Localization of Core Planar Cell Polarity Proteins, PRICKLEs, in Ameloblasts of Rat Incisors: Possible Regulation of Enamel Rod Decussation.

To confirm the possible involvement of planar cell polarity proteins in odontogenesis, one group of core proteins, PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, was examined in enamel epithelial cells and ameloblasts by immunofluorescence microscopy. PRICKLE1 and PRICKLE2 showed similar localization in the proliferation and secretory zones of the incisor. Immunoreactive dots and short rods in ameloblasts and stratum intermedium cells were evident in the proliferation to differentiation zone, but in the secretion zone, cytoplasmic dots decreased and the distal terminal web was positive for PRICKLE1 and PRICKLE2.

Bone formation by human umbilical cord perivascular cells.

We investigated the possibility of employing human umbilical perivascular cells (HUCPVCs) within the context of finding an alternative source of mesenchymal stromal cells (MSC) for bone tissue engineering. Since it has previously been reported that conditioned medium (CM) from osteogenic bone marrow (BM) MSCs can potentiate osteogenic differentiation in a secondary cell population, we also employed BM-MSCs to generate CM to stimulate osteogenesis in the HUCPVCs. The BM-MSCs were a commercially available immortalized human cell line.

Angular distribution of cross-sectioned cell boundaries at the distal terminal web in differentiating preameloblasts, inner enamel secretory ameloblasts and outer enamel secretory ameloblasts.

The cross-sectioned profiles of differentiating preameloblasts, inner enamel secretory ameloblasts and outer enamel secretory ameloblasts at the distal terminal web were quantitatively compared. First, the angles of each line constituting the sectioned cell polygons were measured, and the patterns of angular distribution histograms were compared. Second, all groups of line angles from one differentiating preameloblast population, two inner enamel secretory ameloblast and one outer enamel secretory ameloblast populations at the distal terminal web were compared statistically by the χ(2)-test using the multiple comparison method.

Planar cell polarity protein localization in the secretory ameloblasts of rat incisors.

The localization of the planar cell polarity proteins Vang12, frizzled-3, Vang11, and Celsr1 in the rat incisors was examined using immunocytochemistry. The results showed that Vang12 was localized at two regions of the Tomes' processes of inner enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In contrast, frizzled-3 was localized at adherens junctions of the proximal and distal areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin and β-catenin were localized.

Immunocytochemical localization of claudin-1 in the maturation ameloblasts of rat incisors.

Claudin-1 is a tight junction transmembrane protein. Its localization in the maturation ameloblasts of rat incisors was examined by immunofluorescence microscopy. Distal junction area of ruffle-ended ameloblasts (RA) and the Golgi apparatus of a sub-population of smooth-ended ameloblasts (SA) and RAs stained positive with anti-claudin-1 antibodies.

Histochemistry of nerve fibres double labelled with anti-TRPV2 antibodies and sensory nerve marker AM1-43 in the dental pulp of rat molars.

AM1-43 can label sensory nerve fibres and sensory neurons. Permeation of non-selective cation channels of the nerve cell membrane is suggested to be the mechanism responsible for labelling. To identify these channels, two candidates, TRPV1 and TRPV2 were examined by immunocytochemistry in the dental pulp and trigeminal ganglion of rats injected with AM1-43.

Developmental changes in pulpal sensory innervation of rat incisors and molars shown on a single injection of the fluorescent dye AM1-43.

Developmental changes in pulpal innervation of rat incisors and molars were examined using the fluorescent styryl dye AM1-43, which labels sensory cells and nerves in vivo. From 2 to 40 days after birth, the animals were injected once with a small amount of AM1-43 solution (2 microg/g bodyweight). One day after the injection, the animals were killed and examined.

Systemic labeling and visualization of dental sensory nerves by the novel fluorescent marker AM1-43.

Systemic labeling of sensory nerves was performed by injecting a small amount of the styryl dye AM1-43 subcutaneously to the back skin of 4-week-old mice in order to determine its ability to stain sensory nerves. One or 3 days later, dental tissues were fixed and cryosectioned. Molars showed bright nerve fibers in the periodontal ligament and pulp.

Presence of anti-cystatin C-positive dendritic cells or macrophages and localization of cysteine proteases in the apical bud of the enamel organ in the rat incisor.

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C-positive. Anti-cystatin C-labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure.

Transient increase in anti-p-ATF2 immunoreactivity in the late secretion ameloblasts apical to the transition zone of rat incisors.

Activating transcription factor 2 (ATF2) was localized in the ameloblasts of rat incisors by immunohistochemistry. A specific antibody against phosphorylated ATF2 (p-ATF2), which is an activated form of ATF2, was detected from the proliferation zone to maturation ameloblasts just after the transition. In the secretion zone, a transient increase in p-ATF2 was observed in the late secretion ameloblast nuclei, where a stronger reactivity of p-ATF2 extended from 1 mm apical to the transition to the transition zone, whereas ameloblast nuclei in most of the maturation zone exhibited either weak or no reactivity.

Colchicine-induced apoptosis and anti-Fas localization in rat-incisor ameloblasts.

Apoptosis of ameloblasts were examined by the TdT-mediated dUTP-biotin nick end-labelling method and electron microscopy 8 h after injection of colchicine. The results showed that extensive apoptosis occurred in ameloblasts of secretion to maturation zones. To determine the possible involvement of stimulators in ameloblast apoptosis, Fas, Fas ligand, tumor-necrosis-factor alpha, and tumor-necrosis-factor receptor 1 were examined utilizing immunohistochemistry and Western blotting analysis.

Localization of transcriptional co-activator CBP in the ameloblasts and the other enamel organ-derived cells of the rat incisor.

CREB-binding protein (CBP) was examined in ameloblasts and in other enamel organ-derived cells of the rat incisor, using Western blotting analysis and immunocytochemistry by specific antibodies. Western blotting of labial tissues, including ameloblasts of the incisors, detected a single band with a molecular weight equivalent to the reported value of CBP. In immunocytochemistry, CBP was localized in ameloblast nuclei in the maturation zone but not in the secretion and transition zones.