Department of Anatomy and Physical Anthropology, Nihon University School of Dntistry at Matsudo made a dental anthropological survey of a Chinese minority in Annin in Yunnan Province in 2007. Subjects were young adults of the Miao people historically derived from Southeast Asian area from whom dental impression models were collected. The frequencies of 22 dental non-metric traits of 7 maxillary and 4 mandibular teeth were recorded. Measurements of the teeth were also made by a digital caliper with the precision of 1/100 mm on mesiodistal and buccolingual diameters of each tooth. The results were compared with those of previous studies including Chinese minorities, Dai, Hani and Naxi, Pumi and with other Asian populations. A principal coordinate analysis based on Smith's Mean Measure of Divergence using the frequencies of 17 traits suggested that the Miao belonged to the Sundadont dentition category, which typifies Southeast Asians and positioned close to the Hani. Statistics and multi-variate analysis of the metric study showed that the Miao had relatively small teeth compared with other four minorities in Yunnan Province. Based on our findings, and presumed past and present distributions of Sinodonty and Sundadonty, it was suggested that the Miao in Yunnan Province have genetically inherited traits similar to the Sundadont peoples of Southeast Asian to which other four minorities also belonged. However, different combination of the frequent dental traits and small size of the teeth in Miao might suggest that the Miao occupies a different position of their affinity from other four minorities.

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Three kinds of mutans streptococci ; Streptococcus ferus and two Streptococcus orisuis serovar strains, in the pig oral cavity were identified previously. The ability to form plaque and synthesise insoluble glucan from sucrose was relatively limited in S. ferus and S. orisuis compared with Streptococcus mutans. PCR analysis showed that the two S. orisuis serovar strains possessed a glucosyltransferase (GTase) I gene. S. ferus could not be amplified with GTase B and GTase I primers. Acid production of all test strains was similar and depended on the cell number. In rat caries experiments, S. ferus and the two S. orisuis serovar strains achieved a lower caries score than S. mutans. The total number of bacteria in the rat oral cavity was also lower for these strains than for S. mutans. The S. ferus and two S. orisuis serovar strains from pigs had relatively weak cariogenic potential compared to S. mutans.

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The purpose of this study was to identify and to characterize of Malassezia spp. in the oral cavity of dog. One hundred and seventeen strains of Malassezia spp. were isolated from fresh clinical isolates of fungi from 20 dogs. The isolates grew well on Sabouraud agar, and produced catalase and precipitates on Tween-medium, and hydrolyzed esculin. Colony morphologies and sizes were characteristic on CHROMagar Malassezia (CHROM) : they developed small, violet color, medium, violet color with pink edge, or large, pale pink color colonies. Molecular investigations of 18 isolates in three different morphological colonies on CHROM from 6 subjects were carried out by chitin synthase 2 (CHS2) gene sequence analysis. The results showed that isolates constitute three genetic types (A_C, B_C, and C_C). Type A_C, B_C, and C_C consisted of 3, 8, and 7 isolates, respectively. The three isolates from only one subject were grouped into the same genotype. These results suggest that the isolates in the oral cavity of dog were Malassezia pachydermatis, and the isolates in CHS2 genetic group were diverse. The relationship between colonial morphology and genetic typing was inconsistent.

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We investigated ectopic bone formation in mouse femoral muscles by direct gene transfer of Sonic hedgehog (SHH) cDNA encoding amino-terminal functional domain (SHH-N) using microbubble-enhanced sonoporation. A fragment of mouse SHH cDNA encoding SHH-N was subcloned into the mammalian expression vector pcDNA3.1(+) [yielding pcDNA3.1(+)-cSHH]. We confirmed that mouse fibroblasts would produce the SHH-N protein following transfection of pcDNA3.1(+)-cSHH. Application of sonoporation procedures to mouse femoral muscles involves injection of a DNA-microbubble mixture and exposure to ultrasound. A mixture containing pcDNA3.1(+)-cSHH and microbubbles was divided into four doses. The DNA-microbubble mixture was first injected into gelatin capsules in the left femoral muscles and only pcDNA3.1(+) or microbubbles were injected into those on the right. Immediately after injection, ultrasound irradiation was performed utilizing an ultrasound probe placed adjacent to the injected gelatin capsules in femoral muscles. Soft X-ray images were taken at 10, 14, 21, and 28 days. Fourteen days after transduction at doses of 114.75 μg/0.03 ml and 0.2 ml microbubbles, respectively, we observed a non-penetration image, and gradually the non-penetration image was clear until 28 days. Histological observation showed endochondral ossification tissues at 14, 21, and 28 days after transduction at doses of 114.75 μg DNA/0.03 ml microbubbles and 114.75 μg DNA/0.2 ml microbubbles. In the cartilage matrix of proliferative and mature chondrocytes, much SHH was clearly recognized, while in new bone matrix and hypertrophic chondrocytes, BMP-2 was clearly recognized by immunohistochemical staining with SHH and BMP-2 antibody. This is, to our knowledge, the first report describing direct transduction of a SHH-plasmid vector to the muscle of adult nude mice using microbubble-enhanced sonoporation. We confirmed that it is a promising type of gene transduction judging from its simplicity and small amount of injury.

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Epulis, a relatively common benign polypoid lesion of the gingiva, may be pathologically classified into several subtypes. Although an epulis is thought to be reactive cell proliferation in the gingiva to various stimuli, the mechanism of pathogenesis is unclear. The present study examined histopathological and immunohistochemical analyses to elucidate the cell proliferation in epulis osteoplastica (n=8) using Ki-67 monoclonal antibody MIB-1 as a marker of proliferation and compared that to cell proliferation in epulis fibrosa (n=8). Epulis osteoplastica consisted of massive, trabecular, or woven bone formation surrounded by a proliferation of fibroblasts and blood vessels with minimal inflammatory cell infiltration under the mucosal squamous epithelium. Scanty osteoblastic lining was observed around the bone. Epulis fibrosa was composed of a proliferation of fibroblasts, collagen fibers, and blood vessels with minimal inflammatory cell infiltration ; no evidence of bone formation was found. Immunohistochemically, positive reactivities for MIB-1 were observed in the fibroblasts of epulis osteoplastica and epulis fibrosa, although positive cell rates were higher in epulis osteoplastica than in epulis fibrosa. The findings suggest that epulis osteoplastica has neoplasm-like characteristics unlike epulis fibrosa.

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The dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain osteogenic precursor cells. In this study, we isolated and cultured human dental follicle cells and compared the osteogenic differentiation capacity between human dental follicle cells and human mesenchymal stem cells from bone marrow. Human dental follicle cells were isolated from the dental follicle of extracted third molars by collagenase/dispase digestion. Human dental follicle cells and mesenchymal stem cells were cultured in mesenchymal stem cell osteogenic induction medium (MSCOIM) with (DEX+) or without dexamethasone (DEX-), or in growth medium (MSCGM). The dental follicle cells cultured in MSCGM exhibited fibroblast-like spindle shapes. The ability of osteogenic differentiation in dental follicle cells and mesenchymal stem cells was examined using alizarin red S stain. Alkaline phosphatase activity was also measured. Calcium deposition was observed in dental follicle cells cultured in MSCOIM (DEX+) or MSCOIM (DEX-), but only in mesenchymal stem cells cultured in MSCOIM (DEX+). Alkaline phosphatase activity was higher in dental follicle cells than in mesenchymal stem cells cultured in either MSCOIM (DEX+) or MSCOIM (DEX-). Furthermore, real time-polymerase chain reaction analysis of the mRNA levels of osteomodulin, considered a marker of osteoblast activity, revealed an increased osteomodulin mRNA level in dental follicle cells cultured in MSCOIM (DEX+) or MSCOIM (DEX-) for 7 days. In contrast, the mRNA level of osteomodulin was higher in mesenchymal stem cells cultured in MSCOIM (DEX+) only. We suggest that dental follicle cells, and not mesenchymal stem cells, commit progenitor cells for osteoblasts, and that the dental follicle may serve as a therapeutic cell reservoir for bone regeneration.

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Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells. The cellular differentiation of the invasion site as well as the pattern of invasion is crucial for the tumor behavior. Both resection margins and the pattern of invasions are important predictors of local recurrence and survival in surgically treated patients. Therefore, surgical margins should be free from carcinoma or dysplasia after surgical intervention and the accepted definition of a close margin is tumor within 5 mm of the inked resection margin, but has not so clear yet. Others use less than 2 mm or 3 mm within one high power field or a variable definition related to the pattern of invasion at the tumor host interface. The aim of this study was to determine the immunohistochemical markers for identification of invasion pattern of oral squamous cell carcinoma (OSCC) and detection of epithelial cells of surgical margins. The study analyzed the immunohistochemical characteristics of 32 cases : 8 cases each of carcinoma in situ (CIS), OSCC border, OSCC early invasion and OSCC front using Ki-67, p53, CK 17, CK 13, laminin -5γ2 and type IV collagen. The correlation between the morphological pattern on invasive sites and immunohistochemical staining were studied. The results were obtained as follows :
1)The average number of Ki-67 positive basal cells was 22.7% in CIS, 57.5% in OSCC border, 62.0% in OSCC early invasion and 60.2% in OSCC front, respectively. The percentage of immuno-positive cells in OSCC border, OSCC early invasion and OSCC front are significantly higher than those of CIS (p<0.05). p53 appeared in almost all tumor cells and highest in OSCC front.
2)CK 17 was positive and strong in superficial cells of OSCC border, parabasal cells of OSCC early invasion and pearl nests of OSCC front, although the degree of positivity was variable. It was negative to discrete positive in CIS.
3)CK 13 was positive in superficial and parabasal cells of CIS and pearl nests of OSCC front, negative in basal cells of CIS and OSCC early invasion, and weakly present in variable distribution in OSCC border.
4)The cytoplasmic accumulation of laminin -5γ2 expression was intense in OSCC border, moderate in OSCC early invasion and OSCC front, and discrete in CIS.
5)Type IV collagen was strong in both OSCC border and OSCC early invasion and weak in CIS and OSCC front.
These results suggested that the combined expression of Ki-67 and p53 confirmed the proliferative and mutational activities of these OSCC invasions in contrast to CIS. CK 17 and CK 13 biomarkers detected their histological differentiation and variation in epithelial cells. The migration-promoting activity and role in cancer invasion were regulated by laminin -5γ2 and type IV collagen, and was clearly observed in OSCC border. The present study also revealed high proliferative activity and high invasiveness of epithelial cells in OSCC border similar to cancer and the combined used of the markers for safety margins should be considered important by surgeon.

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Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is considered to be one of the major etiologic agents of adult periodontitis. We previously succeeded in the gene cloning of a 200-kDa antigenic protein (200-kDa-Ag) from P. gingivalis 381, designated pMD101, and identified it as the truncated hemagglutinin A (HagA), which was thought to be a useful vaccine and immunodominant antigen for serodiagnosis. HagA has immunodominant regions encoding VTVPENGK in the N-terminal region and four repeats of the functional motif PVQNLT expressing hemagglutination. Unfortunately, the production rate of 200-kDa-Ag was low and inclusion bodies were made in Escherichia coli, so we established a subclone pMD160 harboring in the N-terminal region with two repeats of the functional motif. In the present study, because the maltose binding protein (MBP) is known as an effective adjuvant with a convenient purification process, we attempted to establish a novel gene clone that encodes the minimum immunodominant antigenic region of the N-terminal with a single unit of functional motif PVQNLT for the development of serodiagnosis and vaccination. The DNA nucleotide sequences of the novel subclone, designated as pMD167, revealed that the gene product was composed of the minimum immunodominant antigenic region of HagA and MBP as a fused protein and it was designated as 75-kDa-HagA/MBP. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the pMD167 clone produced higher amounts of recombinant proteins than the pMD101 clone. Western blot analysis showed that the pMD167 recombinant protein was strongly recognized by the sera of periodontitis patients. These findings suggest that the novel recombinant protein 75-kDa-HagA/MBP from pMD167 may be useful for the development of serodiagnosis and immunotherapy against P. gingivalis infection.

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Enamel prisms are a morphological feature and Hunter-Schreger bands are the main microstructure of mammalian tooth enamel. Subhyracodon is an extinct rhinocerotoid that inhabited North America in the Oligocene geologic period. The Hunter-Schreger bands of some rhinocerotoids run vertically along the axis of the tooth, and the boundary between bands is distinct in the tangential section. In our observation of Subhyracodon tooth enamel, vertical prism decussation along the tooth axis could be observed clearly in the inner three-fourths of the enamel. In this study, the path of the prisms was digitized and reconstructed three-dimensionally. The course of each prism undulated strongly at the border of the bands, and the prisms did not maintain their relative relations but showed positional changes and displacement of adjacent Hunter-Schreger bands.

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Guided bone regeneration is one of the most effective methods to induce osseous regeneration. We assessed the clinical and histopathological features of guided bone regeneration using nonabsorbable membrane in the wound resulting from human tooth extraction. Forty days after the tooth extraction and wound repair, the wound was in the late granulation tissue stage with newly formed trabecular bone where an implant could be placed. Conclusively, it was revealed that polytetrafluoroethylene membrane could be applied for the repair of a wound resulting from tooth extraction with reduced height and width of bone.

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A rare case of cavernous lymphangioma of the lower lip in a 9-year-old girl is reported with clinicopathological, histopathological, and immunohistochemical analyses. Of the benign soft tissue tumors (610 cases) treated at Nihon University School of Dentistry at Matsudo over the past 12 years, angiomas have accounted for about one-third (33.9%), including nine cases of lymphangioma and 198 cases of hemangioma, for a 1 : 23 ratio. Lymphangioma is composed of diffusely arranged lymph vessels lined by a single layer of endothelial cells. On immunostaining with D2-40, endothelial cells of lymph vessels showed positivity, but those of blood vessels were negative. The Ki-67 labeling index (LI) was lower for endothelial cells of lymph vessels (LI : 2.0) than for blood vessels in cavernous hemangioma (LI : 4.0). These results indicate that D2-40 is useful for discriminating between lymphatic vessels and blood vessels, and that cavernous lymphangioma is a hamartoma.

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Aspergillosis of the paranasal sinuses is uncommon ; however, its incidence in recent years has shown a marked increase. The non-invasive form is by far the most prevalent. Three cases of maxillary sinus aspergillosis were retrieved from the archives of the Department of Oral Pathology, Nihon University School of Dentistry at Matsudo, Japan, from 2002 to 2008, and they were clinically and histopathologically reviewed and studied. The specimens were stained with hematoxylin and eosin (HE) and special stains, such as Grocott methenamine silver-nitrate (Grocott) and periodic acid Schiff (PAS) to identify Aspergillus species. Three previous cases of fungal infection of the maxillary sinus reported from 1985 to 1993 were also reviewed. The details of the present cases (cases 4, 5, and 6) are presented. Case 4 : A 24-year-old man with maxillary sinusitis of unknown etiology had left sinus swelling with no acute symptoms. CT revealed complete opacification of the left maxillary sinus with numerous high density particles. On MRI, the mass lesion was shown to have high signal intensity on T2-weighted images and no signal intensity on T1-weighted images as a result of the granulation of the fungal disease. Case 5 : A 30-year-old man had a palatal root of his upper left first molar that extended into the maxillary sinus. CT revealed opacification of the left maxillary sinus. The inner part of the lesion also showed high density particles and mucosal hypertrophy of the sinus wall and nasal membrane. The radiographic findings of Cases 4 and 5 were compatible with aspergillosis associated with calcification. Case 6 : A 70-year-old man with serous discharge from the nasal cavity had a maxillary bony fracture due to a previous automobile accident that was treated with plate fixation. An intra-oral fistula on the buccal side of his upper right first molar was noticed from which pus was discharging. CT and panoramic radiography revealed opacity of the maxillary sinus. CT also revealed maxillary bony resorption on the right side surrounding the plate extending to the nasal cavity, which caused the plate to be exposed in the frontal wall of the nasal cavity. Our 3 previous reported cases were caused by dental procedures and were related to their respective upper first molar ; the 1st and 2nd cases had a root tip that remained in the sinus, while the 3rd case had a perforated sinus after tooth extraction. MRI and CT confirmed fungus infection of the maxillary sinus. Surgical treatment of the sinus revealed the presence of paranasal fungus balls. The typical characteristic branching hyphae of Aspergillus were seen on histopathological examination with the special stains. Although fungal infections of the paranasal sinuses caused by Aspergillus are found uncommonly in our hospital, the significance of its presentation clinically, histopathologically, and on special staining is important to consider, especially since such cases are rare.

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