Recovery of atrophic parotid glands in rats fed a liquid diet by switching to a pellet diet.

OBJECTIVE: In this study, we aimed to clarify how parotid glands, made atrophic by a liquid diet, recover after diet change. DESIGN: Seven-week-old male Wistar rats were fed a pellet (control group) or a liquid diet (experimental group) for the first 14 days. Thereafter, all animals were fed a pellet diet for up to 14 days (days 0-14). The parotid glands were removed, weighed and examined histologically and ultrastructurally. Immunohistochemistry was performed for BrdU, a marker of proliferating cells, and Casp-3, a marker of apoptotic cells. RESULTS: Feeding of a liquid diet for 14 days induced atrophy of the parotid glands. Histologically, acinar cells were small on day 0, compared with the control group. After changing the diet from liquid to pellet form, acinar cells increased in size over time, recovering nearly fully by day 7. Many BrdU-positive acinar cells were observed in the glands in the experimental group on days 1 and 3. Although more acinar cells were Casp-3-positive compared with the control group on day 0, there was no difference between the two groups after the diet change. Ultrastructurally, the cellular organelles did not exhibit a substantial alteration, except for an increase in secretory granules following diet change. CONCLUSIONS: Our findings suggest that atrophic parotid glands are able to recover to their normal size by switching the diet from liquid to pellet form and that an increase in both the size and number of acinar cells plays an important role in this recovery process.

Acinar cell response to liquid diet during rats' growth period differs in submandibular and sublingual glands from that in parotid glands.

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5'-bromo-2-deoxyuridine and cleaved caspase 3), and ultrastructurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5'-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats' growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands.

Changes in collagens and chondrocytes in the temporomandibular joint cartilage in growing rats fed a liquid diet.

The temporomandibular joint (TMJ) of growing rats fed a soft diet is reported to be smaller in size and to have thinner condyle and glenoid fossa cartilage than rats fed a solid diet. The aim of this study was to determine the effect of a soft diet on the collagens and chondrocytes in the growing TMJ cartilage. Forty-eight male Wistar rats were divided into a control group fed a solid diet and an experimental group fed a liquid diet for 1-8 weeks. After the experimental period, the TMJs were harvested and examined histologically, immunohistochemically for collagen types I, II, and X, and with transmission electron microscopy. The condylar cartilage in the experimental rats showed weak immunoreactions for three types of collagens compared with the controls. The ultrastructure had fewer fine collagen fibrils in the experimental rats compared with that of the controls. The glenoid fossa cartilage in the experimental rats showed narrower Alcian blue-positive areas than the control staining. The immunoreactions for three types of collagen in the experimental rats were also weaker than those of the controls. The chondrocytes in the experimental rats appeared dark, had extended thin cytoplasmic processes, and had formed gap junctions, as assessed by transmission electron microscopy. Fewer fine collagen fibrils, but thick bands of collagen fibrils were observed in the glenoid fossa of the experimental cartilage. The results of the present study showed that a liquid diet had deleterious effects on the quality and quantity of collagens and chondrocytes in the TMJ cartilage in growing rats.

Growth of rat parotid glands is inhibited by liquid diet feeding.

This study investigated how liquid diet feeding affects the growth of parotid glands. We weaned 21-day-old rats and thereafter fed them a pellet diet (control group) or a liquid diet (experimental group) for 0, 1, 2, 4, or 8 weeks. Their parotid glands were excised, weighed, examined, and tested for 5-bromo-2'-deoxyuridine (BrdU) and cleaved caspase-3 (Casp-3) as markers of proliferation and apoptosis, respectively. Parotid gland weights were consistently smaller in experimental animals than in controls. Morphometrical analysis showed that control group acinar cells increased in area during the experiment, but experimental group acinar cells were almost unchanged. Labeling indices of BrdU in acinar cells in both groups declined during the experiment, but were consistently lower in the experimental group than in controls. Casp-3-positive acinar cells were rare in both groups, which consistently express significantly similar Casp-3 levels. Ultrastructurally, terminal portions of the experimental parotid glands consisted of a few acinar cells that were smaller than those in controls. Control acinar cells showed mitotic figures within short experimental periods, but not in experimental glands. These observations indicate that liquid diet feeding inhibits growth of parotid glands in growing rats through suppression of growth and proliferation of individual acinar cells, but not through apoptosis.

Effects of a liquid diet on the temporomandibular joint of growing rats.

OBJECTIVE: The aim of the present study was to clarify the effects of a liquid diet on the temporomandibular joint (TMJ) in growing rats. MATERIALS AND METHODS: Twenty-four male Wistar rats were weaned at 21 days and divided into control and experimental groups (12 in each group). Control rats were fed a solid diet and experimental rats were fed a liquid diet from 1 to 8 weeks. After injection with 5-bromo-2'-deoxyuridine (BrdU), the animals were perfused and the heads were removed. Serial coronal sections of the TMJ were stained with hematoxylin and eosin, or BrdU immunohistochemistry was done (12 rats in each group). Three dimensions and the thicknesses of the cartilage layers of the TMJ were measured, and cell proliferation in the TMJ was examined. RESULTS: After 4 weeks, the height and width of the mandibular fossa and the width and length of the mandibular condyle were smaller in the experimental groups than in the control groups. The cartilage layer in these areas was also thinner at 4 weeks. The BrdU levels in the intermediate zone of the mandibular fossa (at 4 weeks) and the mandibular condyle (at 1 and 4 weeks) were lower in the experimental groups than in the controls. CONCLUSION: These findings suggest that the growth of the mandibular fossa and mandibular condyle of rats was inhibited by the low proliferative activity of intermediate zone cells induced by liquid feeding.

Type VII collagen deficiency causes defective tooth enamel formation due to poor differentiation of ameloblasts.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding type VII collagen (COL7), a major component of anchoring fibrils in the epidermal basement membrane zone. Patients with RDEB present a low oral hygiene index and prevalent tooth abnormalities with caries. We examined the tooth enamel structure of an RDEB patient by scanning electron microscopy. It showed irregular enamel prisms, indicating structural enamel defects. To elucidate the pathomechanisms of enamel defects due to COL7 deficiency, we investigated tooth formation in Col7a1(-/-) and COL7-rescued humanized mice that we have established. The enamel from Col7a1(-/-) mice had normal surface structure. The enamel calcification and chemical composition of Col7a1(-/-) mice were similar to those of the wild type. However, transverse sections of teeth from the Col7a1(-/-) mice showed irregular enamel prisms, which were also observed in the RDEB patient. Furthermore, the Col7a1(-/-) mice teeth had poorly differentiated ameloblasts, lacking normal enamel protein-secreting Tomes' processes, and showed reduced mRNA expression of amelogenin and other enamel-related molecules. These enamel abnormalities were corrected in the COL7-rescued humanized mice expressing a human COL7A1 transgene. These findings suggest that COL7 regulates ameloblast differentiation and is essential for the formation of Tomes' processes. Collectively, COL7 deficiency is thought to disrupt epithelial-mesenchymal interactions, leading to defective ameloblast differentiation and enamel malformation in RDEB patients.

Involvement of apoptosis and proliferation of acinar cells in atrophy of rat parotid glands induced by liquid diet.

Parotid glands of experimental animals fed a liquid diet are reported to show atrophy (Hall and Schneyer 1964; Wilborn and Schneyer 1970; Hand and Ho 1981; Scott et al. 1990; Scott and Gunn 1991). To clarify whether apoptosis and proliferation of acinar cells participate in atrophy of rat parotid glands induced by liquid diet, rats were fed a liquid diet and compared to pellet-fed controls. Parotid glands were removed at 3, 7, 14 or 21 days, weighed, and examined using transmission electron microscopy (TEM), and studied immunohistochemically for cleaved-caspase-3 (Casp-3), a marker of apoptotic cells, and 5-bromo-2'-deoxyuridine (BrdU), a marker for proliferating cells. Body weights of experimental rats fed liquid diets were not significantly different from controls fed pellet diets; however weights of experimental parotid glands were smaller than those of controls. In the experimental parotid glands, structures like apoptotic bodies were histologically observed in acini at each time point; more Casp-3-positive acinar cells were identified in experimental parotid glands than in the controls on days 3, 7, and 14. Experimental glands showed fewer BrdU-positive acinar cells at each time point. TEM confirmed typical apoptotic acinar cells in the atrophic glands. These findings suggest that increased acinar cell apoptosis and reduced acinar cell proliferation occur in atrophic parotid glands of rats fed a liquid diet.

Dental pulp can be a good candidate for nerve grafting in a xeno-graft model.

Dental pulp is discarded after extirpation of dental pulp and after tooth extraction. However, it contains nerve tissue abundantly and could be used more effectively. This study was designed to examine whether a dental pulp could be a candidate of donor for nerve grafting in xenografting model. The dental pulp was obtained from a human vital extracted tooth for orthodontic treatment, and treated with freezing and thawing method for reducing antigenicity. The treated sample was inserted into chitosan mesh tube for easy suturing, and then the complex was implanted into transected sciatic nerve in Sprague-Dawley (SD) rats (dental pulp group). As controls, chitosan tubes with and without sciatic nerve harvested from another SD rats were implanted (isograft group and tube group, respectively). As early as 4 weeks after grafting, regenerating axons accompanied by host Schwann cells were found to grout out through basal laminae by electron microscopy. The intact structure of basal laminae at this period suggested that they were derived from the original structure of donor graft. Twelve weeks after grafting, sporadic axonal regeneration was confirmed by light microscopy in the dental pulp group. Thirty-two weeks after implantation, aggregation of axons was observed in this group and matched that in isograft group. The average diameter of axons in dental pulp group was comparable to that in isograft group, whereas number of minifascicles and axon proportion were smaller. It was suggested that some delay occurred in dental pulp group because of the phagocytosis and absorption of tissue debris components remained after the freezing and thawing treatment. These findings clearly demonstrate that even dental pulp can act as conduits for regenerating axons.

The initial process of enamel prism arrangement and its relation to the Hunter-Schreger bands in dog teeth.

The three-dimensional architecture of enamel prisms at early stages of enamel formation and its spatial relationship to the Hunter-Schreger bands were examined in canine tooth germs by light and electron microscopy. In serial semithin sections of demineralized tooth germs tangential to the enamel-dentin junction, a straight row of enamel prisms was depicted along the longitudinal tooth axis at the level of the enamel-dentin junction and then their three-dimensional arrangement was reconstructed using computer software. The spatial arrangement of the groups of enamel rods oriented in specific sideward directions was also reconstructed in deep layers of the enamel. Initially, all enamel prisms were parallel to perpendicular toward the enamel-dentin junction, but at 10µm from the enamel-dentin junction, some small specks, or groups of enamel prisms--tilting to the right or the left--emerged as small islands. In each speck of enamel prism, the inclined prisms were uniformly oriented in a sideward direction and gradually expanded their boundary until merging with the neighboring specks inclined in the same direction. Consequently, at 50µm from the enamel-dentin junction, the group of enamel prisms oriented either to the right or the left formed alternately arranged horizontal belt-like zones, corresponding to the parazone or the diazone of the Hunter-Schreger bands. Reversed images of scanning electron-micrographs of the exposed surfaces of the developing enamel revealed round and bulb-like profiles of Tomes' processes at early amelogenesis and its changes into a characteristic structure combined with flat secretory and enclosing nonsecretory faces that dictated the orientation of corresponding enamel prisms. The results suggest that the groups of enamel prisms oriented in sideward directions first appear as small island-like specks near the enamel-dentin junction, which later merge and form alternating horizontal belt-like zones as a consequence of morphological changes of the Tomes' processes. However, the mechanisms whereby the functional grouping of secretory ameloblasts with similarly oriented Tomes' processes is induced are yet to be determined.

Type XVII collagen is a key player in tooth enamel formation.

Inherited tooth enamel hypoplasia occurs due to mutations in genes that encode major enamel components. Enamel hypoplasia also has been reported in junctional epidermolysis bullosa, caused by mutations in the genes that encode type XVII collagen (COL17), a component of the epithelial-mesenchymal junction. To elucidate the pathological mechanisms of the enamel hypoplasia that arise from the deficiency of epithelial-mesenchymal junction molecules, such as COL17, we investigated tooth formation in our recently established Col17(-/-) and Col17 rescued mice. Compared with wild-type mice, the incisors of the Col17(-/-) mice exhibited reduced yellow pigmentation, diminished iron deposition, delayed calcification, and markedly irregular enamel prisms, indicating the presence of enamel hypoplasia. The molars of the Col17(-/-) mice demonstrated advanced occlusal wear. These abnormalities were corrected in the Col17 rescued humanized mice. Thus, the Col17(-/-) mice clearly reproduced the enamel hypoplasia in human patients with junctional epidermolysis bullosa. We were able to investigate tooth formation in the Col17(-/-) mice because the Col17(-/-) genotype is not lethal. Col17(-/-) mouse incisors had poorly differentiated ameloblasts that lacked enamel protein-secreting Tomes' processes and reduced mRNA expression of amelogenin, ameloblastin, and of other enamel genes. These findings indicated that COL17 regulates ameloblast differentiation and is essential for normal formation of Tomes' processes. In conclusion, COL17 deficiency disrupts the epithelial-mesenchymal interactions, leading to both defective ameloblast differentiation and enamel malformation.

Distribution and organization of lymphatic vessels in the mouse gingiva: An immunohistochemical study with LYVE-1.

OBJECTIVE: This study introduced the usefulness of LYVE-1 immunoreactivity for identification of lymphatic vessels in decalcified tissues, and demonstrated the fine distribution and organization of these vessels in mouse gingiva. DESIGN: After confirming the specificity of anti-mouse LYVE-1, frozen sections of mouse decalcified gingiva were immunostained with the antibody. RESULTS: The LYVE-1-positive lymphatic vessels were clearly found in the connective tissue under the gingival epithelium; these vessels appeared to pass through the lamina propria of the gingiva toward the alveolar crest and run along the external surface of the alveolar bone. The lymphatic vessels were sparse and apart from the oral gingival and sulcular epithelia, while they were dense adjacent to the junctional epithelium. CONCLUSIONS: The dense network of the lymphatic vessels adjacent to the junctional epithelium, which is apparently exposed to foreign antigens, may act as an efficient drainage pathway of the excessive interstitial fluid and immune cells, and play an active role in the immune defense of the gingiva. The present study also revealed the absence of lymphatic connection between gingiva and periodontal ligament.

Apoptosis of odontoclasts under physiological root resorption of human deciduous teeth.

This study was designed to establish the apoptosis of odontoclasts during physiological root resorption of human deciduous teeth. Deciduous teeth were fixed, decalcified, and embedded in paraffin for immunohistochemical (IHC) observations and in Epon for transmission electron microscopy (TEM). Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and then tartrate-resistant acid phosphatase (TRAP) activity was determined on the same sections. Epon-embedded specimens were sectioned serially into 0.5-microm semithin sections; some of these sections were re-embedded in Epon, sectioned into 0.1-microm ultrathin sections, and observed by TEM. IHC revealed that the nuclei of TRAP-positive odontoclasts on the dentine were generally TUNEL-negative. Around these odontoclasts, a few TRAP-positive structures were present together with TUNEL-positive structures, e.g., a TRAP-positive structure with one TUNEL-positive nucleus, a TRAP-positive structure with one TUNEL-positive nucleus plus one or two TUNEL-negative nuclei, or a TRAP-positive structure with no nucleus. By TEM, some odontoclasts showed nuclear fragments including compacted chromatin. The results suggest that, during apoptosis, odontoclasts fragment into variously sized cellular parts including three or fewer nuclei.

Mineralization process during acellular cementogenesis in rat molars: a histochemical and immunohistochemical study using fresh-frozen sections.

This study was designed to detect tissue non-specific alkaline phosphatase (TNSALP) by Azo-dye staining, calcium by glyoxal bis (2-hydroxyanil) (GBHA) staining, bone sialoprotein (BSP) and osteopontin (OPN) by immunoperoxidase staining in developing rat molars, and also to discuss the mineralization process during acellular cementogenesis. To restrain a reduction in histochemical and immunohistochemical reactions, fresh-frozen undemineralized sections were prepared. Where the epithelial sheath was intact, TNSALP reaction was observed in the dental follicle, but not in the epithelial sheath. With the onset of dentin mineralization, the BSP- and OPN-immunoreactive, initial cementum layer appeared. At this point, cementoblasts had shown intense TNSALP reaction and GBHA reactive particles (=calcium-GBHA complex) appeared on the root surface. With further development, the reaction of TNSALP and GBHA became weak on the root surface. Previous studies have shown that the initial cementum is fibril-poor and that matrix vesicles and calciferous spherules appear on the root surface only during the initial cementogenesis. The findings mentioned above suggest that: during the initial cementogenesis, cementoblasts release matrix vesicles which result in calciferous spherules, corresponding to the GBHA reactive particles. The calciferous spherules trigger the mineralization of the initial cementum. After principal fiber attachment, mineralization advances along collagen fibrils without matrix vesicles.

Features of the clear zone of odontoclasts in the Chinook salmon (Oncorhynchus tshawytscha).

This study aims to clarify the features of the clear zone of odontoclasts on shedding teeth of a teleost fish, Chinook salmon, Oncorhynchus tshawytscha (Walbaum), using a light microscope to determine the orientation between a cell body and a resorptive lacuna, followed by transmission electron microscopy. Ultrathin sections of LR White embedded material were incubated in rabbit anti-actin polyclonal antibody and then were incubated with 15 nm gold-conjugated goat anti-rabbit IgG. The clear zones of odontoclasts showed a variable structure with electron-dense structures on sections, but distinct clear zones were not always seen on odontoclasts. In odontoclasts sectioned in the direction perpendicularly to the surface of a resorptive lacuna, some cells showed a wide clear zone, but two types of clear zones were usually observed: a part composed of some cytoplasmic processes and one composed of several complicatedly interwoven processes. Gold particles were localized on the clear zones, especially in electron-dense structures; very few gold particles were detected in ruffled borders. These results show that the clear zone of odontoclasts in Chinook salmon contains actin. Our results suggest that the clear zone of an odontoclast in Chinook salmon is not always a wide annular structure.

Odontoclasts in the Chinook salmon differ from mammalian odontoclasts by exhibiting a great proportion of cells with high nuclei number.

Odontoclasts resorbing teeth are multinucleated cells. Previously, the authors have investigated the distribution of number of nuclei per human odontoclast and showed that the mean number of nuclei per cell is 5.3, the median is 4, and 93.8% of cells have 10 or fewer nuclei. Teleost odontoclasts have features similar to those of mammals; however, the distribution of number of nuclei per cell remains unknown. The present study aimed to examine the distribution of number of nuclei per odontoclast in a teleost fish, Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and to clarify the difference of number of nuclei in odontoclasts between Chinook salmon and humans. The maxillae and mandibles of Chinook salmon were fixed, decalcified, and embedded in Epon 812. Specimens were serially sectioned into 0.5-microm semithin sections and examined by light microscopy. Cells possessing a brush border adjacent to a resorptive lacuna were identified as odontoclasts, and 246 odontoclasts were investigated to determine the distribution of nuclei per cell. The mean number of nuclei per cell was 21.8 and the median was 17; only 24.4% of odontoclasts had 10 or fewer nuclei, and 95.5% had 50 or fewer nuclei. These results suggest that the range for the number of nuclei per odontoclast in Chinook salmon is greater than that in humans.

A crown reconstructing method for resin-embedded transparent teeth.

OBJECTIVE: Resin-embedded transparent teeth are devoid of the original crown morphology, because enamel is lost by demineralization. This study was designed to reproduce artificial enamel and to reconstruct the original crown morphology for resin-embedded transparent teeth. METHOD AND MATERIALS: The impression of the coronal portion of human permanent teeth was taken with a silicone impression material. After demineralization, drawing ink was injected into the pulp cavities. The ink-infiltrated teeth were made transparent with methyl salicylate and embedded with polyester resin. Urethane prepolymer was injected into the impression, and the resin-embedded teeth were reinserted into the impression. After polymerization of the urethane resin, the specimens and the urethane resin were removed from the impression. RESULTS: The original crown morphology of the resin-embedded transparent teeth could be precisely reconstructed with artificial and removable enamel. The resin-embedded teeth showed morphologic details of the black-stained pulp cavities through the transparent dentin and cementum. CONCLUSION: This study established a crown reconstructing method for resin-embedded transparent teeth. The specimens will be useful for demonstration of morphology of teeth and pulp cavities.

Ultrastructural study of cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro.

Many biochemical reports support cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro, however there have been few morphological studies supporting this. Details of cell-cell interaction between osteoclasts and osteoblasts/stroma cells remain unclear. The present study examined cell-cell interaction between osteoclasts and osteoblasts/stroma cells by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Osteoclasts, osteoblasts/stroma cells, and bone marrow cells obtained from 10-day-old ddY mice were cultured on dentin slices for 72 hr. Specimens were fixed, and some were examined by SEM. Specimens were decalcified, embedded in Epon after determination of the tartrate-resistant acid phosphatase activity (TRAP), and TRAP-positive cells for investigation were serially sectioned by alternating semithin and ultrathin sections, and then examined by TEM. By SEM, many cellular contacts were seen between the cells cultured on the dentin, but by TEM there were few special structures on the cell membranes between osteoclasts and osteoblasts/stroma cells, or between osteoclasts and bone marrow cells. A special structure on the cell membranes of osteoclasts was observed between an osteoclast and a cytoplasmic process of osteoblast/stroma cells, and this cell membrane was coated with electron dense or bristle-like structures. These bristle-like structures were very similar to those of coated pits. The present results show that the coated pit-like structure plays an important role in cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro, and suggest that macromolecules binding to the osteoclast-surface receptor via ligands, accumulate in the coated pits, and enter the osteoclast as receptor-macromolecule complexes in endocytic vesicles.