Development of the hypermineralized tissue covering the
tips of the larval urodele (Triturus pyrrhogaster) teeth and
distribution pattern of sulfated glycoconjugates


Yasutoku Kogaya


Department of Oral Anatomy, School of Dentistry
Asahi University, 1851 Hozumi, Hozumi-Cho, Gifu 501-02, Japan


Key words: enameloid/enamel/sulfated glycoconjugates


Abstract:

　 The first tooth substance elaborated between the inner dental epithelium and the dental papilla of the larval urodele (Triturus pyrrhogaster) tooth germs comprised fine fibrils and large amounts of sulfated glycoconjugates, identified as chondroitin sulfates. After the matrix had been deposited in full thickness, coarse collagen fibrils parallel to the tooth surface were newly deposited below. Mineral deposition occurred first associated with the coarse collagen fibrils and then spread into previously formed fine fibril matrix. The fine fibrils became obscure and finally removed with mineralization, indicating that the fine fibrils matrix is enameloid. Chondroitin sulfates in the enameloid tended to decrease as mineralization proceeded. True enamel-like structure which lacked collagen and sulfated glycoconjugates was deposited on the mineralized dentine but not on the tooth tip enameloid.


Introduction

It has been reported that the hypermineralized tissues covering tips of the larval monocuspid and of the adult bicuspid teeth of the urodeles are themselves different: that is, the former is enameloid and the latter true enamel (Smith and Miles, 1971; Kerr, 1960). The general criteria to distinguish developmentally, histologically enamel from enameloid are: 1) enamel is a hypermineralized tissue produced by ameloblasts after mantle dentine has deposited, grows centrifugally, contains no mesoderm-derived collagen but ectoderm-derived proteins (amelogenins and enamelins), almost all of which is finally removed throughout maturation stage, and already includes some minerals even at the formation stage, 2) although enameloid is also a hypermineralized tissue, it is elaborated by both odontoblasts and inner dental epithelial cells, equivalent of ameloblasts, deposited before true dentine is formed, usually includes collagen fibrils, epithelial derived proteins, chondroitin sulfates, and odontoblastic processes, but contains no minerals at least during the matrix formation stage.
Our previous studies (Kogaya, 1989, 1994; Kogaya and Furuhashi, 1988; Kogaya et al., 1992) have demonstrated that there are distinct differences in composition and distribution pattern of sulfated glycoconjugates amongst fish enameloid, amphibian and reptilian aprismatic enamel, and mammalian prismatic enamel: that is, enameloid matrix contains chondroitin sulfates associated with collagen fibrils, aprismatic enamel matrix has no sulfated glycoconjugates, and in the prismatic enamel matrix certain sulfated glycoconjugates whose histochemical properties remain undefined are detected preferentially in the surface layer.
The present study was designed to examine whether the matrix of apical hypermineralized tissue of the larval urodele teeth which has been suggested to be an enameloid actually contains sulfated glycoconjugates. The results provided further evidence indicating that the mineralized tissue is an enameloid.


Materials and Methods

Mandibles and maxillae with tooth germs were dissected from the larval newt (Triturus pyrrhogaster ) and fixed in a mixture of 2% glutaraldehyde /4% paraformaldehyde in 0.1 M cacodylate buffer(pH 7.4) for 12 h. Some specimens were then decalcified with 5% EDTA for 1-3 weeks. Sections about 0.5 mm were cut in buccolingual direction from the jaws. Sections were incubated for 18 h at room temperature in high iron diamine (HID) solution, placed in 2% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated and embedded in Taab 812 resin. Ultrathin-sections were cut and floated onto stainless steel grids, and reacted with thiocarbohydrazide (TCH) for 15 min and then treated with 1% aqueous silver proteinate (SP) for 10 min (see in detail Kogaya and Furuhashi, 1988). For the identification of the precise histochemical properties of the HID-TCH-SP stain deposits, some sections were treated with testicular hyaluronidase (Kogaya and Furuhashi, 1988).


Results

The first formed tooth substance was composed of fine fibrils and large amounts of chondroitin sulfates (Figs. 1, 2). After the fine fibrils matrix had been deposited in full thickness, the second tooth substance appeared below and spread downwards along the distal surface of the inner dental epithelial cells (Figs. 2, 3, 5). This matrix was composed of coarse collagen fibrils oriented in parallel to the tooth surface and matrix vesicles (Fig. 3a). The first mineral　deposition occurred associated with the matrix vesicles and coarse collagen fibrils (Fig. 2) and then spread into previously deposited fine fibrils, which is to constitute in future a highly mineralized tooth tip. As the mineralization proceeded, configuration of fine fibrils became obscure and chondroitin sulfates decreases in amount (Fig. 3b). At the maturation stage of the tooth tip zone, when the matrix components were almostly dissolved with decalcification, distinct basement membrane-like structure was recreated at the interface between the maturing enameloid matrix and the ruffled border, within the infoldings of which considerable amounts of sulfated materials were detected (Fig. 4a). In non-decalcified specimen, the outer most part of enameloid appeared as a thin layer of electron dense zone, suggesting much more heavily mineralized area (Fig. 4b). However, there was no differences in the orientation of enameloid crystallites between the most outer and other zones.
After the first type of dentine matrix, which was composed of corase collagen fibrils parallel to the tooth surface, had been deposited in certain thickness, second type of dentine matrix composed of relatively thin and much more complexly arranged collagen fibrils was newly formed below (Fig. 5). At this stage, a histologically quite different substance, which seemed to be a thin layer of ectodermal true enamel because of lack of collagen fibrils and sulfated glycoconjugates, was deposited on the mineralized dentine matrix (Fig. 6).


Discussion

The first enameloid matrix of the larval urodele is elaborated inside the dental basement membrane and composed of fine fibrils and chondroitin sulfates, which is similar to the initial dentine matrix of the adult urodele teeth. Nontheless, unlike the dentine matrix in which collagen fibrils persist after its mineralization, enameloid matrix finally becomes a highly mineralized tissue with almost completely loss of organic matrix, which has been suggested to be due to enzymatic degradation and absorption of the matrix proteins by inner dental epithelial cells (Kawasaki et al., 1987). So far it is still uncertain if the collagen fibrils of enameloid are physico-chemically different from those of true dentine matrix. Shellis and Miles (1974) have suggested the interaction of epithelial proteins secreted by the inner dental epithelial cells with enameloid collagen, by which the collagen can be solubilized prior to or during mineralization.
In the vertebrates possessing tooth enameloid, enameloid matrix appears first and after full thickness of the enameloid matrix has been deposited, dentine matrix formation commences. As shown in the present study, in the case of larval urodele enameloid matrix appears first, secondly dentine matrix, and subsequently true enamel-like structure is deposited on the mineralized dentine. In all other vertebrates possessing true enamel, dentine matrix called "mantle dentine" is ontogenetically first formed and subsequently true enamel matrix is centrifugally elaborated on the mantle dentine while circumpulpal dentine matrix grows centripetally by addition of new matrix at the surface of the papilla. The mantle dentine is usually histologically characterized by the existence of thicker collagen fibrils referred as Korff's fibrils, membrane bounded matrix vesicles, and large amounts of chondroitin sulfates. Here of particular interesting is the phylogenetic relationships between enameloid and mantle dentine matrices, both of which are ontogenetically first formed in fish and larval urodele teeth and in the teeth of tetrapods, respectively. Poole (1971) has assumed that in mammals the tissue at the junction between enamel and dentine is homologous with enameloid. Maisey (1988) have described that phylogenetically the most primitive hard tissue may be enameloid. Alternatively, in all tetrapods except for larval urodele, the hypermineralized tissues covering the tips of the teeth are ectoderm-derived aprismatic or prismatic enamels but never enameloid. On the basis of ontogeny of the larval urodele tooth hard tissues, it is reasonable to infer that enameloid is as primitive as dentine and that true enamel evolved independently somewhat later. According to fossil records, majority of dentine tubercles in Ordovician agnathans were bare, but some were already covered with enamel or enameloid (Smith and Hall, 1990; Hall, 1990). We speculate that some of the phylogenetically first formed dentinous　matrices as exoskeletal tissues could be converted to a highly mineralized tissue by the simultaneous removal of organic matrix and mineral deposition under the influence of inner dental epithelial cells which specialized to secrete certain proteolytic enzyme capable of degrading enameloid collagen fibrils and/or certain epithelial proteins which may facilitate removal of the enameloid matrix or regulate size of enameloid collagen fibrils. In this sense enameloid can be regarded as a modified dentine, as previously suggested by Schmidt (1958). In fact, there is an interesting highly mineralized tissue (petrodentine) which is elaborated by mesenchymally derived petroblasts (Ishiyama and Teraki, 1990). However, in the adult urodele and other tetrapods what did necessitate enamel instead of enameloid? Although Grady (1970) has implied that the formation of ectodermal enamel may have been an adaptation to air breathing, preventing the dehydration of the underlying dentine, this hypothesis can not explain the presence of ectodermal enamel in scales of some fishes, which are usually covered with epithelial cells (Sire et al., 1987).
There are distinct differences in mineralization pattern between larval urodele enameloid and fish enameloid. It is in general known that fish enameloid matrix mineralization commences in the enameloid collagen fibrils at the junction between dentine and enameloid and proceeds to the outer surface. Enameloid crystallites grow along the collagen fibrils (Inage, 1975; Sasagawa, 1984; Wakita, 1992). In contrast, in the larval urodele first mineral deposition occurred associated with matrix vesicles and coarse collagen fibrils which seem to belong to rather dentine matrix than enameloid one, because even after fully mineralization the coarse collagen fibrils persisted. Subsequently the mineral deposition proceeds into previously deposited fine fibril matrix. It may be that in the larval urodele teeth the timing when enameloid mineralization commences is prolonged.
Sulfated glycoconjugates in the urodele enameloid were identified as chondroitin sulfates like fish enameloid. We have previously demonstrated distinct differences in composition and distribution pattern of sulfated glycoconjugates amongst enameloid, aprismatic enamel, and prismatic enamel (Kogaya, 1989, 1994; Kogaya et al., 1992) and suggested that sulfated glycoconjugates are not a prerequisite at least for aprismatic enamel formation and that for prismatic enamel formation certain sulfated substances other than glycosaminoglycans are newly demanded and hence may be phylogenetically advanced elements (Kogaya et al., 1992; Kogaya, 1994). It was confirmed in the present study that the larval urodele teeth are composed of three elements: that is, 1) enameloid, 2)dentine, both of which contain chondroitin sulfates, and 3) ectoderm-derived true enamel-like structure which contains no sulfated glycoconjugates and collagen fibrils.

In conclusion, the prsent study provided further evidence indicating that the
hypermineralized tissue of the larval urodele tooth tip is an enameloid.


References

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Figure Legends

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Fig. 1.
a: First formed tooth substance stained with HID-TCH-SP. Note stain deposits associated with the fine fibrils (F) and over the dental basement membrane (BM).
b: Specimen treated with testicular hyaluronidase prior to HID staining. The staining associated with fine fibrils has almost disappeared but that over the dental basement membrane (BM) and intercellular spaces between inner dental epithelial cells (IDE) remains.

Fig. 2.
After fine fibril matrix (F) has been deposited in full thickness, coarse collagen fibrils (C) oriented parallel to the tooth surface are elaborated below. First mineral deposition occurs associated with these coarse collagen fibrils.

Fig. 3.
a: Border area between first formed fine fibril matrix (F) and coarse collagen matrix (C) stained with HID-TCH-SP before mineralization occurs. Thestain deposits are observed along the coarse collagen fibrils (C), at the periphery of matrix vesicles (MV) and in the fine fibril matrix (F). b:At maturation stage of tooth tip matrix (asterisk), almost all of the matrix components are removed by decalcification. C: coarse collagen fibrils

Fig. 4.
a: At maturation stage, a basement membrane-like structure (BM) is recreated. Note the stain deposits between cytoplasmic protrusions of well developed ruffled border (arrows).
b: Heavily mineralized tooth tip. Note that the most outer surface layer appears as much more electron dense zone (non-decalcified specimen).

Fig. 5.
A late maturation stage of tooth enameloid. Enameloid matrix (asterisk) is almost completely dissolved by decalcification procedure. Dentine matrix is composed of coarse collagen fibrils (C) parallel to the tooth surface and thinner ones (TC) intermingled in a more complex pattern. Arrow: enamel-like structure on the mineralized dentine (see in detail Fig. 6).

Fig. 6.
Higher magnification of enamel-like structure. Note that it lacks collagen fibrils and sulfated glycoconjugates. D: dentine