Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Fluoridated apatite synthesized using a multi-step fluoride supply system.

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.

Functionally graded fluoridated apatites.

Fluoridated hydroxyapatite was synthesized at 80 +/- 1 degree C and pH 7.4 +/- 0.2 using a gradient fluoride supply system. 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 rod-like crystals similarly to fluorapatite. High-resolution transmission electron microscopy showed electron damage in the core of the crystal. When the apatite pellet was prepared, electron spectroscopy for chemical analysis showed a negative gradient of fluoride concentration with depth in the crystals. The apparent solubility in 0.5 mol/l acetate buffer solution (37 degrees C and pH 4.0) was 9.16 +/- 0.39 mmol/l, much less than that of homogeneous hydroxyapatite 32.3 +/- 1.9 mmol/l, and less than 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. These results suggest that graded fluoridated apatite may be formed by this process and have higher acid resistance than two-layer fluoridated apatite.

Maternal stress induces synaptic loss and developmental disabilities of offspring.

Mild prenatal stress affects the serotonergic system in the hippocampus of rat offspring. Pregnant rats were daily exposed to mild stress treatments (consisting of crowding and saline injection) during days 15 to 21 of pregnancy. Their offspring were assessed by a series of biochemical, histological and behavioral tests. On 35 days after birth, 5-hydroxytryptamine (5-HT) level was decreased by 17% (P < 0.05), whereas 5-hydroxyindolacetic acid (5-HIAA) level was increased by 18% (P < 0.05) in the offspring of prenatally stressed rats. The metabolic rate (5-HIAA/5-HT) was increased by 49% (P < 0.01). Synaptic density in the hippocampus of prenatally stressed offspring was also decreased by 32% (P < 0.0001) on postnatal day 35. There was no significant group difference in the spatial learning acquisition test of the Morris water maze; however, in the reversal task, prenatally stressed 5-week old rats spent more time than control animals searching for the platform of the pool. Escape latency in the cued test showed no significant difference. Together with data in our previous studies, that have shown 5-HT to facilitate synapse formation and maintenance in the central nervous system, synaptic loss is suggested to occur in relation to changes of 5-HT system in the hippocampus of prenatally stressed offspring. This may be associated with reported changes in behavior and learning ability in prenatally stressed offspring.

Effect of solution composition on morphological and structural features of carbonated calcium apatites.

The composition of enamel mineral corresponds to that of a calcium carbonato-apatite. For insight to be gained into the precipitation of carbonato-apatites having specific properties (crystal size, morphology, and carbonate incorporation into the crystal lattice), apatites were prepared at 80 degrees C in aqueous systems having various CO3 concentrations and pH values of around 7.5 or 10.5 (+/- 0.5). The various preparations had a wide range (0.005 to 0.19) of CO3/Ca molar ratios that bracket the ratios found in porcine enamel mineral at various developmental stages. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction analyses showed that the calcium apatites precipitating at neutral pH incorporated the carbonate into both the hydroxyl and phosphate ion sites in their lattices (A,B-types), whereas the preparations made at the alkaline pH (high OH-(-)CO3(2-)-competition) or in the presence of fluoride (F-(-)CO3(2-) competition) yielded only the B-type carbonato-apatite. It was also ascertained that the size and morphology of the carbonato-apatites, assessed by specific surface area determination and high-resolution electron microscopy, were highly dependent on the driving force for precipitation and the presence of regulators (CO3(2-) and F-) in solution. In neutral media, early precipitates were thin-ribbon in appearance, but grew into crystals having flattened-hexagonal cross-sections. In the presence of fluoride or in alkaline media, acicular apatite crystals, precipitated initially, grew into large rod-like carbonato-apatites having a symmetric-hexagonal cross-section. In both neutral and alkaline solutions, carbonate inhibited the growth of apatite crystals along their c axis, leading to the formation of bulkier crystals. The formation of carbonato-apatites at the neutral pH and their properties are consistent with observations made on enamel minerals formed in the early developmental stages.

High-resolution and analytical electron microscopic studies of new crystals induced by a bioactive ceramic (diopside).

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.

Unusual collagen aggregates induced in rat incisor dentin after vinblastine administration.

Young male Wistar rats weighing 100 g were injected intravenously with 0.2 mg of vinblastine sulfate (VBL). At 24, 27, and 36 hours and 2, 4, and 6 days after introduction of the drug, the rats were fixed by means of perfusion and the upper incisors were removed for electron microscopy. Many secretion granules had accumulated in the young odontoblasts 24 hours after VBL administration. Newly formed predentin located between odontoblasts contained small amounts of an unusual collagen aggregate characterized by a fine, symmetrically striated structure, and paired, dense bands, which were present at both ends. After 27 hours, following the disappearance of the secretion granules in the odontoblasts, unusual collagen aggregates had increased in amount. Two days after administration, the predentin layer, which was close to the odontoblasts, was composed largely of unusual collagen aggregates. During dentinogenesis, the layer of unusual collagen aggregates gradually shifted into the dentin, where, 4 days after VBL administration, it was, to a large degree, located. Mineralization of the unusual collagen aggregates began with the deposition of fine, filamentous crystals that later grew into needle-shaped crystals 40 A thick. These findings suggest that unusual collagen aggregates are produced as a result of the secreting function of odontoblasts and are mineralized, as are normal collagen fibrils in dentin.

Electron microscopic study on the effects of vinblastine on young odontoblasts in rat incisor.

Young Wistar male rats were injected intravenously with 2 mg/kg of vinblastine sulfate. After three, six, 12, 24 and 48 hours, the upper incisors were dissected out of the animals, and their young odontoblasts were observed in an electron microscope. Three to six hours after the injection, the regular polarity of the nuclei and the cytoplasmic organelles were dramatically changed following a decrease in the number of microtubules. Disturbance in protein synthesis was suggested at this stage by unusual shapes and the arrangement of the rough endoplasmic reticulum and loss of the polysomal arrangement of the ribosomes attached to the reticulum. After more than 12 hours, accumulation of secretion granules and the occurrence of bodies, containing a few strands of collagen-like fibrils, were frequently seen in various portions of the cells, indicating retarded export of the secretion substance. Occurrence of a number of unusually short fibrils in predentin was another prominent feature noticed in later stages. The fibrils were about 3000A long by 2000A or 600A wide, and many of the 2000A wide fibrils were characterized by a banded structure which resembled the previously reported SLS collagen. The 600A wide fibrils were usually banded with about 600A periodicity.

Epitaxial overgrowth of apatite crystals on the thin-ribbon precursor at early stages of porcine enamel mineralization.

The aim of the present work was to investigate changes in cross-sectional morphologies of enamel crystallites as a function of location in secretory porcine enamel. Enamel tissues were obtained from 5- to 6-month-old slaughtered piglets. For examination by electron microscopy, a portion of the secretory enamel was embedded in resin and ultrathin sections were prepared with a diamond knife. In parallel studies, compositional and structural changes of enamel mineral were assessed by chemical analysis and Fourier transform infrared (FTIR) spectroscopy. For this purpose, two consecutive layers of the outer secretory enamel, each approximately 30 microns thick, were separated from the labial side of permanent incisors. Using high-resolution electron microscopy, early events of enamel crystal growth were characterized as the epitaxial growth of small apatite units on the lateral surfaces of the initially precipitated thin ribbon. These apatite units had regular triangle or trapezoid cross-sections. After fusions of those isolated trapezoids on both lateral sides of the platy template, the resulting enamel crystallites had the well-documented flattened-hexagonal shapes in cross-sections. The initially precipitated thin plate was buried inside the overgrown apatite lamella and then retained as a central dark line. Similar morphological evidence for the epitaxial nucleation and overgrowth of carbonatoapatite on the platy template was obtained in vitro. Chemical and FTIR analyses of the enamel layer samples showed that the characteristics of the youngest enamel mineral were distinct from those of enamel crystals found in older secretory enamel.(ABSTRACT TRUNCATED AT 250 WORDS)