Iron concentration in teeth of Tetra-odontiform fishes and its phylogenetic significance

It is known that iron is deposited in the enameloid of some teleost fishes, although its biological significance has not been clarified. In the present investigation, a quantitative analysis of iron in the enameloid of fishes of a primitive suborder, the Balistoidei, and an advanced suborder, the Tetra-odontoidei, of the Tetra-odontiformes of marine teleosts was performed by means of the electron microprobe. The results indicated that the enameloid of Balistoidei contained from 0.4-13.5% iron at its surface layer, whereas that of Tetra-odontoidei was very low in iron, which could not be discriminated from the background value of the emission intensity. The enameloid of three perciform species belonging to the Acanthuridae--from which the Tetra-odontiformes are considered to have been derived--also contained high iron (2.7-3.9%) throughout its entire layer. The iron concentration in the enameloid seemed to be related to the phylogeny of fishes rather than to their environmental water and feeding habits, and it is believed that the mechanisms of iron concentration into the developing enameloid have been lost during evolution from the Achanthuridae to the Tetra-odontoidei. Since a similar phenomenon has been previously observed with respect to the fluoride concentration in the enameloid of the same fishes (Suga et al., 1981a), it is speculated that the concentrations of iron and fluoride, which have originally no chemical correlation, have some special biological significance, although the timing and distribution pattern of their deposition are completely different.     

Mineral distribution and mineralization pattern in the enameloid of certain elasmobranchs

Undemineralized teeth of eight shark species representing three elasmobranch families were longitudinally and transversely sectioned. By contact microradiography and polarized light microscopy, the mineral distribution and concentration together with crystal orientation were examined. In all species, the mineral concentration in mature enameloid increased centrifugally. In all except one species, the concentration was as high as in human enamel and equalled that of the enameloid in the teleost Gadus. In three species, younger stages of developing teeth were available, and it was found that, primarily, mineral was deposited rapidly in an isolated superficial narrow zone along the cutting edges. Apart from this zone, mineralization had started in the middle and deeper layers of the whole enameloid mantle and progressed in a centrifugal direction before dentine had been formed underneath. Finally, the mature mineral concentration in the outer enameloid layers first occurred basally and then seemed to progress incisally, preceded by dentine formation underneath. In all shark species, the mature enameloid had a highly mineralized thin surface layer where the crystals were oriented circumferentially and transversely to the tooth. Deep to this surface layer, the enameloid contained radial, narrow, longitudinally flattened ribbon-like bands or septa of organic fibres running in the main substance of longitudinally oriented fibres and crystals. In orthodentine, the orientation of the growth lines relative to the enameloid was fundamentally different from that in mammalian teeth, suggesting a mesodermal origin for the enameloid organic matrix. However, it was concluded that dental epithelium contributes to the mineral deposition in developing enameloid.     

The structure, ultrastructure and physicochemical analysis of the hard dental tissues of the Viperidae

The present study, using classical microscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction, infrared spectroscopy, has shown the dental hard tissues of the fangs of Viperidae (poisonous serpents with terrestrial or semi-aquatic habits) to be constituted of: a calcified outer layer, 0.4 microns thick, made of very small needle-like crystals, randomly distributed. The calcified outer layer contains organic invaginations inducing pores at the surface and many collagen fibres incompletely mineralized, which may suggest enameloid. a calcified inner layer, in the wall of the poison canal. The calcified inner layer, 0.6 microns thick, is constituted of very small crystals, which are parallel to each other and perpendicular to the calcified outer layer. It might be the inner layer of enameloid, an orthodentine, whose tubules present a special lateral branching system resembling a fish bone. The TEM data, which show the dentine to be constituted of very small ill-defined crystals and incompletely mineralized collagen fibres are corroborated by chemical analyses which reveal a poorly mineralized apatite with high carbonate content.     

Serum fluoride level and fluoride content of enameloid

We investigated diverse groups of fish species to determine whether the fluorine (F) contents of the dental hard tissues were related to baseline serum F levels. Serum samples, enameloid, dentin, ganoid/enamel, and bone were analyzed for F by either electron microprobe or wet chemistry. Species were categorized into two groups based on the F content of the enameloid. One group contained greater than 2.6 wt% F in enameloid, whereas the other group had less than 0.45 wt% F in enameloid. The dentin and bone from all species (or, in skates, the cartilage), as well as the ganoid/enamel layer of a Holostean fish (alligator gar), showed consistently low F content. In those species whose teeth developed in sequential rows, the F content of enameloid increased with progressive tooth development. The serum F levels of all fish were below 0.05 microgram F/mL (2.63 mumol/L) and were not significantly related to the F content of the enameloid. The results substantiate the idea that F incorporation into enameloid is related to fish phylogeny, not food or habitat. It is suggested that specialized outer dental epithelial cell configurations may facilitate the incorporation of F into enameloid.     

Enameloid formation in two tetraodontiform fish species with high and low fluoride contents in enameloid

Forming teeth of parrotfish and pufferfish were viewed by transmission electron microscopy to correlate cytological features of the enameloid organ with the species' fluoride (F) content in mature enameloid. Secretory-stage inner dental epithelial cells (IDE) of parrotfish (high F) and pufferfish (low F) secreted procollagen granules into the enameloid collagen matrix. The odontoblasts of both species, less numerous than IDE cells, also contained procollagen granules at the enameloid matrix formation stage. After the full thickness of enameloid matrix collagen had been deposited, enameloid crystallites formed parallel to the long axis of the enameloid collagen fibres. Concurrently, the plasma membranes of the outer dental epithelial cells (ODE) became invaginated in both species, but to a much greater extent in parrotfish. Highly undulating parrotfish ODE cells surrounded numerous fenestrated capillaries. In contrast, pufferfish ODE cells remained straight with few adjacent capillaries. Extensive tight junctions formed between ODE and IDE cells of both species, sealing the extracellular space. With increased mineralization, enameloid collagen fibres were no longer discernible. A thin layer of amorphous material, which subsequently mineralized, was secreted on to the enameloid surface by IDE cells in both species. Pufferfish odontoblasts secreted a mineralizing amorphous layer on the pulpal aspect of the enameloid. The results suggest that at the mineralization stage, a triad of cytostructural features, highly invaginated ODE cells, highly vascularized ODE cells, and extensive tight junctions are strongly correlated with high fluoride content of mature enameloid mineral. Species without any one of these features have lower fluoride in the enameloid.     

Scanning electron microscopy of enameloid and dentine in fish teeth

The structure of enameloid and dentine in teeth of pike (Esox lucius), cod (Gadus morhua) and dogfish (Squalus acanthias) was examined by scanning electron microscopy (SEM) in calcified and decalcified states.The surface of the enameloid layer on calcified tooth tips in all three species was smooth and lacked any pattern or imbrication lines such as are found on human enamel surfaces.In dogfish and pike, the surface layer of the enameloid was formed from a terminal membrane composed of a highly calcified non-fibrillar basement membrane (1–2 μm thick) fused to the tooth surface. The similar appearance of the enameloid surface of cod teeth suggests it is formed by a terminal membrane. Terminal membranes are probably formed and calcified by the inner dental epithelial cells of the tooth organ.The bulk of the enameloid tissue (200–300 μm thick) in all three species was subjacent to the terminal membrane and consisted of fibrous-appearing, radially and longitudinally arranged crystals. The whole of the enameloid both terminal membrane and fibrous layer dissolved upon acid decalcification.A distinct dentine-enameloid tissue junction (DEJ) was present in teeth of all three species. Interdigitation of enameloid and dentine at the DEJ provided mechanical stability.The dentine of pike, cod and dogfish consisted largely of bundles of longitudinally arranged coarse collagen fibrils which form the matrix of both osteodentine and primitive orthodentine.     

Fine structure of the organic matrix remaining in the mature cap enameloid in Halichoeres poecilopterus, teleost

Crystals and the organic matrix in both the enameloid and the maturation stage in this teleost fish were viewed by scanning and transmission electron microscopy. For transmission electron microscopy, specimens were demineralized by basic chromium (III) sulphate or phosphotungstic acid. Large numbers of defined compartments, thought to represent the outline of enameloid crystals, were seen in the outer layer of mature cap enameloid after this demineralization. However, the compartments were both less numerous and less well defined in the inner layer. These compartments suggest the existence of an organic enameloid matrix, which may correspond to the enamelins in mammalian enamel. As these defined compartments occurred in the areas of the tooth where large enameloid crystals were present, it is possible that the organic matrix constituting these compartments is involved in the production of large crystals.     

A histological and histochemical study of the matrices of enameloid and dentine in teleost fishes.

Paraffin sections of developing teeth from four species of teleost fish were stained by various histological procedures and by histochemical techniques demonstrating reactive groups associated with proteins. The matrices of dentine and collar enameloid showed weak reactions for protein groups both before and after mineralization. The matrix of cap enameloid showed weak histochemical reactions initially but stained intensely after the onset of mineralization, especially with methods demonstrating tyrosine, tryptophan, sulphydryl and disulphide groups. Mineralizing cap enameloid showed a number of marked histological differences from dentine, whereas, before mineralization commenced, it was histologically similar to dentine. It is suggested that the staining properties of mineralizing cap enameloid are due to the presence of protein secreted into the collagenous matrix by the inner dental epithelium. The histochemical results are interpreted as suggesting that this protein resembles the proteins of developing mammalian enamel in composition. It is suggested that, in collar enameloid and in unmineralized cap enameloid, the epithelial protein is masked by the matrix collagen. Interaction between the collagen and the epithelial protein may play a part in the mineralization of enameloid.     

Molecular evolution of matrix metalloproteinase 20

Dental enamel is a hypermineralized tissue, containing only trace amounts of organic components. During enamel formation, matrix metalloproteinase 20 (MMP20) processes proteins comprising enamel matrix and facilitates hypermineralization. In the human genome, 24 distinct MMP genes have been identified. Among these genes, MMP20 is clustered with eight other genes, including MMP13, and all these clustered genes show phylogenetically close relationships. In this study, we investigated MMP20 and closely related MMP genes in various tetrapods and in a teleost fish, fugu. In the genome of the chicken, a toothless tetrapod, we identified degraded exons of MMP20, which supports the previous proposition that MMP20 is important specifically for enamel formation. Nevertheless, for unknown reasons, we failed to identify MMP20 in the platypus genome. In the opossum, lizard, and frog genomes, MMP20 was found clustered with MMP13. Furthermore, in the fugu genome, we identified an MMP20-like gene located adjacent to MMP13, suggesting that MMP20 arose before the divergence of ray-finned fish and lobe-finned fish. The teleost tooth surface is covered with enameloid, a hypermineralized tissue different from enamel. Thus, we hypothesize that MMP20 could have been used in an ancient hypermineralized tissue, which evolved into enameloid in teleosts and into enamel in tetrapods.     

Ultrastructure of the dental epithelium during enameloid mineralization in a teleost fish, Cichlasoma cyanoguttatum

Secretory-stage inner dental epithelial cells (IDE) of tooth buds deposited an unmineralized, ectodermally-derived, enameloid collagen matrix. Pharyngeal plates bearing tooth buds were fixed: some were demineralized, others treated with guanidine-EDTA, then fixed and post-fixed in osmium tetroxide with potassium ferricyanide. Thin Epon sections were viewed in a Jeol 100B TEM. Nascent enameloid crystals were orientated parallel to the collagen fibres and attained widths of 200 nm. Enameloid collagen was absent in demineralized mature enameloid. The outer dental epithelial plasma membrane was deeply invaginated forming extensive channels associated with elongated fuzzy-coated vesicles. Four configurations of IDE cells were characterized by cellular constituents, including elongated granules, Golgi complexes, multivesicular bodies, large electron-dense granules and extracellular amorphous material which was also adjacent to cells containing few organelles associated with protein synthesis, within infoldings of ruffled apical membranes and multivesicular bodies. This material was considered to be resorbed enameloid collagen, not a secretory product.     

Odontogenic ameloblast-associated protein (ODAM) and amelotin: Major players in hypermineralization of enamel and enameloid

Odontogenic ameloblast-associated protein (ODAM) and amelotin (AMTN) both belong to the secretory calcium-binding phosphoprotein family, which is critical to biomineralization in vertebrates. In mammals, both ODAM and AMTN are expressed by ameloblasts in the maturation stage, when immature enamel grows into a hypermineralized inorganic tissue. At the onset of this stage, ameloblasts produce a specialized basal lamina (BL), over which both ODAM and AMTN are distributed. Enameloid is a different hypermineralized tissue that is found on the tooth surface of most ray-finned fish. Unlike amelogenesis, no such BL is produced during the maturation of enameloid. Nevertheless, ODAM is also found in ray-finned fish, and the expression of this gene has been detected in inner dental epithelial cells, which correspond to ameloblasts, after the enameloid is considerably mineralized. This specific gene expression suggests that ODAM is not a constituent of the BL but is still involved in the hypermineralization of enameloid. Both ODAM and AMTN are unusually rich in Pro and Gln, and they have 1 or 2 clusters of phospho-Ser residues. These characteristics suggest that ODAM and AMTN associate with weak interactions between relatively hydrophobic regions and further bind calcium phosphate via phospho-Ser clusters, similar to milk caseins that are evolutionary descendants of ODAM. Based on these considerations, I hypothesized that ODAM and AMTN generate and maintain the interface between unmineralized and hypermineralizing domains through weak protein–protein interactions and associations with calcium phosphate. This interface presumably facilitates hypermineralization, efficient removal of degraded proteins from the matrix, and the transfer of calcium phosphate to the matrix.     

An electron microprobe investigation of the distribution of iron in rat incisor enamel

abstract – Maxillary incisors from six five-month-old rats were studied with thc electron microprobe. Analysis of enamel in the pigmented portion of the teeth revealed a zone of high iron content extending 15–20 microns from the enamel surface. From a maximum of 10.1% (range 8.8–12.6%) close to the enamel surface, the iron concentration gradually decreased over a distance of 10 microns to less than 0.1%. At the point of maximum iron concentration the calcium content averaged 28.2% and the phosphorus content 15.7%. The concentrations of these elements increased gradually from the enamel surface and reached a plateau of approximately 34% calcium and 17% phosphorus at a point where the concentration of iron had decreased to a minimal Ievel. The mean Ca/P weight ratio in the zone of maximum iron content was 1.80 and differed significantly from the Ca/P ratio of 1.98 of the enamel beneath the pigmented zone. The zone of raised iron concentration coincided with the extension of the pigmented zone of the enamel. Apically to the pigment incorporation zone, the enamel contained less than 0.1% iron in the surface layer as well as in deeper layers.     

A histological and histochemical study of developing teeth in Polypterus (Pisces,Actinopterygii)

The tooth cap matrix contained proteins of ectodermal and mesodermal origin which extended the data already available on teleost teeth and showed that a combined origin for the tooth cap should be regarded as a primitive feature of actinopterygian fish. Furthermore, the dual nature of the Polypterus tooth cap matrix suggested that evolution of tetrapod enamel did not occur within the actinopterygians. The collar tissue was an unmodified dentine, in contrast to its enameloid nature in many other actinopterygian teeth. The presence of a range of developmental states from unmodified dentine to hypermineralized enameloid within Polypterus teeth themselves and also in other parts of the dermal skeleton, as well as the combined origin of the tooth cap, demonstrates that the enamel/enameloid/dentine system forms a continuum of tissues that have diverged from one another by changes in the relative timing of developmental events and matrix production.     

Ultrastructural study and comparative analysis of fluoride content of enameloid in sea-water and fresh-water sharks

The ultrastructural and chemical similarity of enameloid of Carcharhinus leucas (living in fresh water) and sea-water sharks (Prionace glauca, Dalatias lichas) was confirmed by scanning electron microscopy, high resolution electron microscopy, X-ray diffraction analysis, electron microprobe point analysis and direct potentiometric analysis in a iono-analyser with specific electrode. The mean thickness and width measurements of enameloid crystals performed in high resolution TEM were respectively, 47.1 nm (standard deviation 6.8) and 52.3 nm (standard deviation 7.3). The fluoride content of selachian enameloid was nearly as high as that of fluorapatite. However, the fluoride concentration was higher in non-erupted than in erupted teeth of fresh-water sharks, indicating that fluoride content in enameloid is programmed by the ameloblasts and not due to diffusion from the environment.     

Histogenesis of Sea Bream (Pagrus major) enameloid

The enameloid matrix consists of a collagenous matrix, and non collagenous proteins. Mature enameloid is highly mineralised similarly to mammalian enamel a large amount of the protein being removed. This is in contrast with bone, cementum, and dentin, where a large amount of collagen remains in the completed tissue. From the results of a biochemical study it has been suggested that the decrease of a major part of both the collagenous protein in developing enameloid is brought about by proteolytic enzymes (Kawasaki et al. 1987). However, histological and histochemical observations were not made at that time. In this study fourty-five live specimens of Sea Bream (Pagrus major) were used for histological and histochemical observations. Four undemineralised serial cryostat sections were obtained from each tooth garm and the developing enameloid matrix was observed ultrastructurally. The results of this study, show that the proteolytic enzyme activity which could not be detected in the enameloid formative stage, but was demonstrable after mineralisation had spread across the thickness of the matrix. Further more, the stage at which the proteolytic enzyme could be obtained was related to the stage of ALPase activity found in the dental epithelial cells.     

Activity of alkaline and acid phosphatases in dental epithelial cells and enameloid during odontogenesis in two teleost fish,Oreochromis niloticus and Tilapia buttikoferi

The dental epithelial cells and enameloid at the stages of enameloid matrix formation, mineralization and maturation in the teleosts Oreochromis niloticus and Tilapia buttikoferi were investigated by means of enzyme histochemistry in order to identify their functions associated with the structural modification. No marked enzyme activities were found in the inner dental epithelial cells in the stage of enameloid matrix formation, although the outer dental epithelial cells often exhibited moderate alkaline phosphatase (ALPase) activity. In the stages of enameloid mineralization and maturation, the inner dental epithelial cells, which possessed a ruffled border at the distal ends, showed intense ALPase activity at their lateral and proximal cell membranes. At the same time, many acid phosphatase (ACPase)-positive vesicles and granules were localized at the distal cytoplasm of the inner dental epithelial cells. The outer dental epithelial cells, which contained well-developed labyrinthine canalicular spaces, showed neither marked ALPase nor ACPase activity. It is postulated that the dental epithelial cells in these two teleosts are mainly involved in the removal of the organic matrix from the enameloid, and in material transport to the enameloid during the later half of odontogenesis.     

Relationship between mineral distributions in dentine lesions and subsequent remineralization in vitro

Though the mineral distribution of the dentine carious lesion varies largely from tooth to tooth and from patient to patient, there are two main distribution profiles that characterize natural carious lesions in dentine. These profiles include softened and subsurface lesion types. The mineral distribution relationship between the starting profile and the profile after remineralization is not known. In order to study the relational aspects, we have produced demineralized dentine samples in vitro with mineral profiles similar to those of typical natural carious lesions, and subsequently remineralized the samples in a remineralizing solution with various fluoride concentrations (0, 2 and 10 ppm F). The mineral distributions were obtained by using an improved microradiographic technique. In addition, the nature of deposited mineral was analyzed by diamond-coupled total internal reflectance spectroscopy. Definite relationship was observed between the original lesion mineral distribution and the mineral distributions following remineralization. The amount of mineral present in approximately the first 50 microm of the lesion influenced the overall mineral profile after remineralization, possibly through influencing ion transport. If the amount was high (> approximately 10 vol%), the deposited mineral was confined to the surface (0-50 microm). The original mineral at those depths acted like a nucleus of mineral regrowth when the amount of residual mineral was intermediate, and like a transport barrier when the surface layer was well mineralized. If a surface barrier was not present, mineral was deposited at deeper depths in the lesion. Fluoride effect on dentine remineralization was dependent on the original mineral content and its distribution in the lesion. Although a high concentration of fluoride was very effective in low-mineral lesions, it produced hyperremineralization on well-mineralized subsurface lesions so that it prevented effective remineralization especially in deeper lesions.     

上颌中切牙透明层分布的临床分类研究

目的：通过分析人群中上颌中切牙透明层分布特点，为天然牙透明层分布的临床分类工作提供理论依据。方法采用连续整群抽样的方式获取研究样本，利用直接观察法收集目标人群天然牙透明层分布相关信息。结果人群中天然牙透明层分布类型所占比例由大到小分别为“透明层覆盖于整个牙冠，使牙冠整体看上去明亮”（48.39%），“透明层仅覆盖于牙冠切缘”（28.36%），“透明层覆盖于牙冠切缘和邻面”（17.25%），“透明层覆盖于牙冠邻面”（5.99%）；并且，女性天然牙透明层分布在牙冠的切缘和邻接面的概率大于男性；随着年龄增长，天然牙的邻接面有变透明的趋势，46岁以上这种趋势变得更为明显。结论上颌中切牙透明层分布的临床分类具有一定的必要性和科学性。     

Electron microprobe analysis of iron content of incisor enamel in some species of Rodentia

Enamel from maxillary incisors of six species was analysed. In those species where pigmentation was observed in the outermost layer of enamel, a corresponding iron-rich zone was demonstrated by elemental analysis. The enamel of squirrel and beaver contained about 10 per cent of iron close to the enamel surface and the concentration gradually decreased to less than 0.1 per cent within 20–30 μm from the surface. Through the same zone, the calcium and phosphorus concentration profile lines showed a gradual increase until a plateau was reached (about 36 per cent calcium and 17 per cent phosphorus) at the point where iron had decreased below a detectable level. This inverse relationship between iron on one hand and calcium and phosphorus on the other was clearly seen in incisor enamel of hamster and mouse which contained a maximum of 5–6 per cent iron near the enamel surface, but was not observed in the lemming (maximum 3 per cent iron). In the non-pigmented enamel of the guinea-pig, iron was not detected at the 0.1 per cent level, and the calcium and phosphorus profile lines followed a horizontal course through the entire width of the enamel. The sum of iron, calcium and phosphorus was approximately equal in all analysed areas. The results indicate that a clear relationship exists between pigmentation and iron content of rodent incisor.     

The appearance of matrix vesicles and mineralization during tooth development in three teleost fishes with well-developed enameloid and orthodentine

Ultra-thin resin sections of tooth bearing parts from three species were examined by electron microscopy. During enameloid matrix formation, matrix vesicles (MV) and fine aggregations of crystal-like structures probably derived from the MV were found in the enameloid matrix. In enameloid, however, no additional mineralization occurred along the collagen fibrils until mineralization had begun initially at the junction between the enameloid and orthodentine areas. Advance of mineralization along collagen fibrils was inhibited in the enameloid area before initial mineralization despite the presence of MV. Many MV were observed at the initial mineralization site; they appeared in the predentine during odontogenesis after the initial mineralization and were still visible during the stage of basal dentine formation. The odontoblasts continued to produce MV from the early stage of enameloid matrix formation until the late stage of basal dentine formation. However, despite their long appearance, MV are probably involved directly with the mineralization only when the initial mineralization starts.