Takanori Domon

Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland.

Apoptosis and proliferation of parenchymal cells during atrophy of rat submandibular gland induced by double duct ligation were investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) and transmission electron microscopy (TEM). At 2 and 3 days after ligation, increased PCNA positive cells and mitoses were seen in ducts; thereafter PCNA positive cells decreased in number. At 3 and 4 days, the acinar cell population rapidly decreased, with many remaining TUNEL positive acinar cells. During this period, TEM showed typical apoptotic acinar cells that were phagocytosed by adjacent acinar cells or intraepithelial macrophages. After 7 days, most acinar cells had disappeared, leaving prominent residual ducts; a few acinar cells remained, especially at the lobule periphery. Submandibular gland duct ligation thus induced marked depletion of acinar cell by apoptosis and a concurrent short-lived cycle of duct cell proliferation.

Mononuclear odontoclast participation in tooth resorption: The distribution of nuclei in human odontoclasts

Osteoclasts and odontoclasts have been considered multinucleated giant cells which resorb hard tissue by ruffled borders. Recently, the authors reported the presence of a mononuclear osteoclast and odontoclast with a ruffled border. However, the relative frequency of such cells and the distribution of the number of nuclei including mononuclear cells in them have not been elucidated. Six human deciduous teeth were used in this study. After fixation and decalcification, tartrate‐resistant acid phosphatase (TRAP) activity was detected with the azo dye method, and then TRAP‐positive cells were observed on resorbing areas of teeth by light microscopy. The cells for investigation were serially sectioned by semithin sections to observe the presence of resorptive lacuna and the number of nuclei. The TRAP activity was detected in both multinucleated and mononuclear odontoclasts from serial semithin sections, and 242 TRAP‐positive cells which formed lacunae on dentin were investigated to determine the frequency distribution of the number of nuclei. The mean number of nuclei per cell was 5.3, and median was 4. Only 2.9% of odontoclasts were mononucleus and 93.8% had 10 or fewer nuclei. The majority of odontoclasts forming lacunae on the dentin were cells with 10 or fewer nuclei, and mononuclear odontoclasts participated in human deciduous tooth resorption together with multinucleated ones. Anat. Rec. 249:449–457, 1997.

Twisted plywood structure of an alternating lamellar pattern in cellular cementum of human teeth

Human cellular cementum was examined by scanning electron microscopy to elucidate the manner of the alternate lamellar pattern forming the cellular cementum. Specimens were demineralized, trimmed with a freezing microtome, and treated by NaOH-maceration. This procedure was chosen to avoid artifacts in the fibril arrangement, and to study the fibrous architecture in detail. For comparison, non-demineralized, polished and HCl-etched specimens were also prepared. In the NaOH-macerated specimens, the lamellar pattern of the cellular cementum conformed to the twisted plywood principle of bone lamellation with a periodic rotation of matrix fibrils resulting in an alternating lamellar pattern. In contrast, matrix fibrils were irregularly arranged without indication of rotation of matrix fibrils in the polished and etched specimens. Our results suggest that polishing and etching procedures cause damage to fibrils and fibril arrangement.

The roles of apoptosis and mitosis in atrophy of the rat sublingual gland.

The roles of apoptosis and mitosis of acinar and duct cells in the atrophy of the sublingual gland of rat induced by double duct ligation was investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL), and transmission electron microscopy (TEM). Many PCNA-positive duct cells were observed 3 days after duct ligation, and the numbers decreased thereafter. At 3 and 5 days, several TUNEL-positive acinar cells were observed and typical apoptotic acinar cells were identified by TEM. Necrotic acinar cells were also observed ultrastructurally. After 7 days, there were few acini but many ducts, as well as many structures representing transition from acinus to duct. These observations demonstrate that acinar cell loss by apoptosis and duct cell proliferation by mitosis occur in atrophic sublingual glands as well as in other atrophic salivary glands. In addition, it appears that the transition from acinar to duct cell and the necrosis of acinar cells play important roles in the atrophy of the sublingual gland.

Apoptosis and Proliferation of Myoepithelial Cells in Atrophic Rat Submandibular Glands

This study was designed to determine whether apoptosis and proliferation of myoepithelial cells occur in atrophic rat submandibular glands. The excretory duct of the right submandibular gland was doubly ligated with metal clips. The atrophic right submandibular glands removed after 1–28 days of duct ligation were investigated using immunohistochemical double staining for actin as a marker for myoepithelial cells and proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells, double staining for actin immunohistochemistry, nick end-labeling (TUNEL) as a marker for apoptotic cells, and transmission electron microscopy (TEM). A few PCNA- and no TUNEL-positive myoepithelial cells were found in the control submandibular glands taken from animals with no operation. In the experimental glands, PCNA-positive myoepithelial cells were common 2 and 3 days after duct ligation and then decreased in number. TUNEL-positive myoepithelial cells appeared at 2 days and were observed most frequently at 5 days. Apoptotic myoepithelial cells were also identified by TEM. These observations suggest that both apoptosis and proliferation of myoepithelial cells occur, especially in the early phase of atrophy, in the rat submandibular gland.

Immunolocalization of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars

To elucidate the roles of proteoglycans (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGs), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the initial cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal junction were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.

The nature and function of mononuclear cells on the resorbed surfaces of bone in the reversal phase during remodeling

In a reversal phase of bone remodeling many mononuclear cells appear on the resorbed surfaces of bone with characteristic reversal lines as revealed by transmission electron microscopy (TEM). However, these mononuclear cells have been variously hypothesized or reported. The present study examined the TEM features on the resorbed surfaces of three calcified connective tissues, and aimed to clarify the nature and function of the mononuclear cells in a reversal phase. Dentine slices cultured with isolated osteoclasts, human deciduous teeth, and rat mandibles were used in this study. Specimens were fixed, decalcified, and then embedded in Epon 812, and sectioned into 0.1-microm-thick ultrathin sections. The ultrathin sections were stained with uranyl acetate and lead citrate, and then examined by TEM. Many sharply pointed collagen fibrils with striation were observed exposed on the resorbed surfaces of cultured dentine slices, but there were neither cells nor reversal lines. The same features were observed on the root dentine surfaces of human deciduous teeth. Under many mononuclear cells in a reversal phase of remodeling, reversal lines were seen on the resorbed surfaces of rat mandibles, but there were no striated collagen fibrils exposed on the bone surfaces. The alternation of the TEM features on the resorbed surfaces before and after the participation of mononuclear cells in a reversal phase of remodeling suggests the nature and function of these cells: they participate in both degrading the demineralized and disrupted matrix left on the resorbed surfaces and forming reversal lines there.

Some osteocytes released from their lacunae are embedded again in the bone and not engulfed by osteoclasts during bone remodeling

It is generally accepted that osteocytes derive from osteoblasts that have secreted the bone around themselves. Osteocytes are cells embedded in the lacunae in the bone, and they are characteristically in contact with other cells by many slender cytoplasmic processes in canaliculi. During bone remodeling, many osteocytes in the bone are released from their lacunae by osteoclasts; however it remains unclear what happens to these released osteocytes. The cortical bone of the rat mandibular body was used in this study. Mandibles were fixed, decalcified, and then embedded in Epon 812. Specimens were sectioned in the frontal direction into serial 0.5 µm-thick semithin or 0.1 µm-thick ultrathin sections, and then examined by light or transmission electron microscopy. Cells that fitted in the osteocytic lacunae with canaliculi extending to the bone were identified as osteocytes in this study. Among many osteocytes released by osteoclasts in cutting cones, there were osteocytes half-released from their lacunae. These cells fitted in their lacunae with canaliculi extending to the bone and showed developed cell organelles in the cytoplasm. In closing cones, many osteocytes were situated in the bone away from cement lines; however, there were half-embedded osteocytes in the bone formed on cement lines. These cells fitted in their lacunae with canaliculi extending to the bone formed below cement lines and showed developed cell organelles in the cytoplasm. These results show that half-embedded osteocytes in closing cones derive from half-released osteocytes in cutting cones. Osteocytes encircled by osteo- clasts were sometimes observed on one section, but se-rial sections showed that these osteocytes fitted in their remaining lacunae in the bone on other sections. This shows that not all osteocytes released from their lacunae are engulfed by osteoclasts. Consequently, the present results suggests that some osteocytes released from their lacunae are embedded again in the bone and not engulfed by osteoclasts during bone remodeling.

Comparative study of the initial genesis of acellular and cellular cementum in rat molars

The initial genesis of acellular and cellular cementum was examined in rat molars by light and electron microscopy. Before root dentinogenesis, flattened dental follicle cells formed compartments by regularly arranged cellular processes which demarcated collagen fibril bundles oriented in parallel with the root long axis in both of the two kinds of cementum. After this stage, compartments disappeared from the dental follicle cells, which became elongated and polarized, with the cytoplasmic side facing toward the root surface in the acellular cementogenesis. Fibril bundles, oriented in parallel with the root long axis, decreased in number, and principal fibers appeared. Some principal fibers were attached on the first acellular cementum. Observations suggested that the fibril bundles, which had been oriented in parallel with the root long axis, were reoriented to merge into the principal fibers. In cellular cementogenesis, the dental follicle cells continued to hold the fibril bundles in cellular compartments. The regular processes were transformed into randomly oriented, finger-like processes. At the same time, fibers, which may be secreted from the finger-like processes, appeared around the preformed fibril bundles oriented in parallel with the root long axis. The different cellular behavior may result in the different fiber arrangement of acellular and cellular cementum.

Regeneration of myoepithelial cells in rat submandibular glands after yttrium aluminium garnett laser irradiation

The regeneration of myoepithelial cells in rat submandibular salivary gland after partial irradiation with yttrium aluminium garnett (YAG) laser was investigated. The irradiated glands were examined immunohistochemically for actin, histochemically for alkaline phosphatase (ALP), and by transmission electron microscopy (TEM). In control glands, myoepithelial cells were positive for actin and ALP. Electron microscopically, the positive reaction for actin was associated with the myofilaments of myoepithelial cells, and the plasma membrane of myoepithelial cells was positive for ALP. One day after YAG laser irradiation, the irradiated region was necrotic. By 5 days, duct‐like structures and epithelial clusters were observed at the interface between the necrotic zone and the remaining undamaged glands; immature acini appeared after 7 days. No reaction in duct‐like structures or epithelial clusters to actin or ALP was recognizable by 5 days. However, at 7 days, actin and ALP‐positive spindle cells appeared at the periphery of the duct‐like structures and immature acini. After 10 days, both actin‐positive and ALP‐positive cells increased in number. These observations indicate that during regeneration, actin‐positive and ALP‐positive cells regenerate myoepithelial cells, and it is suggested that this differentiation to myoepithelial cells is closely related to that of luminal to acinar cells. In addition, TEM observations indicate that regenerated myoepithelial cells originated from the basal cells of duct‐like structures.