Search

Gars and bichirs develop scales and teeth with ancient actinopterygian characteristics. Their scale surface and tooth collar are covered with enamel, also known as ganoin, whereas the tooth cap is equipped with an enamel-like tissue, acrodin. Here, we investigated the formation and mineralization of the ganoin and acrodin matrices in spotted gar, and the evolution of the scpp5, ameloblastin (ambn), and enamelin (enam) genes, which encode matrix proteins of ganoin. Results suggest that, in bichirs and gars, all these genes retain structural characteristics of their orthologs in stem actinopterygians, presumably reflecting the presence of ganoin on scales and teeth. During scale formation, Scpp5 and Enam were initially found in the incipient ganoin matrix and the underlying collagen matrix, whereas Ambn was detected mostly in a surface region of the well-developed ganoin matrix. Although collagen is the principal acrodin matrix protein, Scpp5 was detected within the matrix. Similarities in timings of mineralization and the secretion of Scpp5 suggest that acrodin evolved by the loss of the matrix secretory stage of ganoin formation: dentin formation is immediately followed by the maturation stage. The late onset of Ambn secretion during ganoin formation implies that Ambn is not essential for mineral ribbon formation, the hallmark of the enamel matrix. Furthermore, Scpp5 resembles amelogenin that is not important for the initial formation of mineral ribbons in mammals. It is thus likely that the evolution of ENAM was vital to the origin of the unique mineralization process of the enamel matrix.

Objective Petrodentine, the core of the lungfish tooth plate, is a well-mineralized tissue similar to mammalian enamel and analogous to enameloid in fish teeth. Petrodentine is formed solely by petroblasts, which are specialized odontoblasts, whereas enameloid is a composite tissue produced by both odontoblasts and dental epithelial cells. To clarify the details of petrodentine formation, petroblasts were investigated using histochemical and immunohistochemical techniques. Methods Extant lungfish (Lepidosiren paradoxa) were used in this study. Tooth plates during the stage of petrodentine formation were observed by means of histochemistry and immunohistochemistry. Commercial kits were used to detect enzyme activity. Correlative sections were immunostained using antibodies against selected peptides. Routine staining such as periodic acid-Schiff (PAS) reaction to identify glycogen and Elastica van Gieson staining for the detection of elastic fibers in histological sections were performed. In addition, conventional transmission electron microscopy was used for observing the fine structure. Results Petroblasts showed marked acid and alkaline phosphatase activities, and positive immunoreactivities against anti-nestin, anti-V-ATPase, and anti-Ca2+-ATPase, during the maturation stage, but in the matrix formation stage, reactions were much weaker than that of the maturation stage. During the maturation stage, petroblasts showed intense PAS reactivity, and glycogen particles were observed in petroblasts by transmission electron microscopy. Glucose transporter 1-immunoreactivity was observed in petroblasts in the matrix formation stage and the initial to mid part of the maturation stage. Conclusions The results in this study suggested that petroblasts have two functional stages, matrix formation and maturation, and glycogen plays an important role in the modulation of petroblasts.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

We investigated the effects of targeted functionalized silica nanoparticles on the radiosensitivity of cancer cells. Better control of the local concentration of silica nanoparticles may facilitate their use as an adjuvant in conjunction with ionizing radiation to target cancer cells while preventing damage to normal cells. Hyperbranched polyamidoamine (PAMAM) was grafted onto the surface of amorphous silica nanoparticles to functionalize them. The PAMAM-coated silica nanoparticles (PCSNs) were then conjugated with fluorescent dyes. Anti-HER2 antibodies were covalently attached to the labeled PCSNs. The HER2-overexpressing SK-BR3 breast cancer cell line was incubated in medium containing the PCSN probes. After incubation; the cells were exposed to X-ray radiation. Cells were counted in all samples using cell proliferation assays; and apoptotic cells were detected. The cell survival results showed that the combination of the targeted PCSN probes and radiation reduced the survival rate of SK-BR3 cells to a greater extent than when either PCSN probes, PCSNs or radiation were applied individually. The results also showed an increase in apoptosis in the SK-BR3 cells that internalized the PCSN probes and were then irradiated. Based on these data, PCSN probes act as specific radiosensitizing agents for HER2-overexpressing cells.

Tooth enameloid in bony fish is a well-mineralized tissue resembling enamel in mammals. It was assumed that the dental epithelial cells are deeply involved in the formation of enameloid. However, unlike enamel matrix which fully consists of several ectodermal enamel matrix proteins (EMPs), whether enameloid matrix contains ectodermal EMPs has been debated for a long time. In the present study, transmission electron microscopy-based immunohistochemical examinations, using the protein A-gold method with antibodies and antiserum against mammalian amelogenin, were performed in order to search for EMP-like proteins in the cap enameloid of basic actinopterygians, Polypterus and gar. Positive immunoreactivity was detected in the cap enameloid matrix just before the appearance of many crystallites along collagen fibrils, indicating that the cap enameloid contains EMP-like proteins. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures. Therefore, it is conceivable that the ectodermal EMP-like proteins in cap enameloid are involved in crystallite formation along collagen fibrils.

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Recent findings regarding the structure and development of jaw teeth and ganoid scales in the basic actinopterygians Polypterus and Lepisosteus have been summarized in the present review. The relationship between cap enameloid, collar enamel in teeth, and ganoine in scales has been studied to understand the evolution of hard tissues in lower vertebrates. Enameloid is a well-mineralized tissue that is formed by both odontoblasts and dental epithelial cells and is found within the outermost layer of dentin. The inner dental epithelial cells surrounding the enameloid maintain the ability to produce amelogenin-like proteins; however, this may be a rudimentary function. The collar enamel of Polypterus and Lepisosteus exhibits marked immunoreactivity to anti-mammalian amelogenin antibodies, suggesting that it accommodates amelogenin-like proteins. It is likely that the collar enamel in basic actinopterygians corresponds to the enamel in sarcopterygians and amphibians. The recent fine structural and developmental studies have shown that the ganoine of ganoid scales in Polypterus and Lepisosteus is an enamel-like tissue. Positive immunoreactivity to the anti-mammalian amelogenin antibodies by the preganoine of both Polypteridae and Lepisosteidae supports that ganoine is a homolog of tooth enamel.

The morphological features of tooth enamel and enameloid in actinopterygian fish are reviewed to provide basic data concerning the biomineralization of teeth in lower vertebrates. Enameloid, which covers the tooth surface, is a unique well-mineralized tissue and usually has the same functions as mammalian tooth enamel. However, the development of enameloid is different from that of the enamel produced by dental epithelial cells. Enameloid is made by a combination of odontoblasts and dental epithelial cells. An organic matrix that contains collagen is provided by odontoblasts, and then dental epithelial cells dissolve the degenerate matrix and supply inorganic ions during advanced crystal growth in enameloid. It is likely that enameloid is a good model for studying the growth of well-mineralized hard tissues in vertebrates. Some actinopterygian fish possess a collar enamel layer that is situated at the surface of the tooth shaft, indicating that the origin of tooth enamel is found in fish. Collar enamel is thought to be a precursor of mammalian enamel, although it is thin and not well mineralized in comparison with enameloid. In Lepisosteus and Polypterus, both of which are living actinopterygians, both enameloid and enamel are found in the same tooth. Therefore, they are suitable materials for examining the developmental processes of enameloid and enamel and the relationship among them.

The fine structure of collar enamel and the cells constituting the enamel organ during amelogenesis in Lepisosteus oculatus was observed by light, scanning electron and transmission electron microscopy. In the enamel, slender crystals were arranged perpendicular to the surface and the stripes that were parallel to the surface were observed, suggesting that the enamel in Lepisosteus shares common morphological features with that in sarcopterygian fish and amphibians. Ameloblasts containing developed Golgi apparatus, rough endoplasmic reticulum (rER) and secretory granules were found in the secretory stage. In the maturation stage, a ruffled border was not seen at the distal end of the ameloblasts, while many mitochondria and lysosome-like granules were obvious in the distal cytoplasm. The enamel organ consisted of the outer dental epithelial cells, stratum reticulum cells and ameloblasts, but there was no stratum intermedium. It is likely that the ameloblasts have less absorptive function in comparison with the inner dental epithelial cells facing cap enameloid.

In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

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 non-specific 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.

An isolated upper fourth premolar (P4) of the extinct red panda Parailurus was recovered from the Pliocene (3–4 Ma) Ushigakubi Formation near Tochio, Niigata Prefecture, Japan. This is the first report of Parailurus from Asia, a genus previously known from Europe and northwestern North America. The Tochio P4 is about 50 percent larger than that of the extant Ailurus fulgens, lacks an entostyle, but has a lingual cingulum, protoloph with paraconule, a marked posterior cingulum with fine wrinkles, and a distal buccolingual groove. The transverse width of the P4 is greater than the anteroposterior length; the reverse is true of Parailurus anglicus from Europe.

This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

Biomineralization is interpreted to be the mineral deposition occurred under the influence of organisms. To study biomineralization, it is quite important to perform biological techniques besides mineralogical ones, because biominerals are closely related to cells and organic materials. This article describes a series of biological techniques for biomineralization study using transmission electron microscopy, and shows some examples.

The mineralogy of sedimentary iron ores from the Gunma iron mine are described to evaluate the role of microorganisms and plants in ore formation. The iron ore is composed of nanocrystalline goethite, well-crystallized jarosite and very small amounts of strengite. The ore characteristically occurs as thick-bands of alternating goethite and jarosite bands, thin-bands of different goethite grain sizes, and fossil-aggregate ore rich in moss and/or leaves. Algal fossils are clearly preserved in the goethite bands in the thick-banded ore. Lattice imaging showed characteristic crystallographic orientations of the goethite nanocrystals. The thin-banded iron ores consist of micrometer-sized chestnut-burr-like goethite aggregates, probably formed by bacterial iron biomineralization. The bands may be attributed to biological or seasonal rhythms. Various products of biomineralization are found in the present-day pH 2‐3, Fe 21 -, and SO -rich streams. Bacterial precipitation had var22 4 iations from amorphous Fe-P-(S) precipitates near the outlet of mineral spring to Fe-P-S precipitates and to Fe-S-(P) (schwertmannite-like) precipitates in the midstream. Mosses and green algae are also collecting Fe precipitates in and around the living and dead cells. Comparison of the processes occurring in the present-day streams and the iron-ore specimens supports the interpretation of these ores as the product of biomineralization.

Ultrastructural and cytochemical studies on the mineralization of enameloid were performed using Heterodontus japonicus, an elasmobranch, and Tilapia buttikoferi, a teleost as materials. The mineralization of the enameloid in the Heterodontus was divided into the following two steps: (1) initial crystallization in the tubular vesicles that originated from the odontoblasts, and (2) crystal growth that was accompanied by the degeneration and removal of the organic matrix around the crystals. In the Tilapia, the mineralization of the cap enameloid followed three steps: (1) initial crystallization at the matrix vesicles, (2) aggregation of fine slender crystals along collagen fibrils, and (3) crystal growth with the degeneration and removal of the organic matrix. The pattern of early mineralization and the composition of organic matrix in enameloid were considerably different between the two species examined, while in both species the odontoblasts were mainly involved in the formation of the organic matrix of enameloid and in the initial mineralization. In the next step, remarkable crystal growth associated with the degeneration and removal of the organic matrix occurred in both the elasmobranch and the teleost species. The absorptive functions of the dental epithelial cells in the later stages of enameloid formation is probably similar in the two types of enameloid, and is essential for the production of well-mineralized enameloid.

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.

Background We propose a new approach to facilitate sentinel node biopsy examination by multimodality imaging in which radioactive and near-infrared (NIR) fluorescent nanoparticles depict deeply situated sentinel nodes and fluorescent nodes with anatomical resolution in the surgical field. For this purpose, we developed polyamidoamine (PAMAM)-coated silica nanoparticles loaded with technetium-99m (99mTc) and indocyanine green (ICG). Methods We conducted animal studies to test the feasibility and utility of this dual-modality imaging probe. The mean diameter of the PAMAM-coated silica nanoparticles was 30 to 50 nm, as evaluated from the images of transmission electron microscopy and scanning electron microscopy. The combined labeling with 99mTc and ICG was verified by thin-layer chromatography before each experiment. A volume of 0.1 ml of the nanoparticle solution (7.4 MBq, except for one rat that was injected with 3.7 MBq, and 1 μg of an ICG derivative [ICG-sulfo-OSu]) was injected submucosally into the tongue of six male Wistar rats. Results Scintigraphic images showed increased accumulation of 99mTc in the neck of four of the six rats. Nineteen lymph nodes were identified in the dissected neck of the six rats, and a contact radiographic study showed three nodes with a marked increase in uptake and three nodes with a weak uptake. NIR fluorescence imaging provided real-time clear fluorescent images of the lymph nodes in the neck with anatomical resolution. Six lymph nodes showed weak (+) to strong (+++) fluorescence, whereas other lymph nodes showed no fluorescence. Nodes showing increased radioactivity coincided with the fluorescent nodes. The radioactivity of 15 excised lymph nodes from the four rats was assayed using a gamma well counter. Comparisons of the levels of radioactivity revealed a large difference between the high-fluorescence-intensity group (four lymph nodes; mean, 0.109% ± 0.067%) and the low- or no-fluorescence-intensity group (11 lymph nodes; mean, 0.001% ± 0.000%, p < 0.05). Transmission electron microscopy revealed that small black granules were localized to and dispersed within the cytoplasm of macrophages in the lymph nodes. Conclusion Although further studies are needed to determine the appropriate dose of the dual-imaging nanoparticle probe for effective sensitivity and safety, the results of this animal study revealed a novel method for improved node detection by a dual-modality approach for sentinel lymph node biopsy.

Tooth germs were examined by light and transmission electron microscopy. Collagen fibrils were relatively dispersed and thin at the early and middle stages of formation of the enameloid matrix, when the enameloid layer was thin. At the late stage, the fibrils became thicker, reaching nearly 30 nm dia, and formed the interwoven thick bundles that are characteristic of teleost cap enameloid. Abundant flocculent and/or fine, network-like material, probably representing glycosaminoglycans or proteoglycans, was located between the collagen fibrils. Tall, columnar, inner dental epithelial cells contained abundant rough endoplasmic reticulum and many mitochondria, and a well-developed Golgi apparatus was seen around the nuclei at the late stage. Elongated vesicles enclosing fine, filamentous material that resembled procollagen granules, and large granules containing fibril-like structures that were 150 nm in thickness and had periodic cross-banding at 32-nm intervals, were usually observed near the Golgi apparatus. The contents of the large granules were well stained with phosphotungstic acid, which suggests that inner dental epithelial cells synthesize collagen fibrils. At this time, odontoblasts also contained abundant rough endoplasmic reticulum and mitochondria, a well-developed Golgi, several kinds of granule including those that probably contained procollagen, and many microtubules. It is proposed that odontoblasts are involved in the formation of a considerable portion of the enameloid matrix, including collagen fibrils.

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middleregion- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.

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.

Tooth germs during cap enameloid formation stages in Polypterus senegalus were investigated by transmission electron microscopy and enzyme histo- and cytochemistry. Enameloid formation was divided into three stages: matrix formation, mineralization, and maturation. The enamel organ consisted of the inner dental epithelial cells, stellate reticulum, and outer dental epithelial cells during cap enameloid formation stages, but no stratum intermedium was found. During the matrix formation stage, the tall inner dental epithelial cells contained well-developed Golgi apparatus, abundant cisternae of rough endoplasmic reticulum and mitochondria. Spindle-shaped vesicles containing a filamentous structure were seen in the distal cytoplasm. During mineralization and maturation stages, many ACPase positive lysosomes were present in the cytoplasm, whereas the organelles were decreased in number. The infoldings of the distal plasma membrane of the inner dental epithelial cells were visible in the mineralization stage but were not marked in the maturation stage. The activity of nonspecific ALPase, Ca-ATPase, and K-NPPase was detected at the plasma membrane of the inner dental epithelial cells during the stages of mineralization and maturation. The results of fine structure and enzyme cytochemistry suggested that the dental epithelial cells were 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 enameloid matrix formation. The results also showed that the structure of the dental epithelial cells in Polypterus was different from that in teleosts and gars, but that the function of the dental epithelial cells was similar to that in teleosts possessing well-mineralized cap enameloid.

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.

This article reviews current findings on the organic matrix and the mineralization patterns in elasmobranchs, including an analysis of the role of the dental epithelial cells and the odontoblasts during odontogenesis. Our electron micrographs demonstrated that tubular vesicles limited by a unit membrane occupied the bulk of the elasmobranch enameloid matrix during the stage of enameloid matrix formation. It is likely that the tubular vesicles originated from the odontoblast processes. Two types of electron-dense fibrils, with cross-striations at intervals of approximately either 17 nm or 55 nm, respectively, were detected in the enameloid matrix. These data suggest that odontoblasts were strongly involved in enameloid matrix formation and in initial enameloid mineralization. Two types of odontoblasts, dark and light cells, were recognized during the stage of dentinogenesis. The light cells contained numerous mitochondria, intermediate filaments, and microtubules that extended their processes into the dentin. The darks cells possessed a well-developed Golgi apparatus and many cisternae in the rough endoplasmic reticulum, which suggests that the dark cells are involved in the formation of dentin. The inner dental epithelial (IDE) cells exhibited a well-developed Golgi apparatus, many mitochondria, cisternae of smooth endoplasmic reticulum, vesicles, vacuoles, and granules during the mineralization and maturation stages. During the stages of mineralization and early maturation, ACPase-positive granules were visible in the IDE cells and ALPase and Ca-ATPase activities were found at the lateral and proximal cell membrane of the IDE cells, suggesting that the IDE cells are involved in the removal of enameloid organic matrix and in the process of mineralization during later stages of enameloid formation. Our data indicate that elasmobranch enameloid is distinct from teleost enameloid, based on its organic content, on the mechanisms of its mineralization, and on the role of IDE cells concerning enameloid formation. Microsc. Res. Tech. 59:396–407, 2002. © 2002 Wiley-Liss, Inc.

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid. J. Morphol. 252:170–182, 2002. © 2002 Wiley-Liss, Inc.

The structural features of the dental epithelial cells and the enameloid in tooth germs of the Japanese Port Jackson shark, Heterodontus japonicus, in the stages of enameloid formation, were investigated by light and transmission electron microscopy. At the enameloid matrix-formation stage, tall columnar inner dental epithelial cells contained large numbers of glycogen particles. At the enameloid mineralization stage, when many sharply outlined crystals appeared throughout the enameloid, the inner dental epithelial cells exhibited well-developed Golgi apparatuses and many mitochondria in the proximal cytoplasm, and abundant vesicles and vacuoles in the distal cytoplasm. Marked interdigitations of the lateral membrane were visible in the inner dental epithelial cells. The outer dental epithelial cells contained many mitochondria, lysosomal bodies, vesicles and microtubules, and the capillaries usually approached the outer dental epithelial cells. At the enameloid maturation stage, large numbers of crystals occupied the enameloid, and most of the organic matrix had disappeared from the enameloid area after demineralization. The organelles in the inner and outer dental epithelial cells decreased in number, but there were still widely distributed Golgi apparatuses, abundant intermediate filaments and granules containing an electron-dense substance in the inner dental epithelial cells. It is probable that the dental epithelial cells are involved in the removal of organic matrix from the enameloid and in the process of mineralization at the later stages of enameloid formation, i.e., the mineralization and the maturation stages.

Morphological features of the cap enameloid and dental epithelial cells were investigated by light and transmission electron microscopy during the various stages of enameloid mineralisation and early maturation in the tilapia. The pattern of mineralisation along collagen fibrils in the enameloid differed from that in the dentine. Many matrix vesicles were found in the predentine and in the enameloid, suggesting that they may be involved in the initial mineralisation in both regions. Most of the organic matrix disappeared from the cap enameloid during mineralisation and maturation. The disappearance of the organic matrix could be divided into 2 stages. Initially a fine network-like matrix, which probably consisted of glycosaminoglycans and extended between collagen fibrils, began to disappear. At the same time, fine crystallites and electron-dense, fine granular material covered the collagen fibrils as mineralisation of the enameloid began. In the second stage, the maturation of the enameloid, the collagen fibrils degenerated completely and disappeared from the cap enameloid, being replaced by large numbers of large crystals. At the mineralisation stage, the numbers of lysosomal bodies tended to increase in the inner dental epithelial (IDE) cells, which contained a well developed Golgi apparatus and rough endoplasmic reticulum (rER). At the early stage of maturation, a ruffled border was noted at the distal ends of the IDE cells, which contained many mitochondria and lysosomal bodies, but less rER. These features suggest that the cells actively absorb the organic matrix, which includes collagen fibrils, in the cap enameloid. The outer dental epithelial (ODE) cells were translucent cells that contained well developed labyrinthine canalicular spaces from the onset of the mineralisation stage to the middle stage of maturation. The IDE and ODE cells were clearly involved in the mineralisation of the cap enameloid at the mineralisation and maturation stages.

In the sting rays Dasyatis akajei and Urolophys aurantiacus, most of the enameloid matrix was occupied by tubular vesicles (TV) wich were limited by a unit membrane. The TV sometimes touches the processes of odontoblasts, and the unit membrane of the TV seemed to be linked to that of the processes of odontoblasts, and the unit membrane of the TV seemed to be linked to that of the processes. The TV in the enameloid matrix probably originate from the processes of the odontoblasts

Histological examinations of erupted teeth and tooth germs in juvenile and adult dog salmon, Oncorhynchus keta, were carried out by light microscopy, scanning and transmission electron microscopy. As results of these observations, it was found that 1) cap enameloid may only be present in the teeth of juveniles and 2) the inner dental epithelial cells (i. d. e. cells) had great influence on the absorption of the majority of the cap enameloid matrix containing collagen fibers rather than on the formation of tooth matrix.In the teeth of juveniles, the tips of teeth were recognized as hypermineralized and collagen free portions by light microscopic, microradiographic, scanning and transmission electron microscopic observations. In the teeth of adults, however, no such features were seen in the tips of teeth. It seems that cap enameloid is absent in adult teeth.In the stage of matrix formation, the features of the inner dental epithelial cells (i. d. e. cells) indicated that the cells had begun their secretory activity. However, the morphological features of i. d. e. cells indicated that activity of the cells in matrix formation was at a low level and that a great quantity of epithelial secretions widely spread out in the tooth matrix was not obtained except around the basal lamina.On the contrary, active absorptive function at the distal margin of i. d. e. cells is expected and it is considered that absorption of organic substances from the cap enameloid area by i. d. e. cells had already begun in the late stage of matrix formation. Therefore, in the case of juvenile dog salmon, it is assumed that the i. d. e. cells play little part in the formation of tooth matrix and that the majority of the tooth matrix originates from the odontoblasts.In the stage of maturation, the taller and clear i. d. e. cells possessing a remarkable ruffled border at the distal margin were found in the portion surrounding the cap enameloid. It is assumed that such cells absorb the organic matrix containing collagen fibers from the cap enameloid area and make it a hypermineralized area. On the contrary, though the i. d. e. cells corresponding to collar enameloid became taller at this stage, the remarkable ruffled border was not found in these cells. The outermost layer of the collar enameloid area, about 0.5μm in thickness, became more electron-dense and the crystals, which appeared to be arranged perpendicular to the tooth surface, were gathered densely. Considerable difference in the formation mechanism may be present between cap and collar enameloid.

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.

Histological examinations of tooth germs in juvenile dog salmon (chum salmon), Oncorhynchus keta, were carried out by transmission electron micoscopy in order to observe fine structures of initial calcification sites in dental matrix and to determine the relationship between electron-dense fibrous substances and matrix vesicles reported by Igarashi (1983). A number of matrix vesicles round or oval in shape and 300-700 A in diameter appeared in the apical dental matrix around the odontoblasts during the stage of matrix formation and about half of them contained more electron-dense crystal-like structures. On the other hand, matrix vesicles relatively decreased in number, but electron-dense fibrous substances consisting of flocculent substances and fine granular substances became well-developed and longer and increased in number in the apical dental matrix distant from the odontoblasts. These fibrous substances also contained more electrondense crystal-like structures. The stereophotographs showed that the fibrous substances exhibited filmy structures in three dimensions and continued to more electron-dense crystal-like structures. Moreover, the electron-dense fine granular substances would seem to attach to these filmy structures in parts. It seems that these filmy structures are the initial form of crystal. A number of matrix vesicles existed near the fibrous substances and some came into contact with the fibrous substances. A state indicative of the unit membrane of these matrix vesicles having broken and part of the contents disappeared was often obtained. It seems that these fibrous substances appeared later than matrix vesicles because of the observation in the early stage of matrix formation. These observations lead us to the conclusion that the fibrous substances derived from the contents of matrix vesicles. Fibrous substances indicated a positive reaction with ruthenium red staining. No fibrous substances appeared in the basal dental matrix during the stage of matrix formation in the basal portion in spite of the appearance of a number of matrix vesicles and small aggregates of crystal-like structures. It seems that this difference is closely concerned with the fact that the apical dental matrix is the initial portion to begin the calcification of tooth.

The tooth plates in the living Holocephalan, Chimaera phantasma were studied by various techniques. They consisted of osteodentin and hypermineralized pleromin (kosmin). The degree of mineralization on the latter was as much as in the enameloid of Chondrichthyes and Osteichthyes. Scanning electron microscope observation indicated that the pleromin consisted of large and granular crystals. X-ray powder diffractometry, electron microprobe analysis and analytical electron microscopy revealed that the inorganic constituent of pleromin included, as an essential element, a beta-Ca3(PO4)2 (whitlockite) structure containing a small amount of Mg.

In this paper, the eleven molars of four Asiatic elephants (Elephas maximus LINNE) in two zoos are described. They were found in the straw where the elephants slept, except one premolar, artificially removed just before shedding.Compared with the molars in function, the shed molars exihibit the following: smaller number of lamellae, shorter crown length, lower crown height by abrasion, resorbed roots, smooth mesial wearing surface with shallow dentin inlets and indistinct enamel foldings and broad distal facette. Root resorption is more evident in the mesial portion than in the distal; the buccal and lingual third parts are more resorbed than the intermediate third portion, on the buccal side in the upper molar and on the lingual side in the lower molar, than the opposite sides in basal aspect.It is shown in specimens no. 6 and 7 that the crown height decreased by 14-15mm through abrasion during three years (P1. I, 1-4). It was noted that the distal lamellae, (specimen no.11) were shed three months earlier than the mesial ones (specimen no.10, P1. I, 5-7)