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Ultrastructure of initial crystal formation in dentin
Copyright © 1972 by Academic Press, Inc. All rights of reprcduction in any form reserved
18
J. ULTRASTRUCTURERESEARCH41, 18-28 (1972)
U l t r a s t r u c t u r e of Initial Crystal F o r m a t i o n in D e n t i n ~ DALE R. EISENMANN a n d PAUL L. GLICK Department o f Histology, University o f Illinois at the Medical Center, Chicago, Illinois 60680
Received January 3, 1972 The present study illustrates a structure of apparent cellular origin in which the first observable mineral crystals of dentin appear. This information supplies a necessary link in the present understanding of the chain of events leading to mineralization of calcified tissues. It is well known that the organic components of tissues such as dentin, bone, and calcified cartilage are synthesized and secreted by closely associated formative cells. Studies of the mineralization process have concentrated on the role of various components of the organic matrix acting independently of the formative cells as templates for initial calcium and phosphate deposition. The direction for this research was initiated by the epitactic theory of Neuman and Neuman (21). They proposed that supersaturated calcium and phosphate in the body fluids interact directly with nucleating templates in the organic matrix of calcifiable tissues to produce hydroxyapatite crystals. Bonucci recently reviewed the history of this work and discussed a new direction for research in this area (8). It is clear that despite innumerable attempts to determine the site upon which nucleation occurs, few widely accepted conclusions were reached which have not been seriously challenged. Attention is now being turned toward the possibility that the formative cells may participate more directly in the mineralization process. Ultrastructural studies of regions of cartilage and bone matrix which are just beginning to mineralize have revealed evidence which supports a cellular role in this process. Membrane-bound structures termed "calcifying globules" or "matrix vesicles" are observed in the appropriate zones of epiphyseal cartilage; they appear to be of a cellular origin (1, 9, 10). The first observable hydroxyapatite crystals of calcifying cartilage are seen within these structures (10). Similar structures termed "osteoblast extrusions" are seen in bone, and they are active in initial crystal formation. The extrusions have been variously described as processes, buds, or extracellnlar vesicles, all of which appear to be derived from the osteoblast (3, 5, 14). 1 This study was supported in part by Grant No. DE-03312 from the National Institute for Dental
Research.
INITIAL DENTIN MINERALIZATION
19
FI~. 1. Zone of beginning dentin formation. Predentin is located between the odontoblasts (Od) and the developing ameloblasts (A), The predentin zone contains abundant collagen (banded fibers), odontoblast processes (P), and small, round bodies (arrows). A basal lamina runs between the predentin and the adjacent developing ameloblasts. x 19 500.
20
EISENMANNAND GLICK
Little if any ultrastructural evidence of structures such as the calcifying globules of cartilage and osteoblast extrusions of bone has been published with regard to dentin mineralization. The abstract of a presentation by Bernard describes initial dentin mineralization as homologous with initial bone formation in that crystals are first observed within buds from the odontoblasts. To our knowledge, ultrastructural illustration of this finding has not been documented (4). InterfibriUar granular masses were described by Bevelander and Nakahara as accumulations of acid mucopolysaccharide extruded by the odontoblast (6). Initial crystal formation of dentin was reported to occur in these granular masses rather than in relation to collagen. It is not possible to determine if any structural detail may have been associated with these clusters of initial crystal formation. This investigation presents ultrastructural evidence of membrane-bound bodies which contain the first observable crystals of developing dentin.
MATERIALS AND METHODS Male white rats weighing 200-250 g were anesthetized with ether and fixed by arterial perfusion with 4% glutaraldehyde in cacodylate buffer. Incisors were dissected free and cut longitudinally with a rotating diamond wheel while being bathed with saline as a coolant. These large but thin (0.5 mm) blocks were further trimmed, postfixed in 1% OsO4, dehydrated in alcohol, and embedded in Araldite in a plane which permitted sectioning through all stages of incisor development. Thin sections were cut with a diamond knife and floated on trough fluid consisting of a saturated solution of calcium phosphate (11). Some sections were floated on water. The sections were stained with uranyl magnesium acetate and lead citrate and examined in Hitachi 7S and l l A electron microscopes at either 50 or 75 kV.
RESULTS The first mineralization of dentin occurs within a zone which is located between the cell bodies of odontoblasts and developing ameloblasts (Fig. 1). This early predentin zone contains numerous branched odontoblast processes, collagen fibers, rather dense, small, round bodies, and an amorphous background material. It is separated from adjacent developing ameloblasts by a distinct basal lamina. Close examination of the various components of the predentin reveals that the first indication of crystalline material is within small (0.1-0.2 ¢~m in diameter) round, membrane-bound bodies (Fig. 2). The "crystal bodies" (as they will be referred to FIG. 2. Predentin at stage of initial crystal formation. Odontoblast processes (P) and banded collagen fibers constitute the bulk of the predentin. A crystal body (arrow) contains several dense, needlelike crystals within a slightly less dense homogeneous interior. A prominent basal lamina (BL) separates the predentin from the developing ameloblasts. × 91 000.
2 ;i~!i
22
EISENMANN A N D G L I C K
here) are surrounded by collagen fibers which contain no visible crystals at this early stage of dentin mineralization. Since the bodies are always rounded in shape, bearing no special relationship with odontoblast processes, it can be assumed that they are discrete entities. They are f o u n d scattered t h r o u g h o u t the entire zone of early predentin but appear more heavily concentrated near the basal lamina. Although the basic f o r m of the crystal bodies (rounded and m e m b r a n e b o u n d ) i s observed consistently, several variations in substructure are seen within the interior of the bodies (Figs. 3-8). The least mature forms contain a rather h o m o g e n e o u s interior and are devoid of crystals (Fig. 3). Some of the crystal bodies contain a similar h o m o g e n e o u s substructure along with several dense, needlelike crystals (Fig. 4). M a n y of the bodies possess a more complex substructure consisting of localizations of dense material (usually in association with the crystals) and other areas of m u c h lower density with varying patterns of organization (Figs. 5-7). The crystals are seen equally as well in sections which were prepared by floating on water rather than on a saturated solution of calcium phosphate (Fig. 7). Higher magnification illustrates more clearly three components of the enclosing m e m b r a n e (Fig. 8). Inner and outer dense layers are separated by an intervening less dense layer. The entire m e m b r a n e is approximately 80-100 ~ in thickness. Further mineralization is evidenced by increasing numbers of crystals within the crystal bodies (Fig. 9). Their membranes and substructure become obscured as crystal formation and growth progress. Eventually the crystals extend b e y o n d the borders of the crystal bodies and join with other such advancing crystal fronts to f o r m small lakes or globules of mineralized dentin (Fig. 10). These expanding globules engulf collagen fibers which lie in their pathway. It is only at this stage that crystals are observed to be associated with collagen. Crystal bodies are observed only in the very earliest stage of dentin mineralization. After a layer of dentin has formed, they are no longer seen. Rather, the advancing front of crystal formation is c o m m o n l y observed to follow the collagen fibers (Fig. l 1).
Flo. 3. Immature crystal bodies. The rounded, membrane-bound crystal bodies are devoid of crystals and possess a rather homogeneous interior, x 139 000. FIG. 4. Crystal body containing several dense, needlelike crystals. The interior substructure is very similar to that of the less mature crystal bodies in Fig. 3. × 99 000. Fro. 5. The substructure of this crystal body includes a dense localized area of crystals and extensive regions of much lower density which appear to be organized in a lacy network, x 188 000. FIG. 6. The crystals in this crystal body are associated with a rounded, elliptically placed dense material. The remainder of the interior is of a lower density. × 149 000. FIG. 7. This crystal body is similar to the one in Fig. 6 with the exception that the dense material is more centrally placed. These crystals also appear to be associated with the dense material. The section used here was prepared by floating on distilled water, x 134 000. FIG. 8. This higher magnification of a crystal body (taken at 75 kV) illustrates the three layers of its enclosing membrane. Several crystals are evident in the central region of this crystal body. × 307 000.
24
EISENMANN AND GLICK
DISCUSSION The present investigation offers further evidence in support of a cellular role in the initial mineralization of dentin. Interest in this general area has centered in recent years around reports of cellular accumulation of calcium and phosphate and descriptions of cell-related bodies involved in initial mineralization of cartilage and bone. The accumulation of labile intracellular calcium and/or phosphate has been demonstrated histochemically and autoradiographically in chondroblasts and osteoblasts (7, 15, 17, 22) and by means of electron microscopic autoradiography in chondroblasts (I9, 20). It was reported recently that odontoblasts contain membrane-bound vesicles which are rich in calcium (13). The calcium was localized by combination with potassium pyronantimonate producing an electron dense deposit, the contents of which were verified using the electron microprobe. Microincineration and electron microscopy have revealed granules of mineral within mitochondria of osteoblasts and chondroblasts (18). It is suggested that these cells accumulate calcium and/or phosphate for later extrusion into the mineralizing medium. The mechanism by which ions are extruded is unknown. Identification of crystal-containing bodies in these calcifying tissues, which appear to be of a cellular origin, supports the view that cellular activity is involved, although it does not explain the mechanism by which transport occurs. The crystal bodies described here are very similar to calcifying globules or matrix vesicles in cartilage (1, 8, 10). The membranes surrounding the crystal bodies are comparable to those of the cartilage vesicles (2). The interior of some crystal bodies consists of a homogeneous granular background similar to that of calcifying globules of cartilage. Variations in the substructure of the crystal bodies include a localized dense area which appears to be in association with the forming crystals. This substructure may be related to the organic framework termed "crystal ghosts" by Bonucci (9) and described as nucleation sites for crystal formation in the calcifying globules of cartilage. Mineralization of cartilage proceeds by a radial growth of crystals outward from the calcifying globule. Early crystal formation is not associated with collagen, but after radiating clusters of crystals have formed, an occasional alignment of crystals with collagen is observed (8). Our results show a similar sequence of events in dentin. Only after crystals radiate out beyond the crystal bodies are they found in FIG. 9. Crystal bodies becoming filled with crystals. Two of the crystal bodies in this field are filled with crystals. Their membranes and substructure are nearly obscured and some crystals appear to be extending beyond the peripheries of the crystal bodies, x 143 000. F:G. 10. This micrograph illustrates growth of crystals beyond the crystal bodies to join with others and form lakes or globules of mineralized dentin. At this point in development, crystals are first observed to be associated with collagen (arrows). × 148 000.
10
"
26
EISENMANN AND GLICK
association with collagen fibers. The lack of crystal bodies along an established mineralizing front of dentin indicates that they are functional only in initiation of dentin mineralization. Once the process of crystal formation has been started it seems to perpetuate itself by advancing along the collagen fibers of the predentin matrix. The osteoblastic extrusions of bone are now generally described as discrete, rounded membrane-bound bodies containing an amorphous background material along with the first observable apatite crystals (5, 8). Their appearance is very similar to that of the crystal bodies of dentin reported here. They too are located between the collagen fibers and are observed only during the very early stages of bone mineralization. Investigation and discussion of cell-related loci of mineralization have concentrated on bone and cartilage giving little attention to other calcified tissues such as dentin. Although a rather obscure reference exists reporting cellular activity in dentin mineralization (4), this area of investigation has gone unnoticed and has not been pursued. It is not known if the ""buds" of odontoblasts described by Bernard were actually shown to be connected to the cells or not. Our finding that the crystal bodies are invariably rounded leads us to believe that they are discrete bodies lacking any direct cellular connection. Two recent ultrastructural investigations of incisor development include reports of relevant structures in the early predentin (adjacent to the developing ameloblasts). Kallenbach (16), studying rat incisors, described circular, membrane-bound profiles of varying sizes and contents resembling certain vesicles found within the odontoblast. N o mention was made of any crystalline material Within these structures. It was stated that the first mineral deposits in dentin occur after the basal lamina is no longer present and with no special relationship to any of the components of the predentin. Illustrations used to demonstrate this point show that a substantial amount of mineral had already been deposited. Our definition of initial mineralization refers to an earlier stage when the basal lamina is still present and the very first crystals are noted. In an investigation of epithelial-mesenchymal interactions in rabbit incisors, Croissant (12) described membrane-bound vesicles with "varying staining characteristics" located in the early predentin matrix. These vesicles were shown to contain RNA-protein complexes. However, no mention was made of any associated crystalline material. In both studies the membrane-bound vesicles in the early predentin are of a similar description to the crystal bodies reported here with the exception that no crystals were noted. It is interesting to speculate that the crystal bodies and Fro. I 1. Mineralization front. This micrograph illustrates the advancing front of dentin mineralization after a layer of dentin has formed. No crystal bodies are seen along the front, x 23 500. Inset: This higher magnification of a similar front illustrates growth of crystals in association with the cNlagen fibers. × 77 000.
i
i
28
EISENMANN AND GLICK
vesicles may be the same structure. The lack of observation of associated crystals may be due either to their loss during tissue processing or inadequate resolution of the internal structure of the vesicles. The authors wish to express their appreciation to Mrs. Elena Baltrusaitis and Mrs. Isabel Stoncius for their excellent technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
ANDERSON,H. C., Y. Cell Biol. 41, 59 (1969). ANDERSON,H. C. and MATSUZAWA,T., Trans. N.Y. Acad. Sci. 32, 619 (1970). ARNOT, H. J. and PAUTARD, F. G. E., Isr. J. Med. Sci. 3, 657 (1967). BERNARD,G. W., Int. Ass. Dent. Res., Abstract No. 305, Houston, Texas (1969). BERNARD,G. W. and PEASE, D. C., Amer. J. Anat. 125, 271 (1969). BEVELANDER,G. and NAKAHARA,H., Anat. Rec. 156, 303 (1966). BOHATmCHUK,F., Amer. J. Anat. 117, 287 (1965). BoNuccI, E., Clin. Orthop. 78, 108 (1971). -J. Ultrastruct. Res. 20, 33 (1967). -Z. Zellforsch. Mikrosk. Anat. 103, 192 (1970). BOOTHROYD,B., J. Cell Biol. 20, 165 (1964). CROISSANT,R. D., J. Dent. Res. 50, 1065 (1971). FRAZIER,P. D., Int. Ass. Dent. Res. Abstract No. 266, Chicago, Illinois (1971). HANCOX,N. M. and BOOTHROYD,B., Clin. Orthop. 40, 153 (1965). JOHNSTON,P. M., J. Biophys. Biochem. Cytol. 4, 163 (1958). KALLENBACH,E., J. Ultrastruct. Res. 35, 508 (1971). KASHIWA,I-I. K., Amer. d. Anat. 129, 459 (1970). MARTIN,J. H. and MATTI-IEWS,J. L., Clin. Orthop. 68, 273 (1970). MATTHEWS,J. L., Amer. J. Anat. 129, 451 (1970). MATTHEWS,J. L., MARTIN, J. H., LYNN, J. A. and COLLINS,E. J., Cal¢. Tiss. Res. 1, 330 (1968). NEUMAN,W. F. and NEtlMAN, M. W., Chem. Rev. 53, 1 (1953). ROLLE, G. K., Calc. Tiss. Res. 3, 142 (1969).
18
J. ULTRASTRUCTURERESEARCH41, 18-28 (1972)
U l t r a s t r u c t u r e of Initial Crystal F o r m a t i o n in D e n t i n ~ DALE R. EISENMANN a n d PAUL L. GLICK Department o f Histology, University o f Illinois at the Medical Center, Chicago, Illinois 60680
Received January 3, 1972 The present study illustrates a structure of apparent cellular origin in which the first observable mineral crystals of dentin appear. This information supplies a necessary link in the present understanding of the chain of events leading to mineralization of calcified tissues. It is well known that the organic components of tissues such as dentin, bone, and calcified cartilage are synthesized and secreted by closely associated formative cells. Studies of the mineralization process have concentrated on the role of various components of the organic matrix acting independently of the formative cells as templates for initial calcium and phosphate deposition. The direction for this research was initiated by the epitactic theory of Neuman and Neuman (21). They proposed that supersaturated calcium and phosphate in the body fluids interact directly with nucleating templates in the organic matrix of calcifiable tissues to produce hydroxyapatite crystals. Bonucci recently reviewed the history of this work and discussed a new direction for research in this area (8). It is clear that despite innumerable attempts to determine the site upon which nucleation occurs, few widely accepted conclusions were reached which have not been seriously challenged. Attention is now being turned toward the possibility that the formative cells may participate more directly in the mineralization process. Ultrastructural studies of regions of cartilage and bone matrix which are just beginning to mineralize have revealed evidence which supports a cellular role in this process. Membrane-bound structures termed "calcifying globules" or "matrix vesicles" are observed in the appropriate zones of epiphyseal cartilage; they appear to be of a cellular origin (1, 9, 10). The first observable hydroxyapatite crystals of calcifying cartilage are seen within these structures (10). Similar structures termed "osteoblast extrusions" are seen in bone, and they are active in initial crystal formation. The extrusions have been variously described as processes, buds, or extracellnlar vesicles, all of which appear to be derived from the osteoblast (3, 5, 14). 1 This study was supported in part by Grant No. DE-03312 from the National Institute for Dental
Research.
INITIAL DENTIN MINERALIZATION
19
FI~. 1. Zone of beginning dentin formation. Predentin is located between the odontoblasts (Od) and the developing ameloblasts (A), The predentin zone contains abundant collagen (banded fibers), odontoblast processes (P), and small, round bodies (arrows). A basal lamina runs between the predentin and the adjacent developing ameloblasts. x 19 500.
20
EISENMANNAND GLICK
Little if any ultrastructural evidence of structures such as the calcifying globules of cartilage and osteoblast extrusions of bone has been published with regard to dentin mineralization. The abstract of a presentation by Bernard describes initial dentin mineralization as homologous with initial bone formation in that crystals are first observed within buds from the odontoblasts. To our knowledge, ultrastructural illustration of this finding has not been documented (4). InterfibriUar granular masses were described by Bevelander and Nakahara as accumulations of acid mucopolysaccharide extruded by the odontoblast (6). Initial crystal formation of dentin was reported to occur in these granular masses rather than in relation to collagen. It is not possible to determine if any structural detail may have been associated with these clusters of initial crystal formation. This investigation presents ultrastructural evidence of membrane-bound bodies which contain the first observable crystals of developing dentin.
MATERIALS AND METHODS Male white rats weighing 200-250 g were anesthetized with ether and fixed by arterial perfusion with 4% glutaraldehyde in cacodylate buffer. Incisors were dissected free and cut longitudinally with a rotating diamond wheel while being bathed with saline as a coolant. These large but thin (0.5 mm) blocks were further trimmed, postfixed in 1% OsO4, dehydrated in alcohol, and embedded in Araldite in a plane which permitted sectioning through all stages of incisor development. Thin sections were cut with a diamond knife and floated on trough fluid consisting of a saturated solution of calcium phosphate (11). Some sections were floated on water. The sections were stained with uranyl magnesium acetate and lead citrate and examined in Hitachi 7S and l l A electron microscopes at either 50 or 75 kV.
RESULTS The first mineralization of dentin occurs within a zone which is located between the cell bodies of odontoblasts and developing ameloblasts (Fig. 1). This early predentin zone contains numerous branched odontoblast processes, collagen fibers, rather dense, small, round bodies, and an amorphous background material. It is separated from adjacent developing ameloblasts by a distinct basal lamina. Close examination of the various components of the predentin reveals that the first indication of crystalline material is within small (0.1-0.2 ¢~m in diameter) round, membrane-bound bodies (Fig. 2). The "crystal bodies" (as they will be referred to FIG. 2. Predentin at stage of initial crystal formation. Odontoblast processes (P) and banded collagen fibers constitute the bulk of the predentin. A crystal body (arrow) contains several dense, needlelike crystals within a slightly less dense homogeneous interior. A prominent basal lamina (BL) separates the predentin from the developing ameloblasts. × 91 000.
2 ;i~!i
22
EISENMANN A N D G L I C K
here) are surrounded by collagen fibers which contain no visible crystals at this early stage of dentin mineralization. Since the bodies are always rounded in shape, bearing no special relationship with odontoblast processes, it can be assumed that they are discrete entities. They are f o u n d scattered t h r o u g h o u t the entire zone of early predentin but appear more heavily concentrated near the basal lamina. Although the basic f o r m of the crystal bodies (rounded and m e m b r a n e b o u n d ) i s observed consistently, several variations in substructure are seen within the interior of the bodies (Figs. 3-8). The least mature forms contain a rather h o m o g e n e o u s interior and are devoid of crystals (Fig. 3). Some of the crystal bodies contain a similar h o m o g e n e o u s substructure along with several dense, needlelike crystals (Fig. 4). M a n y of the bodies possess a more complex substructure consisting of localizations of dense material (usually in association with the crystals) and other areas of m u c h lower density with varying patterns of organization (Figs. 5-7). The crystals are seen equally as well in sections which were prepared by floating on water rather than on a saturated solution of calcium phosphate (Fig. 7). Higher magnification illustrates more clearly three components of the enclosing m e m b r a n e (Fig. 8). Inner and outer dense layers are separated by an intervening less dense layer. The entire m e m b r a n e is approximately 80-100 ~ in thickness. Further mineralization is evidenced by increasing numbers of crystals within the crystal bodies (Fig. 9). Their membranes and substructure become obscured as crystal formation and growth progress. Eventually the crystals extend b e y o n d the borders of the crystal bodies and join with other such advancing crystal fronts to f o r m small lakes or globules of mineralized dentin (Fig. 10). These expanding globules engulf collagen fibers which lie in their pathway. It is only at this stage that crystals are observed to be associated with collagen. Crystal bodies are observed only in the very earliest stage of dentin mineralization. After a layer of dentin has formed, they are no longer seen. Rather, the advancing front of crystal formation is c o m m o n l y observed to follow the collagen fibers (Fig. l 1).
Flo. 3. Immature crystal bodies. The rounded, membrane-bound crystal bodies are devoid of crystals and possess a rather homogeneous interior, x 139 000. FIG. 4. Crystal body containing several dense, needlelike crystals. The interior substructure is very similar to that of the less mature crystal bodies in Fig. 3. × 99 000. Fro. 5. The substructure of this crystal body includes a dense localized area of crystals and extensive regions of much lower density which appear to be organized in a lacy network, x 188 000. FIG. 6. The crystals in this crystal body are associated with a rounded, elliptically placed dense material. The remainder of the interior is of a lower density. × 149 000. FIG. 7. This crystal body is similar to the one in Fig. 6 with the exception that the dense material is more centrally placed. These crystals also appear to be associated with the dense material. The section used here was prepared by floating on distilled water, x 134 000. FIG. 8. This higher magnification of a crystal body (taken at 75 kV) illustrates the three layers of its enclosing membrane. Several crystals are evident in the central region of this crystal body. × 307 000.
24
EISENMANN AND GLICK
DISCUSSION The present investigation offers further evidence in support of a cellular role in the initial mineralization of dentin. Interest in this general area has centered in recent years around reports of cellular accumulation of calcium and phosphate and descriptions of cell-related bodies involved in initial mineralization of cartilage and bone. The accumulation of labile intracellular calcium and/or phosphate has been demonstrated histochemically and autoradiographically in chondroblasts and osteoblasts (7, 15, 17, 22) and by means of electron microscopic autoradiography in chondroblasts (I9, 20). It was reported recently that odontoblasts contain membrane-bound vesicles which are rich in calcium (13). The calcium was localized by combination with potassium pyronantimonate producing an electron dense deposit, the contents of which were verified using the electron microprobe. Microincineration and electron microscopy have revealed granules of mineral within mitochondria of osteoblasts and chondroblasts (18). It is suggested that these cells accumulate calcium and/or phosphate for later extrusion into the mineralizing medium. The mechanism by which ions are extruded is unknown. Identification of crystal-containing bodies in these calcifying tissues, which appear to be of a cellular origin, supports the view that cellular activity is involved, although it does not explain the mechanism by which transport occurs. The crystal bodies described here are very similar to calcifying globules or matrix vesicles in cartilage (1, 8, 10). The membranes surrounding the crystal bodies are comparable to those of the cartilage vesicles (2). The interior of some crystal bodies consists of a homogeneous granular background similar to that of calcifying globules of cartilage. Variations in the substructure of the crystal bodies include a localized dense area which appears to be in association with the forming crystals. This substructure may be related to the organic framework termed "crystal ghosts" by Bonucci (9) and described as nucleation sites for crystal formation in the calcifying globules of cartilage. Mineralization of cartilage proceeds by a radial growth of crystals outward from the calcifying globule. Early crystal formation is not associated with collagen, but after radiating clusters of crystals have formed, an occasional alignment of crystals with collagen is observed (8). Our results show a similar sequence of events in dentin. Only after crystals radiate out beyond the crystal bodies are they found in FIG. 9. Crystal bodies becoming filled with crystals. Two of the crystal bodies in this field are filled with crystals. Their membranes and substructure are nearly obscured and some crystals appear to be extending beyond the peripheries of the crystal bodies, x 143 000. F:G. 10. This micrograph illustrates growth of crystals beyond the crystal bodies to join with others and form lakes or globules of mineralized dentin. At this point in development, crystals are first observed to be associated with collagen (arrows). × 148 000.
10
"
26
EISENMANN AND GLICK
association with collagen fibers. The lack of crystal bodies along an established mineralizing front of dentin indicates that they are functional only in initiation of dentin mineralization. Once the process of crystal formation has been started it seems to perpetuate itself by advancing along the collagen fibers of the predentin matrix. The osteoblastic extrusions of bone are now generally described as discrete, rounded membrane-bound bodies containing an amorphous background material along with the first observable apatite crystals (5, 8). Their appearance is very similar to that of the crystal bodies of dentin reported here. They too are located between the collagen fibers and are observed only during the very early stages of bone mineralization. Investigation and discussion of cell-related loci of mineralization have concentrated on bone and cartilage giving little attention to other calcified tissues such as dentin. Although a rather obscure reference exists reporting cellular activity in dentin mineralization (4), this area of investigation has gone unnoticed and has not been pursued. It is not known if the ""buds" of odontoblasts described by Bernard were actually shown to be connected to the cells or not. Our finding that the crystal bodies are invariably rounded leads us to believe that they are discrete bodies lacking any direct cellular connection. Two recent ultrastructural investigations of incisor development include reports of relevant structures in the early predentin (adjacent to the developing ameloblasts). Kallenbach (16), studying rat incisors, described circular, membrane-bound profiles of varying sizes and contents resembling certain vesicles found within the odontoblast. N o mention was made of any crystalline material Within these structures. It was stated that the first mineral deposits in dentin occur after the basal lamina is no longer present and with no special relationship to any of the components of the predentin. Illustrations used to demonstrate this point show that a substantial amount of mineral had already been deposited. Our definition of initial mineralization refers to an earlier stage when the basal lamina is still present and the very first crystals are noted. In an investigation of epithelial-mesenchymal interactions in rabbit incisors, Croissant (12) described membrane-bound vesicles with "varying staining characteristics" located in the early predentin matrix. These vesicles were shown to contain RNA-protein complexes. However, no mention was made of any associated crystalline material. In both studies the membrane-bound vesicles in the early predentin are of a similar description to the crystal bodies reported here with the exception that no crystals were noted. It is interesting to speculate that the crystal bodies and Fro. I 1. Mineralization front. This micrograph illustrates the advancing front of dentin mineralization after a layer of dentin has formed. No crystal bodies are seen along the front, x 23 500. Inset: This higher magnification of a similar front illustrates growth of crystals in association with the cNlagen fibers. × 77 000.
i
i
28
EISENMANN AND GLICK
vesicles may be the same structure. The lack of observation of associated crystals may be due either to their loss during tissue processing or inadequate resolution of the internal structure of the vesicles. The authors wish to express their appreciation to Mrs. Elena Baltrusaitis and Mrs. Isabel Stoncius for their excellent technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
ANDERSON,H. C., Y. Cell Biol. 41, 59 (1969). ANDERSON,H. C. and MATSUZAWA,T., Trans. N.Y. Acad. Sci. 32, 619 (1970). ARNOT, H. J. and PAUTARD, F. G. E., Isr. J. Med. Sci. 3, 657 (1967). BERNARD,G. W., Int. Ass. Dent. Res., Abstract No. 305, Houston, Texas (1969). BERNARD,G. W. and PEASE, D. C., Amer. J. Anat. 125, 271 (1969). BEVELANDER,G. and NAKAHARA,H., Anat. Rec. 156, 303 (1966). BOHATmCHUK,F., Amer. J. Anat. 117, 287 (1965). BoNuccI, E., Clin. Orthop. 78, 108 (1971). -J. Ultrastruct. Res. 20, 33 (1967). -Z. Zellforsch. Mikrosk. Anat. 103, 192 (1970). BOOTHROYD,B., J. Cell Biol. 20, 165 (1964). CROISSANT,R. D., J. Dent. Res. 50, 1065 (1971). FRAZIER,P. D., Int. Ass. Dent. Res. Abstract No. 266, Chicago, Illinois (1971). HANCOX,N. M. and BOOTHROYD,B., Clin. Orthop. 40, 153 (1965). JOHNSTON,P. M., J. Biophys. Biochem. Cytol. 4, 163 (1958). KALLENBACH,E., J. Ultrastruct. Res. 35, 508 (1971). KASHIWA,I-I. K., Amer. d. Anat. 129, 459 (1970). MARTIN,J. H. and MATTI-IEWS,J. L., Clin. Orthop. 68, 273 (1970). MATTHEWS,J. L., Amer. J. Anat. 129, 451 (1970). MATTHEWS,J. L., MARTIN, J. H., LYNN, J. A. and COLLINS,E. J., Cal¢. Tiss. Res. 1, 330 (1968). NEUMAN,W. F. and NEtlMAN, M. W., Chem. Rev. 53, 1 (1953). ROLLE, G. K., Calc. Tiss. Res. 3, 142 (1969).