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Although oxytalan fibers are known to be a ubiquitous component of the periodontal ligament, little information has been available concerning their organization in the developing periodontal ligament. In the present study, growth and distribution of oxytalan fibers were examined in the developing periodontal ligament of rat molars aged 11, 14, 19, 21 and 28 days. A quantitative analysis of the fibers was made and the spatial relationship between the fibers and blood vessels was studied by means of a three-dimensional reconstruction of serial sections. At the beginning of root formation, oxytalan fibers appeared at first as dot-like structures around the root sheath as well as in areas very close to blood vessels. These structures were resolved in the electron microscope to be made up of 12-nm-wide microfibrils in the vicinity of the surface of the cells of the root sheath. In the process of development, these dot-like structures elongated into entities with helical appearances. As the development further proceeded, longer oxytalan fibers were produced in the apico-occlusal direction along with blood vessels. Quantitative analysis showed that an increase in oxytalan fibers coincided with an increase in the density of the vascular network in the developing periodontal ligament. Based on the results of the present study, the role of oxytalan fibers in the developing periodontal ligament may be in the maintenance of the integrity of the vascular system as previously suggested.

Intracranial germ cell tumors (iGCTs) are the second most common central nervous system tumors in patients under 14 in Japan. The majority of germinomas respond well to combined chemo- and radiotherapy, however non-germinoma germ cell tumors (NGGCTs) may show resistance to therapy and have a poor clinical outcome. Despite their clinical significance, the biology of iGCTs is mostly unknown. The aim of this study is to elucidate the molecular pathogenesis of iGCTs through a comprehensive genomic analysis. A total of 198 germ cell tumors (GCTs) including 133 iGCTs (69 pure germinomas, 56 NGGCTs and 8 metastatic tumors) as well as 65 testicular germ cell tumors (tGCTs) (39 seminomas and 26 non-seminoma GCTs) were collected from 13 centers participating in the Intracranial Germ Cell Tumor Consortium in Japan. Matched normal DNA was available for 70 iGCTs and 4 tGCTs. Somatic mutations in all coding exons were investigated by whole exome sequencing (WES) using SureSelectXT Human All Exon v4 and a GAIIx or HiSeq 2000 system in 41 tumors and the matched normal DNAs. Targeted sequencing with a set of custom made PCR primers was performed using either an IonTorrent PGM or Proton System. The results were integrated with the patients' clinical information that was available for 124 iGCT patients. Mutations in selected candidate genes were further evaluated in an independent tumor cohort using the IonTorrent PGM system. On average, 15.4 non-synonymous somatic mutations were observed in each tumor, ranging from 1 to 140 by WES in 41 iGCTs. Based on the WES data, 41 candidate genes were selected according to the frequency and/or significance of the mutations found. All coding exons of these 41 genes spanning over 160kb were PCR-amplified in a further 157 GCTs using the IonTorrent system. The combined WES and IonTorrent screenings showed that KIT was the most frequently mutated gene in both iGCTs (27%) and tGCTs (18%). MTOR was the second most frequently mutated also in both iGCTs (7%) and tGCTs (6%). RAS mutations (KRAS, HRAS, NRAS) were altogether found in 13% of iGCTs and 12% of tGCTs. These mutations were mutually exclusive to each other and also to KIT mutations. Collectively, the genes involved in the MAPK pathway (e.g., KIT, RAS, NF1) and the PI3K/MTOR pathway (e.g., MTOR, PTEN) were mutated in 44% and 13% of all GCTs. These alterations were significantly more common among pure germinomas than NGGCTs. The mutated MTOR protein was shown to have increased kinase activity, which was suppressed by a specific MTOR inhibitor. Our comprehensive mutational genomic analysis of GCTs revealed that alterations of the MAPK and/or PI3K/MTOR pathways play a critical role in the pathogenesis of both iGCTs and tGCTs, although the extent of their involvement depends on the histopathological subtypes. Our findings will hopefully lead to the development of a targeted therapy for treatment-resistant iGCTs. Citation Format: Koichi Ichimura, Shintaro Fukushima, Yasushi Totoki, Yuko Matsushita, Ayaka Otsuka, Arata Tomiyama, Tohru Niwa, Ryuichi Sakai, Toshikazu Ushijima, Taishi Nakamura, Tomonari Suzuki, Kouhei Fukuoka, Takaaki Yanagisawa, Kazuhiko Mishima, Yoichi Nakazato, Fumie Hosoda, Yoshitaka Narita, Soichiro Shibui, Akihiko Yoshida, Hirokazu Takami, Akitake Mukasa, Koki Aihara, Nobuhito Saito, Toshihiro Kumabe, Masayuki Kanamori, Teiji Tominaga, Keiichi Kobayashi, Saki Shimizu, Motoo Nagane, Toshihiko Iuchi, Masahiro Mizoguchi, Koji Yoshimoto, Kaoru Tamura, Taketoshi Maehara, Kazuhiko Sugiyama, Mitsutoshi Nakada, Keiichi Sakai, Yonehiro Kanemura, Masahiro Nonaka, Kiyotaka Yokogami, Hideo Takeshima, Nobutaka Kawahara, Tatsuya Takayama, Masahiro Yao, Hiromi Nakamura, Natsuko Hama, Masao Matsutani, Tatsuhiro Shibata, Ryo Nishikawa. Whole exome and targeted sequencing identified the MAPK and PI3K pathways as the main targets in intracranial and testicular germ cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4264. doi:10.1158/1538-7445.AM2014-4264

BACKGROUND: The initial symptoms of pineal region tumors might vary according to histology and era. METHODS: The initial symptoms of 39 pineal region tumors who were treated at the International Medical Center, Saitama Medical University and whose initial clinical symptoms were well documented on charts were analyzed. Those symptoms were compared with those of historical control (HC) of 26 "pienalomas" (1), who were treated at the University of Tokyo in pre-CTera (mostly germ cell tumors, but some without histological confirmation). RESULTS: There were 32 germ cell tumors (germinoma 20, non-germinomatous germ cell tumors (NGGCT) 12) and 6 pineal parenchymal tumors (PPT). Increased intracranial pressure (IICP) was observed in 20 cases (51%) which was slightly less than the HC (66.7%) (P = 0.17), The IICP was observed 40% of cases in germinoma, 83.3% in NGCGCT and 33.3% in PPT. Eye movement disorder was observed in 18 of 39 patients (47%) wheres that was only in 12 (33.3%) in the HC (P= 0.26). Abnormality in pupils was observed in 33.3% in the study cohort and in 25% in HC (P = 0.43). Diabetes insipidus, which suggests the presence of multifocal lesion involving hypothalamus was observed in 25% of GCTs of study cohort and in 19.4% in the HC. CONCLUSIONS: With the advancement of modern imaging modalities and accessibility to neurosurgical practice, the clinical pictures of pineal region tumors have been slightly changed but not significantly. Current pineal region tumors tend to be found a little earlier than the pre-CT era. (1) Fujimaki T. Ryoikibetsu-shoukougun series, 2000 SECONDARY CATEGORY: Epidemiology & Cancer Control.

Fluoridated apatite was synthesized at 80 +/- 1 degrees C, pH 7.4 +/- 0.2, using a 5-step fluoride supply system. During the synthesis experiment, 0, 5, 10, 15 and 20 mmol/l of fluoride were each supplied for one-fifth of the experimental period with calcium and phosphate. X-ray diffraction analysis showed a typically apatitic pattern, although the (3 0 0) reflection was broader than that of homogeneous fluorapatite. Scanning electron micrographic observation indicated that the apatite was composed of needle-like crystals similar to hydroxyapatite and fluorapatite. High-resolution transmission electron microscopy showed a slender hexagonal shape similar to homogeneous hydroxyapatite in cross-sections perpendicular to the c-axis and the structural damage in the core of the crystal, although no boundary of step-like layers was observed. The apparent solubility in 0.5 mol/l acetate buffer solution (37 degrees C and pH 4.0) was 12.1 +/- 0.2 mmol/l, much less than that of homogeneous hydroxyapatite 32.3 +/- 1.9 mmol/l, and similar to that of heterogeneous two-layer fluoridated apatite with an outer fluoride-rich layer 12.5 +/- 0.6 mmol/l, which was synthesized previously by supplying fluoride during the latter half of the experimental period.

Two types of heterogeneous fluoridated apatite, H–F–H and F–H–F, were synthesized by supplying fluoride during the middle half (H–F–H) or initial and final quarters (F–H–F) of the experimental period. Although X-ray diffraction patterns and SEM photographs of both H–F–H and F–H–F-type apatites were not significantly different, high-resolution transmission electron microscopy showed quite different features; H–F–H-type apatite crystals were elongated hexagons, while those of F–H–F-type apatite were rather wider hexagons with electron damage in three-quarters of the inner core. These results supported the previous speculations on the two different types of heterogeneous fluoridated hydroxyapatites synthesized with fluoride concentrations stoichiometrically equivalent to that of fluorapatite: hydroxyapatite covered with fluorapatite and fluorapatite covered with hydroxyapatite. F–H–F-type apatite was less soluble than that of H–F–H-type apatite.

Two types of heterogeneous fluoridated apatites, H-F and F-H, were synthesized by supplying fluoride over the whole range of the degree of fluoridation (X = 0-1.0) during the initial or final half of the experimental period. Although X-ray diffraction patterns and scanning electron microscopy (SEM) photographs of both H-F and F-H type apatites were not significantly different, high-resolution transmission electron microscopy (HR-TEM) showed quite different features; H-F type apatites were elongated hexagons with electron beam damage in the core, while F-H type apatites were rather wider hexagons and approached the typical hexagon of fluorapatite. These results supported the previous speculations on the two different types of heterogeneous fluoridated hydroxyapatites synthesized with fluoride concentration stoichiometrically equivalent to that of fluorapatite: hydroxyapatite covered with fluorapatite and fluorapatite covered with hydroxyapatite. The apparent solubility of H-F type apatites decreased with increases in degree of fluoridation, while that of F-H type apatites decreased markedly and then remained almost constant.

Background This study was undertaken to elucidate the type and origin of collagen fibrils which construct the large tubules in deciduous coronal dentin by scanning electron microscope and anti-types I and III collagen antibody procedures. Methods The studies were performed on human deciduous teeth. The teeth were fixed in 4% paraformaldehyde solution and then fractured either mesio-distally parallel to the long axis of the tooth or transversely perpendicular to the long axis of the tooth crown. The specimens were three-dimensionally observed employing the scanning electron microscope to distinguish the content of large tubules. Polyclonal antibodies of anti-type I and anti-type III collagen with 20 nm colloidal gold, and secondary electron imaging and backscatter electron imaging of high-resolution field emission scanning electron microscopy were used to examine the types of collagen fibrils. Results The large tubules extended from the vicinity of the incisal edge of the dentino-enamel junction to the pulp cavity. Inside the large tubules, fibers in compact bundles run parallel to the longitudinal axis of the tubules. The fiber bundles consisted of collagen fibrils which were 50-150 nm in diameter with typical cross striation. Immuno-scanning electron microscopy showed type I collagen-labelling gold particles and type III collagen-labelling gold particles to be abundant on the fibrils. Types I and III collagen-labelling gold particles were present on the banded collagen fibrils regardless of their diameter. Conclusions It was found that type III collagen is present together with type I collagen on the fibrils constructing the large tubules of the human deciduous dentin. This immunohistochemical study suggested that the fibrils constructing the large tubules were derived from the von Korff fibers, and types I and III collagens formed copolymers. Anat. Rec. 248:339-345, 1997. © 1997 Wiley-Liss, Inc.

Diopside has been developed for use in dental root implants and for the filling of bone defects. In previous studies, diopside developed hydroxyapatite (HA) on its surface and achieved a direct bond with bone. The purpose of this study was to investigate the mechanism of crystal formation on the diopside surface. We ultrastructurally evaluated the interface between new diopside-induced crystals and diopside. Specimens were prepared in three experiments: (1) Granular diopside was immersed in simulated body fluid (SBF); (2) granular diopside was implanted into a cavity in rabbit bone; and (3) a diopside dental root implant was implanted into a Japanese monkey. The specimens were examined by contact microradiography, high-resolution transmission electron microscopy, and analytical electron microscopy. In the experiment with SBF, many platelet-like crystals formed in the diopside surface layer. The lattice of diopside and that of the new crystals were very close, but no clear continuation of the lattice was observed. In the experiments which used a rabbit and a monkey, contact microradiography showed close contact between bone and diopside. High-resolution transmission electron microscopy revealed crystal growth from the diopside surface layer, and continuity between the diopside lattice and that of the new crystals. The morphological characteristics of the new crystals and the results of these analyses suggest that these new crystals are HA. With regard to the mechanism by which crystals are formed on the diopside surface layer, it is possible that epitaxial crystal growth could originate as a nucleus on the surface. In this case, epitaxial crystal growth of primarily octacalcium phosphate (OCP) may have occurred, and this may have changed to HA by a phase transition. However, epitaxial growth of OCP on the diopside surface is still highly speculative, since there is no direct supporting evidence.

Background: There have been few detailed transmission electron microscopic studies of the cytoskeleton of osteoclasts to date because of the high electron density of their cytoplasmic matrix, and there is a controversy concerning the presence of stress fibers in these cells. Methods: The cytoskeleton and nuclear matrix of osteoclasts in the rat cranium were studied by transmission electron microscopy after perfusing the organs with a mixture of Triton X-100 and a low concentration of glutaraldehyde to reduce the electron density of the intracellular matrix and by immunoelectron microscopy using anti-actin and anti-vimentin antibodies. Results: In osteoclasts, abundant microfilaments, 5 – 6 nm in diameter, extended across the cell organelles. These filaments were also observed just under the cell membrane facing the bone, but thick bundles of microfilaments were not noted. In the cytoplasm, there were fibers 10–12 nm in diameter, which were considered to be intermediate filaments distributed in the same pattern as microfilaments. Filamentous structures consisting both of the microfilaments and intermediate filaments connected the nuclei. Intermediate filaments linked nuclei at nuclear pores (near the euchromatin), and microfilaments connected nuclei at areas of the nuclear membrane containing nucleolus-associated heterochromatin. In the nuclear matrix after detergent treatment, thin filaments and intermediate -type filaments were found connecting the heterochromatins. Conclusions: Euchromatin is considered to be an active site of DNA and RNA synthesis. Our structural evidence has lead to the proposal that intermediate filaments in the cytoplasm may act as tracks for transmission of information between nuclei and guides for the transit of molecules through the pores. © 1995 Wiley-Liss, Inc.

The present study was performed to examine crystals in salivary calculus by high-resolution electron microscopy (HREM) and electron diffraction analyses. Microradiography (MR) showed that the calculus had three nuclei and showed complex shapes and mineralization. Hydroxyapatite (HA), in a variety of sizes and distributions, and the intermingling of HA and whitlockite (WH) at well-calcified areas, were observed.

Two different types of heterogeneous fluoridated hydroxyapatites previously synthesized at 80 degrees C and pH 7.4, which might be composed of hydroxyapatite covered with fluorapatite (H-FAp) and fluorapatite covered with hydroxyapatite (F-HAp), were observed by high-resolution transmission electron microscopy. The transverse-sectional shapes of both apatites were clearly hexagonal. This confirmed that both apatites were highly crystallized. However, their shapes differed slightly from each other in detail:H-FAp was more slender and F-HAp was typically hexagonal, probably reflecting the shapes of the original core apatites, hydroxyapatite and fluorapatite. The portions thought to be synthesized with fluoride-free solution showed clear electron beam damage. The expanded lattice image observed at the edge of the F-HAp crystal coincided approximately with the a-axis dimension of 0.9443 nm obtained from the X-ray diffraction data of synthetic hydroxyapatite. After incubation in 0.5 mol l-1 acetate buffer solution (37 degrees C, pH 4.0) for one month, the fluorapatite-covered H-FAp maintained its sharp corners, while the hydroxyapatite-covered F-HAp showed rounded corners.

The present study was carried out to investigate the ultrastructure and composition of crystals of pancreatic calculi. Microradiography showed that they were irregularly mineralized with radial or stratified zones of low mineralization. The crystals were identified as calcite composed of CaCO3 in X-ray and electron diffraction studies. Scanning electron microscopy (SEM) revealed that the calculi consist of aggregations of rhombohedral, fang-shaped, botryoidal, foliaceous, and plate-shaped crystals. Some of them showed signs of partial dissolution on their surface. Transmission electron microscopy (TEM) showed crystals of various sizes and shapes either solitary or in clumps. The direct measurement of crystalline lattice intervals obtained from each crystal was 0.386 nm in accordance with the diffraction data. Exocrine pancreatic cell debris are present in pancreatic calculus matrices.

In order to study the effect of F- on tooth enamel-like apatite formation, crystal growth experiments were carried out in the presence of 0.1 approximately 2 ppm F- at 37 degrees C and at pH 6.5 in a model system of enamel formation where octacalcium phosphate (OCP) was stable. Morphology changed from long and thin ribbons to small needle-like plates, and the product changed from OCP to apatite with an increase in F- concentration. In the presence of 0.1-1 ppm F-, apatite-OCP intergrowth took place, and crystals composed of apatite and OCP lamellas were formed. These crystals showed long and thin plate-like morphology and embeded an OCP lamella in the center of the crystal. The OCP lamella and its (100) planes were parallel to the (100) planes of apatite. The thickness of OCP decreased and that of apatite increased with an increase in F- concentration. Some apatite crystals obtained at 1 ppm F- embedded a central plane instead of the distinct OCP lamella. The result indicates that initially formed, thin, plate-like OCP acted as a template for the subsequent epitaxial overgrowth of apatite and, moreover, F- played an important role in regulating the apatite-OCP intergrowth.

The present study was carried out to examine the ultrastructural localization of laminin and type-IV collagen in the junctional epithelium of rat molar gingiva by means of the indirect immunoperoxidase method. Intense laminin reaction occurred in both the internal and the external basal laminae. Laminin reaction products were observed within the cisternae of the rough endoplasmic reticulum of junctional epithelium cells and in concavities formed at the distal plasma membranes adjacent to the basal lamina. Small spherical bodies occurring in the concavities also reacted positively to laminin. Type-IV collagen reaction was intense in the external basal lamina. The internal basal lamina, however, showed no reaction for type-IV collagen. These results indicate that the internal basal lamina contains laminin but no type-IV collagen and that junctional epithelium cells seem very likely to be involved in the production of laminin.

The present study was caried out to observe the process of growth and fusion of apatite crystals in human enamel remineralized in vitro in a solution containing Ca, 1 mM; P, 0.6 mM; and F, 0.05 mM. The growth of newly formed crystals and the regrowth of pre-existing enamel crystals occurred extensively in remineralized enamel. With advancing growth, the crystals came into contact and fused with each other, forming large crystals with hexagonal outlines. Various kinds of crystalline defects, including edge dislocation, low-angle grain boundary, and lattice shifting, were frequently detected between the fusing crystals. X-ray diffraction and electron-probe studies led to the assumption that the regular-hexagonal crystals were fluoroapatite

Cytological changes in the cementoblasts of rats treated with vincristine were investigated. Accumulation of secretion granules indicated a disturbed secretory mechanism in the cells. Conspicuous dilation of the endoplasmic reticulum suggested disturbance in the transport of substances from the rough endoplasmic reticulum to the Golgi elements. These disturbances were interpreted to be a result of disruption of microtubules.

Distribution of α-tubulin in rat-incisor odontoblasts and ameloblasts was observed by means of the immunohistochemical method. Wistar rats were fixed by perfusion with formalin solution or glutar-paraformaldehyde, and their incisors were dissected out, demineralized with either a solution of formic-acid formalin or EDTA, and prepared into 3μm-thick paraffin sections. The sections were incubated with a primary antibody (an anti-α-tubulin mouse monoclonal antibody) and then with a secondary antibody (a horseradish peroxidase-labeled sheep antimouse antibody).Intense and homogeneous α-tubulin immunostaining occurred in the cytoplasm of odontoblasts, inner enamel epithelial cells, and ameloblasts. In the odontoblast processes, however, the staining decreased gradually until it disappeared at a certain level in the dentin. The intense staining was noticed in the distal portions and Tomes' processes of differentiating and secreting ameloblasts. The staining generally decreased in the ameloblasts at an early stage of enamel maturation, and the cells frequently showed either slight or no staining. In reduced ameloblasts, the staining increased and returned to the level of the inner enamel epithelial cells.These results suggest that changes in their α-tubulin distribution reflect functional and morphological changes in odontoblasts and ameloblasts.

Biochemical data generally agree that the organic matrix in enamel is made up mostly of proteins. No agreement has been reached, however, regarding the structure of the organic matrix, its relationship to the enamel crystals, and its role in the mineralization of the enamel. It has been argued that the organic matrix precedes and guides the formation and orientation of the crystals. Conversely, others have described the matrix as a gel; and evidence has been presented to suggest that the organic structures seen in decalcified sections represent, not preformed macromolecular structures, but organic material adsorbed to the surface of enamel crystals.A close relationship between organic matrix and the crystal surface exists from an early stage in the appearance of the first, thin, ribbon-like crystal through all subsequent stages of crystal growth. This is indicated by the finding that, during the maturation stage, organic-matrix structures change parallel with changes of crystal size and shape.Materials identified as amelogenins by their soluble properties make up a relatively small part of the organic materials attached to newly formed enamel crystals. In addition, the amelogenins appear to be diffusely distributed among growing crystals. Materials identified as enamelins, and assumed to account for a major part of crystal-attached material, resist prolonged guanidine-hydrochloride extraction and are tightly bound to the crystal. Extracted from enamel and purified, these two protein groups show a high affinity to enamel crystals in vitro.

Materials used in this work were 13 permanent molars exhibiting dental fluorosis (between 5 and 9 on the Thylstrup-Fejerskov scale, 1978) obtained from adults (aged 20-40 years) living in regions with 3.5 ppm fluoride in the water supplies. Small but deep occlusal caries lesions necessitated extraction. Light and polarized microscopic, microradiographic, electron microscopic, and electron-probe- and ion-micro-analytical studies were made. Enamel surfaces were generally cloudy to opaque, with several pits or defects of various sizes and degrees of brown-staining. An extensively hypomineralized area extended from the inner enamel to the surface layer, which was mineralized to a high degree. The hypomineralized area contained sparsely arranged, flattened, hexagonal crystals with either perforated centers or defects extending from the perimeter and indicating either no or low fluoride content. The highly mineralized surface layer, however, was composed of many large, flattened, hexagonal crystals and extremely small, irregularly shaped crystals. Both types were free of central perforations and defects. A high fluoride concentration was determined in the highly mineralized surface layer. These findings suggest that the hypomineralized area undergoes caries-like changes in terms of crystal dissolution and that the highly mineralized surface layer contains hydroxyapatite and fluoridated-hydroxyapatite, or fluorapatite, or both.

Newly formed osteodentin obtained from the anterior extremities of fetal or young rat incisors was observed by means of electron microscopy and electron probe X-ray microanalysis. Cells related to osteodentin formation frequently showed membrane bound intracellular bodies containing varying amounts of fine, needle-shaped crystals, which were identified as apatite. The intracellular clusters of apatite crystals were extruded from the cells through membrane fusion or cellular degeneration. These extracellular clusters seemed to be gradually incorporated into the mineralizing collagenous matrix, which developed around them. Frequent occurrence of dense, dotshaped or filamentous profiles suggested that the dense bodies seen in the perinuclear regions or in the Golgi area were the sites of crystal formation.

• Young male rats were fed a diet containing cadmium (0, 10, 30, 100, and 300 ppm) so as to morphologically investigate the relationship between bone and kidney lesions caused by experimental cadmium intoxication. In the early stage of the experiment, before the occurrence of kidney lesions, ingested cadmium caused osteoporotic changes in bone. In later stages, slight pathologic changes in the kidney occurred in association with urinary excretion of cadmium. However, there was no evidence of osteomalacic change in bone during the 12-week experimental period. These findings suggest that cadmium may act primarily on bone, rather than secondarily through disturbances of the kidneys, which have some protective ability against cadmium intoxication in the early stage of ingestion of the metal.

The enamel organ of growing rat incisors was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of ouabain-resistant, K+-stimulated p-nitrophenylphosphatase representing the second dephosphorylative step of H-K-ATPase by use of the one-step lead method. Throughout the stages of amelogenesis, the enzymatic activity was found in the plasma membranes, mitochondrial membranes, and lysosomal structures of the cells of stratum intermedium, papillary layer, and ameloblast layer. Gap junctions and desmosomes between these cells were, however, free of reaction product or showed slight precipitates of reaction. The stellate reticulum and the outer enamel epithelium at the stage of enamel secretion were usually negative for reaction. Although secretory, transition, and ruffle-ended maturation ameloblasts showed enzymatic activity at their basolateral cell surfaces, their distal cell surfaces facing the enamel were always free of reaction product. On the other hand, the smooth-ended maturation ameloblasts seldom showed a positive reaction, except in lysosomes and along their basal cell surfaces. An energy-dispersive X-ray microanalysis of reaction products of H-K-ATPase in unosmicated tissue sections demonstrated that they were composed of lead and phosphorus, which had been released during the dephosphorylation of substrate. In cytochemical controls, the enzymatic activity was completely dependent on substrate and potassium ion, resistant to ouabain and levamisole, and inhibited by nolinium bromide, a specific inhibitor of H-K-ATPase. In addition, inorganic trimetaphosphatase as enzymatic marker of lysosome was localized in dark and pale lysosomes, phagosomes, multivesicular bodies, and ferritin-containing vesicles of the ameloblasts and the cells of stratum intermedium and papillary layer. These membrane-bound structures were also positive for H-K-ATPase reaction. These results suggest that: 1) H-K-ATPase functions to maintain an acidic internal pH of lysosomes in the enamel organ cells; and 2) H-K-ATPase localization in the plasma membranes of enamel organ cells is concerned with efflux of protons derived from cytoplasmic water.

The epithelial-mesenchymal junctional area in tooth germs of one- and three-year-old Japanese monkeys was studied with electron microscopy.The plasma membranes at the distal ends of inner enamel epithelium cells were relatively even, and were associated with basement membrane. A large number of filaments, which were 15 nm in diameter and up to 2 μm in length, were present, extending perpendicularly from the basement membrane toward the dental papilla, forming an unique fibrillar layer. The distal cytoplasm of the cell contained rather few vesicles and granules which were positive for the acid phosphatase reaction. The distal ends of differentiating ameloblasts showed irregular undulations and numerous small processes which penetrated through the basement membrane and fibrillar layer. Following an increase of the undulations, the fibrillar layer and the basement membrane were engulfed by the cells and removed from the surface of pre-dentin. Large irregular bodies, which were filled with the filaments of the disintegrating fibrillar layer, were observed frequently. The distal cytoplasm contained a large number of coated pits, coated vesicles, and acid-phosphatase-positive granules. The fibrillar layer then disappeared, being replaced by collagen fibrils in the pre-dentin, which was in the stage of early mineralization.These results indicate that the fibrillar layer is removed by the differentiating ameloblasts and degraded through an engulfing system which is formed by the cell surfaces.

Surface enamel from human subjects with dental fluorosis was studied by means of high-resolution transmission electron microscopy. Immediately below the relatively highly mineralized outermost surface enamel layer was an extensive hypomineralized area. The highly mineralized layer was composed of many large elongated hexagonal crystals and extremely small hexagonal crystals. Frequently the small crystals were attached to the periphery of the large crystals. In the hypomineralized area, large crystals were sparsely arranged; and a few small crystals were seen. Some large crystals showed either perforated centers or defects on their peripheries. These findings suggest that the hypomineralized area undergoes caries-like changes in terms of crystal dissolution and that the highly mineralized surface layer is either formed or modified by remineralization.

A study was undertaken to ascertain whether vitamin D has a direct action on the mineralization of the dentin in rats made rachitic by a diet low in calcium and deficient in vitamin D. Physiological amounts of vitamin D, given orally to rachitic rats, increased their serum calcium from 4.8±0.5 mg/100 ml to 7.5±0.4 mg/100ml. The mineralization of dentin recovered markedly, although osteoporosis occurred in bone trabeculae. The results suggest that vitamin D increases serum calcium by accelerating bone resorption and that the increased serum calcium level acts directly to mineralize the dentin. When calcium was given to rachitic rats by subcutaneous injection, the serum calcium level increased and mineralization of dentin recovered to the same extent as that observed in rats given vitamin D. These results indicate that recovery of mineralization of rachitic dentin depends primarily on recovery of the serum calcium level and that vitamin D is an indirect factor in the mineralization process.