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Cellular influence in the formation of enameloid during odontogenesis in bony fishes
Materials Science and Engineering C 26 (2006) 630 – 634 www.elsevier.com/locate/msec
Cellular influence in the formation of enameloid during odontogenesis in bony fishes Ichiro Sasagawa a,*, Mikio Ishiyama b, Junji Akai c a
Department of Anatomy, School of Dentistry at Niigata, The Nippon Dental University, 1-8 Hamaura-cho, Niigata 951-8580, Japan b Department of Histology, School of Dentistry at Niigata, The Nippon Dental University, Niigata 951-8580, Japan c Department of Geology, Faculty of Science, Niigata University, Niigata 950-2181, Japan Received 1 November 2004; received in revised form 24 March 2005; accepted 24 April 2005 Available online 2 November 2005
Abstract The enameloid covering the surface of the teeth in bony fishes exhibits a very high degree of mineralization, and seems to have functions similar to the enamel in mammals. The aim of this study was to investigate the cellular control system of crystallization during enameloid formation. The enamel organs during the enameloid formation stages in ray-finned fishes were observed by transmission electron microscopy and enzyme cytochemistry, which detects hydrolytic enzyme activities. During the mineralization and maturation stages, many large and long crystals with a clear outline became packed densely in the enameloid. Infoldings of the distal plasma membrane in the inner dental epithelial (IDE) cells were marked, and many acid phosphatase positive lysosomes were present in the cytoplasm. The well-developed Golgi apparatus, and many cisternae of endoplasmic reticulum, secretory granules, mitochondria were visible in the IDE cells during these later stages. The activities of nonspecific alkaline phosphatase, calcium-dependent adenosine triphosphatase and potassium-dependent p-nitrophenylphosphatase were usually detected at the plasma membrane of the IDE cells during the mineralization and maturation stages. These results suggest that the dental epithelial cells are mainly involved in the degeneration and removal of enameloid matrix and in material transportation during the enameloid mineralization and maturation stages, rather than in the formation of enameloid matrix. D 2005 Elsevier B.V. All rights reserved. Keywords: Enameloid; Cellular regulation; Bony fishes; Odontogenesis
1. Introduction Cellular control is essentially important to make biological minerals. To investigate the method by which the cells regulate the formation of large crystals might be useful to analyze principles of biomineralization, and also for biomimetic materials study. The former paper [1] concerning the initial mineralization of both enameloid and dentin mentioned that the unit membrane provided by the odontoblasts probably has a role of template for the initial crystals. Matured enameloid situated at the surface of the teeth in bony fishes is a well-mineralized tissue, and corresponds to the enamel in mammals. It is assumed that the enameloid is an analogue of mammalian enamel, because the origin of enameloid is somewhat different from that of enamel. While amelogen* Corresponding author. Tel.: +81 25 267 1500; fax: +81 25 267 1134. E-mail address: [email protected] (I. Sasagawa). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.04.010
esis in mammals is a major subject of basic dental sciences, enameloid formation in bony fishes is paid little attention, so there are still many unclear aspects of enameloid formation. This article reviews our recent studies regarding the role of the enamel organ that consists of dental epithelial cells during enameloid formation in some fishes. 2. Experimental methods Fishes that belong to actinopterygians, the ray-finned fishes (Table 1) were used in this study. Fine structure of the tooth germs during cap enameloid formation stages was investigated by means of light and transmission electron microscopy. Enzyme cytochemistry for non-specific acid phosphatase (ACPase), non-specific alkaline phosphatase (ALPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase, that represents sodium, potassium-activated adenosine tripho-
I. Sasagawa et al. / Materials Science and Engineering C 26 (2006) 630 – 634
sphatase complex, Na – K-ATPase) was employed to examine the function of the tooth germs. 3. Results and discussion 3.1. Stages of enameloid formation; features of the enameloid matrix in each stage The enameloid formation process is divided into three stages; matrix formation, mineralization and maturation. During the matrix formation stage, the organic matrix of enameloid is produced between the inner dental epithelial (IDE) cells and odontoblasts. Abundant collagen fibrils making bundles are found in the matrix. Many fine network-like materials, probably representing glycosaminoglycans and/or proteoglycans, are located among the collagen fibrils. There are many matrix vesicles and processes of the odontoblasts in the matrix [1,2]. In some species, fine slender crystals appear at the matrix vesicles [1,3]. During the mineralization stage, deposition of fine slender crystals progresses along the collagen fibrils. The mineralization roughly extends from the junction between the enameloid and dentin to the apex of enameloid cap (Fig. 1a). Simultaneously, degeneration of collagen fibrils and fine network-like matrix around the fibrils begins to occur [3,4]. During the maturation stage, abundant large crystals occupy the enameloid area (Fig. 1b). In the demineralized sections, only a small amount of the organic matrix is retained in the enameloid [4]. 3.2. Construction of the enamel organ The enamel organ generally consists of only two layers, the inner dental epithelium and outer dental epithelium (Fig. 1c). Stellate reticulum is present between the two layers in some species, e.g., tilapia, gars, polypterus, amai [5]. The stellate reticulum appears during almost all stages of enameloid formation in gars and polypterus, while in tilapia it is temporarily found in the matrix formation stage [2,6]. The stratum intermedium is found only in gars (Fig. 1d) [7]. 3.3. Fine structure of the enamel organ The IDE cells that correspond to ameloblasts in mammals are generally tall and columnar, while the outer dental epithelial cells situated at the outer periphery of enamel organ are usually cubic (Fig. 1c). Stellate reticulum cells extend many processes and are surrounded by wide intercellular space. In gars, the stratum intermedium cells are larger than the cells of the
631
stellate reticulum, and make a closed cell layer at the distal end of the IDE cells (Fig. 1d). The IDE cells exhibit marked morphological change during enameloid formation, suggesting that the IDE cells are effectively involved in the enameloid formation. The main morphological features of the IDE cells are as follows. During the matrix formation stage, the IDE cells contain welldeveloped Golgi apparatus, abundant cisternae of rough endoplasmic reticulum (ER) and many granules and vesicles. This suggests that the IDE cells are specialized for secreting in the formation stage. Procollagen granules and the granules containing filamentous substance are found in the IDE cells of some species (Fig. 1e) [2,8]. However, it is still unclear whether ectodermal collagen forms part of the enameloid matrix. During the mineralization stage, the IDE cells come to possess a ruffled border at the distal end. Many lysosomal granules, mitochondria and vesicles are found in the IDE cells, although well-developed Golgi apparatus and rough ER are still present. It is implied that absorptive function becomes dominant in the IDE cells [4,6]. In gars, large granules containing amelogenin positive substance are observed in the distal cytoplasm. Amelogenins are a major component of the enamel proteins in mammals. However, there is no such substance in the enameloid. The IDE cells might produce amelogenins, but do not release them during enameloid formation [9]. During the maturation stage, the ruffled border is still present. The IDE cells contain many lysosomal granules and mitochondria, but less rough ER (Fig. 1f). The IDE cells might be engaged in the removal and degeneration of organic matrix from maturing enameloid area, as well as the ruffle-ended ameloblasts (RA) in mammals [4,6]. In tilapia, many ferritin particles/granules appear in the cytoplasm in this stage. It is likely that the ferric iron enters the enameloid through the enamel organ [10]. In tilapia, the outer dental epithelial (ODE) cells situated between the IDE cells and capillaries exhibit particular morphological characteristics in the mineralization and maturation stages. Abundant mitochondria and cisternae of smooth ER occupy the cytoplasm (Fig. 1g), resembling the chloride cells in the gill and mitochondria-rich cells in the inner ear epithelium that are engaged in the ion transport in bony fishes [4]. The morphology of the blood capillaries might reflect the activity of material transport system in tooth germs. During the mineralization and maturation stages in some teleosts, several capillaries enter the enamel organ, so that many deep concavities are made at the surface of the enamel organ [4]. However, in gars and polypterus, no capillaries enter the enamel organ, although they approach the surface of the outer dental epithelium [6]. 3.4. Enzyme cytochemistry of the enamel organ
Table 1 Materials Specimen
Portion
Tilapia: Oreochromis niloticus; Tilapia butttikoferi, Cichlidae, Teleostei, Neopterygii Gars: Lepososteus oculatus, Neopterygii Polypterus: Polypterus senegalus, Chondrostei
Pharyngeal teeth Jaw teeth Jaw teeth
3.4.1. Non-specific ACPase Many ACPase positive lysosomal granules are observed in the IDE cells during the mineralization and maturation stages in tilapia, gars and polypterus (Fig. 1h) [6,11]. The lysosomal granules might be closely related to the absorptive function of the IDE cells.
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3.4.2. Non-specific ALPase Marked activity of the ALPase is usually located at the plasma membrane of the IDE cells during the mineralization and maturation stages (Fig. 1i) [6,11]. This suggests that the IDE cells during these stages are involved in the transport of material and/or phosphate. 3.4.3. Ca-ATPase (calcium pump) Ca-ATPase activity is found at the plasma membrane of the IDE cells during the mineralization and maturation stages in gars and polypterus (Fig. 1j) [6]. The Ca-ATPase activity situates at the plasma membrane, suggesting the presence of
transcellular calcium transport through the IDE cells, which resembles the mechanisms in mammalian ameloblasts and in the inner ear epithelium of fishes. In gars, defined activity is also present at the plasma membrane of SI cells [7]. On the other hand, in tilapia, no Ca-ATPase activity was detected in the enamel organ [unpublished data]. Although many additional studies are needed, it is possible that slightly different mechanisms regarding calcium transport exist in tilapia. 3.4.4. K-NPPase (Na – K-ATPase, sodium pump) The activity of K-NPPase (Na –K-ATPase) that has important roles of cellular ion transport is detected at the plasma
I. Sasagawa et al. / Materials Science and Engineering C 26 (2006) 630 – 634
633
Table 2 Morphological and cytochemical features of the enamel organ during enameloid formation
Table 3 Cellular regulation during enameloid formation in bony fishes Cell, tissue
Function in each stage
Cell/stages Matrix formation
Odontoblasts
Matrix formation stage Produce the organic matrix of enameloid including collagen and GAGs Provide matrix vesicles that are the site of initial mineralization Mineralization stage Involved in the crystal accumulation along the collagen (Supply a part of protease to degenerate the enameloid matrix?) Maturation stage Produce dentin beneath enameloid Extend the processes of odontoblasts into enameloid Matrix formation stage Produce ectodermal collagen (gars, tilapia) Mineralization stage Produce amelogenins (gars) (Supply protease to degenerate the enameloid matrix?) Maturation stage Absorb the degenerated enameloid matrix Provide calcium, phosphate to enameloid Supply iron to enameloid (tilapia)
ODE
IDE
Mineralization
Maturation
Contain many mitochondria and smooth ER Stellate reticulum and/or stratum intermedium are present (gars, polypterus) Well-developed Golgi Well-developed Golgi Abundant rough ER Many mitochondria Procollagen granules Many lysosomal granules (ACPase positive) Amelogenins (gars) Marked lamina densa Ruffled border at the distal end No lamina densa ALPase positive plasma membrane K-NPPase positive plasma membrane Ca-ATPase positive plasma membrane
membrane of the IDE cells during the mineralization and maturation stages in tilapia, gars and polypterus (Fig. 1k) [unpublished data, [7]. It is assumed that the Na –K-ATPase at the IDE cells maintains the local osmotic gradient and is involved in the removal of degenerated organic matrix from enameloid. 4. Roles of the dental epithelial cells during enameloid formation Table 2 shows the morphological and cytochemical characteristics of the dental epithelial cells. The data suggest that the dental epithelial cells are mainly engaged in the degeneration and removal of enameloid matrix and in material transportation during the enameloid formation, rather than the
Dental epithelium
matrix formation of enameloid. The features of the dental epithelial cells in tilapia are somewhat different from those in gars and polypterus. The results also demonstrate that the functions of the dental epithelial cells are similar among the actinopterygians possessing well-mineralized enameloid, although the morphology of the enamel organs is different among the species. Table 3 shows the expected cellular influence during enameloid formation in bony fishes. Morphogenesis to determine the shape of the tooth is another important role of dental epithelial cells, but is not a subject of this paper.
Fig. 1. Light and transmission electron micrographs of the tooth germs. (a) Mineralized enameloid at the late stage of mineralization. Transmission electron micrograph (TEM). Gars. The bundles of slender crystals are interwoven, and formed network-like structure. The non-mineralized sections were cut from an Araldite – Epon block and stained with uranyle acetate and lead citrate (U – Pb). Scale bar = 200 nm. (b) Crystals in the enameloid at the maturation stage. TEM. Tilapia. High resolution electron micrograph shows a regular lattice image in longitudinal section of the enameloid crystal. Non-mineralized section, hydroxyethyl methacrylate (GMA) block, non-stained. Scale bar = 2 nm. (c) Enamel organ at the maturation stage in tilapia. Light micrograph (LM). The enamel organ consists of the outer (ode) and inner (ide) dental epitheliums. Many capillaries (ca) penetrate deeply into the dental epithelial cell layer. In the maturing enameloid (asterisk), most of crystals are dissolved after the treatment of demineralization, and only a few organic matrix remain. The specimen was demineralized in a 5% solution of formic acid. Paraffin sections were stained with hematoxylin – eosin (H – E). Scale bar = 50 Am. (d) Enamel organ at the maturation stage in gars. LM. The outer dental epithelial (ode) cells, stellate reticulum (sr), stratum intermedium (si) and inner dental epithelial (ide) cells constitute the enamel organ. Capillaries (ca) approach the outer dental epithelial cells, but they do not invade the enamel organ. Asterisk shows demineralized enameloid area. The specimen was fixed and demineralized in Bouin’s fluid. Paraffin section, H – E stain. Scale bar = 25 Am. (e) Golgi area in the inner dental epithelial cells at the matrix formation stage. TEM. Gars. Procollagen granules (arrows) containing cross-striations are found in the well-developed Golgi apparatus, suggesting that the cells produce procollagens. Demineralized in a 2.5% solution of EDTA – 2Na, U – Pb. Scale bar = 500 nm. (f) Ruffled border and the distal cytoplasm of the inner dental epithelial cells at the maturation stage. TEM. Tilapia. Many electron-dense lysosomal granules (arrows) are present in the distal cytoplasm. Large vesicles containing electron-dense flocculent substance are found in the cytoplasm near the ruffled border (r). Fine fragments of the organic matrix are scattered in the demineralized enameloid (asterisk). It is implied that the cells actively absorb the organic matrix in the enameloid. Demineralized, U – Pb. Scale bar = 500 nm. (g) Outer dental epithelial cells at the maturation stage. TEM. Tilapia. The outer dental epithelial (ode) cells that are situated between the inner dental epithelial (ide) cells and capillary (ca) contain many mitochondria and labyrinthine canalicular cisternae of smooth ER, suggesting that the cells are involved in the active transport of inorganic ions. Demineralized, U – Pb. Scale bar = 2 Am. (h) ACPase reaction in the distal cytoplasm of the inner dental epithelial cells at the maturation stage. TEM. Tilapia. Many ACPase-positive granules are situated near the ruffled border. Asterisk shows demineralized enameloid. Demineralized. The cryostat sections were incubated in a Gomori’s medium. After the incubation, the sections were embedded in ERL-Quetal 653 resin. U – Pb. Scale bar = 2 Am. (i) ALPase activity in the inner dental epithelial cells at the mineralization stage. TEM. Gars. Marked ALPase lead precipitate is localized at the lateral plasma membrane. Demineralized, Mayahara’s medium, U – Pb. Scale bar = 1 Am. ( j) Ca-ATPase reaction in the distal portion of the inner dental epithelial cells at the maturation stage. TEM. Gars. Ca-ATPase lead precipitate is visible at the complex distal infoldings of plasma membrane. Asterisk shows demineralized enameloid. Demineralized, Ando’s medium, U – Pb. Scale bar = 1 Am. (k) K-NPPase activity at the distal portion of the inner dental epithelial cells at the maturation stage. TEM. Polypterus. The reaction products of K-NPPase are localized at the distal and lateral plasma membrane. Asterisk shows demineralized enameloid. Demineralized, Mayahara’s medium, U – Pb. Scale bar = 1 Am.
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Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research (09671857, 16591844) from the Ministry of Education, Science, Sports and Culture, Japan. References [1] I. Sasagawa, J. Akai, J. Fossil Res. Special Is. 3 (2003) 25. [2] I. Sasagawa, Arch. Oral Biol. 40 (1995) 801.
[3] [4] [5] [6] [7] [8]
I. Sasagawa, Arch. Oral Biol. 33 (1988) 75. I. Sasagawa, J. Anat. 190 (1997) 589. T. Kerr, Proc. R. Soc. Lond., B 133 (1960) 401. I. Sasagawa, M. Ishiyama, Arch. Oral Biol. 50 (2005) 373. I. Sasagawa, M. Ishiyama, Connect. Tissue Res. 43 (2002) 505. K. Prostak, P. Seifert, Z. Skobe, Tooth Enamel V, Florence, Yokohama, 1989, p. 188. [9] M. Ishiyama, T. Inage, H. Shimokawa, Arch. Histol. Cytol. 62 (1999) 191. [10] I. Sasagawa, Jpn. J. Oral Biol. 34 (1992) 620. [11] I. Sasagawa, Eur. J. Oral Sci. 106 (1998) 513.
Cellular influence in the formation of enameloid during odontogenesis in bony fishes Ichiro Sasagawa a,*, Mikio Ishiyama b, Junji Akai c a
Department of Anatomy, School of Dentistry at Niigata, The Nippon Dental University, 1-8 Hamaura-cho, Niigata 951-8580, Japan b Department of Histology, School of Dentistry at Niigata, The Nippon Dental University, Niigata 951-8580, Japan c Department of Geology, Faculty of Science, Niigata University, Niigata 950-2181, Japan Received 1 November 2004; received in revised form 24 March 2005; accepted 24 April 2005 Available online 2 November 2005
Abstract The enameloid covering the surface of the teeth in bony fishes exhibits a very high degree of mineralization, and seems to have functions similar to the enamel in mammals. The aim of this study was to investigate the cellular control system of crystallization during enameloid formation. The enamel organs during the enameloid formation stages in ray-finned fishes were observed by transmission electron microscopy and enzyme cytochemistry, which detects hydrolytic enzyme activities. During the mineralization and maturation stages, many large and long crystals with a clear outline became packed densely in the enameloid. Infoldings of the distal plasma membrane in the inner dental epithelial (IDE) cells were marked, and many acid phosphatase positive lysosomes were present in the cytoplasm. The well-developed Golgi apparatus, and many cisternae of endoplasmic reticulum, secretory granules, mitochondria were visible in the IDE cells during these later stages. The activities of nonspecific alkaline phosphatase, calcium-dependent adenosine triphosphatase and potassium-dependent p-nitrophenylphosphatase were usually detected at the plasma membrane of the IDE cells during the mineralization and maturation stages. These results suggest that the dental epithelial cells are mainly involved in the degeneration and removal of enameloid matrix and in material transportation during the enameloid mineralization and maturation stages, rather than in the formation of enameloid matrix. D 2005 Elsevier B.V. All rights reserved. Keywords: Enameloid; Cellular regulation; Bony fishes; Odontogenesis
1. Introduction Cellular control is essentially important to make biological minerals. To investigate the method by which the cells regulate the formation of large crystals might be useful to analyze principles of biomineralization, and also for biomimetic materials study. The former paper [1] concerning the initial mineralization of both enameloid and dentin mentioned that the unit membrane provided by the odontoblasts probably has a role of template for the initial crystals. Matured enameloid situated at the surface of the teeth in bony fishes is a well-mineralized tissue, and corresponds to the enamel in mammals. It is assumed that the enameloid is an analogue of mammalian enamel, because the origin of enameloid is somewhat different from that of enamel. While amelogen* Corresponding author. Tel.: +81 25 267 1500; fax: +81 25 267 1134. E-mail address: [email protected] (I. Sasagawa). 0928-4931/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2005.04.010
esis in mammals is a major subject of basic dental sciences, enameloid formation in bony fishes is paid little attention, so there are still many unclear aspects of enameloid formation. This article reviews our recent studies regarding the role of the enamel organ that consists of dental epithelial cells during enameloid formation in some fishes. 2. Experimental methods Fishes that belong to actinopterygians, the ray-finned fishes (Table 1) were used in this study. Fine structure of the tooth germs during cap enameloid formation stages was investigated by means of light and transmission electron microscopy. Enzyme cytochemistry for non-specific acid phosphatase (ACPase), non-specific alkaline phosphatase (ALPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase, that represents sodium, potassium-activated adenosine tripho-
I. Sasagawa et al. / Materials Science and Engineering C 26 (2006) 630 – 634
sphatase complex, Na – K-ATPase) was employed to examine the function of the tooth germs. 3. Results and discussion 3.1. Stages of enameloid formation; features of the enameloid matrix in each stage The enameloid formation process is divided into three stages; matrix formation, mineralization and maturation. During the matrix formation stage, the organic matrix of enameloid is produced between the inner dental epithelial (IDE) cells and odontoblasts. Abundant collagen fibrils making bundles are found in the matrix. Many fine network-like materials, probably representing glycosaminoglycans and/or proteoglycans, are located among the collagen fibrils. There are many matrix vesicles and processes of the odontoblasts in the matrix [1,2]. In some species, fine slender crystals appear at the matrix vesicles [1,3]. During the mineralization stage, deposition of fine slender crystals progresses along the collagen fibrils. The mineralization roughly extends from the junction between the enameloid and dentin to the apex of enameloid cap (Fig. 1a). Simultaneously, degeneration of collagen fibrils and fine network-like matrix around the fibrils begins to occur [3,4]. During the maturation stage, abundant large crystals occupy the enameloid area (Fig. 1b). In the demineralized sections, only a small amount of the organic matrix is retained in the enameloid [4]. 3.2. Construction of the enamel organ The enamel organ generally consists of only two layers, the inner dental epithelium and outer dental epithelium (Fig. 1c). Stellate reticulum is present between the two layers in some species, e.g., tilapia, gars, polypterus, amai [5]. The stellate reticulum appears during almost all stages of enameloid formation in gars and polypterus, while in tilapia it is temporarily found in the matrix formation stage [2,6]. The stratum intermedium is found only in gars (Fig. 1d) [7]. 3.3. Fine structure of the enamel organ The IDE cells that correspond to ameloblasts in mammals are generally tall and columnar, while the outer dental epithelial cells situated at the outer periphery of enamel organ are usually cubic (Fig. 1c). Stellate reticulum cells extend many processes and are surrounded by wide intercellular space. In gars, the stratum intermedium cells are larger than the cells of the
631
stellate reticulum, and make a closed cell layer at the distal end of the IDE cells (Fig. 1d). The IDE cells exhibit marked morphological change during enameloid formation, suggesting that the IDE cells are effectively involved in the enameloid formation. The main morphological features of the IDE cells are as follows. During the matrix formation stage, the IDE cells contain welldeveloped Golgi apparatus, abundant cisternae of rough endoplasmic reticulum (ER) and many granules and vesicles. This suggests that the IDE cells are specialized for secreting in the formation stage. Procollagen granules and the granules containing filamentous substance are found in the IDE cells of some species (Fig. 1e) [2,8]. However, it is still unclear whether ectodermal collagen forms part of the enameloid matrix. During the mineralization stage, the IDE cells come to possess a ruffled border at the distal end. Many lysosomal granules, mitochondria and vesicles are found in the IDE cells, although well-developed Golgi apparatus and rough ER are still present. It is implied that absorptive function becomes dominant in the IDE cells [4,6]. In gars, large granules containing amelogenin positive substance are observed in the distal cytoplasm. Amelogenins are a major component of the enamel proteins in mammals. However, there is no such substance in the enameloid. The IDE cells might produce amelogenins, but do not release them during enameloid formation [9]. During the maturation stage, the ruffled border is still present. The IDE cells contain many lysosomal granules and mitochondria, but less rough ER (Fig. 1f). The IDE cells might be engaged in the removal and degeneration of organic matrix from maturing enameloid area, as well as the ruffle-ended ameloblasts (RA) in mammals [4,6]. In tilapia, many ferritin particles/granules appear in the cytoplasm in this stage. It is likely that the ferric iron enters the enameloid through the enamel organ [10]. In tilapia, the outer dental epithelial (ODE) cells situated between the IDE cells and capillaries exhibit particular morphological characteristics in the mineralization and maturation stages. Abundant mitochondria and cisternae of smooth ER occupy the cytoplasm (Fig. 1g), resembling the chloride cells in the gill and mitochondria-rich cells in the inner ear epithelium that are engaged in the ion transport in bony fishes [4]. The morphology of the blood capillaries might reflect the activity of material transport system in tooth germs. During the mineralization and maturation stages in some teleosts, several capillaries enter the enamel organ, so that many deep concavities are made at the surface of the enamel organ [4]. However, in gars and polypterus, no capillaries enter the enamel organ, although they approach the surface of the outer dental epithelium [6]. 3.4. Enzyme cytochemistry of the enamel organ
Table 1 Materials Specimen
Portion
Tilapia: Oreochromis niloticus; Tilapia butttikoferi, Cichlidae, Teleostei, Neopterygii Gars: Lepososteus oculatus, Neopterygii Polypterus: Polypterus senegalus, Chondrostei
Pharyngeal teeth Jaw teeth Jaw teeth
3.4.1. Non-specific ACPase Many ACPase positive lysosomal granules are observed in the IDE cells during the mineralization and maturation stages in tilapia, gars and polypterus (Fig. 1h) [6,11]. The lysosomal granules might be closely related to the absorptive function of the IDE cells.
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I. Sasagawa et al. / Materials Science and Engineering C 26 (2006) 630 – 634
3.4.2. Non-specific ALPase Marked activity of the ALPase is usually located at the plasma membrane of the IDE cells during the mineralization and maturation stages (Fig. 1i) [6,11]. This suggests that the IDE cells during these stages are involved in the transport of material and/or phosphate. 3.4.3. Ca-ATPase (calcium pump) Ca-ATPase activity is found at the plasma membrane of the IDE cells during the mineralization and maturation stages in gars and polypterus (Fig. 1j) [6]. The Ca-ATPase activity situates at the plasma membrane, suggesting the presence of
transcellular calcium transport through the IDE cells, which resembles the mechanisms in mammalian ameloblasts and in the inner ear epithelium of fishes. In gars, defined activity is also present at the plasma membrane of SI cells [7]. On the other hand, in tilapia, no Ca-ATPase activity was detected in the enamel organ [unpublished data]. Although many additional studies are needed, it is possible that slightly different mechanisms regarding calcium transport exist in tilapia. 3.4.4. K-NPPase (Na – K-ATPase, sodium pump) The activity of K-NPPase (Na –K-ATPase) that has important roles of cellular ion transport is detected at the plasma
I. Sasagawa et al. / Materials Science and Engineering C 26 (2006) 630 – 634
633
Table 2 Morphological and cytochemical features of the enamel organ during enameloid formation
Table 3 Cellular regulation during enameloid formation in bony fishes Cell, tissue
Function in each stage
Cell/stages Matrix formation
Odontoblasts
Matrix formation stage Produce the organic matrix of enameloid including collagen and GAGs Provide matrix vesicles that are the site of initial mineralization Mineralization stage Involved in the crystal accumulation along the collagen (Supply a part of protease to degenerate the enameloid matrix?) Maturation stage Produce dentin beneath enameloid Extend the processes of odontoblasts into enameloid Matrix formation stage Produce ectodermal collagen (gars, tilapia) Mineralization stage Produce amelogenins (gars) (Supply protease to degenerate the enameloid matrix?) Maturation stage Absorb the degenerated enameloid matrix Provide calcium, phosphate to enameloid Supply iron to enameloid (tilapia)
ODE
IDE
Mineralization
Maturation
Contain many mitochondria and smooth ER Stellate reticulum and/or stratum intermedium are present (gars, polypterus) Well-developed Golgi Well-developed Golgi Abundant rough ER Many mitochondria Procollagen granules Many lysosomal granules (ACPase positive) Amelogenins (gars) Marked lamina densa Ruffled border at the distal end No lamina densa ALPase positive plasma membrane K-NPPase positive plasma membrane Ca-ATPase positive plasma membrane
membrane of the IDE cells during the mineralization and maturation stages in tilapia, gars and polypterus (Fig. 1k) [unpublished data, [7]. It is assumed that the Na –K-ATPase at the IDE cells maintains the local osmotic gradient and is involved in the removal of degenerated organic matrix from enameloid. 4. Roles of the dental epithelial cells during enameloid formation Table 2 shows the morphological and cytochemical characteristics of the dental epithelial cells. The data suggest that the dental epithelial cells are mainly engaged in the degeneration and removal of enameloid matrix and in material transportation during the enameloid formation, rather than the
Dental epithelium
matrix formation of enameloid. The features of the dental epithelial cells in tilapia are somewhat different from those in gars and polypterus. The results also demonstrate that the functions of the dental epithelial cells are similar among the actinopterygians possessing well-mineralized enameloid, although the morphology of the enamel organs is different among the species. Table 3 shows the expected cellular influence during enameloid formation in bony fishes. Morphogenesis to determine the shape of the tooth is another important role of dental epithelial cells, but is not a subject of this paper.
Fig. 1. Light and transmission electron micrographs of the tooth germs. (a) Mineralized enameloid at the late stage of mineralization. Transmission electron micrograph (TEM). Gars. The bundles of slender crystals are interwoven, and formed network-like structure. The non-mineralized sections were cut from an Araldite – Epon block and stained with uranyle acetate and lead citrate (U – Pb). Scale bar = 200 nm. (b) Crystals in the enameloid at the maturation stage. TEM. Tilapia. High resolution electron micrograph shows a regular lattice image in longitudinal section of the enameloid crystal. Non-mineralized section, hydroxyethyl methacrylate (GMA) block, non-stained. Scale bar = 2 nm. (c) Enamel organ at the maturation stage in tilapia. Light micrograph (LM). The enamel organ consists of the outer (ode) and inner (ide) dental epitheliums. Many capillaries (ca) penetrate deeply into the dental epithelial cell layer. In the maturing enameloid (asterisk), most of crystals are dissolved after the treatment of demineralization, and only a few organic matrix remain. The specimen was demineralized in a 5% solution of formic acid. Paraffin sections were stained with hematoxylin – eosin (H – E). Scale bar = 50 Am. (d) Enamel organ at the maturation stage in gars. LM. The outer dental epithelial (ode) cells, stellate reticulum (sr), stratum intermedium (si) and inner dental epithelial (ide) cells constitute the enamel organ. Capillaries (ca) approach the outer dental epithelial cells, but they do not invade the enamel organ. Asterisk shows demineralized enameloid area. The specimen was fixed and demineralized in Bouin’s fluid. Paraffin section, H – E stain. Scale bar = 25 Am. (e) Golgi area in the inner dental epithelial cells at the matrix formation stage. TEM. Gars. Procollagen granules (arrows) containing cross-striations are found in the well-developed Golgi apparatus, suggesting that the cells produce procollagens. Demineralized in a 2.5% solution of EDTA – 2Na, U – Pb. Scale bar = 500 nm. (f) Ruffled border and the distal cytoplasm of the inner dental epithelial cells at the maturation stage. TEM. Tilapia. Many electron-dense lysosomal granules (arrows) are present in the distal cytoplasm. Large vesicles containing electron-dense flocculent substance are found in the cytoplasm near the ruffled border (r). Fine fragments of the organic matrix are scattered in the demineralized enameloid (asterisk). It is implied that the cells actively absorb the organic matrix in the enameloid. Demineralized, U – Pb. Scale bar = 500 nm. (g) Outer dental epithelial cells at the maturation stage. TEM. Tilapia. The outer dental epithelial (ode) cells that are situated between the inner dental epithelial (ide) cells and capillary (ca) contain many mitochondria and labyrinthine canalicular cisternae of smooth ER, suggesting that the cells are involved in the active transport of inorganic ions. Demineralized, U – Pb. Scale bar = 2 Am. (h) ACPase reaction in the distal cytoplasm of the inner dental epithelial cells at the maturation stage. TEM. Tilapia. Many ACPase-positive granules are situated near the ruffled border. Asterisk shows demineralized enameloid. Demineralized. The cryostat sections were incubated in a Gomori’s medium. After the incubation, the sections were embedded in ERL-Quetal 653 resin. U – Pb. Scale bar = 2 Am. (i) ALPase activity in the inner dental epithelial cells at the mineralization stage. TEM. Gars. Marked ALPase lead precipitate is localized at the lateral plasma membrane. Demineralized, Mayahara’s medium, U – Pb. Scale bar = 1 Am. ( j) Ca-ATPase reaction in the distal portion of the inner dental epithelial cells at the maturation stage. TEM. Gars. Ca-ATPase lead precipitate is visible at the complex distal infoldings of plasma membrane. Asterisk shows demineralized enameloid. Demineralized, Ando’s medium, U – Pb. Scale bar = 1 Am. (k) K-NPPase activity at the distal portion of the inner dental epithelial cells at the maturation stage. TEM. Polypterus. The reaction products of K-NPPase are localized at the distal and lateral plasma membrane. Asterisk shows demineralized enameloid. Demineralized, Mayahara’s medium, U – Pb. Scale bar = 1 Am.
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Acknowledgements This study was supported in part by a Grant-in-Aid for Scientific Research (09671857, 16591844) from the Ministry of Education, Science, Sports and Culture, Japan. References [1] I. Sasagawa, J. Akai, J. Fossil Res. Special Is. 3 (2003) 25. [2] I. Sasagawa, Arch. Oral Biol. 40 (1995) 801.
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