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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Fine structural and cytochemical mapping of enamel organ during the enameloid formation stages in gars, Lepisosteus oculatus, Actinopterygii

During cap enameloid formation in gars (Lepisosteus oculatus), the dental epithelial cells that constitute the enamel organ were observed by means of transmission electron microscopy and enzyme cytochemistry to detect the hydrolytic enzyme activities, alkaline phosphatase (ALPase), acid phosphatase (ACPase), calcium-dependent adenosine triphosphatase (Ca-ATPase) and potassium-dependent p-nitrophenylphosphatase (K-NPPase) (sodium, potassium-activated adenoshine triphosphatase (Na-K-ATPase)). The enameloid formation process in gars was divided into three stages: matrix formation, mineralisation and maturation. The enamel organ consisted of the outer dental epithelial (ODE) cells, stellate reticulum (SR), stratum intermedium (SI) and the inner dental epithelial (IDE) cells during the whole of the cap enameloid formation stages. During the matrix formation stage, many cisternae of rough endoplasmic reticulum and widely distributed Golgi apparatus, in which the procollagen granules containing cross-striations were often found, were remarkable elements in the IDE cells. During the stage of mineralisation, the IDE cells were tall columnar, and infoldings of distal plasma membrane of the IDE cells became marked. The most developed Golgi apparatus was visible at this stage, and large secretory granules containing fine granular or tubular materials were found in the distal cytoplasm that was close to the infoldings of the distal end. Many lysosomes that were ACPase positive were seen near the Golgi apparatus and in the distal cytoplasm of the IDE cells. ACPase positive granules often contained the cross-striation structure resembling procollagen, suggesting that the procollagen is degenerated in the IDE cells. During the maturation stage, the distal infoldings became unclear, and there were no large granules containing tubular materials, but many ACPase positive lysosomes were still present in the IDE cells. Non-specific ALPase was detected at the plasma membrane of the IDE cells at the mineralisation and maturation stages. K-NPPase was markedly detected at the plasma membrane of the IDE cells at the maturation stage. These results demonstrate that the IDE cells might be mainly involved in the removal of degenerated organic matrix from enameloid during the later formation stages. Strong Ca-ATPase activity was observed at the entire plasma membrane of the stratum intermedium cells, and there was slightly weak activity at the plasma membrane of the IDE cells during the mineralisation and maturation stages, implying that these cells are related to the active Ca transport to the maturing enameloid. It is likely that although the structure of the enamel organ is different, the function, especially at the mineralisation and maturation stages, is similar to other actinopterygians having well-mineralized cap enameloid.