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Fine structure of tooth germs during the formation of enameloid matrix in Tilapia nilotica, a teleost fish
Archs oral Biol. Vol. 40, No. 9, pp. 80t 814, 1995
Pergamon
0003-9969(95)00050-X
Copyright G) 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0003-9969/95 $9.50+ 0.00
FINE S T R U C T U R E OF TOOTH G E R M S D U R I N G THE F O R M A T I O N OF E N A M E L O I D M A T R I X IN TILAPIA NILOTICA, A TELEOST FISH I. SASAGAWA Department of Anatomy, School of Dentistry at Niigata, The Nippon Dental University, 1-8 Hamaura-cho, Niigata 951, Japan (Accepted 20 April 1995)
Summary--Tooth germs were examined by light and transmission electron microscopy. Collagen fibrils were relatively dispersed and thin at the early and middle stages of formation of the enameloid matrix, when the enameloid layer was thin. At the late stage, the fibrils became thicker, reaching nearly 30 nm dia, and formed the interwoven thick bundles that are characteristic of teleost cap enameloid. Abundant flocculent and/or fine, network-like material, probably representing glycosaminoglycansor proteoglycans, was located between the collagen fibrils. Tall, columnar, inner dental epithelial cells contained abundant rough endoplasmic reticulum and many mitochondria, and a well-developed Golgi apparatus was seen around the nuclei at the late stage. Elongated vesiclesenclosing fine, filamentous material that resembled procollagen granules, and large granules containing fibril-likestructures that were 150 nm in thickness and had periodic cross-banding at 32-nm intervals, were usually observed near the Golgi apparatus. The contents of the large granules were well stained with phosphotungstic acid, which suggests that inner dental epithelial cells synthesize collagen fibrils. At this time, odontoblasts also contained abundant rough endoplasmic reticulum and mitochondria, a well-developedGolgi, several kinds of granule including those that probably contained procollagen, and many microtubules. It is proposed that odontoblasts are involved in the formation of a considerable portion of the enameloid matrix, including collagen fibrils. Key words: enameloid, teleost, tooth development, fine structure, collagen fibrils.
INTRODUCTION Cap enameloid in teleosts is a highly mineralized tissue that corresponds to the tooth enamel in mammals. Large numbers of collagen fibres occupying the enameloid matrix are a conspicuous feature of the early stage of tooth development in teleosts (Kerr, 1960; Shellis, 1975; Shellis and Miles, 1974, 1976; Yamashita and Ichijo, 1983; Sasagawa, 1984). By contrast, tooth enamel in mammals contains no collagen fibres at any time during its formation. The matrix of cap enameloid appears initially between cells of the inner dental epithelium and the dental papilla in early tooth germs. It has been suggested that the organic matrix in the cap enameloid consists of materials that are provided by both the odontoblasts and the inner dental epithelial cells (Shellis, 1975; Shellis and Miles, 1974, 1976; Meinke, 1982). After the centripetal production of the cap enameloid matrix, the dentine matrix is continuously formed within it. Mineralization begins at the boundary between the cap enameloid and the dentine matrix, and it spreads to the cap enameloid centrifugally and to the dentine centripetally. Matrix vesicles play an important part when the initial mineralization begins, and then crystals are deposited along the collagen fibres in both enameloid and dentine Abbreriation: PAS, periodic acid-Schiff.
(Shellis and Miles, 1976; Sasagawa, 1988). Before the start of defined mineralization, matrix vesicles and fine, crystal-like structures probably derived from them can already be found in the enameloid matrix. In the enameloid, however, no additional mineralization along the collagen fibres occurs until the beginning of mineralization at the boundary between it and dentine. The advance of mineralization along the collagen fibres is inhibited in the enameloid area before the mineralization at the boundary, despite the presence of matrix vesicles (Sasagawa, 1988). It is assumed that removal of the material that inhibits the progress of mineralization might lead to mineralization from the initial site to the surface of the enameloid (Sasagawa, 1988), but the putative material and mechanism remain unknown. Two hypotheses have been proposed for the origin of collagen fibres in the cap enameloid: (a) that the odontoblasts are their main producers (Kerr, 1960; Shellis and Miles, 1974, 1976; Yamashita and Ichijo, 1983) and (b) that inner dental epithelial cells form the bulk of them (Prostak and Skobe, 1984, 1985, 1986; Prostak, Seifert and Skobe, 1993), while the organic matrix of the cap enameloid is supplied by both the inner dental epithelium and odontoblasts. The ratio of ectodermal to mesodermal enameloid matrix collagen would seem to be related to the removal of the collagen fibres in cap enameloid at the maturation stage, and to the size of mature enameloid
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crystals (Prostak, Seifert and Skobe, 1991). Little has been reported about the development of collagen fibres during the formation of the enameloid matrix, the relation between the collagen fibres and other organic matrix in the cap enameloid, and the fine structure of the dental epithelium and the odontoblasts at the early stage of formation of the enameloid matrix. The aim of my study was to examine the fine structure of the cap enameloid matrix and its relation to the cells that are assumed to provide the organic matrix, that is, the dental epithelial cells and the odontoblasts, from the early to the late stage of formation of the enameloid matrix before mineralization.
were then fixed in a 1% solution of osmium tetroxide, buffered with cacodylate (pH 7.4). Some specimens were demineralized with a 2.5% solution o f d i s o d i u m E D T A for 3 weeks before postfixation. After dehydration, the specimens were embedded in Araldite- Epon resin. Semithin sections were cut with glass knives, stained with toluidine blue and then examined under a light microscope. Ultrathin sections were cut with glass or diamond knives, stained with uranyl acetate and lead citrate, or with phosphotungstic acid, and then examined with a transmission electron microscope (H-500; Hitachi, or J E M - 1200EX; JEOL). RESULTS
Light microscopy Early stage of formation of the enameloM matrix.
MATERIALS AND M E T H O D S
Three adult tilapias, Tilapia nilotica, Cichlidae, teleost (total length, 35-37 cm), which possessed large numbers of teeth and tooth germs in their pharyngeal tooth plates, were used in this study. After decapitation, the plates containing the tooth germs were dissected out and placed in buffered 10% formalin (cacodylate buffer, p H 7.4) for 3 days. The specimens were then demineralized with 5% formic acid for 2 weeks. After dehydration and embedding in paraffin wax, serial sections were prepared, stained with haematoxylin-eosin, azan, elastica-van Gieson's stain or PAS-alcian blue, and examined under a light microscope. Some sections were pretreated with human saliva containing salivary amylases for 60 min at 37°C before staining with PAS-alcian blue. Other pharyngeal tooth plates were placed in Karnovsky fixative (cacodylate buffer, pH7.4) overnight at r o o m temperature. These specimens
A small amount of enameloid matrix was produced between the inner dental epithelial cells and the odontoblasts. The short odontoblasts extended a number of processes towards the distal end of the inner dental epithelial cells, which formed a simple layer of low columnar cells facing the enameloid matrix. The other epithelial cells had barely developed at this stage, so that the outer dental epithelial cells were indistinguishable among them. A few PAS-positive particles were found in the dental epithelial cells but not in the odontoblasts. The cells of the tooth germs were haematoxyphilic as a whole at this early stage, which would correspond to the cap stage and/or the early bell stage in mammals, given the morphological resemblance between the features of these stages (Fig. 1).
Middle stage of formation of the enameloid matrix. A layer of enameloid matrix became clearly visible at
(Figs 1 7 opposite) Figures 1-4 are light micrographs. Figures 5 30 are transmission electron micrographs which, unless otherwise stated, are from sections stained with uranyl acetate-lead citrate. Fig. 1. Tooth germ at the early stage of formation of the enameloid matrix. Demineralized in 5% formic acid, stained with haematoxylin-PAS, x 100. Fig. 2. Tooth germ at the middle stage of formation of the enameloid matrix, a layer of which has become visible. An arrow indicates epithelial cells located between the inner and outer dental epithelial cells. Demineralized in 2.5% disodium EDTA, semithin section, toluidine blue. x 233. Fig. 3. Tooth germ at the late stage of formation of the enameloid matrix. A thick enameloid matrix contains many fine fibres stained by eosin. Elongated odontoblasts and inner dental epithelial cells surround the enameloid matrix. The space ('A') is an artefact. Demineralized in 5% formic acid, haematoxylin-eosin, x 100. Fig. 4. Tooth germ at the late stage of formation of the enameloid matrix, which is well stained by PAS-alcian blue. Space (~-) is an artefact. Demineralized, PAS-alcian blue. x I00. Fig. 5. Enameloid matrix and distal portions of inner dental epithelial cells at the early stage of formation of the enameloid matrix. Thin collagen fibrils are visible around the odontoblast processes. Mitochondria, rough endoplasmic reticulum, vesicles and granules are found in the cytoplasm. Demineralized in 2.5% disodium EDTA. x4190. Fig. 6. Golgi apparatus in an odontoblast at the early stage. Several vesicles and vacuoles are visible in the Golgi area. Demineralized. × 15,450. Fig. 7. Odontoblast process containing granules with electron-dense contents at the early stage. Thin collagen fibrils and a fine, network-like material occupy the space around the process. Demineralized. z 19,270.
Enameloid matrix formation in tilapia this stage; in particular, a thick layer was formed at the tooth apex. Fine fibres that were stained with acid dyes (acid fuchsin, aniline blue and eosin) were observed in the matrix. The odontoblasts, with a nucleus at the centre of each, became longer and formed a layer along the unmineralized enameloid matrix. Many odontoblast processes penetrated that matrix. The inner dental epithelial cells were columnar and several cells in the apical region had become
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taller than the others; their nuclei were situated in a wide region from the centre to the proximal margin. The outer dental epithelial cells formed a simple, short columnar or cubic layer. While the bell-shaped enameloid organ usually consisted of only two layers of cells, the inner and outer dental epithelia, a number of epithelial cells were often present between these two layers. In general, the cells in the tooth germs were stained with haematoxylin. A number of
Figs 1-7 (legends opposite).
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Fig. 8. Collagen fibrils showing cross-banding pattern among odontoblast processes at the early stage. Flocculent material is seen around the fibrils and the processes. Demineralized. × 14,610. Fig. 9. Golgi apparatus in an inner dental epithelial cell at the early stage. Several lamellae, vesicles, vacuoles and electron-dense granules are found in the Golgi area. Demineralized. × 13,070. Fig. 10. Odontoblasts at the middle stage of formation of the enameloid matrix. Elongated profiles of rough endoplasmic reticulum, mitochondria, developed Golgi apparatuses and small electron-dense granules are seen in the perinuclear cytoplasm. Demineralized. × 5560. Fig. 11. Golgi apparatus in an odontoblast at the middle stage. A number of long lamellae, vacuoles with flocculent contents and many vesicles are present in the area. Several elongated granules containing fine filamentous material (---~) are found at the centre of the Golgi area. Demineralized. × 13,940. Fig. 12. Enameloid matrix beneath the inner dental epithelial cells at the middle stage. The odontoblast processes are placed close to the basal lamina. The collagen fibrils are thin and dispersed. Many granules and vesicles are seen in the distal cytoplasm of the inner dental epithelial cells. Demineralized. × 6040.
Enameloid matrix formation in tilapia PAS-positive particles were found in the inner dental epithelial cells. This stage would correspond with the middle bell stage in mammals (Fig. 2).
Late stage of formation of the enameloid matrix. The shape of the complete cap enameloid was, for the most part, generated by the organic matrix at this stage, but mineralization had not yet begun. Fine fibres that were stained by acid fuchsin, aniline blue and eosin were densely packed in the matrix. Elongated haematoxyphilic odontoblasts with elliptical nuclei in the distal region were located along the inner surface of the unmineralized enameloid matrix. Many long odontoblast processes were found in the enameloid matrix. The inner dental epithelial cells were relatively translucent, tall columnars, and with their nuclei often central but sometimes distal or proximal; haematoxyphilic ergastoplasm was visible in both the distal and proximal cytoplasm (Fig. 3). There were large numbers of PAS-positive particles in the inner dental epithelial cells. The haematoxyphilic outer dental epithelial cells also contained some PAS-positive particles, which were not seen after pretreatment with human saliva and therefore probably consisted of glycogen. The enameloid matrix was well stained by PAS-alcian blue (Fig. 4).
Transmission electron microscopy Early stage of formation of the enameloid matrix. Odontoblasts were squat and a number of organelles were visible around the nuclei (Figs 5 and 6). The Golgi apparatus consisted of short lamellae, several vesicles and vacuoles near the nucleus (Fig. 6). Mitochondria, short profiles of rough endoplasmic reticulum, small vesicles and granules with electron-dense contents were usually visible in the perinuclear cytoplasm. Many odontoblast processes extended into the enameloid matrix and reached the lamina densa beneath the inner dental epithelial cells. The processes often contained granules with electron-dense contents, mitochondria, short profiles of rough endoplasmic reticulum and small vesicles (Fig. 7). Thin collagen fibrils, which ranged from 12 to 20 nm dia, were found around the odontoblast processes (Fig. 7). Some collagen fibrils that appeared thicker than others showed clear cross-banding at intervals of about 67 nm (Fig. 8). Flocculent or fine, network-like material was seen among the collagen fibrils and the odontoblast processes (Figs 7 and 8). This material often appeared to make contact with the collagen fibrils and sometimes to form a periodic attachment with their cross-banding (Figs 7 and 8). No defined matrix vesicles were seen at this stage, although many cross-sections of odontoblast processes were noted. In the cytoplasms of the squat, inner dental epithelial cells, there were a number of mitochondria, rough endoplasmic reticulum, granules with electron-dense contents and vesicles. The Golgi apparatus, consisting of a number of lamellae, vesicles, vacuoles and electron-dense granules, was situated near the nucleus (Fig. 9). The distal end of each cell was almost straight (Fig. 5). Well-developed interdigitations were often observed in the lateral membranes of the inner dental epithelial cells.
Middle stage of formation of the enameloid matrix. In the odontoblasts, extensive rough endoplasmic
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reticulum, mitochondria, well-developed Golgi apparatus and various kinds of granule were present around the nucleus (Fig. 10). The Golgi apparatus consisted of several long lamellae, many vesicles and condensing vacuoles that contained flocculent material. Elongated granules containing fine, filamentous material were usually found in the Golgi area (Fig. 11). Round granules with electron-dense contents were often seen in the cytoplasm. Desmosomes were often visible among the odontoblasts. Intercellular spaces were usually present distally. There were still many odontoblast processes in the enameloid matrix and some were attached to the lamina densa beneath the inner dental epithelial cells (Figs 12 and 15). The collagen fibrils in the cap enameloid were still thin, being only about 15 nm dia, and there was no difference in their diameters between the outer layer near the inner dental epithelium and the inner layer near the odontoblasts. Some relatively thick collagen fibrils exhibited clear cross-banding. The fibrils tended to form bundles and to be arranged along, and in proximity to, odontoblast processes (Figs 12 and 13). The spaces between the fibrils appeared wide and were filled with abundant flocculent and/or fine, network-like material; periodic attachment of this electron-dense material to the fibril cross-banding was commonly observed in the enameloid matrix (Fig. 13). Beneath the basal lamina, the majority of the odontoblast processes and the collagen fibrils were arranged parallel to the lamina densa (Fig. 12). Small numbers of matrix vesicles (40-80 nm dia) with a clear unit membrane appeared in the cap enameloid matrix (Fig. 13). There were few crystals in the matrix vesicles at this stage. The vesicles often exhibited a serial arrangement along the odontoblast processes and the collagen fibrils. The inner dental epithelial cells became taller and columnar and their nuclei occupied the central to proximal region (Fig. 14). A developed Golgi apparatus was usually seen on the distal side of the nucleus. There were mitochondria and an extensive rough endoplasmic reticulum. Many round or bacilliform granules containing electron-dense material and vesicles were seen in the distal cytoplasm (Fig. 14, inset; Fig. 15). Many electron-dense particles, which were probably glycogen, were found in the cytoplasm. Expanded spaces containing electron-lucent fibrous structures were often present distally between the inner dental epithelial cells. Their distal ends were nearly flat and there was a clear basal lamina beneath them (Figs 12 and 15). Several small epithelial cells were observed between the inner and outer dental epithelial cells at this stage. These resembled stellate reticulum on the apical side (Figs 2 and 16) but were flat near the cervical loop (Figs 2 and 17). The outer dental epithelium comprised a simple layer of short columnar or cubic cells. The nucleus was near to the centre of each cell and there were small numbers of cytoplasmic organelles (Fig. 17).
Lat e stage of formation of the enameloid matrix. The odontoblasts became taller and their nuclei were located proximally. An abundant rough endoplasmic reticulum and well-developed Golgi apparatuses were present in the cytoplasm. The extensive rough endoplasmic reticulum, enclosing fine material, was
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usually arranged almost parallel to the long axis of each cell (Figs 18 and 19). The Golgi consisted of several lamellae, many vesicles and vacuoles. Elongated granules with fine filamentous contents cross-banded at intervals of approx. 300nm were often visible in the Golgi region (Fig. 19, inset). A number of mitochondria, electron-dense granules, small vesicles and microtubules were usually found in the cytoplasm. Desmosomes and short, gap-like junc-
tions were observed between odontoblasts; wide intercellular spaces were scarcely ever seen. Processes penetrating into the enameloid matrix extended from the distal ends of the odontoblasts (Fig. 18). There were large numbers of matrix vesicles with a defined unit membrane in the enameloid matrix near the odontoblasts. The collagen fibrils in the enameloid matrix were mainly of 30-40 nm dia (range, 20-60 nm), that is,
Figs 13--17 (legends opposite).
Enameloid matrix formation in tilapia twice as thick as those at earlier stages (Figs 20-22). Although there was no significant change in their diameter from the apex to the base in the cap enameloid, the fibrils near the odontoblasts, which apparently showed the characteristic arrangement of enameloid matrix, tended to be thicker than those in the apical enameloid. A cross-banding pattern, with a periodicity of about 67 nm, was usually seen at this stage (Fig. 22). These collagen fibrils gathered together to form the thick, interwoven bundles characteristic of cap enameloid (Figs 20 and 21). Odontoblast processes were present in the enameloid matrix near the odontoblasts but they were rarely seen in the apical enameloid matrix. The bundles of collagen fibrils tended to be arranged along the odontoblast processes in the enameloid matrix near the odontoblasts. Matrix vesicles, which usually contained electron-dense, crystal-like structures, were found in the cap enameloid (Fig. 22). Fine aggregates of such structures, which were associated with an electron-dense, fine material that was probably derived from the matrix vesicles, were scattered in the apical enameloid matrix. Fine, moderately electrondense material and electron-dense particles occupied the regions around the collagen fibrils and matrix vesicles (Fig. 22). Tall, columnar, inner dental epithelial cells contained well-developed organelles. Their nuclei were nearly central and there were many well-ordered organelles in the proximal cytoplasm, where mitochondria were gathered (Fig. 23). Large amounts of rough endoplasmic reticulum, arranged nearly parallel to the long axes of the cells, were visible mainly in the proximal cytoplasm. Developed Golgi apparatuses, which consisted of several lamellae, many vesicles, vacuoles and small electron-dense granules, were situated around the nuclei (Figs 24 and 25). Elongated granules with fine, filamentous contents that usually showed cross-banding at intervals of approx. 300 nm were often observed near the Golgi (Fig. 25). In the distal cytoplasm, the organelles were relatively scattered; extensive rough endoplasmic reticulum, several granules, vesicles, vacuoles and lysosomes were usually seen. Several large, irregular granules contained fibrillar structures, 150-nm thick with cross-banding at intervals of about 32 nm and with fine, electron-dense material around them
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(Fig. 26). These large granules were usually found near the Golgi and in the distal cytoplasm. There were many electron-dense particles; probably glycogen, in the cytoplasm of the inner dental epithelial cells (Figs 26-28). Electron-dense, granular or flocculent material was often seen in the expanded spaces between these cells. Desmosomes, gap-like junctions and interdigitations of the lateral cell membranes were still clearly visible at this stage. Fibrils that appeared to cross the basal lamina were occasionally observed (Fig. 27). In sections stained with phosphotungstic acid, several large granules containing positively stained fine filaments were usually visible in the Golgi area and in the distal cytoplasm of the inner dental epithelial cells (Figs 28 and 29). These granules tended to increase in number in cells at the apical side of the tooth germ and to decrease in cells in the cervical region. Although the phosphotungstic acidpositive filaments in the large granules rarely showed periodic cross-banding, they probably corresponded to the fibril-like structures with cross-banding at 32-nm intervals in the sections stained with uranyl acetate and lead citrate. In the granules, fine material usually surrounded the positively stained filaments (Fig. 28). Collagen fibrils in the enameloid matrix were also well stained by phosphotungstic acid (Fig. 29). Outer dental epithelial cells became larger and developed into electron-lucent cells that contained many vesicles and vacuoles; there were mitochondria and lysosomes in the cytoplasm. These cells tended to overlap and to extend projections to the outside at the apical portion of the tooth germs while, in the region near the cervical loop, they still constituted a simple layer. The cells of the outer dental epithelium made direct contact with those of the inner dental epithelium without wide intercellular spaces. Desmosomes and short gap-like junctions were seen between the outer and inner dental epithelial cells (Fig. 30). DISCUSSION
The diameters of the collagen fibrils, 30-40 nm, at the late stage of formation of the enameloid matrix reflect the results obtained in other teleosts (25-50nm, common eel, Shellis and Miles, 1976; 30 nm, Texas cichlid, Prostak and Skobe, 1984, 1985,
(Figs 13-17 opposite) Fig. 13. Thin collagen fibrils, cross-section of odontoblast processes and matrix vesicles (---,) in the enameloid matrix at the middle stage. Flocculent material is situated around the collagen fibrils. Periodic attachment of electron-dense material ( ~ ) is found at the cross-banding of the collagen fibrils (inset). Demineralized. x 30,350. Inset: bar = 100 nm. x 35,580. Fig. 14. Long inner dental epithelial cells situated in the apical area of a tooth germ at the middle stage. Many mitochondria, granules, vesicles, extensive rough endoplasmic reticulum and developed Golgi apparatuses are seen in the distal cytoplasm. Demineralized. x 3790. Inset: enlarged view of the elongated granules with electron-dense contents in the cytoplasm near the Golgi apparatus. Bar = 200 nm. x 20,930. Fig. 15. Distal portion of inner dental epithelial cells and the enameloid matrix at the middle stage. Expanded intercellular spaces containing fine material and a defined basal lamina are seen distally. Demineralized. x 15,180. Fig. 16. Epithelial cells between the inner and outer dental epithelium in the apical part of a tooth germ at the middle stage; these resemble stellate reticulum cells. Demineralized. x 2880. Fig. 17. Columnar outer dental epithelial cells and the fiat intermediate cells ( ~ ) between the inner and outer dental epithelial cells at the middle stage. Demineralized. x 3450.
Fig. 18. Odontoblasts and their processes in the enameloid matrix (M) at the late stage of its formation. A b u n d a n t rough endoplasmic reticulum and well-developed Golgi apparatuses are visible in the odontoblasts. Demineralized. × 5500. Fig. 19. Golgi apparatus and rough endoplasmic reticulum in odontoblasts at the late stage. Arrows indicate vesicles that contain fine filamentous material. Demineralized. x 10,920. Inset: enlarged view of the vesicle containing fine filamentous materials. Bar = 300 nm. x 24,950. Fig. 20. Enameloid matrix at the late stage. Large numbers of matrix vesicles and several odontoblast processes are present. Non-demineralized. x 6060. Fig. 21. Enlarged view of the enameloid matrix including odontoblast processes and matrix vesicles at the late stage. The collagen fibrils, 30~40 n m dia, gather together to form thick bundles. Non-demineralized. × 13,770. Fig. 22. Enlarged view of collagen fibrils in the enameloid matrix at the late stage. Electron-dense fine materials are placed in the space around the collagen fibrils. Non-demineralized. × 59,300. Inset: matrix vesicles containing crystal-like structures at the late stage. Bar = 100 nm. x 57,390. 808
Enameloid matrix formation in tilapia
Fig. 23. C o l u m n a r inner dental epithelial cells and cubic outer dental epithelial cells at the late stage. Mitochondria tend to gather at the proximal side of the inner dental epithelial cells. Demineralized. x 2840. Fig. 24. Portion near the nuclei of inner dental epithelial cells at the late stage. Extensive rough endoplasmic reticulum and well-developed Golgi apparatuses with m a n y granules and vesicles. Demineralized, x 6070. Fig. 25. Golgi apparatus and rough endoplasmic reticulum near the nuclei of inner dental epithelial cells at the late stage. An arrow indicates a large granule that contains a fibril-like structure, and an arrowhead indicates an elongated granule with filamentous contents. Demineralized. × 7590. Inset: enlarged view of the granule with filamentous contents. Bar = 300 nm. x 22,600. Fig. 26. A large granule containing a fibril-like structure with periodic cross-banding at intervals of about 32 n m in an inner dental epithelial cell at the late stage. Demineralized. × 37,940.
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Fig. 27. Distal end of inner dental epithelial cell and basal lamina at the late stage. An arrow indicates a fibril that seems to be passing across the basal lamina. Arrowheads indicate electron-dense glycogen particles in the cytoplasm. Demineralized. × 37,890. Fig. 28. Large granules containing phosphotungstic acid (PTA)-positive fine filaments in inner dental epithelial cells at the late stage. Demineralized, PTA. x 12,120. Inset: enlarged view of the granules. Bar = 100 nm. x 32,300. Fig. 29. Distal portion of inner dental epithelial cells at the late stage. The organelles are relatively scattered in the distal cytoplasm. A number of large granules containing phosphotungstic acid (PTA)-positive fine filaments ( ~ ) are visible at the distal portion. Demineralized, PTA. x 6060. Inset: enlarged view of the gap-like junction at the lateral cell membrane. Bar = 100 nm. x 30,870. Fig. 30. Outer dental epithelial cells at the late stage. These develop into electron-lucent cells that contain m a n y vacuoles and small vesicles. I, inner dental epithelial cells; O, outer dental epithelial cells. Non-demineralized. × 3790. Inset: desmosomes and gap-like junctions between the inner and outer dental epithelial cells. Demineralized. Bar = 1 p m . x 7380.
Enameloid matrix formation in tilapia 1986). The collagen fibrils of 30-40 nm dia in the late-stage enameloid matrix formed the thick bundles characteristic of teleost cap enameloid. However, the fibrils at the early and middle stages were thin, of approx. 15 nmdia, and dispersed. At the late stage each fibril was almost twice as thick as at the earlier stages. It appears that the collagen fibrils are dispersed and thin at the early and middle stages before the matrix has attained its final thickness. It has been reported that the density of collagen fibrils in cap enameloid increases in tooth buds that have nearly achieved their full thickness (Prostak and Skobe, 1986). The increase in thickness of collagen fibrils during the formation of the enameloid matrix is probably involved in the increase in density of the collagen fibrils in cap enameloid. In general, collagen fibrils in loose connective tissue increase in diameter after they are first polymerized outside fibroblasts, suggesting that each fibril grows by apposition, with further collagen molecules joining those already assembled in the same staggered fashion (Ham and Cormack, 1979). In hamster and rat incisors, collagen fibrils had a small diameter on the cell-body side of the predentine, and increased in thickness through the predentine, achieving a maximum thickness at the dentine mineralization front (Watson and Avery, 1954; Reith, 1968). It is assumed that appositional growth of collagen fibrils occurs during the formation of enameloid matrix. Simultaneously, in my material, the number of odontoblast processes decreased in the cap enameloid at the late stage. Matrix vesicles began to appear at the middle stage and, at the late stage, large numbers were visible in the cap enameloid. If the odontoblast processes are involved in providing procollagen molecules for the enameloid matrix, the morphological changes in those processes and in the matrix vesicles might be related to the growth in thickness of the collagen fibrils in the cap enameloid. In Polypterus, rather thick collagen fibrils, of 100-120nmdia, were reported in the area near the odontoblasts when the enameloid layer was thin, while the collagen fibrils at the surface were of 3 0 - 5 0 n m d i a (Kogaya, 1989). This discrepancy suggests variations among species or among portions of the samples examined. At the middle and the later stages of formation of the enameloid matrix, the odontoblasts contained much rough endoplasmic reticulum and many mitochondria, well-developed Golgi apparatuses, several kinds of granule and many microtubules. Several elongated granules containing fine filaments were observed in the Golgi area. The morphological appearance of the elongated granules was similar to that of the secretory granules containing procollagen molecules in mammalian odontoblasts, in which the synthesis, migration and release of precursor collagen have been clearly demonstrated by autoradiography (Frank, 1970; Weinstock and Leblond, 1974). In particular, the fine filaments in the granules exhibited cross-banding at intervals of approx. 300 nm, which resembled the length of procollagen molecules (Weinstock, 1972). Consequently, the elongated granules are probably procollagen granules. It is suggested that the odontoblasts synthesise procollagen molecules. Prostak and Skobe (1985, 1986) and
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Prostak et al. (1993) noted that odontoblasts in Texas cichlid, parrotfish and pufferfish probably provide only small quantities of enameloid collagen because they contain small numbers of secretory granules, and they showed little or no signs of secretory activity during the formation of the enameloid matrix in experiments using colchicine. In Tilapia nilotica, however, the morphological features of the odontoblasts imply that they produce considerable amounts of collagen fibrils as part of the enameloid matrix, although the exact proportion of the collagen that originates from odontoblasts in that matrix cannot be determined from the present data. There were large numbers of matrix vesicles, which presumably originated from odontobtasts, in the enameloid matrix near the cells (Sasagawa, 1988). Odontoblast processes were observed in the cap enameloid during the matrix-formation stage. At the early to middle stage, many odontoblast processes were visible in the enameloid matrix from the surface to the lower part. It is possible that odontoblast processes influence the arrangement of collagen fibrils in the cap enameloid (Wakita, Takahashi and Hanaizumi, 1993) because the fibrils near the processes tend to lie in the same direction as the processes. Therefore, the evidence supports the hypothesis that odontoblasts play an important part in the formation of cap enameloid matrix, and of the collagen fibrils in particular (Kvam, 1946; Kerr, 1960; Shellis and Miles, 1974, 1976). It is also likely that the contribution of odontoblasts to the formation of the enameloid matrix varies among species of fish (Prostak et al., 1991). Prostak and Skobe (1986) observed interfibrillar material among collagen fibrils in EDTA-treated specimens and they interpreted the material as a partially solubilized collagen. In my study, similar fine material was observed in both demineralized and non-demineralized specimens. The staining of the enameloid matrix by PAS-alcian blue implies that large amounts of glycosaminoglycans are present. Kogaya (1989, 1994) detected large quantities of chondroitin sulphates in the developing enameloid of Polypterus senegalus and Hoplognathus fasciatus, and these were in close association with the enameloid collagen. Proteoglycan filaments tend to cross the collagen fibrils at intervals of about 67 nm (Scott, 1980; Furuhashi, K o g a y a and Yoshioka, 1984: Ruggeri and Benazzo, 1984). The presence of fine material forming a network-like structure and periodic attachment to the collagen fibrils in the tilapia enameloid suggests that large amounts of glycosaminoglycans and/or proteoglycans are located among the collagen fibrils, while further histochemical and immunohistochemical studies at the electron-microscopic level should provide exact details of the structural relation. The existence of non-collagenous proteins that are of odontoblast origin cannot be clarified until much more is known about the histochemical features of the developing enameloid. In teleosts, Golgi apparatuses and rough endoplasmic reticulum generally develop in the distal cytoplasm of the inner dental epithelial cells at the late stage of formation of the enameloid matrix (Yamashita and Ichijo, 1983). In Tilapia nilotica.
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well-developed Golgi apparatuses, abundant rough endoplasmic, reticulum, mitochondria and several kinds of vesicle mainly occupied the proximal cytoplasm, as is the case in eels (Shellis and Miles, 1976). This finding again suggests differences among species. The presence of a well-developed Golgi, rough endoplasmic reticulum and many vesicles strongly indicates that the inner dental epithelial cells engage in secretion at this stage. Prostak and Skobe (1984, 1985, 1986) proposed that the bulk of collagen fibres in the teleost enameloid arises from the epithelial cells. Wakita (1974) reported the presence of fibrous structures with cross-banding in inner dental epithelial cells. Sasagawa (1984) suggested that these cells might secrete collagen fibres because of morphological similarities between their Golgi and that of the odontoblasts. Enameloid collagen-like fibrils have been observed in the spaces between inner dental epithelial cells (Kogaya, 1989). In the present study, large granules that contained fibril-like structures, which often had periodic stripes at about 32-nm intervals (about half the length of a single D-period; 67 nm) were found in the inner dental epithelial cells, and the contents of these granules were stained by phosphotungstic acid as fine filaments. It is likely that the contents were a kind of procollagen. Moreover, several elongated granules containing fine filaments were seen in the Golgi area and appeared to be secretory granules. These fine filaments in the elongated granules usually had cross-banding at intervals of approx. 300 nm. This also suggests that procollagen granules are produced in the Golgi. These two types of granule in the inner dental epithelial cells resembled the secretory granules that have been observed in the odontoblasts of mammals (Nagai, 1970; Weinstock and Leblond, 1974) and described in the reports of Prostak and Skobe (1984, 1985, 1986). Hence, it is certain that the inner dental epithelial cells in Tilapia nilotica can synthesize collagen. Ectodermal collagen fibres have often been found in other normal organs (chick corneal epithelium, Trelstad, 1971; Hay and Dodson, 1973; chick notochordal epithelium, Carlson, 1973), so it seems likely that the epithelial cells of tooth germs in teleosts produce some type of collagen fibre. It is unclear whether the bulk of collagen fibres in cap enameloid is of ectodermal origin because of many problems associated with interpreting the migl:ation of ectodermal collagen into the enameloid matrix. It is still unclear how ectodermal procollagen molecules and/or collagen fibrils might move into the enameloid matrix through the basal lamina. In this study, the collagen fibrils seemed to penetrate the basal lamina, as shown in Fig. 27. Similar evidence that fibrous substances might cross the basal lamina of inner dental epithelial cells was found at the late stage of formation of enameloid matrix in the dog salmon (Sasagawa, 1984). There are probably pores, through which macromolecules including collagen fibrils can pass, in the basal lamina at the late stage. However, I rarely saw material that appeared to be passing through the basal lamina under the transmission electron microscope even at the late stage. Substances that consisted of electron-dense, fine particles or flocculent material were often observed
between the inner dental epithelial cells and the basal lamina, implying that material of high molecular weight was stored in that region because of obstruction by the basal lamina. The inner dental epithelial cells that surrounded the apex of the enameloid contained many granules that included ectodermal procollagen, but not all cells facing the enameloid matrix had an abundance of such granules. In the present study, the collagen fibrils of nearly 30 nm dia and with 67-nm periodic cross-banding, which were seen in the enameloid matrix, were not found in the spaces between the inner dental epithelial cells at the late stage. In the avian cornea the epithelium produces type I collagen and primary corneal stroma in the early stages of embryogenesis. However, after fibroblasts have invaded the cornea, its total synthesis of collagen increases enormously because of their important contribution (Hay, 1980). It is likely that the inner dental epithelial cells in Tilapia nilotica actually secrete ectodermal collagen but that it is not the only or major component of the enameloid matrix. It has been proposed that the collagen produced by the odontoblasts provides a scaffold from which the collagen of the inner dental epithelial cells can assemble and the fibrils can lengthen (Prostak et al., 1993). It is also possible that the ectodermal collagen is associated with the increase in thickness of collagen fibrils during the formation of the enameloid matrix. It is reported that the electrophoretic patterns of acetic acid-soluble proteins obtained from teleost enameloid matrix showed characteristic type I collagen bands, and additional 33- and 30-kDa bands, which were thought to be degradation peptides of enameloid collagen itself (Prostak, Seifert and Skobe, 1992). However, neither morphological nor histochemical data concerning the differences between the ectodermal collagen and the collagen from odontoblasts were obtained from the cap enameloid matrix. Unresolved issues of interest in relation to the collagen in the cap enameloid of teleosts are: the proportion and distribution of the ectodermal collagen in the enameloid matrix; the role of ectodermal collagen during formation of the enameloid; and the interaction between the ectodermal collagen and the collagen from the odontoblasts. It is probable that the inner dental epithelial cells in teleosts secrete not only collagen fibrils but also other proteins, such as enamelin (Slavkin et al., 1983; Herold, Rosenbloom and Granovsky, 1989) and proteolytic enzymes (Kawasaki, Shimoda and Fukae, 1987). Immunohistochemistry together with transmission electron microscopy might reveal details of the distribution of the proteins from these cells in enameloid matrix. Stellate reticulum is generally found between the inner and outer epithelial cells in tooth germs in higher vertebrates such as reptiles and mammals. However, the corresponding cells show variations among different groups of fish (Kerr, 1960; Yoshitani, 1959). Here, in Tilapia nilotica, some stellate cells that resemble those of the stellate reticulum in mammals were noticed between the inner and outer dental epithelia just at the middle stage of formation of the enameloid matrix. It is likely that cells resembling stellate reticulum are temporarily
Enameloid matrix formation in tilapia present a m o n g the dental epithelial cells d u r i n g t o o t h d e v e l o p m e n t in this teleost. Large n u m b e r s of glycogen particles have been reported in the inner dental epithelial cells from the stage o f f o r m a t i o n o f the enameloid matrix to the beginning of the mineralization in several fishes, including sharks a n d sting-rays ( G a r a n t , 1970; Ono, 1974; W a k i t a , 1974; G o t o , 1976; Y a m a s h i t a a n d Ichijo, 1983; Sasagawa a n d Akai, 1992). In Tilapia nilotica, great n u m b e r s of electron-dense particles, p r o b a b l y glycogen, were f o u n d in the inner dental epithelial cells at the late stage of f o r m a t i o n of the enameloid matrix, but I have preliminary evidence that they disappeared rapidly from these cells at the s u b s e q u e n t mineralization stage. A characteristic m o r p h o l o g i c a l feature of the t o o t h germs of Tilapia nilotica is the scattered r a t h e r t h a n aggregated distrib u t i o n of glycogen particles in the cytoplasm of inner dental epithelial cells. The role of glycogen in these cells remains to be clarified. However, it is possible that glycogen m i g h t have a role w h e n these cells later c h a n g e into absorptive cells at the mineralization and m a t u r a t i o n stages. A cknowledgements--I thank the staff members of Hokuetsu Mizugiken Inc. for their kindness in offering their facilities for the collection of specimens.
REFERENCES
Carlson E. C. (1973) Periodic fibrillar material in membrane-bounded bodies in notochordal epithelium of the early chick embryo. J. ultrastruct. Res. 42, 287-297, Frank R. M. (1970) Etude autoradiographique de la dentinogen~se en microscopie 61ectronique 5. l'aide de la proline triti6e chez le chat. Archs oral Biol. 15, 583-596. Furuhashi K., Kogaya Y. and Yoshioka Y. (1984) Ruthenium hexammine trichloride-positive materials in developing mouse molar predentine and intercellular spaces between preodontoblasts. J. electron. Microsc. 33, 384-387. Garant P. R. (1970) Observation on the ultrastructure of the ectodermal component during odontogenesis in Helostoma temmincki. Anat. Rec. 166, 167-188. Goto M. (1976) Histogenetic studies on the teeth of leopard shark (Triakis scyllia). J. Stomatol. Soc. Jpn 45, 527 584. Ham A. W. and Cormak D. H, (1979) Histology, 8th edn, p. 233. Lippincott, Toronto. Hay E. D. (1980) Development of the vertebrate cornea. Int. Rev. Cytol. 63, 263-322. Hay E. D. and Dodson J. W. (1973) Secretion of collagen by corneal epithelium. I. Morphology of the collagenous products produced by isolated epithelia grown on frozenkilled lens. J. cell Biol. 57, 190-213. Herold R. C., Rosenbloom J. and Granovsky M. (1989) Phylogenetic distribution of enamel proteins: immunohistochemical localization with monoclonal antibodies indicates the evolutionary appearance of enamelins prior to amelogenins. Calc. Tiss. Int. 45, 88-94. Kawasaki K., Shimoda S. and Fukae M. (1987) Histological and biochemical observations of developing enameloid of the sea bream. Adv. dent. Res. 1, 191 195. Kerr T. (1960) Development and structure of some actinopterygian and urodele teeth. Proc. zool. Soe. Lond. 133, 401-422. Kogaya Y. (1989) Histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus. Histochemistry 91, 185 190. AOB 40/9-- B
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Kogaya Y. (1994) Sulfated glycoconjugates in amelogenesis. Prog. Histochem. Cytoehem. 29, 1-108. Kvam T. (1946) Comparative study of the ontogenetic and phylogenetic development of dental enamel. Norsk. Tannlaegeforen. Tid. 56, Suppl; 1 198. Meinke D. K. (1982) A histological and histochemical study of developing teeth in Polypterus (Pisces, Actinopterygii). Archs oral Biol. 27, 197 206. Nagai N. (1970) Electron microscopy of the cytoplasmic bodies in the odontoblasts of young rat incisors. Bull. Tokyo dent. Coll. 11, 47 83. Ono T, (1974) Electron microscopic studies on the ultrastructure of ameloblasts during amelogenesis in Oplegnathus fasciatus. Jpn J. oral Biol. 16, 407-464. Prostak K. and Skobe Z. (1984) Effects of colchicine on fish enameloid matrix formation. In Tooth Enamel IV (Eds Fernhead R. W. and Sug S.), 1st edn, p. 525. Elsevier, Amsterdam. Prostak K. and Skobe Z. (1985) The effects of colchicine on the ultrastructure of the dental epithelium and odontoblasts of teleost tooth buds. J. eranio/ac. Genet. derl Biol. 5, 75-88. Prostak K. and Skobe Z. (1986) Ultrastructure of the dental epithelium and odontoblasts during enameloid matrix deposition in cichlid teeth. J. Morphol. 187, 159 172. Prostak K., Seifert P. and Skobe Z. (1991) Tooth matrix formation and mineralization in extant fishes. In Mechanisms and Phylogeny of Mineralization in Biological Systems (Eds Suga S. and Nakahara H.), 1st edn. p. 465. Springer, Tokyo. Prostak K., Seifert P. and Skobe Z. (1992). Fish tooth formation: an assessment of biological factors affecting the fluoride content of enameloid. In Hard Tissue Mineralization and Demineralization (Eds Suga S. and Watabe N.), 1st edn, p. 33. Springer, Tokyo. Prostak K., Seifert P. and Skobe Z. (1993) Enarneloid formation in two tetraodontiform fish species with high and low fluoride contents in enameloid. Archs oral Biol. 38, 1031 1044. Reith E. J. (1968) Ultrastructural aspects of dentinogenesis. In Dentine and Pulp: Their Structure and Reactions (Ed. Symons N. B. B.), 1st edn, p. 19. Thomson, Dundee. Ruggeri A. and Benazzo F. (1984) Collagen- proteoglycan interaction. In Ultrastrueture of the Connectit,e Tissue Matrix (Eds Ruggeri A. and Motta P. M.), 1st edn, p. 113. Martinus Nijhoff, Boston. Sasagawa I. (1984) Formation of cap enameloid in the jaw teeth of dog salmon, Oncorhynchus keta. Jap. J. oral Biol. 26, 477-495. Sasagawa I. (1988) The appearance of matrix vesicles and mineralization during tooth development in three teleost fishes with well-developed enameloid and orthodentine. Archs oral Biol. 33, 75 86. Sasagawa I. and Akai J. (1992) The fine structure of the enameloid matrix and initial mineralization during tooth development in the sting rays, Dasyatis akajei and Urolophus aurantiacus. J. electron. Microse. 41, 242 252. Scott J. E. (1980) Collagen-proteoglycan interactions. Localization of proteoglycans in tendon by electron microscopy. Biochem. J. 187, 887-891. Shellis R. P. (1975) A histological and histochemical study of the matrices of enameloid and dentine in teleost fishes. Archs oral Biol. 20, 183 187. Shellis R. P. and Miles A. E. W. (1974) Autoradiographic study of the formation of enameloid and dentine matrices in teleost fishes using tritiated amino acids. Proc. R. Soc. Lond. B 185, 51 72. Shellis R. P. and Miles A. E. W. (1976) Observations with the electron microscope on enameloid formation in the common eel (Anguilla anguilla; Teleostei). Proc. R. Soc. Lond. B 194, 253 269. Slavkin H. C., Zeichner-David M., Snead M. L.,
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Graham E. E., Samuel N. and Ferguson M. W. J. (1983) Amelogenesis in reptilia: evolutionary aspects of enamel gene products. In The Structure, Development and Evolution of Reptiles (Ed. Ferguson M. W. J.), 1st edn, p. 275. Academic Press, London. Trelstad R. L. (1971) Vacuoles in the embryonic chick corneal epithelium, an epithelium which produces collagen. J. cell Biol. 48, 689~94. Wakita M. (1974) Studies on the ultrastructure of ameloblasts during amelogenesis in Prionurus mierolepidotus Lac6pdde. Jap, J. oral Biol. 16, 129-185. Wakita M., Takahashi S. and Hanaizumi Y. (1993) Studies on the matrix of enameloid in Paralichtys olivaceus. Arch, comp. Biol. Tooth Enamel 3, 7 18. Watson M. L. and Avery J. K, (1954) The development of
the hamster lower incisor as observed by electron microscopy. Am. J. Anat. 95, 109. Weinstock M. (1972) Collagen formation--Observations on its intracellular packaging and transport. Z. Zell/'orsch. 129, 455470. Weinstock M. and Leblond C. P. (1974) Synthesis, migration, and release of precursor collagen by odontoblasts as visualized by radioautography after [3H]proline administration. J. cell Biol. 60, 92-127. Yamashita Y. and Ichijo T. (1983) Comparative studies on the structure of the ameloblasts. In Mechanisms ~/ Tooth Enamel Formation (Ed. Suga S.), 1st edn, p. 91. Quintessence PuNishing, Tokyo. Yoshitani M. (1959) Histogenetic studies on the teeth of sea breams. J. stomatol. Soc. Jpn. 26, 811 834.
Pergamon
0003-9969(95)00050-X
Copyright G) 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0003-9969/95 $9.50+ 0.00
FINE S T R U C T U R E OF TOOTH G E R M S D U R I N G THE F O R M A T I O N OF E N A M E L O I D M A T R I X IN TILAPIA NILOTICA, A TELEOST FISH I. SASAGAWA Department of Anatomy, School of Dentistry at Niigata, The Nippon Dental University, 1-8 Hamaura-cho, Niigata 951, Japan (Accepted 20 April 1995)
Summary--Tooth germs were examined by light and transmission electron microscopy. Collagen fibrils were relatively dispersed and thin at the early and middle stages of formation of the enameloid matrix, when the enameloid layer was thin. At the late stage, the fibrils became thicker, reaching nearly 30 nm dia, and formed the interwoven thick bundles that are characteristic of teleost cap enameloid. Abundant flocculent and/or fine, network-like material, probably representing glycosaminoglycansor proteoglycans, was located between the collagen fibrils. Tall, columnar, inner dental epithelial cells contained abundant rough endoplasmic reticulum and many mitochondria, and a well-developed Golgi apparatus was seen around the nuclei at the late stage. Elongated vesiclesenclosing fine, filamentous material that resembled procollagen granules, and large granules containing fibril-likestructures that were 150 nm in thickness and had periodic cross-banding at 32-nm intervals, were usually observed near the Golgi apparatus. The contents of the large granules were well stained with phosphotungstic acid, which suggests that inner dental epithelial cells synthesize collagen fibrils. At this time, odontoblasts also contained abundant rough endoplasmic reticulum and mitochondria, a well-developedGolgi, several kinds of granule including those that probably contained procollagen, and many microtubules. It is proposed that odontoblasts are involved in the formation of a considerable portion of the enameloid matrix, including collagen fibrils. Key words: enameloid, teleost, tooth development, fine structure, collagen fibrils.
INTRODUCTION Cap enameloid in teleosts is a highly mineralized tissue that corresponds to the tooth enamel in mammals. Large numbers of collagen fibres occupying the enameloid matrix are a conspicuous feature of the early stage of tooth development in teleosts (Kerr, 1960; Shellis, 1975; Shellis and Miles, 1974, 1976; Yamashita and Ichijo, 1983; Sasagawa, 1984). By contrast, tooth enamel in mammals contains no collagen fibres at any time during its formation. The matrix of cap enameloid appears initially between cells of the inner dental epithelium and the dental papilla in early tooth germs. It has been suggested that the organic matrix in the cap enameloid consists of materials that are provided by both the odontoblasts and the inner dental epithelial cells (Shellis, 1975; Shellis and Miles, 1974, 1976; Meinke, 1982). After the centripetal production of the cap enameloid matrix, the dentine matrix is continuously formed within it. Mineralization begins at the boundary between the cap enameloid and the dentine matrix, and it spreads to the cap enameloid centrifugally and to the dentine centripetally. Matrix vesicles play an important part when the initial mineralization begins, and then crystals are deposited along the collagen fibres in both enameloid and dentine Abbreriation: PAS, periodic acid-Schiff.
(Shellis and Miles, 1976; Sasagawa, 1988). Before the start of defined mineralization, matrix vesicles and fine, crystal-like structures probably derived from them can already be found in the enameloid matrix. In the enameloid, however, no additional mineralization along the collagen fibres occurs until the beginning of mineralization at the boundary between it and dentine. The advance of mineralization along the collagen fibres is inhibited in the enameloid area before the mineralization at the boundary, despite the presence of matrix vesicles (Sasagawa, 1988). It is assumed that removal of the material that inhibits the progress of mineralization might lead to mineralization from the initial site to the surface of the enameloid (Sasagawa, 1988), but the putative material and mechanism remain unknown. Two hypotheses have been proposed for the origin of collagen fibres in the cap enameloid: (a) that the odontoblasts are their main producers (Kerr, 1960; Shellis and Miles, 1974, 1976; Yamashita and Ichijo, 1983) and (b) that inner dental epithelial cells form the bulk of them (Prostak and Skobe, 1984, 1985, 1986; Prostak, Seifert and Skobe, 1993), while the organic matrix of the cap enameloid is supplied by both the inner dental epithelium and odontoblasts. The ratio of ectodermal to mesodermal enameloid matrix collagen would seem to be related to the removal of the collagen fibres in cap enameloid at the maturation stage, and to the size of mature enameloid
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crystals (Prostak, Seifert and Skobe, 1991). Little has been reported about the development of collagen fibres during the formation of the enameloid matrix, the relation between the collagen fibres and other organic matrix in the cap enameloid, and the fine structure of the dental epithelium and the odontoblasts at the early stage of formation of the enameloid matrix. The aim of my study was to examine the fine structure of the cap enameloid matrix and its relation to the cells that are assumed to provide the organic matrix, that is, the dental epithelial cells and the odontoblasts, from the early to the late stage of formation of the enameloid matrix before mineralization.
were then fixed in a 1% solution of osmium tetroxide, buffered with cacodylate (pH 7.4). Some specimens were demineralized with a 2.5% solution o f d i s o d i u m E D T A for 3 weeks before postfixation. After dehydration, the specimens were embedded in Araldite- Epon resin. Semithin sections were cut with glass knives, stained with toluidine blue and then examined under a light microscope. Ultrathin sections were cut with glass or diamond knives, stained with uranyl acetate and lead citrate, or with phosphotungstic acid, and then examined with a transmission electron microscope (H-500; Hitachi, or J E M - 1200EX; JEOL). RESULTS
Light microscopy Early stage of formation of the enameloM matrix.
MATERIALS AND M E T H O D S
Three adult tilapias, Tilapia nilotica, Cichlidae, teleost (total length, 35-37 cm), which possessed large numbers of teeth and tooth germs in their pharyngeal tooth plates, were used in this study. After decapitation, the plates containing the tooth germs were dissected out and placed in buffered 10% formalin (cacodylate buffer, p H 7.4) for 3 days. The specimens were then demineralized with 5% formic acid for 2 weeks. After dehydration and embedding in paraffin wax, serial sections were prepared, stained with haematoxylin-eosin, azan, elastica-van Gieson's stain or PAS-alcian blue, and examined under a light microscope. Some sections were pretreated with human saliva containing salivary amylases for 60 min at 37°C before staining with PAS-alcian blue. Other pharyngeal tooth plates were placed in Karnovsky fixative (cacodylate buffer, pH7.4) overnight at r o o m temperature. These specimens
A small amount of enameloid matrix was produced between the inner dental epithelial cells and the odontoblasts. The short odontoblasts extended a number of processes towards the distal end of the inner dental epithelial cells, which formed a simple layer of low columnar cells facing the enameloid matrix. The other epithelial cells had barely developed at this stage, so that the outer dental epithelial cells were indistinguishable among them. A few PAS-positive particles were found in the dental epithelial cells but not in the odontoblasts. The cells of the tooth germs were haematoxyphilic as a whole at this early stage, which would correspond to the cap stage and/or the early bell stage in mammals, given the morphological resemblance between the features of these stages (Fig. 1).
Middle stage of formation of the enameloid matrix. A layer of enameloid matrix became clearly visible at
(Figs 1 7 opposite) Figures 1-4 are light micrographs. Figures 5 30 are transmission electron micrographs which, unless otherwise stated, are from sections stained with uranyl acetate-lead citrate. Fig. 1. Tooth germ at the early stage of formation of the enameloid matrix. Demineralized in 5% formic acid, stained with haematoxylin-PAS, x 100. Fig. 2. Tooth germ at the middle stage of formation of the enameloid matrix, a layer of which has become visible. An arrow indicates epithelial cells located between the inner and outer dental epithelial cells. Demineralized in 2.5% disodium EDTA, semithin section, toluidine blue. x 233. Fig. 3. Tooth germ at the late stage of formation of the enameloid matrix. A thick enameloid matrix contains many fine fibres stained by eosin. Elongated odontoblasts and inner dental epithelial cells surround the enameloid matrix. The space ('A') is an artefact. Demineralized in 5% formic acid, haematoxylin-eosin, x 100. Fig. 4. Tooth germ at the late stage of formation of the enameloid matrix, which is well stained by PAS-alcian blue. Space (~-) is an artefact. Demineralized, PAS-alcian blue. x I00. Fig. 5. Enameloid matrix and distal portions of inner dental epithelial cells at the early stage of formation of the enameloid matrix. Thin collagen fibrils are visible around the odontoblast processes. Mitochondria, rough endoplasmic reticulum, vesicles and granules are found in the cytoplasm. Demineralized in 2.5% disodium EDTA. x4190. Fig. 6. Golgi apparatus in an odontoblast at the early stage. Several vesicles and vacuoles are visible in the Golgi area. Demineralized. × 15,450. Fig. 7. Odontoblast process containing granules with electron-dense contents at the early stage. Thin collagen fibrils and a fine, network-like material occupy the space around the process. Demineralized. z 19,270.
Enameloid matrix formation in tilapia this stage; in particular, a thick layer was formed at the tooth apex. Fine fibres that were stained with acid dyes (acid fuchsin, aniline blue and eosin) were observed in the matrix. The odontoblasts, with a nucleus at the centre of each, became longer and formed a layer along the unmineralized enameloid matrix. Many odontoblast processes penetrated that matrix. The inner dental epithelial cells were columnar and several cells in the apical region had become
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taller than the others; their nuclei were situated in a wide region from the centre to the proximal margin. The outer dental epithelial cells formed a simple, short columnar or cubic layer. While the bell-shaped enameloid organ usually consisted of only two layers of cells, the inner and outer dental epithelia, a number of epithelial cells were often present between these two layers. In general, the cells in the tooth germs were stained with haematoxylin. A number of
Figs 1-7 (legends opposite).
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Fig. 8. Collagen fibrils showing cross-banding pattern among odontoblast processes at the early stage. Flocculent material is seen around the fibrils and the processes. Demineralized. × 14,610. Fig. 9. Golgi apparatus in an inner dental epithelial cell at the early stage. Several lamellae, vesicles, vacuoles and electron-dense granules are found in the Golgi area. Demineralized. × 13,070. Fig. 10. Odontoblasts at the middle stage of formation of the enameloid matrix. Elongated profiles of rough endoplasmic reticulum, mitochondria, developed Golgi apparatuses and small electron-dense granules are seen in the perinuclear cytoplasm. Demineralized. × 5560. Fig. 11. Golgi apparatus in an odontoblast at the middle stage. A number of long lamellae, vacuoles with flocculent contents and many vesicles are present in the area. Several elongated granules containing fine filamentous material (---~) are found at the centre of the Golgi area. Demineralized. × 13,940. Fig. 12. Enameloid matrix beneath the inner dental epithelial cells at the middle stage. The odontoblast processes are placed close to the basal lamina. The collagen fibrils are thin and dispersed. Many granules and vesicles are seen in the distal cytoplasm of the inner dental epithelial cells. Demineralized. × 6040.
Enameloid matrix formation in tilapia PAS-positive particles were found in the inner dental epithelial cells. This stage would correspond with the middle bell stage in mammals (Fig. 2).
Late stage of formation of the enameloid matrix. The shape of the complete cap enameloid was, for the most part, generated by the organic matrix at this stage, but mineralization had not yet begun. Fine fibres that were stained by acid fuchsin, aniline blue and eosin were densely packed in the matrix. Elongated haematoxyphilic odontoblasts with elliptical nuclei in the distal region were located along the inner surface of the unmineralized enameloid matrix. Many long odontoblast processes were found in the enameloid matrix. The inner dental epithelial cells were relatively translucent, tall columnars, and with their nuclei often central but sometimes distal or proximal; haematoxyphilic ergastoplasm was visible in both the distal and proximal cytoplasm (Fig. 3). There were large numbers of PAS-positive particles in the inner dental epithelial cells. The haematoxyphilic outer dental epithelial cells also contained some PAS-positive particles, which were not seen after pretreatment with human saliva and therefore probably consisted of glycogen. The enameloid matrix was well stained by PAS-alcian blue (Fig. 4).
Transmission electron microscopy Early stage of formation of the enameloid matrix. Odontoblasts were squat and a number of organelles were visible around the nuclei (Figs 5 and 6). The Golgi apparatus consisted of short lamellae, several vesicles and vacuoles near the nucleus (Fig. 6). Mitochondria, short profiles of rough endoplasmic reticulum, small vesicles and granules with electron-dense contents were usually visible in the perinuclear cytoplasm. Many odontoblast processes extended into the enameloid matrix and reached the lamina densa beneath the inner dental epithelial cells. The processes often contained granules with electron-dense contents, mitochondria, short profiles of rough endoplasmic reticulum and small vesicles (Fig. 7). Thin collagen fibrils, which ranged from 12 to 20 nm dia, were found around the odontoblast processes (Fig. 7). Some collagen fibrils that appeared thicker than others showed clear cross-banding at intervals of about 67 nm (Fig. 8). Flocculent or fine, network-like material was seen among the collagen fibrils and the odontoblast processes (Figs 7 and 8). This material often appeared to make contact with the collagen fibrils and sometimes to form a periodic attachment with their cross-banding (Figs 7 and 8). No defined matrix vesicles were seen at this stage, although many cross-sections of odontoblast processes were noted. In the cytoplasms of the squat, inner dental epithelial cells, there were a number of mitochondria, rough endoplasmic reticulum, granules with electron-dense contents and vesicles. The Golgi apparatus, consisting of a number of lamellae, vesicles, vacuoles and electron-dense granules, was situated near the nucleus (Fig. 9). The distal end of each cell was almost straight (Fig. 5). Well-developed interdigitations were often observed in the lateral membranes of the inner dental epithelial cells.
Middle stage of formation of the enameloid matrix. In the odontoblasts, extensive rough endoplasmic
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reticulum, mitochondria, well-developed Golgi apparatus and various kinds of granule were present around the nucleus (Fig. 10). The Golgi apparatus consisted of several long lamellae, many vesicles and condensing vacuoles that contained flocculent material. Elongated granules containing fine, filamentous material were usually found in the Golgi area (Fig. 11). Round granules with electron-dense contents were often seen in the cytoplasm. Desmosomes were often visible among the odontoblasts. Intercellular spaces were usually present distally. There were still many odontoblast processes in the enameloid matrix and some were attached to the lamina densa beneath the inner dental epithelial cells (Figs 12 and 15). The collagen fibrils in the cap enameloid were still thin, being only about 15 nm dia, and there was no difference in their diameters between the outer layer near the inner dental epithelium and the inner layer near the odontoblasts. Some relatively thick collagen fibrils exhibited clear cross-banding. The fibrils tended to form bundles and to be arranged along, and in proximity to, odontoblast processes (Figs 12 and 13). The spaces between the fibrils appeared wide and were filled with abundant flocculent and/or fine, network-like material; periodic attachment of this electron-dense material to the fibril cross-banding was commonly observed in the enameloid matrix (Fig. 13). Beneath the basal lamina, the majority of the odontoblast processes and the collagen fibrils were arranged parallel to the lamina densa (Fig. 12). Small numbers of matrix vesicles (40-80 nm dia) with a clear unit membrane appeared in the cap enameloid matrix (Fig. 13). There were few crystals in the matrix vesicles at this stage. The vesicles often exhibited a serial arrangement along the odontoblast processes and the collagen fibrils. The inner dental epithelial cells became taller and columnar and their nuclei occupied the central to proximal region (Fig. 14). A developed Golgi apparatus was usually seen on the distal side of the nucleus. There were mitochondria and an extensive rough endoplasmic reticulum. Many round or bacilliform granules containing electron-dense material and vesicles were seen in the distal cytoplasm (Fig. 14, inset; Fig. 15). Many electron-dense particles, which were probably glycogen, were found in the cytoplasm. Expanded spaces containing electron-lucent fibrous structures were often present distally between the inner dental epithelial cells. Their distal ends were nearly flat and there was a clear basal lamina beneath them (Figs 12 and 15). Several small epithelial cells were observed between the inner and outer dental epithelial cells at this stage. These resembled stellate reticulum on the apical side (Figs 2 and 16) but were flat near the cervical loop (Figs 2 and 17). The outer dental epithelium comprised a simple layer of short columnar or cubic cells. The nucleus was near to the centre of each cell and there were small numbers of cytoplasmic organelles (Fig. 17).
Lat e stage of formation of the enameloid matrix. The odontoblasts became taller and their nuclei were located proximally. An abundant rough endoplasmic reticulum and well-developed Golgi apparatuses were present in the cytoplasm. The extensive rough endoplasmic reticulum, enclosing fine material, was
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usually arranged almost parallel to the long axis of each cell (Figs 18 and 19). The Golgi consisted of several lamellae, many vesicles and vacuoles. Elongated granules with fine filamentous contents cross-banded at intervals of approx. 300nm were often visible in the Golgi region (Fig. 19, inset). A number of mitochondria, electron-dense granules, small vesicles and microtubules were usually found in the cytoplasm. Desmosomes and short, gap-like junc-
tions were observed between odontoblasts; wide intercellular spaces were scarcely ever seen. Processes penetrating into the enameloid matrix extended from the distal ends of the odontoblasts (Fig. 18). There were large numbers of matrix vesicles with a defined unit membrane in the enameloid matrix near the odontoblasts. The collagen fibrils in the enameloid matrix were mainly of 30-40 nm dia (range, 20-60 nm), that is,
Figs 13--17 (legends opposite).
Enameloid matrix formation in tilapia twice as thick as those at earlier stages (Figs 20-22). Although there was no significant change in their diameter from the apex to the base in the cap enameloid, the fibrils near the odontoblasts, which apparently showed the characteristic arrangement of enameloid matrix, tended to be thicker than those in the apical enameloid. A cross-banding pattern, with a periodicity of about 67 nm, was usually seen at this stage (Fig. 22). These collagen fibrils gathered together to form the thick, interwoven bundles characteristic of cap enameloid (Figs 20 and 21). Odontoblast processes were present in the enameloid matrix near the odontoblasts but they were rarely seen in the apical enameloid matrix. The bundles of collagen fibrils tended to be arranged along the odontoblast processes in the enameloid matrix near the odontoblasts. Matrix vesicles, which usually contained electron-dense, crystal-like structures, were found in the cap enameloid (Fig. 22). Fine aggregates of such structures, which were associated with an electron-dense, fine material that was probably derived from the matrix vesicles, were scattered in the apical enameloid matrix. Fine, moderately electrondense material and electron-dense particles occupied the regions around the collagen fibrils and matrix vesicles (Fig. 22). Tall, columnar, inner dental epithelial cells contained well-developed organelles. Their nuclei were nearly central and there were many well-ordered organelles in the proximal cytoplasm, where mitochondria were gathered (Fig. 23). Large amounts of rough endoplasmic reticulum, arranged nearly parallel to the long axes of the cells, were visible mainly in the proximal cytoplasm. Developed Golgi apparatuses, which consisted of several lamellae, many vesicles, vacuoles and small electron-dense granules, were situated around the nuclei (Figs 24 and 25). Elongated granules with fine, filamentous contents that usually showed cross-banding at intervals of approx. 300 nm were often observed near the Golgi (Fig. 25). In the distal cytoplasm, the organelles were relatively scattered; extensive rough endoplasmic reticulum, several granules, vesicles, vacuoles and lysosomes were usually seen. Several large, irregular granules contained fibrillar structures, 150-nm thick with cross-banding at intervals of about 32 nm and with fine, electron-dense material around them
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(Fig. 26). These large granules were usually found near the Golgi and in the distal cytoplasm. There were many electron-dense particles; probably glycogen, in the cytoplasm of the inner dental epithelial cells (Figs 26-28). Electron-dense, granular or flocculent material was often seen in the expanded spaces between these cells. Desmosomes, gap-like junctions and interdigitations of the lateral cell membranes were still clearly visible at this stage. Fibrils that appeared to cross the basal lamina were occasionally observed (Fig. 27). In sections stained with phosphotungstic acid, several large granules containing positively stained fine filaments were usually visible in the Golgi area and in the distal cytoplasm of the inner dental epithelial cells (Figs 28 and 29). These granules tended to increase in number in cells at the apical side of the tooth germ and to decrease in cells in the cervical region. Although the phosphotungstic acidpositive filaments in the large granules rarely showed periodic cross-banding, they probably corresponded to the fibril-like structures with cross-banding at 32-nm intervals in the sections stained with uranyl acetate and lead citrate. In the granules, fine material usually surrounded the positively stained filaments (Fig. 28). Collagen fibrils in the enameloid matrix were also well stained by phosphotungstic acid (Fig. 29). Outer dental epithelial cells became larger and developed into electron-lucent cells that contained many vesicles and vacuoles; there were mitochondria and lysosomes in the cytoplasm. These cells tended to overlap and to extend projections to the outside at the apical portion of the tooth germs while, in the region near the cervical loop, they still constituted a simple layer. The cells of the outer dental epithelium made direct contact with those of the inner dental epithelium without wide intercellular spaces. Desmosomes and short gap-like junctions were seen between the outer and inner dental epithelial cells (Fig. 30). DISCUSSION
The diameters of the collagen fibrils, 30-40 nm, at the late stage of formation of the enameloid matrix reflect the results obtained in other teleosts (25-50nm, common eel, Shellis and Miles, 1976; 30 nm, Texas cichlid, Prostak and Skobe, 1984, 1985,
(Figs 13-17 opposite) Fig. 13. Thin collagen fibrils, cross-section of odontoblast processes and matrix vesicles (---,) in the enameloid matrix at the middle stage. Flocculent material is situated around the collagen fibrils. Periodic attachment of electron-dense material ( ~ ) is found at the cross-banding of the collagen fibrils (inset). Demineralized. x 30,350. Inset: bar = 100 nm. x 35,580. Fig. 14. Long inner dental epithelial cells situated in the apical area of a tooth germ at the middle stage. Many mitochondria, granules, vesicles, extensive rough endoplasmic reticulum and developed Golgi apparatuses are seen in the distal cytoplasm. Demineralized. x 3790. Inset: enlarged view of the elongated granules with electron-dense contents in the cytoplasm near the Golgi apparatus. Bar = 200 nm. x 20,930. Fig. 15. Distal portion of inner dental epithelial cells and the enameloid matrix at the middle stage. Expanded intercellular spaces containing fine material and a defined basal lamina are seen distally. Demineralized. x 15,180. Fig. 16. Epithelial cells between the inner and outer dental epithelium in the apical part of a tooth germ at the middle stage; these resemble stellate reticulum cells. Demineralized. x 2880. Fig. 17. Columnar outer dental epithelial cells and the fiat intermediate cells ( ~ ) between the inner and outer dental epithelial cells at the middle stage. Demineralized. x 3450.
Fig. 18. Odontoblasts and their processes in the enameloid matrix (M) at the late stage of its formation. A b u n d a n t rough endoplasmic reticulum and well-developed Golgi apparatuses are visible in the odontoblasts. Demineralized. × 5500. Fig. 19. Golgi apparatus and rough endoplasmic reticulum in odontoblasts at the late stage. Arrows indicate vesicles that contain fine filamentous material. Demineralized. x 10,920. Inset: enlarged view of the vesicle containing fine filamentous materials. Bar = 300 nm. x 24,950. Fig. 20. Enameloid matrix at the late stage. Large numbers of matrix vesicles and several odontoblast processes are present. Non-demineralized. x 6060. Fig. 21. Enlarged view of the enameloid matrix including odontoblast processes and matrix vesicles at the late stage. The collagen fibrils, 30~40 n m dia, gather together to form thick bundles. Non-demineralized. × 13,770. Fig. 22. Enlarged view of collagen fibrils in the enameloid matrix at the late stage. Electron-dense fine materials are placed in the space around the collagen fibrils. Non-demineralized. × 59,300. Inset: matrix vesicles containing crystal-like structures at the late stage. Bar = 100 nm. x 57,390. 808
Enameloid matrix formation in tilapia
Fig. 23. C o l u m n a r inner dental epithelial cells and cubic outer dental epithelial cells at the late stage. Mitochondria tend to gather at the proximal side of the inner dental epithelial cells. Demineralized. x 2840. Fig. 24. Portion near the nuclei of inner dental epithelial cells at the late stage. Extensive rough endoplasmic reticulum and well-developed Golgi apparatuses with m a n y granules and vesicles. Demineralized, x 6070. Fig. 25. Golgi apparatus and rough endoplasmic reticulum near the nuclei of inner dental epithelial cells at the late stage. An arrow indicates a large granule that contains a fibril-like structure, and an arrowhead indicates an elongated granule with filamentous contents. Demineralized. × 7590. Inset: enlarged view of the granule with filamentous contents. Bar = 300 nm. x 22,600. Fig. 26. A large granule containing a fibril-like structure with periodic cross-banding at intervals of about 32 n m in an inner dental epithelial cell at the late stage. Demineralized. × 37,940.
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Fig. 27. Distal end of inner dental epithelial cell and basal lamina at the late stage. An arrow indicates a fibril that seems to be passing across the basal lamina. Arrowheads indicate electron-dense glycogen particles in the cytoplasm. Demineralized. × 37,890. Fig. 28. Large granules containing phosphotungstic acid (PTA)-positive fine filaments in inner dental epithelial cells at the late stage. Demineralized, PTA. x 12,120. Inset: enlarged view of the granules. Bar = 100 nm. x 32,300. Fig. 29. Distal portion of inner dental epithelial cells at the late stage. The organelles are relatively scattered in the distal cytoplasm. A number of large granules containing phosphotungstic acid (PTA)-positive fine filaments ( ~ ) are visible at the distal portion. Demineralized, PTA. x 6060. Inset: enlarged view of the gap-like junction at the lateral cell membrane. Bar = 100 nm. x 30,870. Fig. 30. Outer dental epithelial cells at the late stage. These develop into electron-lucent cells that contain m a n y vacuoles and small vesicles. I, inner dental epithelial cells; O, outer dental epithelial cells. Non-demineralized. × 3790. Inset: desmosomes and gap-like junctions between the inner and outer dental epithelial cells. Demineralized. Bar = 1 p m . x 7380.
Enameloid matrix formation in tilapia 1986). The collagen fibrils of 30-40 nm dia in the late-stage enameloid matrix formed the thick bundles characteristic of teleost cap enameloid. However, the fibrils at the early and middle stages were thin, of approx. 15 nmdia, and dispersed. At the late stage each fibril was almost twice as thick as at the earlier stages. It appears that the collagen fibrils are dispersed and thin at the early and middle stages before the matrix has attained its final thickness. It has been reported that the density of collagen fibrils in cap enameloid increases in tooth buds that have nearly achieved their full thickness (Prostak and Skobe, 1986). The increase in thickness of collagen fibrils during the formation of the enameloid matrix is probably involved in the increase in density of the collagen fibrils in cap enameloid. In general, collagen fibrils in loose connective tissue increase in diameter after they are first polymerized outside fibroblasts, suggesting that each fibril grows by apposition, with further collagen molecules joining those already assembled in the same staggered fashion (Ham and Cormack, 1979). In hamster and rat incisors, collagen fibrils had a small diameter on the cell-body side of the predentine, and increased in thickness through the predentine, achieving a maximum thickness at the dentine mineralization front (Watson and Avery, 1954; Reith, 1968). It is assumed that appositional growth of collagen fibrils occurs during the formation of enameloid matrix. Simultaneously, in my material, the number of odontoblast processes decreased in the cap enameloid at the late stage. Matrix vesicles began to appear at the middle stage and, at the late stage, large numbers were visible in the cap enameloid. If the odontoblast processes are involved in providing procollagen molecules for the enameloid matrix, the morphological changes in those processes and in the matrix vesicles might be related to the growth in thickness of the collagen fibrils in the cap enameloid. In Polypterus, rather thick collagen fibrils, of 100-120nmdia, were reported in the area near the odontoblasts when the enameloid layer was thin, while the collagen fibrils at the surface were of 3 0 - 5 0 n m d i a (Kogaya, 1989). This discrepancy suggests variations among species or among portions of the samples examined. At the middle and the later stages of formation of the enameloid matrix, the odontoblasts contained much rough endoplasmic reticulum and many mitochondria, well-developed Golgi apparatuses, several kinds of granule and many microtubules. Several elongated granules containing fine filaments were observed in the Golgi area. The morphological appearance of the elongated granules was similar to that of the secretory granules containing procollagen molecules in mammalian odontoblasts, in which the synthesis, migration and release of precursor collagen have been clearly demonstrated by autoradiography (Frank, 1970; Weinstock and Leblond, 1974). In particular, the fine filaments in the granules exhibited cross-banding at intervals of approx. 300 nm, which resembled the length of procollagen molecules (Weinstock, 1972). Consequently, the elongated granules are probably procollagen granules. It is suggested that the odontoblasts synthesise procollagen molecules. Prostak and Skobe (1985, 1986) and
8ll
Prostak et al. (1993) noted that odontoblasts in Texas cichlid, parrotfish and pufferfish probably provide only small quantities of enameloid collagen because they contain small numbers of secretory granules, and they showed little or no signs of secretory activity during the formation of the enameloid matrix in experiments using colchicine. In Tilapia nilotica, however, the morphological features of the odontoblasts imply that they produce considerable amounts of collagen fibrils as part of the enameloid matrix, although the exact proportion of the collagen that originates from odontoblasts in that matrix cannot be determined from the present data. There were large numbers of matrix vesicles, which presumably originated from odontobtasts, in the enameloid matrix near the cells (Sasagawa, 1988). Odontoblast processes were observed in the cap enameloid during the matrix-formation stage. At the early to middle stage, many odontoblast processes were visible in the enameloid matrix from the surface to the lower part. It is possible that odontoblast processes influence the arrangement of collagen fibrils in the cap enameloid (Wakita, Takahashi and Hanaizumi, 1993) because the fibrils near the processes tend to lie in the same direction as the processes. Therefore, the evidence supports the hypothesis that odontoblasts play an important part in the formation of cap enameloid matrix, and of the collagen fibrils in particular (Kvam, 1946; Kerr, 1960; Shellis and Miles, 1974, 1976). It is also likely that the contribution of odontoblasts to the formation of the enameloid matrix varies among species of fish (Prostak et al., 1991). Prostak and Skobe (1986) observed interfibrillar material among collagen fibrils in EDTA-treated specimens and they interpreted the material as a partially solubilized collagen. In my study, similar fine material was observed in both demineralized and non-demineralized specimens. The staining of the enameloid matrix by PAS-alcian blue implies that large amounts of glycosaminoglycans are present. Kogaya (1989, 1994) detected large quantities of chondroitin sulphates in the developing enameloid of Polypterus senegalus and Hoplognathus fasciatus, and these were in close association with the enameloid collagen. Proteoglycan filaments tend to cross the collagen fibrils at intervals of about 67 nm (Scott, 1980; Furuhashi, K o g a y a and Yoshioka, 1984: Ruggeri and Benazzo, 1984). The presence of fine material forming a network-like structure and periodic attachment to the collagen fibrils in the tilapia enameloid suggests that large amounts of glycosaminoglycans and/or proteoglycans are located among the collagen fibrils, while further histochemical and immunohistochemical studies at the electron-microscopic level should provide exact details of the structural relation. The existence of non-collagenous proteins that are of odontoblast origin cannot be clarified until much more is known about the histochemical features of the developing enameloid. In teleosts, Golgi apparatuses and rough endoplasmic reticulum generally develop in the distal cytoplasm of the inner dental epithelial cells at the late stage of formation of the enameloid matrix (Yamashita and Ichijo, 1983). In Tilapia nilotica.
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well-developed Golgi apparatuses, abundant rough endoplasmic, reticulum, mitochondria and several kinds of vesicle mainly occupied the proximal cytoplasm, as is the case in eels (Shellis and Miles, 1976). This finding again suggests differences among species. The presence of a well-developed Golgi, rough endoplasmic reticulum and many vesicles strongly indicates that the inner dental epithelial cells engage in secretion at this stage. Prostak and Skobe (1984, 1985, 1986) proposed that the bulk of collagen fibres in the teleost enameloid arises from the epithelial cells. Wakita (1974) reported the presence of fibrous structures with cross-banding in inner dental epithelial cells. Sasagawa (1984) suggested that these cells might secrete collagen fibres because of morphological similarities between their Golgi and that of the odontoblasts. Enameloid collagen-like fibrils have been observed in the spaces between inner dental epithelial cells (Kogaya, 1989). In the present study, large granules that contained fibril-like structures, which often had periodic stripes at about 32-nm intervals (about half the length of a single D-period; 67 nm) were found in the inner dental epithelial cells, and the contents of these granules were stained by phosphotungstic acid as fine filaments. It is likely that the contents were a kind of procollagen. Moreover, several elongated granules containing fine filaments were seen in the Golgi area and appeared to be secretory granules. These fine filaments in the elongated granules usually had cross-banding at intervals of approx. 300 nm. This also suggests that procollagen granules are produced in the Golgi. These two types of granule in the inner dental epithelial cells resembled the secretory granules that have been observed in the odontoblasts of mammals (Nagai, 1970; Weinstock and Leblond, 1974) and described in the reports of Prostak and Skobe (1984, 1985, 1986). Hence, it is certain that the inner dental epithelial cells in Tilapia nilotica can synthesize collagen. Ectodermal collagen fibres have often been found in other normal organs (chick corneal epithelium, Trelstad, 1971; Hay and Dodson, 1973; chick notochordal epithelium, Carlson, 1973), so it seems likely that the epithelial cells of tooth germs in teleosts produce some type of collagen fibre. It is unclear whether the bulk of collagen fibres in cap enameloid is of ectodermal origin because of many problems associated with interpreting the migl:ation of ectodermal collagen into the enameloid matrix. It is still unclear how ectodermal procollagen molecules and/or collagen fibrils might move into the enameloid matrix through the basal lamina. In this study, the collagen fibrils seemed to penetrate the basal lamina, as shown in Fig. 27. Similar evidence that fibrous substances might cross the basal lamina of inner dental epithelial cells was found at the late stage of formation of enameloid matrix in the dog salmon (Sasagawa, 1984). There are probably pores, through which macromolecules including collagen fibrils can pass, in the basal lamina at the late stage. However, I rarely saw material that appeared to be passing through the basal lamina under the transmission electron microscope even at the late stage. Substances that consisted of electron-dense, fine particles or flocculent material were often observed
between the inner dental epithelial cells and the basal lamina, implying that material of high molecular weight was stored in that region because of obstruction by the basal lamina. The inner dental epithelial cells that surrounded the apex of the enameloid contained many granules that included ectodermal procollagen, but not all cells facing the enameloid matrix had an abundance of such granules. In the present study, the collagen fibrils of nearly 30 nm dia and with 67-nm periodic cross-banding, which were seen in the enameloid matrix, were not found in the spaces between the inner dental epithelial cells at the late stage. In the avian cornea the epithelium produces type I collagen and primary corneal stroma in the early stages of embryogenesis. However, after fibroblasts have invaded the cornea, its total synthesis of collagen increases enormously because of their important contribution (Hay, 1980). It is likely that the inner dental epithelial cells in Tilapia nilotica actually secrete ectodermal collagen but that it is not the only or major component of the enameloid matrix. It has been proposed that the collagen produced by the odontoblasts provides a scaffold from which the collagen of the inner dental epithelial cells can assemble and the fibrils can lengthen (Prostak et al., 1993). It is also possible that the ectodermal collagen is associated with the increase in thickness of collagen fibrils during the formation of the enameloid matrix. It is reported that the electrophoretic patterns of acetic acid-soluble proteins obtained from teleost enameloid matrix showed characteristic type I collagen bands, and additional 33- and 30-kDa bands, which were thought to be degradation peptides of enameloid collagen itself (Prostak, Seifert and Skobe, 1992). However, neither morphological nor histochemical data concerning the differences between the ectodermal collagen and the collagen from odontoblasts were obtained from the cap enameloid matrix. Unresolved issues of interest in relation to the collagen in the cap enameloid of teleosts are: the proportion and distribution of the ectodermal collagen in the enameloid matrix; the role of ectodermal collagen during formation of the enameloid; and the interaction between the ectodermal collagen and the collagen from the odontoblasts. It is probable that the inner dental epithelial cells in teleosts secrete not only collagen fibrils but also other proteins, such as enamelin (Slavkin et al., 1983; Herold, Rosenbloom and Granovsky, 1989) and proteolytic enzymes (Kawasaki, Shimoda and Fukae, 1987). Immunohistochemistry together with transmission electron microscopy might reveal details of the distribution of the proteins from these cells in enameloid matrix. Stellate reticulum is generally found between the inner and outer epithelial cells in tooth germs in higher vertebrates such as reptiles and mammals. However, the corresponding cells show variations among different groups of fish (Kerr, 1960; Yoshitani, 1959). Here, in Tilapia nilotica, some stellate cells that resemble those of the stellate reticulum in mammals were noticed between the inner and outer dental epithelia just at the middle stage of formation of the enameloid matrix. It is likely that cells resembling stellate reticulum are temporarily
Enameloid matrix formation in tilapia present a m o n g the dental epithelial cells d u r i n g t o o t h d e v e l o p m e n t in this teleost. Large n u m b e r s of glycogen particles have been reported in the inner dental epithelial cells from the stage o f f o r m a t i o n o f the enameloid matrix to the beginning of the mineralization in several fishes, including sharks a n d sting-rays ( G a r a n t , 1970; Ono, 1974; W a k i t a , 1974; G o t o , 1976; Y a m a s h i t a a n d Ichijo, 1983; Sasagawa a n d Akai, 1992). In Tilapia nilotica, great n u m b e r s of electron-dense particles, p r o b a b l y glycogen, were f o u n d in the inner dental epithelial cells at the late stage of f o r m a t i o n of the enameloid matrix, but I have preliminary evidence that they disappeared rapidly from these cells at the s u b s e q u e n t mineralization stage. A characteristic m o r p h o l o g i c a l feature of the t o o t h germs of Tilapia nilotica is the scattered r a t h e r t h a n aggregated distrib u t i o n of glycogen particles in the cytoplasm of inner dental epithelial cells. The role of glycogen in these cells remains to be clarified. However, it is possible that glycogen m i g h t have a role w h e n these cells later c h a n g e into absorptive cells at the mineralization and m a t u r a t i o n stages. A cknowledgements--I thank the staff members of Hokuetsu Mizugiken Inc. for their kindness in offering their facilities for the collection of specimens.
REFERENCES
Carlson E. C. (1973) Periodic fibrillar material in membrane-bounded bodies in notochordal epithelium of the early chick embryo. J. ultrastruct. Res. 42, 287-297, Frank R. M. (1970) Etude autoradiographique de la dentinogen~se en microscopie 61ectronique 5. l'aide de la proline triti6e chez le chat. Archs oral Biol. 15, 583-596. Furuhashi K., Kogaya Y. and Yoshioka Y. (1984) Ruthenium hexammine trichloride-positive materials in developing mouse molar predentine and intercellular spaces between preodontoblasts. J. electron. Microsc. 33, 384-387. Garant P. R. (1970) Observation on the ultrastructure of the ectodermal component during odontogenesis in Helostoma temmincki. Anat. Rec. 166, 167-188. Goto M. (1976) Histogenetic studies on the teeth of leopard shark (Triakis scyllia). J. Stomatol. Soc. Jpn 45, 527 584. Ham A. W. and Cormak D. H, (1979) Histology, 8th edn, p. 233. Lippincott, Toronto. Hay E. D. (1980) Development of the vertebrate cornea. Int. Rev. Cytol. 63, 263-322. Hay E. D. and Dodson J. W. (1973) Secretion of collagen by corneal epithelium. I. Morphology of the collagenous products produced by isolated epithelia grown on frozenkilled lens. J. cell Biol. 57, 190-213. Herold R. C., Rosenbloom J. and Granovsky M. (1989) Phylogenetic distribution of enamel proteins: immunohistochemical localization with monoclonal antibodies indicates the evolutionary appearance of enamelins prior to amelogenins. Calc. Tiss. Int. 45, 88-94. Kawasaki K., Shimoda S. and Fukae M. (1987) Histological and biochemical observations of developing enameloid of the sea bream. Adv. dent. Res. 1, 191 195. Kerr T. (1960) Development and structure of some actinopterygian and urodele teeth. Proc. zool. Soe. Lond. 133, 401-422. Kogaya Y. (1989) Histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus. Histochemistry 91, 185 190. AOB 40/9-- B
813
Kogaya Y. (1994) Sulfated glycoconjugates in amelogenesis. Prog. Histochem. Cytoehem. 29, 1-108. Kvam T. (1946) Comparative study of the ontogenetic and phylogenetic development of dental enamel. Norsk. Tannlaegeforen. Tid. 56, Suppl; 1 198. Meinke D. K. (1982) A histological and histochemical study of developing teeth in Polypterus (Pisces, Actinopterygii). Archs oral Biol. 27, 197 206. Nagai N. (1970) Electron microscopy of the cytoplasmic bodies in the odontoblasts of young rat incisors. Bull. Tokyo dent. Coll. 11, 47 83. Ono T, (1974) Electron microscopic studies on the ultrastructure of ameloblasts during amelogenesis in Oplegnathus fasciatus. Jpn J. oral Biol. 16, 407-464. Prostak K. and Skobe Z. (1984) Effects of colchicine on fish enameloid matrix formation. In Tooth Enamel IV (Eds Fernhead R. W. and Sug S.), 1st edn, p. 525. Elsevier, Amsterdam. Prostak K. and Skobe Z. (1985) The effects of colchicine on the ultrastructure of the dental epithelium and odontoblasts of teleost tooth buds. J. eranio/ac. Genet. derl Biol. 5, 75-88. Prostak K. and Skobe Z. (1986) Ultrastructure of the dental epithelium and odontoblasts during enameloid matrix deposition in cichlid teeth. J. Morphol. 187, 159 172. Prostak K., Seifert P. and Skobe Z. (1991) Tooth matrix formation and mineralization in extant fishes. In Mechanisms and Phylogeny of Mineralization in Biological Systems (Eds Suga S. and Nakahara H.), 1st edn. p. 465. Springer, Tokyo. Prostak K., Seifert P. and Skobe Z. (1992). Fish tooth formation: an assessment of biological factors affecting the fluoride content of enameloid. In Hard Tissue Mineralization and Demineralization (Eds Suga S. and Watabe N.), 1st edn, p. 33. Springer, Tokyo. Prostak K., Seifert P. and Skobe Z. (1993) Enarneloid formation in two tetraodontiform fish species with high and low fluoride contents in enameloid. Archs oral Biol. 38, 1031 1044. Reith E. J. (1968) Ultrastructural aspects of dentinogenesis. In Dentine and Pulp: Their Structure and Reactions (Ed. Symons N. B. B.), 1st edn, p. 19. Thomson, Dundee. Ruggeri A. and Benazzo F. (1984) Collagen- proteoglycan interaction. In Ultrastrueture of the Connectit,e Tissue Matrix (Eds Ruggeri A. and Motta P. M.), 1st edn, p. 113. Martinus Nijhoff, Boston. Sasagawa I. (1984) Formation of cap enameloid in the jaw teeth of dog salmon, Oncorhynchus keta. Jap. J. oral Biol. 26, 477-495. Sasagawa I. (1988) The appearance of matrix vesicles and mineralization during tooth development in three teleost fishes with well-developed enameloid and orthodentine. Archs oral Biol. 33, 75 86. Sasagawa I. and Akai J. (1992) The fine structure of the enameloid matrix and initial mineralization during tooth development in the sting rays, Dasyatis akajei and Urolophus aurantiacus. J. electron. Microse. 41, 242 252. Scott J. E. (1980) Collagen-proteoglycan interactions. Localization of proteoglycans in tendon by electron microscopy. Biochem. J. 187, 887-891. Shellis R. P. (1975) A histological and histochemical study of the matrices of enameloid and dentine in teleost fishes. Archs oral Biol. 20, 183 187. Shellis R. P. and Miles A. E. W. (1974) Autoradiographic study of the formation of enameloid and dentine matrices in teleost fishes using tritiated amino acids. Proc. R. Soc. Lond. B 185, 51 72. Shellis R. P. and Miles A. E. W. (1976) Observations with the electron microscope on enameloid formation in the common eel (Anguilla anguilla; Teleostei). Proc. R. Soc. Lond. B 194, 253 269. Slavkin H. C., Zeichner-David M., Snead M. L.,
814
I. Sasagawa
Graham E. E., Samuel N. and Ferguson M. W. J. (1983) Amelogenesis in reptilia: evolutionary aspects of enamel gene products. In The Structure, Development and Evolution of Reptiles (Ed. Ferguson M. W. J.), 1st edn, p. 275. Academic Press, London. Trelstad R. L. (1971) Vacuoles in the embryonic chick corneal epithelium, an epithelium which produces collagen. J. cell Biol. 48, 689~94. Wakita M. (1974) Studies on the ultrastructure of ameloblasts during amelogenesis in Prionurus mierolepidotus Lac6pdde. Jap, J. oral Biol. 16, 129-185. Wakita M., Takahashi S. and Hanaizumi Y. (1993) Studies on the matrix of enameloid in Paralichtys olivaceus. Arch, comp. Biol. Tooth Enamel 3, 7 18. Watson M. L. and Avery J. K, (1954) The development of
the hamster lower incisor as observed by electron microscopy. Am. J. Anat. 95, 109. Weinstock M. (1972) Collagen formation--Observations on its intracellular packaging and transport. Z. Zell/'orsch. 129, 455470. Weinstock M. and Leblond C. P. (1974) Synthesis, migration, and release of precursor collagen by odontoblasts as visualized by radioautography after [3H]proline administration. J. cell Biol. 60, 92-127. Yamashita Y. and Ichijo T. (1983) Comparative studies on the structure of the ameloblasts. In Mechanisms ~/ Tooth Enamel Formation (Ed. Suga S.), 1st edn, p. 91. Quintessence PuNishing, Tokyo. Yoshitani M. (1959) Histogenetic studies on the teeth of sea breams. J. stomatol. Soc. Jpn. 26, 811 834.