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A dental implant is used to replace a missing tooth. Fixing the implant in its natural position requires the engineering of a substantial amount of conformal bone growth inside the implant socket, osseointegration. However, this conventional implant attachment does not include the periodontal ligament (PDL), which has a fundamental role in cushioning high mechanical loads. As a result, tooth implants have a shorter lifetime than the natural tooth and have a high chance of infections. We have engineered a “bio-implant” that provides a living PDL connection for titanium implants. The bio-implant consists of a hydroxyapatite coated titanium screw, ensheathed in cell sheets made from immortalized human periodontal cells. Bio-implants were transplanted into the upper first molar region of a tooth-extraction mouse model. Within 8 weeks the bio-implant generated fibrous connective tissue, a localised blood vessel network and new bone growth fused into the alveolar bone socket. The study presents a bio-implant engineered with human cells, specialised for the root connection, and resulted in the partial reconstruction of a naturalised tooth attachment complex (periodontium), consisting of all the principal tissue types, cementum, PDL and alveolar bone.

Background: The purpose of this study was to investigate the differentiation and maturation of osteoblasts on surfaces with different roughnesses. Methods: Blasting a culture dish surface with Al2O3 particles of 25 μm, 50 μm or 180 μm diameter produces mean surface a roughness (Ra) of 0.48 ± 0.07, 1.02 ± 0.14 or 3.10 ± 0.36, respectively. Morphological changes of cells were compared, and the expression of mRNAs of osteopontin and osteocalcin were evaluated. Further, the ratio of alizarin red positive area was calculated. Results: Cells cultured on the 180 μm blasted surface had almost the same morphology as those cultured on the 50 μm blasted surface; however, cellular bridges tended to be much wider and more spread out. The expressions of osteopontin and osteocalcin mRNAs in cells cultured on the 180 μm blasted surface have greater up-regulation than the other cells. Further, the ratio of alizarin red positive area in180 μm blasted surface was significantly higher than those of others. Conclusions: These results suggest that the 180 μm blasted surface, with a surface roughness of approximately 3 μm in width, is the best surface for MC3T3-E1 cells to differentiate and mature in vitro.

A 13-month-old Japanese boy presented with painless swelling in a left mandible and cheek. Intraoral examination revealed swelling in the left mandible and hemorrhage of oral mucosa due to biting. CT images revealed a wide osteolytic lesion of the left mandible with floating teeth. Biopsy was carried out and histopathological diagnosis was discussed.

Oral Diseases (2010) 16, 263–268 Objective: The aim of this study was to investigate the proliferation, migration and death of periodontal ligament (PDL) cells after tooth replantation. Materials and methods: Maxillary first molars were extracted from 4-week-old male (n = 28) Sprague–Dawley rats and immediately replanted, after which, proliferation, migration and death of PDL cells were investigated. Results: At 3 days after tooth replantation, many proliferative cell nuclear antigen (PCNA)-positive PDL cells were observed on the alveolar bone side, but fewer on the root side. However, while a gradual decrease was observed in number of PCNA-positive PDL cells on the alveolar bone side until 7 days, an increase was seen on the root side. At 3 weeks, cells labeled with PKH26 (fluorescent dye into plasma membrane) were located in the middle of the PDL space. However, these PKH26-labeled cells did not spread to the surface of the cementum or the alveolar bone. TUNEL-positive cells were observed on both the bone and root sides at 3 days. Number of apoptotic cells increased until 7 days on the bone sides, but decreased on root sides. Conclusion: These results suggest that both cell proliferation and apoptosis occur in different patterns and at different times to maintain regular spacing of the PDL after tooth replantation.

Background and Objective: The 4-META/MMA-TBB [4-(2-methacryloxyethyl)trimellitic anhydride/methyl methacrylate-tributylborane] resin is widely used as a dental adhesive. It has also been applied in the dressing of gingival wound surfaces following periodontal surgery. However, its effect on the regeneration and/or cell attachment of the oral epithelium remains to be clarified. To evaluate the effect of the resin applied as a wound dressing, we investigated expression of laminin 5, integrin β4 and cytokeratin 14 in regenerating oral epithelium treated with this resin following gingivectomy from the viewpoint of cell attachment and differentiation. Material and Methods: The resin was applied to the entire wound surface in rats after gingival surgery, and regenerating epithelium was examined immediately and at 1, 3, 5, 7 and 14 days later. The resin was removed 2 weeks after application in some animals and tissue further examined at 1, 3, 5 and 7 days later. Results: Regenerating epithelium under the resin was not keratinized, but became keratinized immediately after removal of the resin. Laminin 5 and integrin β4 were immunolocalized in the basal lamina, the internal basal lamina, in marginal cells of the regenerating epithelium and at the resin–regenerating epithelium interface. Cytokeratin 14 localized in the regenerating epithelium underneath the resin, as well as in healthy and regenerated junctional epithelial cells. Conclusion: These results suggest that this resin covers the wound surface and that the regenerating epithelium biologically adheres to the resin during the initial process of its regeneration.

Background and objective The junctional epithelium attaches to the enamel surface with hemidesmosomes (of which laminin-5 and integrin-alpha(6)beta(4) are the main components) in the internal basal lamina. Laminin-5 is also involved in cell motility with integrin-alpha(3)beta(1), although their functions have not yet been clarified.The purpose of this study was to determine the functions of those adhesive components between the tooth and the junctional epithelium during cell migration.Because an idea has been proposed that directly attached to tooth cells (DAT cells) may not contribute to cell migration, 5-bromo-2-deoxyuridine staining was performed to confirm cell migration. Material and methods We investigated laminin-gamma(2) (contained only in laminin-5), integrin-beta(4) (involved in cell-extracellular matrix contact) and integrin-alpha(3) (inducing cell migration) in the junctional epithelium, oral gingival epithelium and gingival sulcus epithelium of 6-wk-old ICR mice using laser microdissection, quantitative real-time reverse transcription-polymerase chain reaction, immunofluorescence and 5-bromo-2-deoxyuridine staining. Results Laminin and integrins were clearly immuno-localized in the basal lamina of all epithelium. Quantitative analysis of laminin and integrin mRNAs by laser microdissection showed that they were more highly expressed in DAT cells than in basal cells in the oral gingival epithelium. In particular, a 12-fold higher expression of laminin-5 was observed in the junctional epithelium compared with the oral gingival epithelium. 5-Bromo-2-deoxyuridine staining showed rapid coronal migration of DAT cells. Conclusion These results suggest that the abundant expression of laminin-5 and integrin-alpha(6)beta(4) is involved in the attachment of DAT cells to teeth by hemidesmosomes. Abundant expression of laminin-5 and integrin-alpha(3)beta(1) might assist in DAT cell migration, confirmed by 5-bromo-2-deoxyuridine staining during the turnover of junctional epithelium.

Temporomandibular disorders are a group of chronic and painful conditions in the masticatory musculature and temporomandibular joint. The pathological mechanisms underlying temporomandibular disorders remain to be clarified; however, it is known that disturbances in neurophysiological, cognitive, behavioral and neuromuscular functions are involved in the development and persistence of such disorders. In addition, it has been suggested that neuropathological changes in the central nervous system are involved in the development of temporomandibular disorders. This review describes changes in the cortical machinery in patients with temporomandibular disorders by comparing neuromagnetic signals between healthy subjects and patients elicited by observing jaw opening movements made by another person.

The regeneration of wounded stratified epithelium is accomplished via the migration of keratinocytes from the margins of the wound. However, the process of keratinocyte migration on the wound surface and the role of epithelial stem cells during re-epithelialization remain to be elucidated. Therefore, we administered BrdU to embryonic mice and generated epithelial defects on the buccal mucosa of these mice at two weeks after birth, using CO(2) laser irradiation, with which we removed the entire thickness of the epithelium. In the unwounded epithelium, cytokeratin 14, p63, and BrdU were localized within the basal layer of the epithelium, but the majority of cells within the regenerated epithelium were immunopositive for these proteins. PCNA-negative and BrdU-positive basal keratinocytes, which evidence a slow cell cycle, were localized solely within the basal layer of the unwound epithelium facing the tips of dermal papillae. After laser irradiation, these basal keratinocytes facing the tips of the papillae evidenced positive immunoreactivity for PCNA, in addition to BrdU. These results indicate that epithelial stem cells of oral mucosa may be localized in the basal layer of the epithelium facing the tips of dermal papillae, and may migrate laterally with other basal keratinocytes in response to external stimuli.

The submandibular gland (SMG) is a tissue that can be regenerated in a tissue injury model and that has adult stem cells capable of self-renewal and differentiation into functional cells. We have analyzed the localization of label-retaining cells (LRCs), which are putative progenitor cells, by using the BrdU-labeling method. 5-Bromo-2'-deoxyuridine (BrdU) injection followed by a long chasing period permitted the identification of LRCs based on the slow-cycling characteristic. In order to confirm the accurate localization of LRCs, BrdU and SMG-specific markers, including aquaporin5, cytokeratin, and smooth muscle actin, were examined by double-immunofluorescence staining. We found that LRCs were distributed in the acinus, duct, myoepithelium, and connective tissue. Moreover, ABCG2 (a known stem cell marker) was used for the characterization of LRCs and the localization of cells as putative stem/progenitor cells. ABCG2-expressing cells were distributed in various regions of the SMG but did not co-localize with LRCs. We suggest that putative progenitor cells exist in various regions of the SMG and have diverse capacities to differentiate into specific cells.

A rare case of hepatocellular carcinoma (HCC) that metastasized to the posterior mandible of a 74-year-old Japanese female is described. Computed tomography (CT) revealed buccolingual osteolytic masses. Histopathologically, the solid tumor nests were composed of cells with granular eosinophilic cytoplasm, oval nuclei and enlarged nucleoli. Immunohistochemically, the tumor cells were strongly positive for hepatocyte paraffin 1 (Hep Par 1) and cytokeratin 18 (CK18). Ultrastructurally, abundant mitochondria, rough endoplasmic reticulum, lysosome-like granules, ductal structures and intercellular junctional complexes were distinct in the tumor cells. At autopsy, multinodular tumor masses and cirrhosis were detected in the liver. Microscopically, the tumor cells had trabecular, solid and sarcomatous appearances with severe atypism. Metastatic foci were found in the spleen, stomach, lymph nodes and ribs, but not in the lungs. We finally diagnosed this as mandibular metastasis of HCC, which is a very rare cancer, 1 of only 5 known cases in a female.

Background: Osteopontin (OPN) expression in squamous cell carcinoma (SCC) of the tongue has not been clearly elucidated. Methods: We selected 46 cases of tongue SCC and investigated the expression of OPN by immunohistochemical staining. The immunopositive reaction and score for each case were semiquantitatively evaluated. Results: Scores were significantly higher in carcinoma nests than in neighboring normal epithelium or epithelial dysplasia. The OPN was expressed clearly in the cytoplasm of carcinoma cells. In cases of early invasive carcinoma, in particular, expression of OPN showed a remarkable increase at the invasion front compared with the non-invaded regions. However, there was no significant correlation between expression of OPN in the primary tumor nest and lymphatic metastasis, recurrence, or survival rate. Conclusion: This suggests that OPN is a useful biomarker of early invasion by SCC in tongue.

Background and Objective: It is known that epithelial islands are embedded in the cementum during tooth root formation, but details of this process remain unknown. The purpose of this study was to investigate the dynamic characteristics of Malassez's epithelial rest cells in the cementum during tooth root formation in pigs in vivo. Material and Methods: The first molars of 6-mo-old pigs were used in this study. Specimens were decalcified before being embedded in paraffin. Paraffin sections were investigated using TdT-mediated dUTP-biotin nick end labeling (TUNEL), immunohistochemical, and ultrastructural techniques. Results: Malassez's epithelial rest cells were located close to the root surface at the apical one-third of the periodontal ligament, and epithelial clusters surrounded by distinct lamina cementia were sometimes observed in the cementum. TUNEL-positive cells were detected only in the cementum. Malassez's epithelial rest cells in the periodontal ligament were completely surrounded by basement membranes, but epithelial clusters in the cementum were only intermittently surrounded by such membranes. Cytokeratin-positive cells in the superstratum of the cementum were directly connected by cementocytes and by desmosome-like structures. However, organelles were scarce in the cytokeratin-positive cells in the substratum of the cementum, and the matrix of the cementum was deposited in the cells. Conclusion: These results suggest that the majority of the fragmented Hertwig's root sheath remains in the periodontal ligament and that some cells, which are connected to cementoblasts, are embedded in the cementum and progress to apoptosis.

We examined expression of syndecan-1 in squamous cell carcinoma (SCC) of tongue using immunohistochemistry. Forty-three cases of SCC arising in lateral border of tongue were investigated. From the immunohistochemical staining pattern, the cases were divided into two groups based on expression of syndecan-1 at the supra-peripheral cells of the tumor nest: Group A, completely or mainly positive; Group B, sporadically positive or negative. Most poorly differentiated SCC cases were classified into Group B (81.8%). The number of Group B cases in T1-2 was different from that in T3-4. The number of cases where syndecan-1 expression was reduced was much greater in T3-4, and represented the majority of Group B (86.7%). More than 80% of Grade 4D cases were in Group B (83.3%) based on the Yamamoto-Kohama criteria. These results indicate that reduction of syndecan-1 correlates to histological grade, tumor size and mode of invasion in tongue SCC.

Objectives The aim of this study was to investigate the differential immunolocalization of laminin gamma(2) and integrin beta(4) in primary cultures of the rat gingival epithelium. Methods The gingival epithelium was obtained from Sprague-Dawley rats and was cultured in serum-free keratinocyte growth medium (DK-SFM). Western blotting analysis, immunofluorescence, confocal laser scanning microscopy (CLSM), and immuno-gold labeling for laminin gamma(2) and integrin beta(4) were employed. CLSM images for laminin and integrin were analyzed in horizontal (x-y axis) and in vertical (x-z axis) sections. Results Both laminin gamma(2) and integrin beta(4) were detected by Western blot analysis in the gingival epithelium. Immunolocalization of laminin gamma(2) was distinct in the cytoplasm to form one or two irregular rings in gingival epithelial cells. By contrast, integrin beta(4) was localized diffusely in the cytoplasm. F-actin (indicating actin filaments) was clearly discernible at the periphery of the cytoplasm to form a cellular fringe. In x-z axis images obtained by CLSM, laminin gamma(2) was recognized as large foci in the most inner portion just above the basal plasma membrane. Integrin beta(4) existed in the area where F-actin was labeled surrounding the membrane. Immuno-electron microscopy showed that 10nm colloidal gold particles indicating laminin gamma(2) were mainly localized at the extracellular portion and in the peripheral cytoplasm, whereas integrin beta(4) was distributed in the cytoplasm close to the basal plasma membrane but not in extracellular regions. Conclusions In primary cultures of the rat gingival epithelium, both laminin gamma(2) and integrin beta(4) may be produced by the epithelium, and irregular rings of laminin gamma(2) are formed in areas where gingival cells adhere to the extracellular matrix.

Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE–connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 ( γ 2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant–PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE–connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant–PIE interface.

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.

Previous reports have shown the expression of several mechanosensitive ionic channels on the plasma membrane in odontoblasts, which are the cells responsible for dentin formation. The membrane characteristics of odontoblasts imply that they could play critical roles in the mechano-transduction of fluid displacement within dentinal tubules into the electrical cell signals, to carry dentin sensation to the central nervous system. However, the direct ionic mechanism underlying such a dentin nociceptive function remains unclear. In the present study, we investigated the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1) channel--which essentially contributes to the detection of pain sensation--in rat odontoblasts by immunohistochemical and nystatin perforated patch-clamp techniques. Immunohistochemical observation showed the localization of TRPV1-immunoreactions on the distal regions of odontoblast membranes. In the patch-clamp experiments, we observed capsaicin-induced inward currents that were inhibited by capsazepine, a TRPV1 channel antagonist. Our results indicate a significant expression of TRPV1 channels in odontoblasts, suggesting that odontoblasts may directly respond to noxious stimuli such as a thermal-heat stimulus, and point to the necessity for a reconsideration of the cellular mechanisms of dentin sensation based on the transmembrane ionic signals in odontoblasts.

We examined expression and function of osteopontin (OPN) in oral cancer cell lines using antisense oligonucleotide (AS). Quantitative real-time RT-PCR showed that expression in BSC-OF cells was significantly higher (10-fold) than that in KB cell. AS-study showed that foci of AS-treated BSC-OF cells possessed thin processes and radiated morphologically, although BSC-OF cells showed round foci. Cell growth in AS-group was lower (

The purpose of the current study was to evaluate the bone formation when beta-tricalcium phosphate (TCP) was implanted in bone defects of rat femurs. beta-TCP granules were applied to defects created in the femurs of 65 male rats who were sacrificed 3, 7, 10, 14 or 30 days later. Bone tissues were embedded in paraffin, serial sections were cut and then stained with hematoxylin-eosin. Histomorphometric analyses were also conducted. Furthermore, total mRNAs were extracted, homogenized, and reverse transcribed, after which quantitative PCR assays were conducted with a LightCycler using the double-stranded DNA dye Syber Green I with primers for either rat osteopontin or osteocalcin. Tissues in defects without beta-TCP were used as controls. The amount of newly formed bone tissue in the beta-TCP implanted group was significantly greater in both the side areas and the central area of defects than in the control group. Expressions of osteopontin and osteocalcin mRNAs of cells in the defects of the experimental group were up-regulated compared with the control group at all time periods. Taken together, these results prove that beta-TCP is an appropriate material for osteoconduction and promotes bone formation in bone defects.

A 57-year-old man was referred to our hospital because of a lump of his lateral neck region. Aspiration and intraoperative biopsies were performed to confirm the pathological diagnosis. The tumor showed a neoplastic epithelium arranged in a ductal or follicular pattern with papillary proliferation, and tumor cells were immunohistochemically positive for thyroglobulin. The pathological diagnosis was papillary thyroid carcinoma metastasis to a lymph node in the lateral neck region. Metastases were also observed in other lymph nodes in the submandibular region, and multiple metastatic foci were later found in the lungs and brain. Heavy ion radiotherapy was performed to extend the patient's life.

A group of neurons in the monkey premotor cortex discharges both when the monkey is performing a given action as well as when it is observing the experimenter performing a similar action (mirror neuron system; action execution/observation matching system). Several brain imaging studies indicate that this system also exists in humans. The present study aimed to compare activation patterns of the cortical areas activated during observation of jaw movements between healthy normal subjects and patients with temporomandibular disorders (TMD). Whole-scalp neuromagnetic responses were recorded from five healthy volunteers and six patients with TMD. The subjects were instructed to carefully observe a pre-recorded video of bilaterally symmetrical jaw-opening movements performed by another individual. During the observation task in healthy subjects, we found magnetic signals generated by the region of the occipitotemporal region near the inferior temporal sulcus and maxillofacial area of the primary motor cortex. In patients, however, we found the attenuation of magnetic response from primary motor cortex. These findings suggest that the maxillofacial area of the primary motor cortex is included in the jaw movement-related action execution/observation matching system in humans, and provide new neuropathological evidence that TMD patients exhibit dysfunction of the jaw movement-related action execution/observation matching system.

The aim of the present study was to reveal the spatiotemporal relations among cortical regions involved in the initiation of voluntary swallowing in humans, using magnetoencephalography (MEG). The swallowing-related activity was distributed widely for 2000 ms before the electromyogram (EMG) onset of the right suprahyoid muscle. The cingulate cortex, the insula and the inferior frontal gyrus were the main loci active prior to swallowing. These cortical loci coincide with those suggested by previous human brain mapping studies that investigated the brain mechanism, which controls swallowing. Activation in the cingulate cortex was registered in the early stage of swallowing and could be related to the cognitive process regarding the food being safe to swallow. The activation in the insula lasted for a long period of time before the initiation of swallowing. This suggests that the long-lasting activation in the insula prior to swallowing is essential for the initiation of swallowing.

Background and objective: It is still an open question why long junctional epithelium can proliferate and occupies the root surface following periodontal surgery or experimentally produced periodontitis, and why the epithelium repopulated once on the root surface is replaced by the connective tissue. The aim of this study is to investigate the proliferative activity of the newly formed regenerative connective tissue and long junctional epithelium during wound healing by staining argyrophilic proteins of the nucleolar organizer regions (AgNORs). Methods: Regenerative connective tissue and long junctional epithelium were experimentally created by insertion of a rubber piece between maxillary molars of rats for 1 week. After removal of the rubber, AgNORs parameters including nuclear area (NA), AgNORs area (AA), AgNORs percentage nuclear area (APNA), AgNORs number (AN) and nuclear number (NN) in regenerative connective tissue and long junctional epithelium were measured and analyzed statistically. Results: APNA in long junctional epithelium after 1 and 4 weeks was over two times greater than that in the regenerative connective tissue. AA in long junctional epithelium was significantly higher than in regenerative connective tissue at 1 and at 4 weeks post-treatment. AN was higher in the central portion than at the root surface except at 20 weeks. APNA and AA decreased remarkably in long junctional epithelium at 12 weeks post-treatment (approximately half at 4 weeks), whereas in regenerative connective tissue, they did not change distinctly. Conclusions: These results imply that long junctional epithelium cannot supply sufficient epithelial cells because of their significantly low rates of proliferation, consequently long junctional epithelium becomes shorter after 12 weeks, whereas the proliferative activity of regenerative connective tissue maintains the same level of proliferation, and ultimately long junctional epithelium is replaced by regenerative connective tissue.

The purpose of this study was to investigate the behavior of L929 fibroblasts on substrates of different pore sizes and to find an appropriate pore size that allows them to differentiate into type 1 collagen synthesizing cells. The behavior of cultured fibroblasts on Millipore filters of 4 different pore sizes (1.2 μm, 3.0 μm, 5.0 μm and 8.0 μm) was compared morphologically. The expression of type 1 collagen mRNA was also analyzed by RT-PCR. The cell length was the greatest in the control group (without a filter) compared to the experimental groups cultured on filters at 3 days, however there were no significant differences among any of the groups at 7 days. At the ultrastructural level, small and fine cell processes had invaded the pores of filter membranes with pore sizes of 1.2 μm or 3.0 μm, while long and thick cell processes containing many organelles (some of them degenerated) had invaded the pores of filter membranes with pore sizes of 5.0 μm or 8 μm. The expression of type 1 collagen mRNA was the highest in cells cultured on membranes with 1.2 μm pores at 3 days but was not significantly changed among any of the groups at 7 days. This suggests that Millipore filters with a 1.2 μm pore size are the most suitable to allow L929 fibroblasts to express the phenotype for type 1 collagen synthesis at early time periods.

Property modification of an implant surface is known to influence the behavior of cells surrounding implant material. This study examined the osteogenic cell behavior on protein immobilized surface in vitro. Following plasma surface modification, fibronectin or bone morphogenetic protein-2 (BMP-2) was immobilized. The number of cells adhering to fibronectin-immobilized surface increased after 1 and 2 h of incubation compared with non-immobilized surface. Alkaline phosphatase activity and osteocalcin mRNA expression of the osteogenic cells on the BMP-2 immobilized surface was greater than that on the non-immobilized surface. This study demonstrated that protein can be immobilized to a polystyrene surface after treatment with O2 plasma and that the osteogenic cells surrounding a biomaterial can be controlled by the immobilization of protein to the biomaterial.

Different types of osteoblast behavior result from external stimulation in the early and in the late stages of cell differentiation. The purpose of this study was to investigate the influence of a diode laser on osteoblasts and to compare any effects between the proliferating and the differentiating phases. In the proliferating phases, the numbers of cells greatly increased in the laser irradiated group as compared with that of the control group. The increases in the numbers of cells in the differentiating phase were no greater than were those in the proliferating phase. The alkaline phosphatase activity of cells in the proliferating phase was highest 5 days after the laser irradiation, however, the activity in the differentiating phase increased day by day. Although osteopontin mRNA levels in the proliferating phase increased in the first 3 days of culture and then decreased in both groups, this decrease of osteopontin mRNA was small in the irradiated group. In contrast, osteopontin mRNA expression in the differentiating phase increased at day 1 and then decreased in the control group, while osteopontin mRNA levels in the irradiated group did not decrease.

The purpose of this study was to investigate pulp cell responses during hypoxia and reoxygenation. Pulp tissues obtained from beagle dogs were cultured. In the control group, pulp cells were incubated in normoxic conditions (20% O2) for 1-4 d. In the hypoxia group, pulp cells were incubated under hypoxic conditions (2% O2) for 1-4 d. In the reoxygenation group, pulp cells were first incubated under hypoxic conditions for 24 h, and were then incubated in normoxic conditions (20% O2) for one to three additional days. Cell viability, MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay, cellular proliferation, and alkaline phosphatase (ALPase) activity were determined. Expression of heat shock protein 70 (HSP70) and vascular endothelial growth factor (VEGF) was analysed by Western blotting. Hypoxia inducible factor-1alpha (HIF-1alpha) in pulp cells was analysed by reverse transcriptase polymerase chain reaction (RT-PCR). The cell growth rate and ALPase activity were significantly higher in the hypoxia group than in the control group. After reoxygenation, cellular proliferation and ALPase activity decreased to the level of the control group while HSP70 expression increased. Hypoxia inducible factor-1alpha expression was detected in pulp cells, and VEGF expression (which is regulated by HIF-1alpha) increased under hypoxic conditions. These results suggest that dynamic responses to hypoxia and reoxygenation occur in pulp cells.

We report a case of oncocytic carcinoma arising in submandibular gland. The tumour occurred in the left submandibular gland of an 82-year-old Japanese man. Histologically, the tumour was mostly composed of large cells with eosinophilic granules in the cytoplasm and they were arranged in the solid sheets, islands with duct-like structure and cords. The tumour cells had aggressively invaded muscles and perineural tissues, and cervical lymphatic metastasis was frequently observed. Histochemically, the tumour cells were strongly positive for phosphotungstic acid-hematoxylin (PTAH) stain, and we diagnosed this malignant tumour as oncocytic carcinoma. Immunohistochemically, the tumour cells reacted positively for cytokeratin 7, 8, 19, epithelial membrane antigen (EMA), alpha-1-antichymotrypsin and carcinoembryonic antigen (CEA), but negative for cytokeratin 13, 14, smooth muscle actin (HHF35) and S-100 protein (S-100). Tumour was diagnosed as oncocytic carcinoma in submandibular gland. Its characteristics are discussed in term of its histopathological and immunohistochemical features.

We report a rare case of a papillary cystadenoma arising from the upper lip. This tumor was not distinctly encapsulated and had proliferated replacing the ductal epithelium. Mast cells were found not only in the stroma but also in the oncocytic epithelial layer. There was a strong immunoreaction with mitochondrial antibody in the epithelial layer. Only one case (0.9%) of papillary cystadenoma has occurred among the 110 benign intraoral salivary gland tumors seen in our hospital from 1966 through September 2003.

The osteogenic capability of cells derived from the bone marrow of young (4 week old) or adult (10 month old) male Wistar rats was investigated in vitro. The proliferation ratio of osteogenic cells from the young and adult animals increased until 9 days of culture, and then rapidly decreased. Alkaline phosphatase (ALP) activity in both types of cells increased in intensity from 5 days and peaked at 11 days, and ALP activity in the young cells was significantly higher than in the adult cells at all time periods. Calcified nodules were first detected after 7 days in both types of cells. The Ca/P ratio of calcified nodules increased until 13 days in both types of cells, and was significantly higher in the young cells than that in the adult cells at all time periods, except at 7 days. Connexin 43-immunoreactive spots were detected at cell-to-cell junctions at early times in the young cells, but in the adult cells, only a few connexin 43 immunoreactive spots were observed at early times, and these gradually increased at later times. These results suggest that cells derived from the bone marrow of young rats differentiate into osteogenic cells and synthesize calcified nodules at earlier time periods in vitro compared with cells derived from adult rats.

The purpose of this study was to investigate the immunohistochemical and the ultrastructural features of the implant circumference epithelium of the beagle dog using various types of antibodies. The peri-implant epithelium was at an acute-angle from the gingival epithelium and was arranged in parallel to the implant surface. With immunohistochemical staining, the peri-implant epithelium was strongly positive for KL-1, and weakly positive for CK4, CK8 and CK19. These positive reactions for keratins and also for PCNA and BM-1 were similar to those seen in the oral mucosa. In the peri-implant epithelium, a plentitude of microvilli were observed at the periphery of cells at the implant sites, and bacteria were observed between the implant and the peri-implant epithelium without the formation of half desmosomes. There were many lipid-like vacuoles or lysosome-like granules. The intercellular space was wider than the junctional epithelium, and random migrations of large numbers of neutrophils could be seen. Taken together, the peri-implant epithelium is similar to that seen in the oral mucosa, and it is structurally different from the junctional epithelium.

Purpose : The purpose of this study was to investigate Merkel cell behavior under the condition of continuous pressure by a denture base.Materials and Methods : A denture base made of 4-META/MMA-TBB adhesive resin was applied on the palatal rugae of golden hamsters, and the animals were sacrificed 2 and 3 weeks later.Results : Immunohistochemically, an aggregation of CK 20-positive cells was seen at the tip of the rugae in the control groups. However, a significant decrease in CK 20-positive cells was observed in the experimental groups at each of the time periods.Conclusion : These results suggest that the Merkel cells became apoptotic under continuous pressure and that the sensory system might have changed.

Clear findings relative to where in the brain the starting point of voluntary swallowing is controlled were obtained in the present magnetoencephalographic study. Namely, the cerebral activity was observed in the cingulate gyrus and supplementary motor area for about 80 ms between 1,000 and 1,500 ms before swallowing in all test subjects. Thus, it is clear that this type of central control mechanism also plays an important role in complicated swallowing movements.

The behavior of epithelial cells during tooth root formation in porcine molars was investigated using TUNEL, immunohistochemical and transmission electron microscopic methods. Cells of Hertwig's epithelial root sheath (HERS) expressed bcl-2, but were TUNEL-negative. Epithelial cells of HERS were tightly connected and surrounded by a continuous basement membrane. When dental papilla cells were attached to the inner HERS, they became positive for osteopontin (OPN). The inner basement membrane was intermittent immediately after those cells started to synthesize dentin matrix, and the intercellular spaces of the HERS became wider. Dental sac cells then migrated between the fragmented HERS and were directly connected to epithelial cells by desmosome-like structures. After the cementum was synthesized by these mesenchymal cells, epithelial cells aggregated to form cell clusters, which were again surrounded by a continuous basement membrane. These results suggest that fragmentation of the HERS is due to the invasion of dental sac cells, but is not related to cell death. Furthermore, the differentiation of dental papilla cells into odontoblasts occurred following their connection to the HERS through the basement membrane. Moreover, it is suggested that the differentiation of dental sac cells into cementoblasts occurred upon their direct connection to epithelial cells of the HERS.

Because dental implants contact many different tissues, the implant material must have optimum surface compatibility with the host bone tissue, subepithelial connective tissue, and epithelial tissue. In addition, dental implant surfaces exposed to the oral cavity must remain plaque-free. Such materials can be created under well-controlled conditions by modifying the surfaces of metals that contact those tissues. “Tissue-compatible implants,” which are compatible with all host tissues, must integrate with bone tissue, easily form hemidesmosomes, and prevent bacterial adhesion. This research was aimed at developing such tissue-compatible implants by modifying titanium surfaces using a dry process for closely adhering to the titanium substrate and ensuring good wear resistance. The process includes ion beam dynamic mixing (thin calcium phosphates), ion implantation (Ca+, N+, F+), titania spraying, ion plating (TiN, alumina), and ion beam mixing (Ag, Sn, Zn, Pt) with Ar+. At the bone tissue/implant interface, a thin calcium phosphate coating and rapid heating with infrared radiation were effective in controlling the dissolution without cracking the coating. This thin calcium phosphate coating may directly promote osteogenisis, but it may also enable immobilization of functional proteins or drugs. At the oral fluid/implant interface, an alumina coating and F+ implantation were responsible for inhibiting the adhesion of microbial plaque. In conclusion, dry-process surface modification is useful in controlling the physicochemical nature of surfaces, including the surface energy and the surface electrical charge, and in developing tissue-compatible implants.

It has been reported that swallowing is a rhythmic movement, in which the onset of the oro-pharyngeal stage of swallowing starts from the mylohyoid muscle, followed by movement of the oral and pharyngeal muscles, and reaching the superior esophageal sphincter muscle. This is defined as the oro-pharyngeal stage of swallowing. It has also been reported that along with this movement, the larynx elevates in an antero-superior direction. To investigate the swallowing movement, it would be useful to be able to detect the start of swallowing movements from the body surface. Such a device was designed in this study to investigate the relationships between the onset of laryngeal movement and the EMG initiation of the anterior digastric muscle. Although experimental conditions must be further examined, we were able to record the reproducible movement and the position of larynx using our device provides another tool for studying the swallowing movement.

Since dental implants are used in contact with many different tissues, it is necessary to have optimum surface compatibility with the host bone tissues and soft tissues. Furthermore, dental implant surfaces exposed to the oral cavity must remain plaque-free. Such materials can be created under well-controlled conditions by modifying the surfaces of metals that contact those tissues. “Tissue-compatible implants,” which are compatible with all host tissues, must integrate with bone tissue, easily form hemidesmosomes, and prevent bacterial adhesion. This research was aimed at developing such tissue-compatible implants by modifying titanium surfaces using a dry process for closely adhering to the titanium substrate and ensuring good wear resistance. The process includes ion beam dynamic mixing (thin calcium phosphates), ion implantation, titania spraying, ion plating and ion beam mixing. At the bone tissue/implant interface, a thin calcium phosphate coating and rapid heating with infrared radiation was effective in controlling the dissolution without cracking the coating. This thin calcium phosphate coating may directly promote osteogenisis, but also enable immobilization of functional proteins or drugs such as bisphosphonate for dug delivery system. At the oral fluid/implant interface, an alumina coating and F + -implantation were responsible for inhibiting the adhesion of microbial plaque. In conclusion, dry-process surface modification is useful in controlling the physicochemical nature of surfaces, including the surface energy and the surface electrical charge, and in developing tissue-compatible implants.

We have examined the expression and localization of connexin 43 (CX43) in rat incisor odontoblasts using reverse transcriptase polymerase chain reaction, in situ hybridization and immunohistochemistry. The CX43 gene was expressed in odontoblasts, and levels of gene expression increased throughout the course of development. In contrast, CX43 was down-regulated at an incisal segment. In situ hybridization analysis showed no positive signal for CX43 RNA in the cytoplasm of differentiating dental papilla cells, but faint positive signals for CX43 RNA were observed in early pre-odontoblasts. Those signals were more intense in young and in old odontoblasts, but were less in short odontoblasts. CX43 could not be detected in differentiating dental papilla cells or in early pre-odontoblasts by immunohistochemical localization, but a positive reaction was found in the late pre-odontoblast stage where predentin had been produced. The positivity gradually increased during odontoblast maturation, and was highest in the layer of old odontoblasts. These results indicate that odontoblasts that secrete actively dentin matrix components are tightly in contact with each other by gap junctions as suggested by the intense CX43.

This paper revealed the changes occurred on the cylindrical cytoplasmic processes in Merkel cells after single cell isolation at hourly intervals observed under scanning electron microscopy (SEM). After 1 h of single Merkel cell isolation, the cylindrical cytoplasmic processes on the cell surface were bent and other numerous vestiges were observed parallely in contact with the cell surface. After 2 h, a decrease on the cylindrical cytoplasmic processes as well as agglutination to the cell membrane was observed. After 5 h, almost no cylindrical cytoplasmic processes were observed, thus the cell surface became smooth similar to a balloon's surface. The cylindrical cytoplasmic processes, which are considered to be receptors of mechanical stimulation, disappeared after isolation with the passing of time, leading to a Merkel cell with a spherical appearance. When mechanical stimulation by mean of Krebs solution perfusion was carried out, an absence of activity of the mechanically-gated channels, which are present on the cell membrane, was observed. It was thought that this might be due to the disappearance of the above mentioned cylindrical cytoplasmic processes. Then, we considered that the perfusion fluid was inadequate for showing the reception of mechanical stimulation of isolated single Merkel cells.

This case report describes an ameloblastic fibro-odontoma arising from a calcifying odontogenic cyst (COC) in the mandible of a twenty-three-year old male. The patient was referred to the Department of Oral Surgery, Tokyo Dental College, on March 30th, 2000, complaining of a painful swelling, which had appeared three weeks earlier on his left mandibular molar region. In a pathological view, the lesion was a round cyst the size of a chicken-egg, dark red in color, and surrounded by a thick membrane. The cyst had an epithelium of varying thickness which included many ghost cells and an enamel-like structure on the inside, and a thick wall of connective tissue with an ameloblastic fibro-odontoma on the outside. Enamel organ-like epithelial islands were structured radially in the form of strands with immature dentin. Cytokeratin 19 was strongly immunoreactive in the epithelium of the lesion; osteopontin and osteocalcin reacted in the mesenchymal cells and weakly in the epithelial element of this tumor.

The aim of this study was to elucidate the distribution and morphological changes of tight junctions during secretion in parotid gland acinar cells. Localization of tight junction-associated polypeptide ZO-1, and of tight junction transmembrane protein Occludin, was examined in rat parotid gland by immunofluorescence and immunogold labelling of ultrathin sections. Adult male Sprague-Dawley rats were intraperitoneally injected with IPR and, after 10 and 30 minutes, parotid glands were extirpated. In control specimens, positive immunoreaction for ZO-1 and Occludin was observed on the adluminal side between adjacent cells in the form of narrow elongated profiles corresponding to intercellular canaliculi. After IPR injection, canaliculi became dilated and fluorescence was no longer seen as a continuous line but appeared as an aggregation of separate bright particles. ZO-1 was more widely distributed and was recognized in other areas of the cytoplasm as well. Concurrently, omega-shaped concavities, marked by actin fluorescence, appeared along the intercellular canaliculi. We concluded that, during exocytosis, the selective permeability barrier to the paracellular pathway, based on tight junctions, becomes more leaky, owing to segregation of Occludin caused by intracellular ZO-1 distributional changes associated with actin filaments.

The role played by the internal basal lamina (IBL) and hemidesmosomes between an implant and the peri-implant epithelium (PIE) in the adherence of the epithelium to the implant is controversial. This study used rat maxilla implantation models to clarify the ultrastructure of the PIE-implant interface.Ti-6Al-4V implants were inserted either immediately or 2 weeks after the extraction of the upper left first molar of 6- or 4-week-old rats, respectively. The junctional epithelium (JE) of the upper right molars in the same animals was used as a control. Four weeks after implantation, the animals were sacrificed to prepare specimens for light and immunoelectron microscopy.Under light microscopy, the PIE appeared to attach to the implant surface. Ultrastructurally, IBL, consisting of the lamina densa and lamina lucida, and hemidesmosomes were formed only in the lower region, and rarely in the middle region, of the PIE-implant interface. In control teeth, the IBL and hemidesmosomes formed throughout the dento-JE interface. Laminin-1 was found in the IBL and also in the vesicles and vacuoles of the PIE and JE cells. Statistical analysis showed that there was also a significant difference in the amount of IBL between the PIE-implant and dento-JE interfaces.PIE attached to the implant via hemidesmosomes and IBL in the lower region of the PIE-implant interface. Although PIE cells may secrete laminin-1, which contributes to epidermal cell adhesion, the PIE which attaches to implants only in the lower region of the interface is considered to be the poorly adhered epithelium.

We investigated the occurrence of apoptosis and other types of cell death around the crown during tooth eruption of the rat upper molar. The TdT-mediated-dUTP-biotin nick end labeling (TUNEL) method and transmission electron microscopy (TEM) were employed. Apoptosis was detected by both TUNEL and TEM in part of the reduced enamel epithelium and connective tissue in the resorbing bony crypt of the pre-erupted tooth. In TEM, a large number of cells showed condensed chromatin and membrane-bound small bodies (apoptotic bodies). Macrophages that phagocytosed apoptotic bodies could be detected. Based upon the distance between bone surface and these apoptotic cells, and the characteristics of their organelles, we suggested that the apoptotic cells might be osteocytes, bone-lining cells (osteoblasts), and macrophages. We surmised that the osteoclasts had also died. Cells which contained autophagic vacuoles and autophagosomes, and others whose cytoplasm had dissolved, were also frequently observed. No progressive cell death was found in the oral epithelium or the fibrous connective tissue over the crown. These results suggest that apoptosis gives rise to some cell death during tooth eruption, but that other types of cell death also occur in various cells.

In the present study, we examined to identify down-regulated genes between pre- and post dental papillae by microarray analysis in order to identify genes involved in tooth morphogenesis.The dental papilla tissues obtained from E16, E18, P03 and P10 of ICR mice were used for microarray analysis. Microarray analysis showed that the number of up-regulated genes ranged from 860 to 2214 from E16 to P10 and was decreased following the tooth morphogenesis. On the other hand, the number of down-regulated genes ranged from 1337 to 2924, and number of down-regulated genes was the highest (2924 genes) during E18 and P3. The results suggest that the reduced or disappeared genes in dental papilla between P0 and P3 are associated with forming and calcifying the dentin of tooth crown and regeneration of tooth.

A rare case of clear cell odontogenic carcinoma was investigated using histochemical and immunohistochemical methods. The tumor occurred in the anterior mandible of a 69-year-old Japanese man. Histologically, the tumor was composed mostly of large clear cells and squamous cells. Columnar-shaped cells with basophilic nuclei polarized away from the basement membrane were observed at the periphery of the tumor foci. The tumor cells had aggressively invaded muscle and perineural tissues. The tumor cells were positive for PAS staining. Immunohistochemically, tumor cells reacted positively to keratin, cytokeratin19, epithelial membrane antigen, and S-100 protein. The tumor was diagnosed as a clear cell odontogenic carcinoma. Its characteristics are discussed in term of its histopathological, histochemical and immunohistochemical features.

We report a rare case of a completely isolated hemangioma with the histological findings of an arteriovenous hemangioma which occurred in the tongue of a 42-year-old female. This lesion was completely encapsulated by fibrous connective tissue and had focally proliferated with extensive vascular development. The tumor showed a lobular arrangement of vessels of generally uniform wall thickness. Vessel walls were stained by EVG and were immunohistochemically stained with SMA. Mast cells reactive for toluidine blue staining were frequently identified in the stroma and in the surrounding vascular area.