in Figure 5). All spectra were obtained at determination of molar Ca/P ratios from X-ray intensity 25 keV with a stationary beam spot, beam current of 1 x lO-"A, and counts of calcium and phosphorus obtained by probe 360 sec integrated detecting time. The abscissa is calibrated relative microanalysis was made by interpolation from a stanto X-ray energy (20 eV/channel). Characteristic lines are phosphorus, chlorine, calcium, and silicon. Chlorine originates from the Epon dard calibration curve (Landis, 1980; Landis and Glimcher, 1982). For calculation of Ca/P intensity ratios embedment, and silicon from electron-beam-inducedcontamination. (Landis and Glimcher, 1982),X-ray spectra from off-peak Fig. 5. Scanning transmission image (25 keV) of Figure 2 following regions of the tissues were taken over organic matrices electron probe microanalysis. The center of the contamination spots that arise during data acquisition indicates the spatial location of the adjacent to enamel. Such spectra were generally unreelectron beam. X-ray intensities for Ca and P generated from the markable and were considered negligible for computaindividual points noted on the micrograph are presented in Table 1. tional purposes. Fig. 1. Conventional transmission image (60 keV) of an unstained incisor thin section of immature enamel in the zone of secretion from a normal male Sherman rat, perfused with 100%ethylene glycol. The exact regions of the tooth from which electron diffraction and electron probe microanalysis data were obtained are enclosed and shown at higher magnifications in other figures. The ultrastructural features illustrated are secretory ameloblasts (A), Tomes' processes WP), enamel ( E l dentin (D). and Dredentin (PD). x2.100. The sites examined were selected to sample enamel deposits near the ameloblast-enamel junction, the dentin-enamel junction, and the area between those two regions. ~7,000. Fig. 6. The region of developing interrod enamel elements corresponding to Figure 3 following electron probe microanalysis. The scan- ning transmission image shows the sites examined along individual interrods from interrod growth regions to the dentin-enamel junction. Selected corresponding data are given in Table 2. ~3,400. RESULTS The portion of the rat incisor examined was that along the labial tooth surface within the zone of secretion, where immature enamel is formed (Leblond and Warshawsky, 1979; Smith, 1979; Warshawsky et al., 1981). Figure 1 illustrates this area at relatively low magnifi- 236 W.J. LANDIS ET AL. indicate a specific trend with respect to changes in calcium or phosphorus. Nor was there an apparent pattern of change measured with respect to distance (Fig. 6) along individual interrod or forming rod elements in inner enamel secretion regions (Table 2), although a suggestion of slightly increasing Ca/P may be inferred 1 Initial enamel 1.23 1.04 from the average of points such as A, B, and C. 1.56 1.28 2 Selected area electron diffraction of the same regions 1.63 1.34 3 from which electron probe data were generated yielded 1.49 1.23 4 diffraction patterns with few reflections (Figs. 7, 81, but 1.78 1.46 5 among those evident yere lattice spacings identified as 1.69 1.39 6 d = 3.44 and -2.78 A , corresponding to the two major 1.63 1.34 7 1.38 1.15 reflections of hydroxyapatite. The absence of other 8 1.44 1.19 9 prominent reflections and the indistinct resFlution of 1.15 0.98 10 the triplet of lines d = 2.81, 2.78, and 2.72 A indicate Average 1.24 + 0.15 that enamel is a poorly crystalline hydroxyapatite at Analytical conditions are as described for Figure 4 and in Materials this stage of its development. There were only minor and Methods. Each site was examined once. Sites correspond to those differences in the intensity of most of the observed renumbered in Figure 5. The exact progression of site number with flections over the entire zone of enamel examined, and probe location, either transversely or longitudinally along the enamel, no changes in either the number or sharpness of those must be followed in Figure 5 . reflections were apparent. TABLE 1. CalP measurements by high-spatial-resolution electron probe microanalysis of initial enamel deposits in the rat incisor (Fig. 5) Ca/P X-ray Ca/P Site Location intensity ratio molar ratio cation in an undecalcified, unstained thin tissue section treated anhydrously. The precise regions studied by probe microanalysis and electron diffraction were those between initial and inner enamel secretion (Leblond and Warshawsky, 1979; Warshawsky et al., 1981), in which flattened, wide proximal portions of ameloblast Tomes’ processes become apparent and the first enamel deposits are evident as small, irregularly shaped masses. The enamel is much less electron-dense than the adjacent layer of relatively mature dentin (Fig. 2). Within the initial enamel masses are narrow, Cibbon-like crystals whose thickness varies up to 400 A (inset, Fig. 2). As they are elaborated, the first interrod enamel elements alternate with the short Tomes’ processes in this portion of the rat incisor, and enamel rods are formed between interrod partitions as development of an enamel matrix proceeds (Figs. 2, 3). The results of electron probe X-ray microanalysis of immature enamel are given in Tables 1and 2. A typical X-ray spectrum is presented in Figure 4. After 360-sec counting times, the predominant peaks are those of calcium and phosphorus, silicon, and chlorine. The sites examined are identified in Figures 5 and 6, scanning transmission images corresponding to Figures 2 and 3, but illustrating the enamel regions of interest following microanalysis. The small, dense spots in Figures 5 and 6 are the result of electron beam-induced contamination in this dose-sensitive tissue region; the centers of tbese spots serve to mark the exact areas probed (- 100 A in diameter). The underlying sources of such contamination or the causes of the observed sensitivity are unknown. At points analyzed from initial enamel, the data (Table 11, corrected for background, show a highly variable calcium and phosphorus content and Ca/P molar ratios between 0.98 and 1.46. Nearly all enamel values were lower than those of adjacent dentin (Ca/P = 1.50 k 0.05 to 1.55 0.04; manuscript in preparation). The presence of other elements, such as magnesium and sodium, was not detected in these spectra. Comparison of results obtained from regions of initial enamel adjacent t o ameloblasts, regions adjacent t o the dentinenamel junction, and regions between the two did not - + DISCUSSION On the basis of electron probe measurements, Glick (1979) and Halse and Selvig (1974)have demonstrated a gradient in the content of calcium and phosphorus with respect to location in rat incisor teeth. Their work showed an increase in calcium and phosphorus concentration transversely from the ameloblast-enamel junction to the dentin-enamel junction in the zone of secretion as well as longitudinally from the apical to the incisal region of the incisor. Thus, the most recently deposited enamel in these teeth exists at the apical ameloblast-enamel junction (zone of secretion). In the present study, within this particular region of initial and inner enamel secretion of the incisor, the progressive increases in calcium and phosphorus reported by Glick (1979) and Halse and Selvig (1974) over the whole incisor were also suggested by increasing Ca/P ratios along some individual forming rod and interrod elements extending both transversely and longitudinally as defined above. It appears, then, that small calcium and phosphorus gradients occur even over relatively limited portions of the rat tooth, here restricted to 15 pm in thickness and -40 pm in length in the zone of secretion and representing a very short transit time for the ameloblasts, calculated as about 80 min (based on the data of Leblond and Warshawsky, 1979, Fig. 1, p. 951). In addition, with respect t o characterization of the initial development of enamel, the present data indicate a solid mineral phase having a variable but consistently low Ca/P molar ratio that does not correspond to the known stoichiometry of a single specific calcium phosphate species. The enamel Ca/p values are generally higher than ratios obtained from the lowest-density fractions of embryonic chick bone [molar Ca/P = 0.90-1.04 (Roufosse et al., 1979)],brushite and monetite standards [Ca/p = 1.03 and 1.00, respectively (Roufosse et al., 197911, and earliest deposits of an extracellular solid mineral phase measured in situ in epiphyseal growth plate cartilage from rat tibiae [Ca/P = 0.88-1.21 (Landis and Glimcher, 198211. On the other hand, some Ca/P ratios of the early enamel are similar to the values determined from thin tissue sections of the mineral deposits in osteoid regions of embryonic chick bone [Ca/P - 237 ELECTRON OPTICS OF RAT INCISOR ENAMEL TABLE 2. CalP measurements by high-spatial-resolution electron probe microanalysis of sites of earliest rod and interrod enamel in the rat incisor (Fig. 6) C a P X-ray C a P molar intensity ratio ratio Site Location (range) (average f SD) A B C D E F G H I 1- 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Interrod 1(growth region) Interrod 1(midzone) Interrod 1(near dentin-enamel junction) Newly forming rod Interrod-rod transition Interrod (near dentin-enamel junction) Interrod 2 (growth region) Interrod 2 (midzone) Interrod 2 (near dentin-enamel junction) Interrod progressively analyzed Adjacent to ameloblast Adjacent to dentin - enamel junction 1.23 - 1.82 1.31 - 1.79 1.26 - 1.84 1.24 - 1.87 1.37 - 2.01 1.64 - 1.95 1.44 - 1.84 1.42 - 1.83 1.25 - 1.73 1.96 1.61 1.79 1.31 1.50 1.65 1.31 1.61 1.58 1.57 1.55 1.60 1.69 1.54 1.34 1.48 1.67 1.73 1.85 1.88 1.94 1.66 1.57 1.82 1.45 1.64 1.99 n n n n n n n n n = = = = = = = = = 10* 10 10 10 10 10 10 10 10 1.24 f 0.12 1.29 & 0.13 1.34 0.14 1.26 f 0.14 1.38 f 0.14 1.40 f 0.07 1.31 f 0.08 1.33 f 0.10 1.28 f 0.12 1.58 1.33 1.45 1.10 1.25 1.37 1.14 1.33 1.31 1.30 1.28 1.34 1.39 1.27 1.16 1.24 1.38 1.43 1.52 1.55 1.58 1.37 1.30 1.48 1.21 1.35 1.59 *n = the number of individual sites microanalyzed at the particular location described. Analytical conditions are as described for Figure 4 and in Materials and Methods. Each site was examined once. Sites correspond to those numbered in Figure 6. The exact progression of site number with probe location, either transversely or longitudinally along the enamel, must be followed in Figure 6. - 1.2-1.3 (Landis and Glimcher, 1978)], a n octacalcium phosphate standard [CaR 1.34 (Landis and Glimcher, 1978)], and a calcium phosphate containing poorly crystalline hydroxyapatite [CaJP = 1.45 (Roufosse et al., 1979). Most of the rat enamel CaJP ratios are lower than density fractions of embryonic bovine enamel [ C d P = 1.48-1.82 (Landis and Navarro, 1983; Landis et al., 1984)l and a well-crystallized synthetic hydroxyapatite [ C a = 1.62 (Roufosse et al., 197911. In terms of electron diffraction, the reflections of initial and inner enamel are characteristic of those of a very poorly crystalline hydroxyapatite. Similar electron diffraction patterns have also been identified under comparable conditions in all embryonic bovine enamel density fractions (Landis et al., 1984), intermediate and higher-density fractions of embryonic chick bone (Roufosse et al., 1979), whole unfractionated embryonic and postnatal (14 week) chicken bone (Roufosse et al., 19791, - and thin tissue sections of early extracellular mineral deposits in rat growth plate cartilage (Landis and Glimcher, 1982) and embryonic chick tibiae (Landis and Glimcher, 1978). The present enamel diffraction patterns may resemble as well that generated from young rat incisor enamel prepared elsewhere by anhydrous means (Bishop and Warshawsky, 1982); but because exact lattice spacings were not published (Bishop and Warshawsky, 1982), direct comparison is not possible. The electron diffraction data reported here show no evidence of brushite or octacalcium phosphate, a result consistent with earlier studies of embryonic bovine enamel (Landis and Navarro, 1983; Landis et al., 1984). Magnesium found in the same bovine enamel fractions (Landis and Navarro, 1983) and in sections of rat incisors (Casciani et al., 1979) does not appear in electron probe spectra, possibly because its concentration is below instrument sensitivity. Nor could the presence of 238 W.J. LANDIS ET AL. carbonate, suggested in recent enamel work (Casciani et al., 1979; Driessens and Verbeeck, 1982; Landis and Navarro, 1983; Landis et al., 1984), be detected by the methods employed here. Auger electron and X-ray photoelectron spectroscopies (Landis et al., 1982)are continuing in order to examine more carefully the possibility of carbonate phases in enamel. To describe fully the mineralization process in enamel, it is important to relate present data from electron probe microanalysis and electron diffraction of the extracellular calcium phosphate in the rat incisor with similar studies of the organic matrices in this model. With respect to the latter, reports from other laboratories have already provided some significant results from secretory and maturation ameloblasts (Boyde and Reith, 1977, 1978; Reith and Boyde, 1978,1979), and most recently a detailed study of the role of the organic matrix in mineralization of enamel from the rat and other species has been completed (Jodaikin, 1986). In the latter, the nature of the organic and mineral phases of the tissues of interest has been elegantly described in large part by Xray crystallography and electron diffraction means. The mineral was determined to be apatitic, as shown here, and was found to interact with a protein and lipid moeity of enamel at the molecular structure level (Jodaikin, 1986). Additional studies of inorganic-organic matrix interrelations in rat incisor enamel treated anhydrously are in progress in this laboratory to extend current results. Glimcher, M.J., and S.M. Krane (1968) The organization and structure of bone, and the mechanism of calcification. In: A Treatise on Collagen. B.S. Could and G.N. Ramachandran, eds. Academic Press, New York, Vol. 2B, pp. 68-251. Hake, A., and K.A. Selvig (1974) Mineral content of developing rat incisor enamel. Scand. J. Dent. Res., 8240-46. Hodge, A.J., and J.A. Petruska (1963) Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In: Aspects of Protein Structure. G.N. Ramachandran, ed. Academic Press, New York, pp. 289-300. Jodaikin, A. (1986) The role of the organic matrix in enamel mineralization. Doctoral Thesis, Weizmann Institute of Science, Rehovot, Israel. Katchburian, E. (1973) Membrane-bound bodies as initiators of mineralization in dentine. J. Anat., 116:285-302. Landis, W.J. (1980) X-ray microanalysis of calcium phosphate solids prepared anhydrously as calibration standards for mineralized tissues. In: Eighth International Congress on X-ray Optics and Microanalysis. D.R. Beaman, R.E. Ogilvie, and D.B. Wittry, eds. Pendell Publishing Co., Midland, MI., pp. 497-500. Landis, W.J. (1985) Temporal sequence of mineralization in calcifying turkey leg tendon. In: The Chemistry and Biology of Mineralized Tissues. W.T. Butler, ed. EBSCO Media, Birmingham, AL., pp. 360-363. Landis, W.J. (1986) A study of calcification in the leg tendons from the domestic turkey. J. Ultrastruct. Res., 94:217-238. Landis, W.J., and M.J. Glimcher (1978) Electron diffraction and electron probe microanalysis of the mineral phase of bone tissue prepared by anhydrous techniques. J. Ultrastruct. Res., 63:188-223. Landis, W.J., and M.J. Glimcher (1982) Electron optical and analytical observations of rat growth plate cartilage prepared by ultracryomicrotomy. J. Ultrastruct. Res., 78:227-268. Landis, W.J., and M. Navarro (1983) Correlated physicochemical and age changes in embryonic bovine enamel. Calcif. Tissue Int., 35:4855. Landis, W.J., M.C. Paine, and M.J. Glimcher (1977) Electron microscopic observations of bone tissue prepared anhydrously in organic ACKNOWLEDGMENTS solvents. J. Ultrastruct. Res., 59:l-30. Landis, W.J., M.D. Grynpas, R.M. Latanision, and J.R. Martin (1982) This work was supported by research grants from the Mineralized biological tissues studied by Auger electron and x-ray National Institute of Dental Research, National Instiphoto-electron spectroscopy. In: Microbeam Analysis. K.F.J. Heinrich, ed. San Francisco Press, San Francisco, CA, pp. 121-127. tutes of Health (grant DE 05351 to W.J.L.), and from the W.J., M. Navarro, J.R. Neuringer, and K. Kurz (1984) Single Medical Research Council of Canada (to H.W. and A.N.). Landis, enamel particles examined by electron optics. J. Dent. Res., 63:629634. Leblond, C.P., and H. Warshawsky (1979) Dynamics of enamel formaLITERATURE CITED tion in the rat incisor tooth. J. Dent. Res., 58@3):950-975. Bishop, M.A., and H. Warshawsky (1982) Electron microscopic studies Reith, E.J., and A. Boyde (1978) Histochemical and electron probe analysis of secretory ameloblasts of developing rat molar teeth. on the potential loss of crystallites from routinely processed secHistochemistry, 55:17-26. tions of young enamel in the rat incisor. Anat. Rec., 202177-186. Boyde, A,, and E.J. Reith (1977) Qualitative electron probe analysis of Reith, E.J., and A. Boyde (1979) The enamel organ, a control gate for calcium influx into the enamel. J. Dent. Res., 580:980. secretory ameloblasts and odontoblasts in the rat incisor. HistoRoufosse, A.H., W.J. Landis, W.K. Sabine, and M.J. Glimcher (1979) chemistry, 50:347-354. Identification of brushite in newly deposited bone mineral from Boyde, A,, and E.J. Reith (1978) Electron probe analysis of maturation embryonic chicks. J. Ultrastruct. Res., 68:235-255. ameloblasts of the rat incisor and calf molar. Histochemistry,55:41Smith, C.E. (1979) Ameloblasts: Secretory and resorptive functions. J. 48. Dent. Res., 58@):695-706. Casciani, F.W., E.S. Etz, D.E. Newbury, and S.B. Doty (1979) Raman microprobe studies of two mineralizing tissues: Enamel of the rat Warshawsky, H., K. Josephsen, A. Thylstrup, and 0. Fejerskov (1981) The development of enamel structure in rat incisors as compared incisor and the embryonic chick tibia. In: Scanning Electron Mito the teeth of monkey and man. Anat. Rec., 200:371-399. croscopy. 0. Johari, ed. SEM, Inc., AMF O’Hare, IL., Vol 2, pp. Weiner, S., and W. Traub (1984) Macromolecules in mollusk shells and 383-391. their functions in biomineralization. Phil. Trans. R. SOC.Lond., Driessens, F.C.M., and R.M.H. Verbeeck (1982) The probable phase 304(B):421-438. composition of the mineral in sound enamel and dentine. Bull. SOC. Weiner, S., Y. Talmon, and W. Traub (1983) Electron diffraction of Chim. Belg., 91573-596. mollusk shell organic matrices and their relationship to the minGlick, P.L. (1979) Patterns of enamel maturation. J. Dent. Res., eral phase. Int. J. Biol. Macromol., 5:325-328. 58(B):883-892.