We report two bacterial strains isolated from individual persistent apical periodontitis lesions where the presence of bacteria was noted in root canals after numerous treatments. Following sampling from the root canals, a representative single colony was randomly selected from each case for further study. These strains were identified as Rothia mucilaginosa by 16S ribosomal RNA gene sequencing. Scanning electron microscopy observation revealed that these isolates possessed unique phenotypic characteristics by exhibiting dense meshwork-like structures on their cell surfaces, which had a similar appearance of structure typically possessed by a number of biofilm-forming bacterial species. These results suggest that Rothia mucilaginosa isolated from persistent apical periodontitis lesions, which possess dense meshwork-like structures on their cell surface, might form biofilms to survive in host environments.

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We have previously reported that clinical isolates of Escherichia hermannii (E. hermannii) from a persistent apical periodontitis lesion had the capacity to form biofilm containing mannose rich exopolysaccharide. We generated a biofilm-defective mutant strain 455 from E. hermannii strain YS-11 by random transposon insertion mutagenesis and demonstrated that a mutant lacking wzt (one of the ABC-transporter genes) was incapable of producing the viscous materials necessary for biofilm formation. In this study, we employed a quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to learn how the transcriptional level of this gene fluctuates along with the growth of this organism. Strain YS-11 showed high transcriptional levels of wzt from the early exponential to the stationary phase of growth, and the peak was seen at 6hr of culture. When a plasmid pWZT carrying a wild type wzt ORF was introduced to strain 455, a recombinant strain designated as strain 455-LM showed denser meshwork structures around cells as well as higher transcriptional levels than those of the parent strain. These results may suggest that wzt is involved in the formation of E. hermannii biofilm.

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A prototype surface reaction-type pre-reacted glass ionomer filler-containing root canal sealer (FS) was developed for its potential to strengthen teeth and to inhibit microbial propogation by releasing multielements including fluoride. We investigated the direct effect of FS on human periodontal ligament cells (HPLCs) and compared its effect to that of the zinc oxide eugenol Endosealer^[○!R] (ES) by quantifying the gene expression responsible for inflammation and bone resorption. We used real-time reverse-transcliptase polymerase chain reaction to quantify the mRNA expression of IL-1β, IL-6, TNF-α, osteoprote-gerin(OPG) and receptor activator NF-kB ligand (RANKL) in HPLC in contact with FS. TNF-α mRNA expression induced by FS was significantly greater immediately after setting (FS0) and at 4 hours after setting (FS4). The mRNA expression of TNF-α and IL-6 induced by ES increased significantly at 4 hours after setting (ES4). Although the prototype S-PRG filler-containing root canal sealer (FS) showed a mild direct proinflammatory property, it did not directly affect bone resorption.

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Cancer testis antigens (CTA) are a group of normal testicular proteins. Although their expression appears in cancer cells, it does not appear in somatic cells with the exception of male germ cells. Consequently CTAs are immunogenic in cancer patients, and considered promising target molecules for cancer vaccines. In CTAs, brother of the regulator of imprinted sites (BORIS), which is also known as CCCTC-binding factor-like (CTCFL), is an epigenetic-acting oncogene that supresses the tumor inhibitor functions of CTCF. For this reason, BORIS is thought to be an ideal molecular target for drugs against various cancers. In order to detect CTAs in oral cancer, we used the methylation pattern to select six genes containing BORIS, ACTL7B, PPP3R2, SYCP1, DAZL, and TAF7L. We then analyzed the expression of these genes in oral squamous cell carcinoma cell lines and various mice organs. Expression of only BORIS in these genes was detectable in SAS, HSC-3 and HSC-4. Boris expression in adult mice was restricted to the testes. These findings imply that BORIS might be the target of immunotherapy for oral cancer.

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We investigated the morphology of ferret connective tissue papillae and the microvascular architecture of the filiform papillae. Connective tissue papillae were basically composed of an elliptical base with between 10 and 17 processes arranged in an elliptical pattern. The microvascular architecture had essentially between 11 and 18 capillary loops arranged in an elliptical pattern. The number of processes of connective tissue papillae and capillary loops were approximately equal, at between 10 and 18 per papilla from the lingual apex region to the posterior region. Thus, the composition of connective tissue papillae and capillary loops did not change in number or distribution by region.

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It is well established that osteoclast differentiation/activation is involved in orthodontic tooth movement at compression sites. However, the mechanism of osteoclast differentiation/activation in orthodontic tooth movement remains unclear. Nitric oxide (NO) has been reported to play a crucial role in bone remodeling induced by mechanical loading, and several reports have shown that inhibition of NO syntheses prevents orthodontic tooth movement in animal models. We investigated whether osteoclast differentiation can be induced by NO in vitro. We found that sustained release of NO by NOC-18 induces osteoclast differentiation of not only RAW264 cells but also bone marrow macrophages, and that the inhibition of ERK and activation of p38 MAPK may be involved in NOC-18-induced osteoclast differentiation.

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Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors when compared to normal tissue, its contribution to tumorigenicity is still not known. We analyzed the effects of HB-EGF on the invasion activity of a cultured oral cancer cell line, HSC3, using small interfering RNA (siRNA) vector. An oral squamous carcinoma cell line, HSC3, was transfected with siRNA vector targeting HB-EGF (HSC3-siRNA). Expression of HB-EGF was analyzed by real-time polymerase chain reaction. The invasiveness of the transfected cells was determined using Matrigel Invasion Assay, and MMP-9 production was measured by reverse transcriptional polymerase chain reaction (RT-PCR) and gelatin zymography. The expression of HB-EGF was reduced in HSC3-siRNA cells. The Matrigel Invasion Assay demonstrated that invasiveness of HSC3-siRNA cells was also reduced. RT-PCR demonstrated that in HSC3-siRNA cells, MMP-9 mRNA was decreased. These findings suggest that HB-EGF expression is related to the invasion activity of oral cancer, particularly via regulation of MMP-9.

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Although the application of calcium hydroxide in the root canal is reportedly effective for the prevention of external root resorption, details of its mechanism of action are unclear. We studied the effect of calcium hydroxide on the expression of Iipopolysaccharide (LPS) induced hard tissue resorption-related genes in human osteoblastic cells (Saos-2) to evaluate how it prevented hard tissue resorption. The effects of calcium hydroxide on the expression of genes of TNF-α, IL-6, OPG and RANKL expressed by LPS-stimulated human osteoblastic cells were evaluated using the real-time reverse transcriptase-polymerase chain reaction. The data were compared employing Student's t-test (n=6) at the significance level of 0.05. Calcium hydroxide inhibited the expression of proinflammatory cytokines induced by LPS. It also inhibited the expression of RANKL, which is always expressed in human osteoblastic cells. Calcium hydroxide appeared to inhibit LPS-induced inflammatory hard tissue resorption.

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Three-dimensional (3D) data has recently been used in clinical orthodontics to determine facial soft-tissue morphology. We extracted crosssectional images from a 3D facial model, quantified characteristics of the contour curve using the radius of curvarure, and studied the relationship between the radius of curvature and the maxillofacial morphology. In order to obtain 3D facial data for each subject, 3D facial images were exposed of 31 patients with individual normal occlusion using a non-contact 3D laser scanner. Four cross sections were extracted from the 3D facial data and the radius of curvature was calculated for five sections on each cross section. Cephalometric radiography was performed on each subject, and maxillofacial morphology was analyzed. Correlation analysis was performed between the radius of curvature and maxillofacial morphology at each section in each subject. We found that in subjects with individual normal occlusion, the 3D soft-tissue morphology around the median of the lips strongly reflected the anteroposterior position of the maxillary and mandibular incisors and the position of the mandible. Three-dimensional analysis of soft tissue morphology in association with maxillofacial morphology appears potentially useful in the diagnosis and management of orthodontic treatment.

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During denture fabrication, the shape of the palatal plate is similar to that of the patient's palate, and its thickness is uniform in most cases. However, clinically, patients wearing new dentures often complain of discomfort during swallowing. We evaluated how the shape of the palatal plate affected food transport in healthy subjects using various foods, and attempted to clarify the best shape to facilitate swallowing. A plaster model of the maxilla of each subject was fabricated using hard stone, New Plastone LE^[○!R] (GC, Tokyo, Japan). Duran^[○!R] 3.0mm (Scheu-Dental, Iserlohn, Germany) was softened using a compression molding machine for dental use, and formed to the palate of the working model. This was used as a base plate. In addition four experimental palatal plates were fabricated based on the following criteria by adding self-curing resin. Plate 1 had a uniform thickness of 1.5mm. In Plate 2, the thickness of the palate was increased to 1/3 the distance from palate to the occlusal surface. In Plate 3, the thickness of the anterior region of the palate from the incisive papilla to the first premolars was gradually increased to 5mm. In Plate 4, the thickness of the left and right lateral palatal regions of the plate were increased by 5mm. Fifteen tapioca pearls (Youki Food, Tokyo, Japan) were used as the test food. The subjects assumed a routine sitting position for eating with a natural posture for swallowing. The examiner placed the test food on the anterior dorsal region of the tongue using a spoon, and the subjects were instructed to swallow it as they would normally. The examiner then confirmed the amount of food remaining and recorded this information. The subjects were classified into two groups based on the amount of food that remained in their mouth. We found that swallowing tended to be easier with Plates 2 or 4 and more difficult with Plate 3. These results suggest that the posterior palatal area of the plate has only a slight influence on food bolus transport.

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p120-catenin (p120) is a very critical element in the cadherin/catenin cell-cell adhesion complex. We previously reported that phosphorylated β-catenin disrupts the regulation of E- and P-cadherin stability and adherens junction formation in cancer tissues. In order to study the relationship between p120 phosphorylation and malignancy in a human gingival squamous cancer cell line (BICR78), we investigated the expression of p120 in BICR 78 treated with ZD1839 (Gefitinib), which inhibits epithelial growth factor receptor (EGFR) phosphorylation. BICR78 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 4μg/mL hydrocortisone and 10% fetal bovine serum. After nearly confluent cells were cultured in serum-free DMEM overnight, they were incubated with 10ng/mL EGF and 1μM ZD1839 for one hour. After cultivation, we performed Western blotting and immunocytochemical analyses to investigate the expression of EGFR, Src, Akt and p120, as well as their phosphorylated forms. We observed a decrease in the number of BrdU-labeled BICR78 cells after treatment with ZD1839. Although the expression of Akt and p120 showed no changes, the phosphorylation of EGFR, Akt and p120 decreased in BICR78 treated with ZD1839. Expression of Src and phosphorylated Src also decreased after treatment with ZD1839. EGFR, p120 and phosphorylated p120 were observed to be distributed on the cellular membrane, while phosphorylated EGFR, both forms of Src and both forms of Akt were localized diffusely in cellular plasma. These results indicate that the inhibition of phosphorylation in EGFR and the downregulation of Src by interception of the Akt-PKB signaling pathway inhibit p120 phosphorylation, thereby controlling the growth of BICR78 cells.

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Although there are many reports about the preferred chewing side, there are few conclusions. We objectively investigated the relationship between the preferred chewing side and the temperature of the masseter muscle using a deep body thermometer. Eight persons were selected as the normal subjects. The deep temperature (DT) of the masseter muscle was recorded for 15 minutes during free-sided gum chewing and for 10 minutes after chewing. After the measurements, we questioned the subject on his/her preferred chewing side. The results indicated the increase in the deep temperature (ΔDT) of the masseter muscle was greatest on the preferred chewing side after 15 minutes of free-sided gum chewing. Within the limitations of this study, we concluded that measuring the deep temperature of the masseter muscle was a useful method for the objective determination of the preferred chewing side.

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