Sumio Nishikawa

DNA LOCALIZATION IN NUCLEAR FRAGMENTS OF APOPTOTIC AMELOBLASTS USING ANTI-DNA IMMUNOELECTRON MICROSCOPY : PROGRAMMED CELL DEATH OF AMELOBLASTS

Ameloblasts responsible for tooth enamel formation are classified into two different phases: secretion and maturation. At the transition between these secretion and maturation stages, a considerable number of cells die. In this study, we examined the morphology of degenerating ameloblasts by conventional electron microscopy, and DNA cleavage in degenerating ameloblast nuclei by the in situ terminal transferase assay. The results suggest that apoptosis (programmed cell death) in ameloblasts, including DNA ligation is induced at the transitional stage. The nuclear fragments, chromatin condensation and DNA relocation in apoptotic nuclei were examined quantitatively by post-embedding anti-DNA immunogold electron microscopy and the in situ terminal transferase assay combined with electron microscopy. Numerical analysis revealed that immunogold labeling density in the condensed chromatin of apoptotic nuclei was comparable on the average to that in the perinuclear heterochromatin of normal nuclei, and that individual apoptotic nuclear fragments exhibited highly variable gold particle density, from fragments with lower density to that of normal heterochromatin, to fragments with densities twice as high as that of normal heterochromatin. The in situ terminal transferase assay combined with electron microscopy detected DNA ends exposed by ultrathin sectioning as well as DNA cleavage by a putative endonuclease. In conclusion, the state of the DNA, including its ligation and degeneration, changes gradually during chromatin condensation and nuclear fragmentation of apoptosis.

Apoptosis of Dental Pulp Cells and Their Elimination by Macrophages and MHC Class II-expressing Dendritic Cells

Apoptosis of dental pulp cells of rat incisors was investigated by the TUNEL method and electron microscopy. The results showed that a considerable amount of apoptosis occurred in the pulp, increasing in extent with incisal direction. OX6, ED1, and ED2 antibodies were used to detect macrophages and dendritic cells in combination with immunoelectron microscopy. Apoptotic fragments were eliminated mainly by MHC Class II-expressing cells, including dendritic cells positive for the OX6 antibody, and by MHC Class II-negative macrophages. Macrophages and dendritic cells positive for OX6, ED1, or ED2 increased from the apical to incisal direction of the incisor. These results indicate that apoptosis contributes to normal pulp formation and maintenance.

Localization of Transcription Factor AP-1 Family Proteins in Ameloblast Nuclei of the Rat Incisor

We examined by immunocytochemistry the localization of the AP-1 family proteins c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2 in rat incisor ameloblasts. Most of the antibodies against AP-1 family proteins, except for c-Fos-specific antibody, labeled ameloblast nuclei. The labeling intensity of the c-Jun, JunD, and Fra-2 antibodies was stronger than that of JunB, FosB, and Fra-1. Antibody reactivities of c-Jun, JunD, and Fra-2 were greatly enhanced during or after the transition zone. Furthermore, c-Jun antibodies labeled maturation ameloblasts in a cyclic pattern, which was correlated with ameloblast modulation. Disruption of ameloblast modulation by colchicine injection resulted in greatly decreased reactivity of the c-Jun antibody in the ameloblast nuclei of the maturation zone. Phospho-specific antibodies to c-Jun labeled ameloblast nuclei only weakly throughout the secretion, transition, and maturation zones. These results suggest that the stage-specific localization of AP-1 in ameloblasts is closely related to tooth enamel formation.

Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis

Xenopus embryonic epidermis changes its cellular composition during development: the appearance of ciliated epidermal cells before hatching is a remarkable characteristic. In this study, the functional change of ciliated cells to mucus-secreting cells was examined with immunocytochemistry using anti-tubulin and anti-chondroitin 6-sulfate (C6S). Before hatching, most epidermal cells were labeled with anti-C6S in a granular fashion. Immunoelectron microscopy revealed that the anti-C6S-positive structure was the mucus granule. Ciliated epidermal cells lacked anti-C6S staining, but were strongly labeled with anti-tubulin. After hatching, most ciliated cells in the surface of the embryo disappeared. During their disappearance, some ciliated cells exhibited anti-C6S-positive granular labeling. This strongly suggests that the disappearance of ciliated cells is a functional conversion to mucus-secreting cells instead of shedding through cell death.

Detection of Immature Dendritic Cells in the Enamel Organ of Rat Incisors by Using Anti-cystatin C and Anti-MHC Class II Immunocytochemistry

Dendritic cells in the enamel organ of rat incisors were examined with immunocytochemistry using an anti-cystatin C antibody for immature dendritic cells and macrophages, OX6 for MHC Class II, ED1 for macrophages and dendritic cells, and ED2 for macrophages. Single cells positive for anti-cystatin C appeared in the enamel organ in zones at which ameloblasts secrete enamel matrix proteins. They were also present in transition and enamel maturation zones. In addition, ameloblasts, osteocytes, and osteoclasts were labeled by anti-cystatin C. ED1 and ED2 immunocytochemistry revealed that there was no macrophage population in the enamel organ of secretion, transition, or enamel maturation zone. A double labeling study showed that most anti-cystatin C-positive cells in the enamel maturation zone were also positive for OX6, whereas anti-cystatin C-positive and OX6-negative cells were prevalent in the secretion zone. The results suggest that immature dendritic cells penetrate the enamel organ of the secretion zone and begin to mature in the zones of transition and enamel maturation.

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. frizzled-3 was also localized in differentiating inner enamel epithelial cells. Vang11 was localized sparsely in differentiating preameloblasts and extensively at the cell boundary of stratum intermedium. Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively. These results suggest the involvement of planar cell polarity proteins in odontogenesis.

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. In vitro assays showed negligible levels of osteogenic gene expression in HUCPVCs compared to BM-MSC, but alkaline phosphatase was detected when HUCPVC were cultured in osteogenic medium in the presence of CM from BM-MSC. An in vivo assay employing a rat calvarial osteotomy defect, together with a collagen sponge scaffold, showed that HUCPVCs provided statistically significant bony repair compared to controls. BM-MSC loaded scaffolds were not statistically different from either controls or HUCPVCs. The addition of BM-MSC CM to HUCPVCs also produced no statistically significant difference to the bone formed by HUCPVCs alone. Our results demonstrate that the in vitro assays employed did not predict in vivo outcomes, and that the BM-MSC cell line employed, or CM from such cells, provided no osteogenic advantage over the use of HUCPVCs alone.

INTERNALIZATION OF AMELOGENIN BY DENDRITIC CELLS OF THE PAPILLARY LAYER DURING TRANSITION AND EARLY MATURATION STAGES

Abstract The existence of amelogenin in the papillary layer facing transition and early maturation zones of ameloblasts of rat incisors and the role of dendritic cells were examined by light microscopic immunocytochemistry and immunoelectron microscopy using anti-porcine 25 kDa amelogenin antibodies and anti-MHC class II antibodies. At the light microscopic level, no overall relationship was observed between anti-class II-positive dendritic cells and anti-amelogenin-positive materials located intercellularly: anti-amelogenin-positive dots were scattered in papillary layers and ameloblasts. Anti-MHC class II-positive cells were present in the papillary layer at the transition and maturation stages. Under electron microscopy, however, the dendritic cells occasionally endocytosed anti-amelogenin-positive materials and formed small vesicles. The results suggest that the dendritic cells play a role in eliminating amelogenin from the enamel organ.

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. The nuclei of the other enamel organ-derived cells were also positive. Because this protein is suggested to take part in c-Jun-mediated transcription, the present study and the results of a previous report showing c-Jun localization in the nuclei of enamel organ-derived cells suggest that the enamel organ-derived cells, including maturation ameloblasts, undergo active transcriptional regulation.

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. PRICKLE3 and PRICKLE4 showed cytoplasmic labeling in ameloblasts and other enamel epithelial cells. Double labeling of PRICKLE2 with VANGL1, which is another planar cell polarity protein, showed partial co-localization. To examine the transport route of PRICKLE proteins, PRICKLE1 localization was examined after injection of a microtubule-disrupting reagent, colchicine, and was compared with CX43, which is a membrane protein transported as vesicles via microtubules. The results confirmed the retention of immunoreactive dots for PRICKLE1 in the cytoplasm of secretory ameloblasts of colchicine-injected animals, but fewer dots were observed in control animals. These results suggest that PRICKLE1 and PRICKLE2 are transported as vesicles to the junctional area, and are involved in pattern formation of distal junctional complexes and terminal webs of ameloblasts, further implying a role in the formed enamel rod arrangement.