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Ultrastructural characterization of extracellular matrix vesicles in the mineralizing fronts of apical cementum in cats
Archs oral Bid. Vol. 30, No. 5, pp. 44549, Printed in Great Britain. All rights reserved
1985 Copyright 0
SHORT
0003-9969/85 53.00 + 0.00 1985 Pergamon Press Ltd
COMMUNICATION
ULTRASTRUCTURAL CHARACTERIZATION OF EXTRACELLULAR MATRIX VESICLES IN THE MINERALIZING FRONTS OF APICAL CEMENTUM IN CATS Y. HAYASHI Department
of Conservative
Dentistry,
Faculty
of Dentistry,
Kyushu
University
61, Fukuoka
812, Japan
Summary-Fine structure and the organic-inorganic relationship in mineralized fronts of cementurn was studied with post-embedding demineralization and staining method. Typical matrix vesicles were observed between apical cementum and cementoblasts. Some vesicles were enclosed by a unit membrane; others contained needle-like needle-like strulztures
crystals. Cementum which disappeared
crystals were found on unstained sections as filament- and completely on EDTA treatment; similarly-shaped structures
re-appeared following uranyl and lead staining. These findings suggest that matrix vesicles might act as initiators of additional cementogenesis and that crystal ghosts seen in cementum consist of organic materials.
Matrix vesicles were first discovered by Bonucci (1967) and Anderson (1969) in epiphyseal cartilage. They are involved in the early stages of the mineralization process, and it now seems to be generally agreed that they are sites of initial crystal formation in many normally and pathologically-mineralizing tissues (Bonucci, 1984a). The presence of a peripheral membrane, and its morphological (Bonucci, 1967, 1970; Anderson, 1969) and biochemical (Peress, Anderson and Sajdera, 1974; Wuthier, 1975, 1976; Glaser and Corad, 1981) fsimilarity with the membrane of chondrocytes, suggest that matrix vesicles are of cellular origin. Rabinovitch and .4nderson (1976) hypothesized that there are four different ways in which matrix vesicles may be formed by: 1. Budding from cells and cellular processes. 2. Extrusion of preformed structures. 3. Disintegration of degenerate cells. 4. Extracellular assembly of secreted subunits. Many morphological findings support hypotheses 1 and 3. On the other hand, Listgarten and Shapiro (1974) considered that matrix vesicles act as initiators of coronal cementogenesis in guinea-pig molars. However, the coronal cementum of the guinea-pig is a cartilage-like unlike human cementum. Though structure, fine-structure studies of the regeneration of cementum and periodontal connective tissue have been widely carried out (Nalbandian and Frank, 1980; Frank, Fiore-Donno and Cimasoni, 1983), there are no reports describing the extracellular matrix vesicles in the mineralizing fronts of apical cementum. Eight fully-formed canine teeth from two adult cats (weight about 3 kg) were used. Anaesthesia was obtained by intraperitoneal injection of 25.6 mg/kg pentobarbital sodium. The animals were perfused through the left ventricle with 2 per cent pin formaldehyde and 21.5 per cent glutaraldehyde 0.1 M phosphate buffer at pH 7.3. The teeth were carefully extracted. Pieces of apical cementum about
1 mm in width were excised with a rotating diamonddisk under a stream of Ringer solution and then immersed in the same fixative for 3 h. They were post-fixed in 2.5 per cent osmium tetroxide in 0.1 M phosphate buffer at pH 7.3 for 1 h. The blocks were then dehydrated in increasing concentrations of ethanol and embedded in epoxy resin. Sections, about 1 pm thick, were stained with toluidine blue for light-microscopy. Ultra-thin sections were collected in a specimen boat containing a saturated solution of human dentine powder to prevent demineralization of the sections. Ultra-thin sections were mounted on Formvar-coated 150-mesh copper grids and stained with uranyl acetate and lead citrate and examined in a Hitachi H-500 electron microscope at 100 kV. To reveal organic-inorganic relationships in the mineralizing fronts of cementum, the free-floating method of Marinozzi (1961) was used to demineralize the ultra-thin sections using 10 per cent EDTA at pH 7.2. These demineralized sections were examined in the electron microscope with and without staining. Apical cementurn was extremely irregular under the electron microscope (Fig. 1). Furthermore, the various irregular island-like structures infiltrated with crystals were scattered in unmineralized matrix/apical periodontium (Figs 1 and 4). The larger islands presumably formed by coalescence (Fig. 4). Cememtoblast processes and numerous vesicular elements were observed between cementoblast and cementum (Fig. 1). Close examination of these vesicles (60-70 nm in diameter) revealed a marked variation in shape and content. Their contents exhibited varying electron densities; some vesicles with electronlucent contents were enclosed by a unit membrane and had a smooth outline; others contained needlelike crystals and had an irregular outline (Fig. 2). Cementoblasts had large nuclei, well-formed rough-surfaced endoplasmic reticulum with long interconnecting cisternae and mitochondria. They were 445
446
Y. HAYASHI
of irregular shapes and had numerous cell processes free ribosomes and (Fig. 3) which contained microfilaments. Cementum crystals in unstained sections were filament- and needle-like (Fig. 4); they disappeared completely following EDTA treatment for 15 min; similarly-shaped structures resembling crystal ghosts (Bonucci, 1967) re-appeared following uranyl and lead staining (Fig. 5). These ghosts were found at the peripheries of island-like structures; and centres of the ghosts were electron-lucent and amorphous (Fig. 5). Island-like structures were present in the peripheral part of apical cementum and showed signs of being formed by coalescence, as in circumpulpal dentinogenesis (Hayashi, 1984). In initial dentinogenesis, the matrix vesicles act as initiators of mineralization (Bernard, 1972; Eisenmann and Glick, 1972; Sisca and Provenza, 1972; Larsson and Bloom, 1973; Sela et al., 1981; Hayashi, 1982, 1983); they were never observed in circumpulpal dentinogenesis (Hayashi, 1984). Typical matrix vesicles were found in the apical periodontium in the cat, indicating that matrix vesicles act as initiators of additional cementogenesis. The difference between circumpulpal dentinogenesis and cementogenesis may be assoicated with the fact that cementum like alveolar bone, is part of the periodontal ligament (Dougherty, 1978; Bab, Muhlrad and Sela, 1979) Resorption and the addition of hard tissue occur physiologically. Matrix vesicles are indispensable in rapid mineralization; they are less important in tissues which mineralize slowly (Bonucci, 1984b). In accord with this, the presence of matrix vesicles in late cementogenesis indicates that apical cementum mineralizes rapidly. Matrix vesicles seen near odontoblasts and osteoblasts are believed to originate from the budding of cell membrane (Almuddaris and Dougherty, 1979; Dougherty, 1978). I found vesicular structures in connection with cell membrane of cementoblast (Fig. 3) which indicates that matrix vesicles seen near cementoblast also originate from the budding of cell membrane. After post-embedding demineralization and staining (Bonucci, 1984b), the ultrastructure of the organic components of the bone nodules is well preserved, and most of the crystal ghosts appear at the periphery of nodules; central part of the nodules consists of a finely granular, amorphous, and irregular filamentous matrix. Any collagen fibrils which may have been trapped in the nodules are masked by this matrix (Bonucci and Reurink, 1978). I believe that the filamentous structures (Fig. 5) are crystal ghosts like those in the bone nodules, which Bonucci (1981) suggests consist of proteo-glycolipid complexes which might derive from solubilized vesicles and might be capable of nucleating hydroxyapatite. Furthermore, the concentrations of 0-phosphoserine and 0-phosphothreonine and the amino-acid composition of the EDTA-extractable proteins of bovine cementum are more similar to those found in bone than in dentine or enamel (Glimcher, Lefteriou and Kossiva, 1979). Thus the crystal ghosts of cementum probably also consist of proteo-glycolipid complexes.
Acknowledgement-I am grateful to Professor H. Nagasawa for his critical reading of the manuscript. REFERENCES Almuddaris M. F. and Dougherty W. J. (1979) The association of amorphous mineral deposits with the plasma membrane of pre-young odontoblasts and their relationship to the origin of dental matrix vesicles in rat incisor teeth. Am. J. Anat. 155, 223-244. Anderson H. C. (1969) Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41, 59-72. Bab I. A., Muhlrad A. and Sela J. (1979) Ultrastructural and biochemical study of extracellular matrix vesicles in normal alveolar bone of rats. Cell Tissue Res. 202, l-7. Bernard G. W. (1972) Ultrastructural observations of initial calcification in dentin and enamel. J. Ultrastrucf. Rex 41, 1-17. Bonucci E. (1967) Fine structure of early cartilage calcification. J. Ultrasfruct. Rex 20. 33-50. Bonucci E. (1970) Fine structure and histochemistry of “calcifying globules” in epiphyseal cartilage. Z. ZeNforsch. 103, 192-2 17. Bonucci E. and Reurink J. (1978) The fine structure of decalcified cartilage and bone: A comparison between decalcification procedures performed before and after embedding. Ca&$ Tissue Res. 25, 179-190. Bonucci E. (1981) Intra-vs. extra-vesicles calcification in epiphyscal cartilage. In: Matrix Vesicles (Edited by Ascenzi A., Bonucci E. and Bernard B. de) pp. 167-172. Wichtig Editore srl, Milano. Bonucci E. (1984a) Matrix vesicles: Their role in calcification. In: Dentin and Denfinogenesis (Edited by Linde A.) Vol I, pp. 135-154. CRC Press, Florida. Bonucci E. (1984b) The structural basis of calcification. In: Ultrastructure of The Connecfiw Tissue Matrix (Edited by Ruggeri A. and Motta P. M.) pp. 165-191. Martinus Nijhoff, Boston. Dougherty W. J. (1978) The occurrence of amorphous mineral deposits in association with the plasma membrane of active osteoblasts in rat and mouse alveolar bone. Metab. Bone Dis. Ret. Res. 1, 119-123. Eisenmann D. R. and Glick P. L. (1972) Ultrastructure of initial crystal formation in dentin. J. Ultrastruct. Res. 41, 18-28. Frank R. M., Fiore-Donno G. and Cimasoni G. (1983) Cementogenesis and soft tissue attachment after citric acid treatment in a human: An electron microscopic study. J. Periodont. 54, 389-401. Glaser J. H. and Conrad H. E. (1981) Formation on matrix vesicles by cultured chick embryo chondrocytes. J. biol. Chem. 256, 12607-1261 I. Glimcher M. J., Lefteriou B. and Kossiva D. (1979) Identification of O-phosphoserine, O-phosphothreonine and y-carboxyglutamic acid in the non-collagenous proteins of bovine cementum; Comparison with dentine, enamel and bone. Calc$ Tissue Int. u1, 83-86. Hayashi Y. (1982) Ultrastructure of initial calcification in wound healing following pulpotomy. J. oral Path. 11, 174180. Hayashi Y. (1983) Crystal growth in matrix vesicles of permanent tooth germs in kittens. Acta anat. 116, 62-68. Hayashi Y. (1984) Crystal growth in calcifvinpl fronts durine. dentinogenesis: Acia a&t. 118, 13-17.Larsson A. and Bloom G. D. (1973) Studies on dentinogenesis in the rat: Fine structure of developing odontoblasts and predentin in relation to the mineralization process. Z. Anar. EnrwGesch. 139, 227-246. Listgarten M. A. and Shapiro I. M. (1974) Fine structure and composition of coronal cementum in guinea-pig molars. Archs oral Biol. 19, 679-696. Marinozzi V. (1961) Silver impregnations of ultrathin sec-
Matrix
vesicles in periodontium
tions for electron microscopy. J. biophys. biochem. Cytol. 9, 121-133. Nalbandian J. and Frank R. M. (1980) Electron microscopic study of the regeneration of cementum and periodontal connective tissue attachment in the cat. J. periodont. Rex 15, 71-89. Peress N. S., Anderson H. C. and Sajdera S. W. (1974) The lipids of matrix vesicles from bovine fetal epiphyseal cartilage. Culcif. Tissue Res. 14, 275-283. Rabinovitch A. L. and Anderson H. C. (1976) Biogenesis of matrix vesicles in cartilage growth plates. Fedn Proc. Fedn Am. Sots exp. Biol. 35, I 12-l 16.
Plate
447
Sela J., Tamari I., Hirschfeld Z. and Bab I. (1981) Transmission electron microscopy of reparative dentin in rat molar pulps. Primary mineralization via extracellular matrix vesicles. Acta anat. 109, 247-251. Sisca R. F. and Provenza D. V. (1972) Initial dentin formation in human deciduous teeth: An electron microscope study. Calcif. Tissue Res. 9, l-16. Wuthier R. E. (1975) Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles. Biochim. biophys. Acta 409, 128-143. Wuthier R. E. (1976) Lipids of matrix vesicles. Fedn Proc. Fedn Am. Sots exp. Biol. 35, 117-121.
1 overleaf.
Y. HAYASHI
448
Plate
1.
Fig. I. Mineralizing fronts (upper part) and apical periodontium. Note cementoblast process (Cp) and matrix vesicles (arrows) in periodontium. Uranyl acetate and iead-citrate staining. x 22,500 Fig. 2. High magnification of matrix vesicles. Some vesicles (arrow heads) contain electron¢ and others (arrows) contain electron-dense needle-like crystals. Uranyl acetate and lead-citrate x 90,000 Fig. 3. Cementoblast with numerous protruding processes. Arrow R = free ribosomes. Uranyl acetate and lead-citrate Fig. 4. Mineralizing
fronts consisting
of various
irregular island-like x 45,000
indicates staining. structures.
the budding x 45,000 Without
material staining.
of a vesicle.
electron-staining.
Fig. 5. Ultra-thin section demineralized with EDTA for 15 min and stained with uranyl acetate and lead citrate. Note filament-like structures (arrow) at periphery of island-like structures. x 90,000
Matrix
vesicles in periodontium
Plate
I
449
1985 Copyright 0
SHORT
0003-9969/85 53.00 + 0.00 1985 Pergamon Press Ltd
COMMUNICATION
ULTRASTRUCTURAL CHARACTERIZATION OF EXTRACELLULAR MATRIX VESICLES IN THE MINERALIZING FRONTS OF APICAL CEMENTUM IN CATS Y. HAYASHI Department
of Conservative
Dentistry,
Faculty
of Dentistry,
Kyushu
University
61, Fukuoka
812, Japan
Summary-Fine structure and the organic-inorganic relationship in mineralized fronts of cementurn was studied with post-embedding demineralization and staining method. Typical matrix vesicles were observed between apical cementum and cementoblasts. Some vesicles were enclosed by a unit membrane; others contained needle-like needle-like strulztures
crystals. Cementum which disappeared
crystals were found on unstained sections as filament- and completely on EDTA treatment; similarly-shaped structures
re-appeared following uranyl and lead staining. These findings suggest that matrix vesicles might act as initiators of additional cementogenesis and that crystal ghosts seen in cementum consist of organic materials.
Matrix vesicles were first discovered by Bonucci (1967) and Anderson (1969) in epiphyseal cartilage. They are involved in the early stages of the mineralization process, and it now seems to be generally agreed that they are sites of initial crystal formation in many normally and pathologically-mineralizing tissues (Bonucci, 1984a). The presence of a peripheral membrane, and its morphological (Bonucci, 1967, 1970; Anderson, 1969) and biochemical (Peress, Anderson and Sajdera, 1974; Wuthier, 1975, 1976; Glaser and Corad, 1981) fsimilarity with the membrane of chondrocytes, suggest that matrix vesicles are of cellular origin. Rabinovitch and .4nderson (1976) hypothesized that there are four different ways in which matrix vesicles may be formed by: 1. Budding from cells and cellular processes. 2. Extrusion of preformed structures. 3. Disintegration of degenerate cells. 4. Extracellular assembly of secreted subunits. Many morphological findings support hypotheses 1 and 3. On the other hand, Listgarten and Shapiro (1974) considered that matrix vesicles act as initiators of coronal cementogenesis in guinea-pig molars. However, the coronal cementum of the guinea-pig is a cartilage-like unlike human cementum. Though structure, fine-structure studies of the regeneration of cementum and periodontal connective tissue have been widely carried out (Nalbandian and Frank, 1980; Frank, Fiore-Donno and Cimasoni, 1983), there are no reports describing the extracellular matrix vesicles in the mineralizing fronts of apical cementum. Eight fully-formed canine teeth from two adult cats (weight about 3 kg) were used. Anaesthesia was obtained by intraperitoneal injection of 25.6 mg/kg pentobarbital sodium. The animals were perfused through the left ventricle with 2 per cent pin formaldehyde and 21.5 per cent glutaraldehyde 0.1 M phosphate buffer at pH 7.3. The teeth were carefully extracted. Pieces of apical cementum about
1 mm in width were excised with a rotating diamonddisk under a stream of Ringer solution and then immersed in the same fixative for 3 h. They were post-fixed in 2.5 per cent osmium tetroxide in 0.1 M phosphate buffer at pH 7.3 for 1 h. The blocks were then dehydrated in increasing concentrations of ethanol and embedded in epoxy resin. Sections, about 1 pm thick, were stained with toluidine blue for light-microscopy. Ultra-thin sections were collected in a specimen boat containing a saturated solution of human dentine powder to prevent demineralization of the sections. Ultra-thin sections were mounted on Formvar-coated 150-mesh copper grids and stained with uranyl acetate and lead citrate and examined in a Hitachi H-500 electron microscope at 100 kV. To reveal organic-inorganic relationships in the mineralizing fronts of cementum, the free-floating method of Marinozzi (1961) was used to demineralize the ultra-thin sections using 10 per cent EDTA at pH 7.2. These demineralized sections were examined in the electron microscope with and without staining. Apical cementurn was extremely irregular under the electron microscope (Fig. 1). Furthermore, the various irregular island-like structures infiltrated with crystals were scattered in unmineralized matrix/apical periodontium (Figs 1 and 4). The larger islands presumably formed by coalescence (Fig. 4). Cememtoblast processes and numerous vesicular elements were observed between cementoblast and cementum (Fig. 1). Close examination of these vesicles (60-70 nm in diameter) revealed a marked variation in shape and content. Their contents exhibited varying electron densities; some vesicles with electronlucent contents were enclosed by a unit membrane and had a smooth outline; others contained needlelike crystals and had an irregular outline (Fig. 2). Cementoblasts had large nuclei, well-formed rough-surfaced endoplasmic reticulum with long interconnecting cisternae and mitochondria. They were 445
446
Y. HAYASHI
of irregular shapes and had numerous cell processes free ribosomes and (Fig. 3) which contained microfilaments. Cementum crystals in unstained sections were filament- and needle-like (Fig. 4); they disappeared completely following EDTA treatment for 15 min; similarly-shaped structures resembling crystal ghosts (Bonucci, 1967) re-appeared following uranyl and lead staining (Fig. 5). These ghosts were found at the peripheries of island-like structures; and centres of the ghosts were electron-lucent and amorphous (Fig. 5). Island-like structures were present in the peripheral part of apical cementum and showed signs of being formed by coalescence, as in circumpulpal dentinogenesis (Hayashi, 1984). In initial dentinogenesis, the matrix vesicles act as initiators of mineralization (Bernard, 1972; Eisenmann and Glick, 1972; Sisca and Provenza, 1972; Larsson and Bloom, 1973; Sela et al., 1981; Hayashi, 1982, 1983); they were never observed in circumpulpal dentinogenesis (Hayashi, 1984). Typical matrix vesicles were found in the apical periodontium in the cat, indicating that matrix vesicles act as initiators of additional cementogenesis. The difference between circumpulpal dentinogenesis and cementogenesis may be assoicated with the fact that cementum like alveolar bone, is part of the periodontal ligament (Dougherty, 1978; Bab, Muhlrad and Sela, 1979) Resorption and the addition of hard tissue occur physiologically. Matrix vesicles are indispensable in rapid mineralization; they are less important in tissues which mineralize slowly (Bonucci, 1984b). In accord with this, the presence of matrix vesicles in late cementogenesis indicates that apical cementum mineralizes rapidly. Matrix vesicles seen near odontoblasts and osteoblasts are believed to originate from the budding of cell membrane (Almuddaris and Dougherty, 1979; Dougherty, 1978). I found vesicular structures in connection with cell membrane of cementoblast (Fig. 3) which indicates that matrix vesicles seen near cementoblast also originate from the budding of cell membrane. After post-embedding demineralization and staining (Bonucci, 1984b), the ultrastructure of the organic components of the bone nodules is well preserved, and most of the crystal ghosts appear at the periphery of nodules; central part of the nodules consists of a finely granular, amorphous, and irregular filamentous matrix. Any collagen fibrils which may have been trapped in the nodules are masked by this matrix (Bonucci and Reurink, 1978). I believe that the filamentous structures (Fig. 5) are crystal ghosts like those in the bone nodules, which Bonucci (1981) suggests consist of proteo-glycolipid complexes which might derive from solubilized vesicles and might be capable of nucleating hydroxyapatite. Furthermore, the concentrations of 0-phosphoserine and 0-phosphothreonine and the amino-acid composition of the EDTA-extractable proteins of bovine cementum are more similar to those found in bone than in dentine or enamel (Glimcher, Lefteriou and Kossiva, 1979). Thus the crystal ghosts of cementum probably also consist of proteo-glycolipid complexes.
Acknowledgement-I am grateful to Professor H. Nagasawa for his critical reading of the manuscript. REFERENCES Almuddaris M. F. and Dougherty W. J. (1979) The association of amorphous mineral deposits with the plasma membrane of pre-young odontoblasts and their relationship to the origin of dental matrix vesicles in rat incisor teeth. Am. J. Anat. 155, 223-244. Anderson H. C. (1969) Vesicles associated with calcification in the matrix of epiphyseal cartilage. J. Cell Biol. 41, 59-72. Bab I. A., Muhlrad A. and Sela J. (1979) Ultrastructural and biochemical study of extracellular matrix vesicles in normal alveolar bone of rats. Cell Tissue Res. 202, l-7. Bernard G. W. (1972) Ultrastructural observations of initial calcification in dentin and enamel. J. Ultrastrucf. Rex 41, 1-17. Bonucci E. (1967) Fine structure of early cartilage calcification. J. Ultrasfruct. Rex 20. 33-50. Bonucci E. (1970) Fine structure and histochemistry of “calcifying globules” in epiphyseal cartilage. Z. ZeNforsch. 103, 192-2 17. Bonucci E. and Reurink J. (1978) The fine structure of decalcified cartilage and bone: A comparison between decalcification procedures performed before and after embedding. Ca&$ Tissue Res. 25, 179-190. Bonucci E. (1981) Intra-vs. extra-vesicles calcification in epiphyscal cartilage. In: Matrix Vesicles (Edited by Ascenzi A., Bonucci E. and Bernard B. de) pp. 167-172. Wichtig Editore srl, Milano. Bonucci E. (1984a) Matrix vesicles: Their role in calcification. In: Dentin and Denfinogenesis (Edited by Linde A.) Vol I, pp. 135-154. CRC Press, Florida. Bonucci E. (1984b) The structural basis of calcification. In: Ultrastructure of The Connecfiw Tissue Matrix (Edited by Ruggeri A. and Motta P. M.) pp. 165-191. Martinus Nijhoff, Boston. Dougherty W. J. (1978) The occurrence of amorphous mineral deposits in association with the plasma membrane of active osteoblasts in rat and mouse alveolar bone. Metab. Bone Dis. Ret. Res. 1, 119-123. Eisenmann D. R. and Glick P. L. (1972) Ultrastructure of initial crystal formation in dentin. J. Ultrastruct. Res. 41, 18-28. Frank R. M., Fiore-Donno G. and Cimasoni G. (1983) Cementogenesis and soft tissue attachment after citric acid treatment in a human: An electron microscopic study. J. Periodont. 54, 389-401. Glaser J. H. and Conrad H. E. (1981) Formation on matrix vesicles by cultured chick embryo chondrocytes. J. biol. Chem. 256, 12607-1261 I. Glimcher M. J., Lefteriou B. and Kossiva D. (1979) Identification of O-phosphoserine, O-phosphothreonine and y-carboxyglutamic acid in the non-collagenous proteins of bovine cementum; Comparison with dentine, enamel and bone. Calc$ Tissue Int. u1, 83-86. Hayashi Y. (1982) Ultrastructure of initial calcification in wound healing following pulpotomy. J. oral Path. 11, 174180. Hayashi Y. (1983) Crystal growth in matrix vesicles of permanent tooth germs in kittens. Acta anat. 116, 62-68. Hayashi Y. (1984) Crystal growth in calcifvinpl fronts durine. dentinogenesis: Acia a&t. 118, 13-17.Larsson A. and Bloom G. D. (1973) Studies on dentinogenesis in the rat: Fine structure of developing odontoblasts and predentin in relation to the mineralization process. Z. Anar. EnrwGesch. 139, 227-246. Listgarten M. A. and Shapiro I. M. (1974) Fine structure and composition of coronal cementum in guinea-pig molars. Archs oral Biol. 19, 679-696. Marinozzi V. (1961) Silver impregnations of ultrathin sec-
Matrix
vesicles in periodontium
tions for electron microscopy. J. biophys. biochem. Cytol. 9, 121-133. Nalbandian J. and Frank R. M. (1980) Electron microscopic study of the regeneration of cementum and periodontal connective tissue attachment in the cat. J. periodont. Rex 15, 71-89. Peress N. S., Anderson H. C. and Sajdera S. W. (1974) The lipids of matrix vesicles from bovine fetal epiphyseal cartilage. Culcif. Tissue Res. 14, 275-283. Rabinovitch A. L. and Anderson H. C. (1976) Biogenesis of matrix vesicles in cartilage growth plates. Fedn Proc. Fedn Am. Sots exp. Biol. 35, I 12-l 16.
Plate
447
Sela J., Tamari I., Hirschfeld Z. and Bab I. (1981) Transmission electron microscopy of reparative dentin in rat molar pulps. Primary mineralization via extracellular matrix vesicles. Acta anat. 109, 247-251. Sisca R. F. and Provenza D. V. (1972) Initial dentin formation in human deciduous teeth: An electron microscope study. Calcif. Tissue Res. 9, l-16. Wuthier R. E. (1975) Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles. Biochim. biophys. Acta 409, 128-143. Wuthier R. E. (1976) Lipids of matrix vesicles. Fedn Proc. Fedn Am. Sots exp. Biol. 35, 117-121.
1 overleaf.
Y. HAYASHI
448
Plate
1.
Fig. I. Mineralizing fronts (upper part) and apical periodontium. Note cementoblast process (Cp) and matrix vesicles (arrows) in periodontium. Uranyl acetate and iead-citrate staining. x 22,500 Fig. 2. High magnification of matrix vesicles. Some vesicles (arrow heads) contain electron¢ and others (arrows) contain electron-dense needle-like crystals. Uranyl acetate and lead-citrate x 90,000 Fig. 3. Cementoblast with numerous protruding processes. Arrow R = free ribosomes. Uranyl acetate and lead-citrate Fig. 4. Mineralizing
fronts consisting
of various
irregular island-like x 45,000
indicates staining. structures.
the budding x 45,000 Without
material staining.
of a vesicle.
electron-staining.
Fig. 5. Ultra-thin section demineralized with EDTA for 15 min and stained with uranyl acetate and lead citrate. Note filament-like structures (arrow) at periphery of island-like structures. x 90,000
Matrix
vesicles in periodontium
Plate
I
449