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Ultrastructural observations of initial calcification in dentine and enamel
Copyright © 1972 by Academic Press, lnc. All rights of reproduction in any form reserved
J. ULTRASTRUCTURERESEARCH41, 1-17 (1972)
Ultrastructural Observations of Initial Calcification in Dentine and Enamel GEORGE W. BERNARD Division of Oral Biology, School of Dentistry, and Department of Anatomy, School of Medicine, University of California, Los Angeles 90024 Received July 31, 1969, and in revised form April 12, 1972 An electron microscopic study of developing mandibular molars of SwissWebster mice indicates that the pattern of initial hydroxyapatite formation in dentine is identical with woven bone. Initial calcification loci are the cellular extensions or "buds" of odontoblasts into the predentine. Many of these cellular elements have intact plasma membranes when crystallization has just begun. These membranes disappear when calcification is more extensive. Hydroxyapatite growing radially from these initial calcification loci becomes spheroidal. These spheroids coalesce to form seams of the first formed dentine, mantle dentine. Later circumpulpal calcification develops from the previously formed territorial zone of calcified mantle dentine. When mantle dentine forms a continuous layer, the basement lamina underlying the ameloblasts disappears. Pre-enamel protein is secreted which almost immediately calcifies. The first enamel crystals are coextensive with hydroxyapatite of dentine at the dentinoenamel junction. The dentinal hydroxyapatitic territorial domain appears to be the source of secondary crystallization growth centers for enamel crystals. The calcification of dentine and enamel has been investigated from many points of view. As in bone, very little has been reported on the very earliest in vivo apatite crystal formation (5). Rather, most investigators have concentrated on the mineralization of the organic matrix after the original initiation of crystal formation. In vitro studies have eliminated the cellular components of mineralizing tissues and so have oversimplified a rather complex biological process. Glimcher (9) discusses the nucleation of hydroxyapatite crystals only in relationship with the "hole zones" of collagen. This approach emphasizes the role of the organic matrix while diminishing the role of the cell in the process. This has led to the assumption that initial and subsequential calcification follow the same obligatory process of nucleation. Since this approach is apparently not valid in the process of ossification for immature bone, which forms differently from maturing bone (3, 5) it seemed reasonable to assume that dentine and enamel would also calcify by different processes in the initial and maturing phases.
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In order to investigate this possibility, developing mouse molar tooth buds were prepared and examined by electron microscopy to demonstrate initial calcification in dentine and enamel. METHODS AND MATERIALS Developing mandibular molar tooth germs from newborn Swiss-Webster mice were fixed in 2.5 % glutaraldehyde buffered in 0.1 M cacodylate and post fixed in 1.67% osmium tetroxide with identical buffer. After dehydration in a graded series of alcohols, the tissue was embedded in Epon, sectioned on a Porter-Blum MT1 ultramicrotome, stained with uranyl acetate and lead citrate (15) and viewed for electron microscopy with a Siemens IA electron microscope. OBSERVATIONS
Dentine When predentine begins to calcify, the first crystals of hydroxyapatite are demonstrated in, and adjacent to, cellular "buds" which are probably shed from odontoblasts (Fig. 1). These are the initial calcification loci and are located at some distance from the apical end of the cells towards the presumptive dentinoenamel junction. Randomly oriented collagen fibrils enmesh the cellular "buds," some of which have become initial calcification loci. At the time of initial calcification the basement lamina underlying the ameloblasts is still relatively intact (Figs. I and 3). The cellular extensions or pseudopodal "buds" have a variable structure. Some are filled with an amorphous electron dense substance (Figs. 2 and 5), others are granular (Figs. 3 and 5), still others have frank apatitic crystals associated with them (Figs. 2-6). In the earliest stages of crystal formation, portions of the plasma membrane are still intact around the "buds" (Figs. 2-4). Later, when crystal growth is more abundant, the plasma membrane disappears (Figs. 5 and 6). Crystals grow randomly from these initial calcification loci and in time become arranged into a spheroidal form (Fig. 7). These spheroids are initial calcification nodules and are homologous to the bone nodules of immature bone. Hydroxyapatite crystals from these extend into, onto, and between collagenous fibrils (Fig. 7). Some nodules coalesce, and with calcified collagen form seams of calcified dentine (Fig. 7). This early dentine is mantle dentine and is characterized by irregularities and structural unevenness due to the randomness of the calcified collagen and to the abundance of initial calcification nodules. After the initiation of calcification, hydroxyapatite crystals grow epitaxially into the predentine. Here the collagenous fibrils orient and help structure crystal growth (Fig. 8). The structural irregularities caused by an abundance of calcification and irregularly FIG. 1. A zone of initial calcification of predentine with numerous cellular extensions from odontoblasts (off the field) and several differentiatingameloblasts with underlyingbasement lamina, x 16 500.
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FIG. 2. Predentine with numerous initial calcification loci ("buds"). A few, containing hydroxyapatite crystals, still have an intact portion of their plasma membranes (arrows). x 40 000. FIG. 3. Predentine with several " b u d s " from odontoblastic extensions, at least two of which contain hydroxyapatite crystals (arrows). x 40 000. FI~. 4. Two initial calcification loci with different stages of calcification, each with intact plasma membranes (arrows). x 44 000.
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F ~ . 5. Initial calcification loci with various stages of calcification, one has granular inclusions (arrow). Another area has hydroxyapatite crystals growing randomly with no apparent plasma membrane (2 arrows), x 40 000.
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FIG. 6. Several crystals are related to one initial calcification locus (I arrow) while one "bud" has a single crystal internal to the plasma membrane (2 arrows). × 40 000.
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arranged collagen are the distinguishing features of mantle dentine. After 3-4/z of growth, mantle dentine nodules develop into circumpulpal dentine which is characterized by few calcification nodules, large randomly distributed, calcified collagenous fibrils and a tightly woven meshwork of hydroxyapatite crystals (Fig. 9).
Enamel Prior to the complete formation of mantle dentine, the basement lamina underlying the ameloblasts disappears (12, 13). This is coincident with the formation of enamel protein in the pre-enamel (Fig. 10). Continuity is therefore established between calcified mantle dentine and the protein of the pre-enamel (Fig. 12). Into this organic framework, crystals of hydroxyapatite appear to grow from already formed crystals in the dentine (Figs. 11-13). Enamel crystals, although hydroxyapatite, can be readily distinguished from hydroxyapatite crystals of dentine by their elongated shape, which is possibly due to the special orientation of enamel protein (Figs. 12 and 13).
DISCUSSION Pautard rather prophetically stated in 1965, "... the cell creates the image of the mineral." Since then, observations of initial calcification in mammalian tissue has been demonstrated to be a cellularly related event in immature or woven bone (3-5); and in calcified cartilage (i, 6, 11). The initial calcification loci "vesicles" or " b u d s " are almost never connected to cartilage cells (1, 6) or to bone cells (5) yet are cellularly derived from the cells in juxtaposition to the calcifying front. In this study, the first calcification seen in dentine is also within or on a cellular process. It is interesting to note that in each of these mineralized tissues there is no precedent calcification; that is, calcification is a de novo event in a previously unmineralized matrix. Contrasted with this, the mineralization of lamellar bone (3) and, as the present study shows, the mineralization of circumpulpal dentine as well as enamel are subsequential events to a previously formed territorial area of calcification. This would indicate that there are two predominant modes in the calcification process; the initial immature process FIG. 7. Mantle dentine with a spheroidal calcification nodule. A partially decalcified nodule contains an initial calcification locus in the center (arrow). x 66 000. FIG. 8. Beginning circumpulpal dentine. Collagenous fibrils orient and help structure hydroxyapatitic crystal growth. Note that the primary growth is from the previously formed territorial zone of calcification x 66 000. FIG. 9. An area of circumpulpal dentine with the newest crystals formed at the interface of the territorial zone of calcification and the predentine, x 55 000. FIG. 10. Regularlyarranged enamel protein is secreted by the ameloblast (A) in juxtaposition to the calcified dentine (D). This begins to calcify almost immediately, x 50 000. FIG. 11. Crystals of enamel hydroxyapatite appear to grow from the dentinaI crystals, probably oriented by enamel protein, x 50 000.
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F~G. 13. Enamel crystals appearing to take origin in the dentine, x 72 000.
FIo. 12. Elongated enamel crystals appearing to grow into the pre-enamel from the territorial calcification zone of dentine. × 66 000. 2 - 721836 J . Ultrastructure Research
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and the subsequent mature one. Immature calcification begins in "buds" from the cellular extensions of osteoblasts, chondrocytes, and odontoblasts. It would appear that nucleation of the youngest crystals of hydroxyapatite takes place intracellularly rather than within the organic matrix. It is only after the crystals have grown beyond the initial cellular calcification loci that the matrix becomes involved in crystal growth. It is Glimcher's view (8) that each crystal has its own nucleation site within the "hole zones" of collagen and that calcification is primarily dependent upon the concentration of calcium and phosphate ions in the vicinity of the "hole zones." With this theory, it is necessary to postulate a reason for the lack of calcification in the organic matrix near the cells, since the combination of ion concentration and "holes" should cause nucleation to occur wherever this combination exists. Glimcher (8), in fact, speculates about this by suggesting that mucopolysaccharides act as cationic binders to inhibit calcification in the uncalcified matrix. The current study, as well as preceding ones (3, 5), suggests that the involvement of structural protein is secondary to initial crystallization and that protein orients and structures the size of crystals rather than organizes their nucleation. In fact, Takuma (14) has stated, "There is some indication that the morphological nature of crystals is related to the situation in which they are deposited." This is not to say that nucleation of crystals does not occur in collagen or in enamel protein. However, our observations indicate that the calcification process originates in initial calcification loci, which subsequently grow by secondary crystallization from already formed crystals, in, on, and between collagen and enamel protein. According to our view there is no need to postulate an inhibitory mechanism in osteoid, predentine, or preenamel during active, early mineralization. Rather, crystallization proceeds from the already formed territorial domain of hydroxyapatite as new crystals grow into the organic matrix by secondary crystallization from the previously crystallized hydroxyapatite. Growth of the crystals in bone and dentine would follow mucosubstance zones found within and surrounding the collagen filaments since the space between these filaments in always filled with mucosubstances (7). These spaces would make a dimensionally and conceptually appropriate pathway for growth of mineral crystals. The precalcification zones are areas of newly secreted matrix awaiting crystallization from the territorial domain of hydroxyapatite. The question of why there is always an uncalcified zone adjacent to mature resting cells of bone and dentine can best be understood by the effects of constant turnover of crystals and matrix which are activated by enzymes secreted by mature cells (2). Mantle dentine follows a similar pattern of cellularly initiated immature calcification to that of woven bone and calcified cartilage. In circumpulpal dentine, the organic matrix calcifies in much the same way as lamellar bone (4). The pattern of enamel calcification follows an interesting temporal pattern. When ameloblasts line
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up at the presumptive dentine-enamel junction a thick basement lamina is always present. The dentine begins to calcify first. Dentinogenesis begins with initial calcification loci in the presence of an intact basement lamina, but this disappears prior to the formation of a discernible layer of mantle dentine (12, 13). Enamel protein appears to be secreted simultaneously with the disappearance of the basement lamina. This protein calcifies almost immediately, the first crystals being oriented in juxtaposition with, and apparently growing from, the territorial domain of hydroxyapatite of the dentine. Of considerable importance is Glimcher's (10) finding that young calcification of enamel always gives the X-ray diffraction pattern of hydroxyapatite without going through an amorphous stage. This would substantiate, at least in part, the point of view that enamel hydroxyapatite grows by secondary crystallization of mature hydroxyapatite already formed in dentine. Enamel follows a pattern of subsequential calcification similar to that of lamellar bone and circumpulpal dentine. REFERENCES 1. ANDERSON,H. C., J. Cell Biol. 41, 59 (1969). 2. B~LANGER,L., SEMBA,T., TOLNAI,S., CoPP, D. H., KROOK,L. and GRIES,C., in FLEISCH, H., BLACKWOOD,J. J. J. and OWENM. (Eds.), Calcified Tissues 1965, p. 1. SpringerVerlag, Berlin and New York, 1966. 3. BERNARD,G. W., J. Dent. Res. 48, Suppl. 5 (1969). 4. BERNARD,G. W. and PEASE,n . C., d. Appl. Phys. 37, 3932 (1966). 5. - Amer. J. Anat. 125, 3 (1969). 6. BONUCCI,E. J., d. Ultrastruct. Res. 20, 33 (1967). 7. BOUTEILLE,M. and PEASE,D. C., Anat. Rec. 163, 157 (1969). 8. GLIMCHER,M. J., Rev. Nod. Phys. 31, 359 (1959). 9. - Clin. Orthop. 61, 16 (1968). 5th Syrnp. on Biology of Hard Tissue, March 9-12, 1969, Santa Ynez, Caiifornia. 10. - Interdisciplinary Communications. Program. 11. MATHEWS,J. L., MARTIN,J. H. and COLLINS,E. J., Clin. Orthop. 58, 213 (1968). 12. PANNESE,E., d. Ultrastruct. Res. 6, 186 (1962). 13. REITrI, E., or. Ultrastruct. Res. 17, 503 (1967). 14. TAKUMA,S., in MILES, A. E. W. (Ed.), Structural and Chemical Organization of Teeth, Vol. 1, p. 325. Academic Press, New York, 1967. 15. VENABLE,J. H. and COGaESHALL,R., J. Ceil Biol. 25, 407 (1965).
J. ULTRASTRUCTURERESEARCH41, 1-17 (1972)
Ultrastructural Observations of Initial Calcification in Dentine and Enamel GEORGE W. BERNARD Division of Oral Biology, School of Dentistry, and Department of Anatomy, School of Medicine, University of California, Los Angeles 90024 Received July 31, 1969, and in revised form April 12, 1972 An electron microscopic study of developing mandibular molars of SwissWebster mice indicates that the pattern of initial hydroxyapatite formation in dentine is identical with woven bone. Initial calcification loci are the cellular extensions or "buds" of odontoblasts into the predentine. Many of these cellular elements have intact plasma membranes when crystallization has just begun. These membranes disappear when calcification is more extensive. Hydroxyapatite growing radially from these initial calcification loci becomes spheroidal. These spheroids coalesce to form seams of the first formed dentine, mantle dentine. Later circumpulpal calcification develops from the previously formed territorial zone of calcified mantle dentine. When mantle dentine forms a continuous layer, the basement lamina underlying the ameloblasts disappears. Pre-enamel protein is secreted which almost immediately calcifies. The first enamel crystals are coextensive with hydroxyapatite of dentine at the dentinoenamel junction. The dentinal hydroxyapatitic territorial domain appears to be the source of secondary crystallization growth centers for enamel crystals. The calcification of dentine and enamel has been investigated from many points of view. As in bone, very little has been reported on the very earliest in vivo apatite crystal formation (5). Rather, most investigators have concentrated on the mineralization of the organic matrix after the original initiation of crystal formation. In vitro studies have eliminated the cellular components of mineralizing tissues and so have oversimplified a rather complex biological process. Glimcher (9) discusses the nucleation of hydroxyapatite crystals only in relationship with the "hole zones" of collagen. This approach emphasizes the role of the organic matrix while diminishing the role of the cell in the process. This has led to the assumption that initial and subsequential calcification follow the same obligatory process of nucleation. Since this approach is apparently not valid in the process of ossification for immature bone, which forms differently from maturing bone (3, 5) it seemed reasonable to assume that dentine and enamel would also calcify by different processes in the initial and maturing phases.
2
BERNARD
In order to investigate this possibility, developing mouse molar tooth buds were prepared and examined by electron microscopy to demonstrate initial calcification in dentine and enamel. METHODS AND MATERIALS Developing mandibular molar tooth germs from newborn Swiss-Webster mice were fixed in 2.5 % glutaraldehyde buffered in 0.1 M cacodylate and post fixed in 1.67% osmium tetroxide with identical buffer. After dehydration in a graded series of alcohols, the tissue was embedded in Epon, sectioned on a Porter-Blum MT1 ultramicrotome, stained with uranyl acetate and lead citrate (15) and viewed for electron microscopy with a Siemens IA electron microscope. OBSERVATIONS
Dentine When predentine begins to calcify, the first crystals of hydroxyapatite are demonstrated in, and adjacent to, cellular "buds" which are probably shed from odontoblasts (Fig. 1). These are the initial calcification loci and are located at some distance from the apical end of the cells towards the presumptive dentinoenamel junction. Randomly oriented collagen fibrils enmesh the cellular "buds," some of which have become initial calcification loci. At the time of initial calcification the basement lamina underlying the ameloblasts is still relatively intact (Figs. I and 3). The cellular extensions or pseudopodal "buds" have a variable structure. Some are filled with an amorphous electron dense substance (Figs. 2 and 5), others are granular (Figs. 3 and 5), still others have frank apatitic crystals associated with them (Figs. 2-6). In the earliest stages of crystal formation, portions of the plasma membrane are still intact around the "buds" (Figs. 2-4). Later, when crystal growth is more abundant, the plasma membrane disappears (Figs. 5 and 6). Crystals grow randomly from these initial calcification loci and in time become arranged into a spheroidal form (Fig. 7). These spheroids are initial calcification nodules and are homologous to the bone nodules of immature bone. Hydroxyapatite crystals from these extend into, onto, and between collagenous fibrils (Fig. 7). Some nodules coalesce, and with calcified collagen form seams of calcified dentine (Fig. 7). This early dentine is mantle dentine and is characterized by irregularities and structural unevenness due to the randomness of the calcified collagen and to the abundance of initial calcification nodules. After the initiation of calcification, hydroxyapatite crystals grow epitaxially into the predentine. Here the collagenous fibrils orient and help structure crystal growth (Fig. 8). The structural irregularities caused by an abundance of calcification and irregularly FIG. 1. A zone of initial calcification of predentine with numerous cellular extensions from odontoblasts (off the field) and several differentiatingameloblasts with underlyingbasement lamina, x 16 500.
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FIG. 2. Predentine with numerous initial calcification loci ("buds"). A few, containing hydroxyapatite crystals, still have an intact portion of their plasma membranes (arrows). x 40 000. FIG. 3. Predentine with several " b u d s " from odontoblastic extensions, at least two of which contain hydroxyapatite crystals (arrows). x 40 000. FI~. 4. Two initial calcification loci with different stages of calcification, each with intact plasma membranes (arrows). x 44 000.
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F ~ . 5. Initial calcification loci with various stages of calcification, one has granular inclusions (arrow). Another area has hydroxyapatite crystals growing randomly with no apparent plasma membrane (2 arrows), x 40 000.
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FIG. 6. Several crystals are related to one initial calcification locus (I arrow) while one "bud" has a single crystal internal to the plasma membrane (2 arrows). × 40 000.
BERNARD
arranged collagen are the distinguishing features of mantle dentine. After 3-4/z of growth, mantle dentine nodules develop into circumpulpal dentine which is characterized by few calcification nodules, large randomly distributed, calcified collagenous fibrils and a tightly woven meshwork of hydroxyapatite crystals (Fig. 9).
Enamel Prior to the complete formation of mantle dentine, the basement lamina underlying the ameloblasts disappears (12, 13). This is coincident with the formation of enamel protein in the pre-enamel (Fig. 10). Continuity is therefore established between calcified mantle dentine and the protein of the pre-enamel (Fig. 12). Into this organic framework, crystals of hydroxyapatite appear to grow from already formed crystals in the dentine (Figs. 11-13). Enamel crystals, although hydroxyapatite, can be readily distinguished from hydroxyapatite crystals of dentine by their elongated shape, which is possibly due to the special orientation of enamel protein (Figs. 12 and 13).
DISCUSSION Pautard rather prophetically stated in 1965, "... the cell creates the image of the mineral." Since then, observations of initial calcification in mammalian tissue has been demonstrated to be a cellularly related event in immature or woven bone (3-5); and in calcified cartilage (i, 6, 11). The initial calcification loci "vesicles" or " b u d s " are almost never connected to cartilage cells (1, 6) or to bone cells (5) yet are cellularly derived from the cells in juxtaposition to the calcifying front. In this study, the first calcification seen in dentine is also within or on a cellular process. It is interesting to note that in each of these mineralized tissues there is no precedent calcification; that is, calcification is a de novo event in a previously unmineralized matrix. Contrasted with this, the mineralization of lamellar bone (3) and, as the present study shows, the mineralization of circumpulpal dentine as well as enamel are subsequential events to a previously formed territorial area of calcification. This would indicate that there are two predominant modes in the calcification process; the initial immature process FIG. 7. Mantle dentine with a spheroidal calcification nodule. A partially decalcified nodule contains an initial calcification locus in the center (arrow). x 66 000. FIG. 8. Beginning circumpulpal dentine. Collagenous fibrils orient and help structure hydroxyapatitic crystal growth. Note that the primary growth is from the previously formed territorial zone of calcification x 66 000. FIG. 9. An area of circumpulpal dentine with the newest crystals formed at the interface of the territorial zone of calcification and the predentine, x 55 000. FIG. 10. Regularlyarranged enamel protein is secreted by the ameloblast (A) in juxtaposition to the calcified dentine (D). This begins to calcify almost immediately, x 50 000. FIG. 11. Crystals of enamel hydroxyapatite appear to grow from the dentinaI crystals, probably oriented by enamel protein, x 50 000.
INITIAL C A L C I F I C A T I O N OF D E N T I N E A N D ENAMEL
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F~G. 13. Enamel crystals appearing to take origin in the dentine, x 72 000.
FIo. 12. Elongated enamel crystals appearing to grow into the pre-enamel from the territorial calcification zone of dentine. × 66 000. 2 - 721836 J . Ultrastructure Research
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and the subsequent mature one. Immature calcification begins in "buds" from the cellular extensions of osteoblasts, chondrocytes, and odontoblasts. It would appear that nucleation of the youngest crystals of hydroxyapatite takes place intracellularly rather than within the organic matrix. It is only after the crystals have grown beyond the initial cellular calcification loci that the matrix becomes involved in crystal growth. It is Glimcher's view (8) that each crystal has its own nucleation site within the "hole zones" of collagen and that calcification is primarily dependent upon the concentration of calcium and phosphate ions in the vicinity of the "hole zones." With this theory, it is necessary to postulate a reason for the lack of calcification in the organic matrix near the cells, since the combination of ion concentration and "holes" should cause nucleation to occur wherever this combination exists. Glimcher (8), in fact, speculates about this by suggesting that mucopolysaccharides act as cationic binders to inhibit calcification in the uncalcified matrix. The current study, as well as preceding ones (3, 5), suggests that the involvement of structural protein is secondary to initial crystallization and that protein orients and structures the size of crystals rather than organizes their nucleation. In fact, Takuma (14) has stated, "There is some indication that the morphological nature of crystals is related to the situation in which they are deposited." This is not to say that nucleation of crystals does not occur in collagen or in enamel protein. However, our observations indicate that the calcification process originates in initial calcification loci, which subsequently grow by secondary crystallization from already formed crystals, in, on, and between collagen and enamel protein. According to our view there is no need to postulate an inhibitory mechanism in osteoid, predentine, or preenamel during active, early mineralization. Rather, crystallization proceeds from the already formed territorial domain of hydroxyapatite as new crystals grow into the organic matrix by secondary crystallization from the previously crystallized hydroxyapatite. Growth of the crystals in bone and dentine would follow mucosubstance zones found within and surrounding the collagen filaments since the space between these filaments in always filled with mucosubstances (7). These spaces would make a dimensionally and conceptually appropriate pathway for growth of mineral crystals. The precalcification zones are areas of newly secreted matrix awaiting crystallization from the territorial domain of hydroxyapatite. The question of why there is always an uncalcified zone adjacent to mature resting cells of bone and dentine can best be understood by the effects of constant turnover of crystals and matrix which are activated by enzymes secreted by mature cells (2). Mantle dentine follows a similar pattern of cellularly initiated immature calcification to that of woven bone and calcified cartilage. In circumpulpal dentine, the organic matrix calcifies in much the same way as lamellar bone (4). The pattern of enamel calcification follows an interesting temporal pattern. When ameloblasts line
INITIAL CALCIFICATIONOF DENTINEAND ENAMEL
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up at the presumptive dentine-enamel junction a thick basement lamina is always present. The dentine begins to calcify first. Dentinogenesis begins with initial calcification loci in the presence of an intact basement lamina, but this disappears prior to the formation of a discernible layer of mantle dentine (12, 13). Enamel protein appears to be secreted simultaneously with the disappearance of the basement lamina. This protein calcifies almost immediately, the first crystals being oriented in juxtaposition with, and apparently growing from, the territorial domain of hydroxyapatite of the dentine. Of considerable importance is Glimcher's (10) finding that young calcification of enamel always gives the X-ray diffraction pattern of hydroxyapatite without going through an amorphous stage. This would substantiate, at least in part, the point of view that enamel hydroxyapatite grows by secondary crystallization of mature hydroxyapatite already formed in dentine. Enamel follows a pattern of subsequential calcification similar to that of lamellar bone and circumpulpal dentine. REFERENCES 1. ANDERSON,H. C., J. Cell Biol. 41, 59 (1969). 2. B~LANGER,L., SEMBA,T., TOLNAI,S., CoPP, D. H., KROOK,L. and GRIES,C., in FLEISCH, H., BLACKWOOD,J. J. J. and OWENM. (Eds.), Calcified Tissues 1965, p. 1. SpringerVerlag, Berlin and New York, 1966. 3. BERNARD,G. W., J. Dent. Res. 48, Suppl. 5 (1969). 4. BERNARD,G. W. and PEASE,n . C., d. Appl. Phys. 37, 3932 (1966). 5. - Amer. J. Anat. 125, 3 (1969). 6. BONUCCI,E. J., d. Ultrastruct. Res. 20, 33 (1967). 7. BOUTEILLE,M. and PEASE,D. C., Anat. Rec. 163, 157 (1969). 8. GLIMCHER,M. J., Rev. Nod. Phys. 31, 359 (1959). 9. - Clin. Orthop. 61, 16 (1968). 5th Syrnp. on Biology of Hard Tissue, March 9-12, 1969, Santa Ynez, Caiifornia. 10. - Interdisciplinary Communications. Program. 11. MATHEWS,J. L., MARTIN,J. H. and COLLINS,E. J., Clin. Orthop. 58, 213 (1968). 12. PANNESE,E., d. Ultrastruct. Res. 6, 186 (1962). 13. REITrI, E., or. Ultrastruct. Res. 17, 503 (1967). 14. TAKUMA,S., in MILES, A. E. W. (Ed.), Structural and Chemical Organization of Teeth, Vol. 1, p. 325. Academic Press, New York, 1967. 15. VENABLE,J. H. and COGaESHALL,R., J. Ceil Biol. 25, 407 (1965).