Calcified Tissue Research
Calcif. Tiss. Res. 24, 271-274 (1977)
9 by Springer-Verlag 1977
The Early Mineralization of Enamel Fine Structural Observations on the Cellular Localization of Calcium with the Potassium Pyroantimonate Technique
D.A. Deporter Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario M5G 1G6, Canada
Summary. The potassium pyroantimonate technique was used to study the cellular distribution of calcium during the early mineralization of enamel in rat molar tooth germs at the electron microscope level. Differing patterns of calcium distribution were observed in the ameloblast seemingly associated with the appearance of Tomes' process. In the early secretory ameloblast calcium pyroantimonate deposits were observed within the Golgi apparatus, within coated vesicles, within mitochondria and associated with the inner aspect of the cell membrane. However, with the development of Tomes' process the ameloblasts no longer demonstrated these discrete deposits of calcium pyroantimonate. Instead they showed a diffuse cytoplasmic staining pattern with no preference for any particular organelle. Key words: Enamel - - Mineralization - - Calcium.
Introduction It has recently been reconfirmed that the mineralization of rat enamel occurs in two distinct stages [8]. The greatest quantities of mineral in absolute terms are deposited by the post-secretory or reduced ameloblasts after the full thickness of enamel matrix has been formed [3, 15, 16]. However, during the formative phase of amelogenesis some mineral ( 3 0 - 3 5 % of the final mineral content) is added to the organic matrix of enamel as soon as it is secreted by the secretory ameloblasts [7], and, considering the high appositional rate of enamel matrix formation in the rat, the requirement for calcium per unit time must be very high during this phase of amelogenesis as well. Send offprint requests to D.A. Deporter at the above address
Little information exists in the literature on the cellular mechanisms involved in the early mineralization phase. Therefore, as part of an ongoing study of mechanisms of mineralization, it was decided to investigate the fine structural distribution of calcium deposits during early amelogenesis of rat molars using the potassium pyroantimonate staining procedure [19].
Materials and Methods Four-day-old Wistar rats were killed by decapitation. The maxillary first molar tooth germs were dissected out and fixed for 90 min at 4 ~ C in an aqueous mixture of 0.05 M potassium pyroantimonate (Fisher Scientific) and 1% osmium tetroxide, pH 7.5 [19]. Controls were fixed in an aqueous mixture of 0.05 M potassium pyrophosphate and 1% osmium tetroxide. As an additional control some tooth germs were pretreated at 4 ~ C with 5 mM EGTA ((ethylenebis (oxyethylenenitrilo) tetra-acetic acid)) in 0.05 M Tris buffer, pH 7.4 for 15 to 30 rain and washed in Tris buffer without EGTA before being fixed with the potassium pyroantimonateosmium tetroxide mixture. Following fixation the specimens were dehydrated through graded alcohols and propylene oxide and embedded in Epon. Ultrathin sections were examined unstained and, after staining with 70% methanolic uranyl acetate, using a Phillips 200 electron microscope.
Results Electron dense calcium pyroantimonate deposits were observed in several locations depending upon the state of differentiation of the ameloblast. There were no deposits observed in the undifferentiated cells of the internal enamel epithelium. However, as these cells differentiated into ameloblasts, distinct and differing patterns of calcium localization were found seemingly associated with the development of Tomes' process. Before the development of Tomes' process, in what will be called the early secretory ameloblasts, calcium pyroantimonate deposits were observed in distinct locations (see Figs. 1 and 2). First, deposits were
272
D.A. Deporter: Cellular Localizationof Calcium During Early Amelogenesis
Fig. 1. Early secretory ameloblasts. Calcium pyroantimonate deposits are localized to the plasma membrane, the Golgi region (arrows) and to most mitochondria. Dentine (D) x5236
Fig. 3. Fully differentiated ameloblasts with characteristic Tomes' processes (TP) to show diffuse cytoplasmic staining pattern of calcium pyroantimonatedeposits. Enamel (E) x 8640
vesicles in the apical or secretory pole of the cells. Many of the coated vesicles seemed to be in the process' of releasing their calcium content into the stippled material. Finally, calcium pyroantimonate deposits were observed within most, if not all, of the mitochondria of the early secretory ameloblasts. These deposits were localized to the intracristal spaces of the mitochondria. As the cells became fully differentiated into secretory ameloblasts with well recognizable Tomes' processes the pattern of calcium pyroantimonate deposits changed. The precise localization described above was no longer observed but was replaced by a diffuse cytoplasmic staining pattern which was primarily localized to the secretory pole of the ameloblasts (Fig. 3). None of the deposits described above were observed in the control specimens.
Fig. 2. Early secretory ameloblast~s. Some cytoplasmic vesicles (arrows) are discharging their calcium content into the stippled material (S). Dentine (D) x 20,328
localized to the inner or cytoplasmic aspect of the plasma membrane including the apical plasma membrane adjacent to the newly secreted stippled material of the pre-enamel matrix [18]. Second, deposits were found within the Golgi apparatus and within coated
Discussion It is not known how the secretory ameloblast mediates partial mineralization of the newly deposited enamel matrix. Ultrastructural, autoradiographic and biochemical studies of other calcium secreting epithelial cells, such as the mucosal cells of the calciferous glands of the earthworm [13] and the mucosal ceils of the avian shell gland [9], have demonstrated that the mechanism of calcium secretion probably involves the
D,A. Deporter: Cellular Localization of Calcium During Early Amelogenesis
packaging and secretion of calcium within cytoplasmic vesicles of Golgi origin. Thus, it may be that, as argued by Deporter and Ten Cate [6], calcium is secreted by secretory ameloblasts via Golgi vesicles, possibly within the same vesicles that contain the organic matrix of enamel. The results of the present study provide some support for this hypothesis of early mineralization of enamel in that it was possible to demonstrate calcium deposits within the Golgi apparatus and within cytoplasmic vesicles of the early secretory ameloblasts. However, as the ameloblasts became fully differentiated into secretory ameloblasts with well developed Tomes' processes it was no longer possible to demonstrate these sites of calcium localization. This finding suggests that the early mineralization of enamel may involve two distinct mechanisms. The initial mineralization, that is that mediated by the early secretory ameloblasts, may involve the packaging of calcium by the Golgi apparatus into secretory vesicles which then discharge their calcium content by reverse endocytosis at the secretory pole of the cell. Thereafter, once the initial mineralization of the first formed enamel matrix has occurred and the secretory ameloblasts have become fully differentiated, the mechanism of enamel mineralization may change. Calcium may then diffuse freely through the cytoplasm of the secretory ameloblasts as suggested by previous workers [1, 11, 12]. It should be noted that Nagai and Frank [12], in their electron microscope autoradiographic study of 45Ca distribution during amelogenesis in the cat, suggested that large quantities of calcium also diffused to the mineralization front of enamel between the secretory ameloblasts. Although calcium has been demonstrated extracellularly between odontoblasts using the potassium pyroantimonate technique [14;] (Deporter and Belzycki, in preparation), calcium deposits were never observed between the secretory ameloblasts in the present study. It may be that the observations of Nagai and Frank are due to the imprecise localization of silver grains inherent in electron microscope autoradiographic technique. The plasma membrane localization of calcium in the early secretory ameloblasts suggests that these cells store large quantities of calcium at this membrane site for later use after they have differentiated into mature secretory ameloblasts. The absence of calcium pyroantimonate deposits on the exterior plasma membrane surface of the early secretory ametoblasts indicates that the calcium does not diffuse across the cell membrane. The demonstration of calcium pyroantimonate deposits within the mitochondria of early secretory ameloblasts is of special interest in view of the proposed role for mitochondria in mineralization of
273
various hard tissues. Similar deposits have previously been reported in the mitochondria of hypertrophic cartilage cells just before the onset of mineralization of epiphyseal cartilage [5]. Moreover, results from numerous workers using a variety of different techniques have suggested that mitochondria store calcium in preparation for the onset of a mineralization process [2, 4, 5, 9, 10, 17]. Thus, for example, Hohman and Schraer [9] demonstrated that 4~Ca accumulated in the mitochondria of mucosal cells of the avian shell gland before mineralization of the egg but was later discharged from mitochondria with the onset of mineralization. The present demonstration of calcium deposits within mitochondria of early secretory ameloblasts but not in the mitochondria of fully differentiated secretory ameloblasts provides further evidence for this proposed role for mitochondria in mineralization. Acknowledgments. The author is a Scholar of the Medical Research Council of Canada. He wishes to thank Mr. George Pudy and Mrs. Amy Shiga for their expert technical assistance, Mrs. Orrie Warren for typing the manuscript and Professor A.R. Ten Care for his critical evaluation of the text.
References 1. Boyd, A., Reith, E.J.: Qualitative electron probe analysis of secretory ameloblasts and odontoblasts in the rat incisor. Histochemistry 50, 347-354 (1977) 2. Bechtel, D.B., Horner, H.T.: Calcium excretion and deposition during sporogenesis in Physarella oblonga. Calcif. Tiss. Res. 18, 195-213 (1975) 3. Beynon, A.D.: Enzymes in enamel maturation. Proc. Roy. Soc. Med. 65, 911-912 (1972) 4. Brighton, C.T., Hunt, R.M.: Mitochondrial calcium and its role in calcification. Histochemical localization of calcium in electron micrographs of the epiphyseal growth plate with Kpyroantimonate. Clin. Orthop. Rel. Res. 100, 406-416 (1976) 5. Brighton, C.T., Hunt, R.M.: Histochemical localization of calcium in growth plate mitochondria and matrix vesicles. Fed. Proc. 35, 143-147 (1976) 6. Deporter, D.A., Ten Cate, A.R.: Fine structural localization of alkaline phosphatase in relation to enamel formation in the mouse molar. Archs. Oral Biol.21, 7-12 (1976) 7. Frank, R.M., Nalbandian, J.: Ultrastructure of amelogenesis. In: Structural and chemical organization of teeth (A.E.W. Miles, ed.), pp. 399-466. New York: Academic Press 1967 8. Glick, P.L., Edie, J.W.: Mineralization of rat enamel: a c o m bined electron-microprobe, electron microscopic and his tologic study. Jour. Dent. Res. 54A, abstract 348, 1975 9. Hohman, W., Schraer, H.: The intracellular distribution of calcium in the mucosa of the avian shell gland. J. Cell Biol. 30, 317-331 (1966) 10. Mathews, J.L., Martin, J.H., Arsenis, C., Eisenstein, R., Kuettner, K . : The role of mitochondria in intracellular calcium regulation. In: Cellular mechanisms for calcium transfer and homeostasis (G. Nichols and R.H. Wasserman, eds.), pp. 239-255. New York: Academic Press 1971
274
D.A. Deporter: Cellular Localization of Calcium During Early Amelogenesis
11. Munhoz, C.O.G., Leblond, C.P.: Deposition of calcium phosphate into dentine and enamel as shown by radiography of sections of incisor teeth following injection of 45Ca into rats. Calcif. Tiss. Res. 15, 221-235 (1974) 12. Nagai, N., Frank, R.M.: Tranfert du 45Ca par autoradiographic en microscopic electronique au cours de l'amelogenese. Calcif. Tiss. Res. 19, 211-221 (1975) 13. Nakahara, H., Bevelander, G.: An electron microscope and autoradiographic study of the calciferous glands of the earthworm, Lumbricus terrestris. Calcif. Tiss. Res. 4, 193-201 (1969) 14. Reith, E.J.: The binding of calcium within the Golgi saccules of the rat odontoblast. Amer. J. Anat. 147, 267-272 (1976) 15. Reith, E.J., Cotty, V.F.: Autoradiographic studies on calcification of enamel. Archs. Oral Biol. 7, 365-372 (1962)
16. Reith, E.J., Cotty, V.F.: The absorptive activity of ameloblasts during the maturation of enamel. Anat. Rec. 157, 577588 (1967). 17. Shapiro, I.M., Greenspan, J.S.: Are mitochondria directly involved in biological mineralization? Calcif. Tiss. Res. 3, 100-102 (1969) 18. Slavkin, H.C., Mino, W., Bringas, P.: The biosynthesis and secretion of precursor enamel protein by ameloblasts as visualized by autoradiography after tryptophan administration. Anat. Rec. 185, 289-312 (1976) 19. Spicer, S.S., Hardin, J.H., Green, W.B.: Nuclear precipitates in pyroantimonate-osmium tetroxide-fixed tissues. J. Cell Biol. 39, 216-221 (1968)
Received May 19 / Revised August 19 / Accepted August 22, 1977
Calcif. Tiss. Res. 24, 271-274 (1977)
9 by Springer-Verlag 1977
The Early Mineralization of Enamel Fine Structural Observations on the Cellular Localization of Calcium with the Potassium Pyroantimonate Technique
D.A. Deporter Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Ontario M5G 1G6, Canada
Summary. The potassium pyroantimonate technique was used to study the cellular distribution of calcium during the early mineralization of enamel in rat molar tooth germs at the electron microscope level. Differing patterns of calcium distribution were observed in the ameloblast seemingly associated with the appearance of Tomes' process. In the early secretory ameloblast calcium pyroantimonate deposits were observed within the Golgi apparatus, within coated vesicles, within mitochondria and associated with the inner aspect of the cell membrane. However, with the development of Tomes' process the ameloblasts no longer demonstrated these discrete deposits of calcium pyroantimonate. Instead they showed a diffuse cytoplasmic staining pattern with no preference for any particular organelle. Key words: Enamel - - Mineralization - - Calcium.
Introduction It has recently been reconfirmed that the mineralization of rat enamel occurs in two distinct stages [8]. The greatest quantities of mineral in absolute terms are deposited by the post-secretory or reduced ameloblasts after the full thickness of enamel matrix has been formed [3, 15, 16]. However, during the formative phase of amelogenesis some mineral ( 3 0 - 3 5 % of the final mineral content) is added to the organic matrix of enamel as soon as it is secreted by the secretory ameloblasts [7], and, considering the high appositional rate of enamel matrix formation in the rat, the requirement for calcium per unit time must be very high during this phase of amelogenesis as well. Send offprint requests to D.A. Deporter at the above address
Little information exists in the literature on the cellular mechanisms involved in the early mineralization phase. Therefore, as part of an ongoing study of mechanisms of mineralization, it was decided to investigate the fine structural distribution of calcium deposits during early amelogenesis of rat molars using the potassium pyroantimonate staining procedure [19].
Materials and Methods Four-day-old Wistar rats were killed by decapitation. The maxillary first molar tooth germs were dissected out and fixed for 90 min at 4 ~ C in an aqueous mixture of 0.05 M potassium pyroantimonate (Fisher Scientific) and 1% osmium tetroxide, pH 7.5 [19]. Controls were fixed in an aqueous mixture of 0.05 M potassium pyrophosphate and 1% osmium tetroxide. As an additional control some tooth germs were pretreated at 4 ~ C with 5 mM EGTA ((ethylenebis (oxyethylenenitrilo) tetra-acetic acid)) in 0.05 M Tris buffer, pH 7.4 for 15 to 30 rain and washed in Tris buffer without EGTA before being fixed with the potassium pyroantimonateosmium tetroxide mixture. Following fixation the specimens were dehydrated through graded alcohols and propylene oxide and embedded in Epon. Ultrathin sections were examined unstained and, after staining with 70% methanolic uranyl acetate, using a Phillips 200 electron microscope.
Results Electron dense calcium pyroantimonate deposits were observed in several locations depending upon the state of differentiation of the ameloblast. There were no deposits observed in the undifferentiated cells of the internal enamel epithelium. However, as these cells differentiated into ameloblasts, distinct and differing patterns of calcium localization were found seemingly associated with the development of Tomes' process. Before the development of Tomes' process, in what will be called the early secretory ameloblasts, calcium pyroantimonate deposits were observed in distinct locations (see Figs. 1 and 2). First, deposits were
272
D.A. Deporter: Cellular Localizationof Calcium During Early Amelogenesis
Fig. 1. Early secretory ameloblasts. Calcium pyroantimonate deposits are localized to the plasma membrane, the Golgi region (arrows) and to most mitochondria. Dentine (D) x5236
Fig. 3. Fully differentiated ameloblasts with characteristic Tomes' processes (TP) to show diffuse cytoplasmic staining pattern of calcium pyroantimonatedeposits. Enamel (E) x 8640
vesicles in the apical or secretory pole of the cells. Many of the coated vesicles seemed to be in the process' of releasing their calcium content into the stippled material. Finally, calcium pyroantimonate deposits were observed within most, if not all, of the mitochondria of the early secretory ameloblasts. These deposits were localized to the intracristal spaces of the mitochondria. As the cells became fully differentiated into secretory ameloblasts with well recognizable Tomes' processes the pattern of calcium pyroantimonate deposits changed. The precise localization described above was no longer observed but was replaced by a diffuse cytoplasmic staining pattern which was primarily localized to the secretory pole of the ameloblasts (Fig. 3). None of the deposits described above were observed in the control specimens.
Fig. 2. Early secretory ameloblast~s. Some cytoplasmic vesicles (arrows) are discharging their calcium content into the stippled material (S). Dentine (D) x 20,328
localized to the inner or cytoplasmic aspect of the plasma membrane including the apical plasma membrane adjacent to the newly secreted stippled material of the pre-enamel matrix [18]. Second, deposits were found within the Golgi apparatus and within coated
Discussion It is not known how the secretory ameloblast mediates partial mineralization of the newly deposited enamel matrix. Ultrastructural, autoradiographic and biochemical studies of other calcium secreting epithelial cells, such as the mucosal cells of the calciferous glands of the earthworm [13] and the mucosal ceils of the avian shell gland [9], have demonstrated that the mechanism of calcium secretion probably involves the
D,A. Deporter: Cellular Localization of Calcium During Early Amelogenesis
packaging and secretion of calcium within cytoplasmic vesicles of Golgi origin. Thus, it may be that, as argued by Deporter and Ten Cate [6], calcium is secreted by secretory ameloblasts via Golgi vesicles, possibly within the same vesicles that contain the organic matrix of enamel. The results of the present study provide some support for this hypothesis of early mineralization of enamel in that it was possible to demonstrate calcium deposits within the Golgi apparatus and within cytoplasmic vesicles of the early secretory ameloblasts. However, as the ameloblasts became fully differentiated into secretory ameloblasts with well developed Tomes' processes it was no longer possible to demonstrate these sites of calcium localization. This finding suggests that the early mineralization of enamel may involve two distinct mechanisms. The initial mineralization, that is that mediated by the early secretory ameloblasts, may involve the packaging of calcium by the Golgi apparatus into secretory vesicles which then discharge their calcium content by reverse endocytosis at the secretory pole of the cell. Thereafter, once the initial mineralization of the first formed enamel matrix has occurred and the secretory ameloblasts have become fully differentiated, the mechanism of enamel mineralization may change. Calcium may then diffuse freely through the cytoplasm of the secretory ameloblasts as suggested by previous workers [1, 11, 12]. It should be noted that Nagai and Frank [12], in their electron microscope autoradiographic study of 45Ca distribution during amelogenesis in the cat, suggested that large quantities of calcium also diffused to the mineralization front of enamel between the secretory ameloblasts. Although calcium has been demonstrated extracellularly between odontoblasts using the potassium pyroantimonate technique [14;] (Deporter and Belzycki, in preparation), calcium deposits were never observed between the secretory ameloblasts in the present study. It may be that the observations of Nagai and Frank are due to the imprecise localization of silver grains inherent in electron microscope autoradiographic technique. The plasma membrane localization of calcium in the early secretory ameloblasts suggests that these cells store large quantities of calcium at this membrane site for later use after they have differentiated into mature secretory ameloblasts. The absence of calcium pyroantimonate deposits on the exterior plasma membrane surface of the early secretory ametoblasts indicates that the calcium does not diffuse across the cell membrane. The demonstration of calcium pyroantimonate deposits within the mitochondria of early secretory ameloblasts is of special interest in view of the proposed role for mitochondria in mineralization of
273
various hard tissues. Similar deposits have previously been reported in the mitochondria of hypertrophic cartilage cells just before the onset of mineralization of epiphyseal cartilage [5]. Moreover, results from numerous workers using a variety of different techniques have suggested that mitochondria store calcium in preparation for the onset of a mineralization process [2, 4, 5, 9, 10, 17]. Thus, for example, Hohman and Schraer [9] demonstrated that 4~Ca accumulated in the mitochondria of mucosal cells of the avian shell gland before mineralization of the egg but was later discharged from mitochondria with the onset of mineralization. The present demonstration of calcium deposits within mitochondria of early secretory ameloblasts but not in the mitochondria of fully differentiated secretory ameloblasts provides further evidence for this proposed role for mitochondria in mineralization. Acknowledgments. The author is a Scholar of the Medical Research Council of Canada. He wishes to thank Mr. George Pudy and Mrs. Amy Shiga for their expert technical assistance, Mrs. Orrie Warren for typing the manuscript and Professor A.R. Ten Care for his critical evaluation of the text.
References 1. Boyd, A., Reith, E.J.: Qualitative electron probe analysis of secretory ameloblasts and odontoblasts in the rat incisor. Histochemistry 50, 347-354 (1977) 2. Bechtel, D.B., Horner, H.T.: Calcium excretion and deposition during sporogenesis in Physarella oblonga. Calcif. Tiss. Res. 18, 195-213 (1975) 3. Beynon, A.D.: Enzymes in enamel maturation. Proc. Roy. Soc. Med. 65, 911-912 (1972) 4. Brighton, C.T., Hunt, R.M.: Mitochondrial calcium and its role in calcification. Histochemical localization of calcium in electron micrographs of the epiphyseal growth plate with Kpyroantimonate. Clin. Orthop. Rel. Res. 100, 406-416 (1976) 5. Brighton, C.T., Hunt, R.M.: Histochemical localization of calcium in growth plate mitochondria and matrix vesicles. Fed. Proc. 35, 143-147 (1976) 6. Deporter, D.A., Ten Cate, A.R.: Fine structural localization of alkaline phosphatase in relation to enamel formation in the mouse molar. Archs. Oral Biol.21, 7-12 (1976) 7. Frank, R.M., Nalbandian, J.: Ultrastructure of amelogenesis. In: Structural and chemical organization of teeth (A.E.W. Miles, ed.), pp. 399-466. New York: Academic Press 1967 8. Glick, P.L., Edie, J.W.: Mineralization of rat enamel: a c o m bined electron-microprobe, electron microscopic and his tologic study. Jour. Dent. Res. 54A, abstract 348, 1975 9. Hohman, W., Schraer, H.: The intracellular distribution of calcium in the mucosa of the avian shell gland. J. Cell Biol. 30, 317-331 (1966) 10. Mathews, J.L., Martin, J.H., Arsenis, C., Eisenstein, R., Kuettner, K . : The role of mitochondria in intracellular calcium regulation. In: Cellular mechanisms for calcium transfer and homeostasis (G. Nichols and R.H. Wasserman, eds.), pp. 239-255. New York: Academic Press 1971
274
D.A. Deporter: Cellular Localization of Calcium During Early Amelogenesis
11. Munhoz, C.O.G., Leblond, C.P.: Deposition of calcium phosphate into dentine and enamel as shown by radiography of sections of incisor teeth following injection of 45Ca into rats. Calcif. Tiss. Res. 15, 221-235 (1974) 12. Nagai, N., Frank, R.M.: Tranfert du 45Ca par autoradiographic en microscopic electronique au cours de l'amelogenese. Calcif. Tiss. Res. 19, 211-221 (1975) 13. Nakahara, H., Bevelander, G.: An electron microscope and autoradiographic study of the calciferous glands of the earthworm, Lumbricus terrestris. Calcif. Tiss. Res. 4, 193-201 (1969) 14. Reith, E.J.: The binding of calcium within the Golgi saccules of the rat odontoblast. Amer. J. Anat. 147, 267-272 (1976) 15. Reith, E.J., Cotty, V.F.: Autoradiographic studies on calcification of enamel. Archs. Oral Biol. 7, 365-372 (1962)
16. Reith, E.J., Cotty, V.F.: The absorptive activity of ameloblasts during the maturation of enamel. Anat. Rec. 157, 577588 (1967). 17. Shapiro, I.M., Greenspan, J.S.: Are mitochondria directly involved in biological mineralization? Calcif. Tiss. Res. 3, 100-102 (1969) 18. Slavkin, H.C., Mino, W., Bringas, P.: The biosynthesis and secretion of precursor enamel protein by ameloblasts as visualized by autoradiography after tryptophan administration. Anat. Rec. 185, 289-312 (1976) 19. Spicer, S.S., Hardin, J.H., Green, W.B.: Nuclear precipitates in pyroantimonate-osmium tetroxide-fixed tissues. J. Cell Biol. 39, 216-221 (1968)
Received May 19 / Revised August 19 / Accepted August 22, 1977
