We examined the adherence to epithelial cells, hemagglutinating activity, production of hydrolytic enzymes and enterotoxins in methicillin-resistant, coagulase-negative staphylococci (MRCNS).
     A significant number of MRCNS strains tested adhered to epithelial cell. The average number of attached bacteria per epithelial cell in mannitol-positive MRCNS was approximately 332. In more than half of the mannitol-negative MRCNS strains, the average number of attached bacteria exceeded 500 per epithelial cell. Strong adherence to epithelial cells (more than 1,000/cell) was seen in 15.6% of the MRCNS, indicating that they may adhere better to human buccal epithelial cells than methicillin-resistant Staphylococcus aureus (MRSA). Some strains agglutinated weakly with rabbit erythrocytes, although many showed no adhesion. No correlation was found between adherence to epithelial cells and hemagglutination activity. These results suggest that different adhesins against both functions may exist in MRCNS.
     Many strains produced lipase, lecithinase, DNase, β-lactamase or hyaluronidase. Mannitol-positive strains produced hyaluronidase but not lecithinase. While more than half the mannitol-negative strains produced lecithinase, none produced hyaluronidase.
     These results indicate that the pathogenic potential of MRCNS may be equivalent to that of MRSA.

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     We studied the normal in vitro ultrastructure of Prevotella intermedia cells to understand what changes occur in this ultrastructure as a result of host infection. Two standard and six clinical strains of P. intermedia and one standard strain of Porphyromonas gingivalis were used. TEM samples were prepared from bacterial cells in broth, by spreading the broth on agar plates, and from colonies grown on agar plates. These samples were fixed by GA-OsO₄, Kellenberger-Ryter solution (KR), GA-OsO₄ containing tannic acid (TA), GA-OsO₄ containing ruthenium red (Ru) and freeze substitution using OsO₄-acetone (FS).
     All standard strains had an outer layer ultrastructure consisting of cytoplasmic membrane, periplasmic space, peptidoglycan layer, periplasmic space, outer membrane, electron dense layer and fibrous structure from the cytoplasm by GA-OsO₄ and KR fixation. The periplasmic space of all strains used was filled with periplasmic gel fixed by FS. Ru, TA and FS were effective in fixing the electron dense layer and fibrous structure, while FS and KR were effective in fixing the cytoplasm.
     The outer layer of P. intermedia cells consisted of seven layers, and many vesicles were formed by the outer membrane. The width of each layer could accurately be measured on electron micrographs fixed by FS. The appearance of the periplasmic gel suggests that the peptidoglycan of the P. intermedia and P. gingivalis cells broke up in the periplasmic space.

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     I investigated the competitive adsorption of fluorosurfactant (FSA) and protein to synthetic hydroxyapatite (HAp) measuring surface tension and zeta potential of HAp placed in a solution of FSA and protein. Six types of FSA and two proteins were used. At an FSA concentration of 1×10^-4 vol%, the surface tension of the mixed solution was similar to that for protein alone at the different protein concentrations. However, in 1×10^-2 vol%, three types of changes in surface tension were observed as the protein concentration was varied. The surface tension was either very close to that for protein alone, it decreased and then was stable for higher concentrations, or it increased and then was stable. The zeta potential of HAp in the mixed solution was similar to that for protein alone.
     It was found that the protein or new FSA-complexed protein dominated the competitive adsorption to HAp in any solution of FSA and protein.

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     We investigated age-related changes in the cranial bone collagen of senescence-accelerated mice (SAMP8) using SAMR1 mice as controls. Collagen were extracted from cranial bone at 7, 17, 27 and 37 weeks of age. The extractability of soluble collagen by neutral salt solvent decreased with age, especially in the experimental group at 37 weeks. The amount of hydroxyproline in insoluble collagen decreased in both groups with age. Although the hydroxylation of proline was very similar in the experimental and control groups, hydroxylation of lysine decreased with age, and this decrease was particularly marked at 17 weeks in the experimental group. The pyridinoline content in both groups increased with age. In the experimental group this value was either higher than or essentially the same as that in age-matched controls.
     The above findings relating to collagen suggest that these changes are characteristic of the acceleration of normal maturation and aging.

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     I investigated changes in force vectors in the human mandibular first molar as mastication progressed. Masticatory forces were measured by a three-dimensional transducer placed in the mandibular first molar; mandibular movements were recorded at the same time. Peanuts and raisins were used as the test materials in four subjects with normal stomatognathic function.
     As mastication progressed, the duration, peak and impulse of the vertical and resultant forces decreased, while the lateral forces, and in particular their peak values, showed no significant change. Also, the impulse ratio of lateral to resultant forces increased. The angle of the resultant force with vertical increased over time when this force peaked.
     During a single masticatory stroke, the vector of resultant forces was vertical in the closing phase, and varied considerably in the occluding phase. Directional changes in the vector decreased as mastication progressed.
     These results clearly show that masticatory force vectors have characteristic patterns during crushing and grinding.

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We studied the relation between the resistance to β-lactam antibiotics and β-lactamase activity, and the role of the outer membrane permeability of β-lactam antibiotic resistant Prevotella in the saliva of elementary school children. Strains which had a higher antibiotic minimum inhitory concentration (MIC) also had high β-lactamase activity. The number of strains was calculated that had both on MIC of 50 μg/ml or greater and β-lactamase activity of 30 mU/mg or more. The percentage of these strains was 17-57% for penicillin G, 0-61% for ampicillin, 50-83% for cefaclor, 0-57% for cefazolin and 0-50% for aztreonam. However, the percentages for cefmetazole, latamoxef and cefteram were low, and the value for imipenem was zero. Nine of the 58 strains tested had a cefaclor MIC with EDTA that was 1/4 or less the cefaclor MIC without EDTA. These results suggest that β-lactamase plays an important role in the mechanism of β-lactam antibiotic resistance in the Prevotella tested, and that outer membrane permeability is related to this mechanism in some of the strains.

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We compared the antibiotic sensitivity and β-lactamase activity of Capnocytophaga and Prevotella in saliva of children with infantile acute lymphatic leukemia. The minimum inhibitory concentration of β-lactam antibiotics such as penicillin G (PCG), ampicillin (ABPC), cefazolin (CEZ), cefaclor, cefmetazole, cefteram, latamoxef and aztreonam was high to PCG resistant Capnocytophaga and Prevotella. All strains tested showed resistance to β-lactam antibiotics, and had varying β-lactamase activities. The value for Capnocytophaga was 2〜33 mU/mg for ABPC and 2〜94 mU/mg for CEZ, while these figures for Prevotella were 5〜149 mU/mg and 5〜3, 135 mU/mg, respectively. Ofloxacin showed strong antibacterial activity against β-lactam antibiotic resistant organisms. These results suggest that multi-resistant strains were isolated from oral cavities of children with infantile acte lymphatic leukemia, and β-lactamase was closely related to the mechanism of β-latam antibiotic resistance in these organisms.

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I investigated the effect of the antifibrinolytic agent tranexamic acid on tumor growth and vacularization. VX2 carcinoma cells were implanted into the ears of rabbits. Tranexamic acid (2,000 mg/kg) was administered daily by femoral intramuscular injection in the experimental group. Tumor growth was suppressed in the experimental group compared with the controls which received no injection. Examination of the microvascular structure revealed that tumor angiogenesis was delayed in the experimental group. Further capillary networks were constructed throughout the entire tumor region after two weeks, but were loose compared with the controls. These results suggest suppression of tumor growth in the VX2 carcinoma resulted from inhibition of tumor angiogenesis by the tranexamic acid.

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The purpose of this study was to investigate the spread of methicillin-resistant staphylococci (MRS) in Osaka Dental University Hospital. We examined 65 locations in the hospital wards and tested the antimicrobial susceptibility of the isolates. MRS were isolated from 55 hospital ward locations. About half the MRS strains were isolated from locations in contact with the medical staff or inpatients, and the others from non-contact locations. MRS were classified into various types according to antibiotic sensitivity. All strains tested were susceptible to vancomycin. These results suggest that nosocomial infections were occurring in the hospital wards.

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We attempted to isolate hemagglutinins from P. intermedia strain E18 and to characterize their active fractions. Hemagglutination activities were found in three different fractions (designated as A, B and C) by sucrose density gradient ultracentrifugation. Electron microscopic study revealed that fraction A was an amorphous structure, while extracellular vesicles were a major constituent of fractions B and C. All fractions were sensitive to heat and proteolytic enzymes. However, the sensitivity pattern of fraction A was different from that of B and C. D-Glucosamine, D-galactosamine and N-acety-neuraminic acid caused hemagglutination inhibition of all fractions. In addition, fraction A was sensitive to L-arginine. These results indicate that at least two different hemagglutinins, which are proteins found in nature, may exist in P. intermedia strain E18.

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We did SEM studies of sequential changes in microvasculature during osseous healing after apicoectomy utilizing bone-vascular microcorrosion casts. After one week, newly-formed sinusoidal capillaries sprouted inside the lingual cortical bone of the mandible. At two weeks, primary spongy trabeculae were observed around the ectomized root wound orifice and the inferior alveolar canal. At four weeks, a bone trabecular bridge was formed beneath the ectomized root. By 8 to 12 weeks, the trabeculae had matured and filled the wound to the level of the orifice with a well-developed capillary plexus beneath the ectomized root.

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In order to understand the process of carcinogenesis in rat tongue carcinomas induced by 4-nitroquinoline 1-oxide (4NQO), I compared keratin expression with localization of the epidermal growth factor (EGFR) and its ligands. The tumor growth site was detected with anti-PCNA antibody. EGFR was expressed in the prickle cell layer of normal, hyperplastic, and dysplastic epithelium. This pattern was also observed in malignant epithelium. Highly differentiated areas in the central region of the tumor nest were intensively stained. EGF/TGFα and keratin expression was observed together with that of EGFR. I concluded that EGFR carried out an autocrine reaction with EGF/TGFα, that it was most likely involved in cellular keratin production, and that it closely correlated with squamous cell differentiation rather than tumor growth.

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We investigated the expression of p53 and the localization of Ki-67-positive cells in oral squamous cell carcinomas (SCCs), and the correlation between these factors and tumor differentiation and invasion. p53 showed expression in 28 of 30 cases, the degree being higher in moderate and poorly differentiated than in well differentiated carcinoma. The average of observation of Ki-67-positive cells in well, moderate and poorly differentiated carcinomas was 27.2, 31.7 and 36.5%, respectively. In contrast, the corresponding rates were 23.2% in grade 2, 28.3% in grade 3, 30.5% in grade 4C and 38.7% in grade 4D, according to the classification of Yamamoto et al. These results indicate that p53 is responsible for carcinogenesis in oral SCC, and that tumor proliferative activity is higher in poorly differentiated and grade 4 carcinomas.

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We carried out histochemical investigations of lectin binding patterns in order to elucidate the characteristics of junctional epithelium (JE) regenerated from oral gingival epithelium (OGE). The maxillae and associated soft tissues were removed from rats under ether anesthesia immediately, 5, 10, 15 and 20 days after gingivectomy of the upper left palatal gingivae. Formalin-fixed, paraffin-embedded sections were reacted histochemically with Ulex europaeus agglutinin-I, peanut agglutinin, soybean agglutinin or wheatgerm agglutinin labeled with peroxidase. The lectin binding patterns in the regenerated epithelium were different from those in the OGE after the regenerated epithelium had developed morphologically into the JE, even though the latter was derived from the former. These reactions were the same as those in the reduced enamel epithelium. These results indicate that the expression of glycoconjugates in regenerated epithelium derived from the OGE has a tendency to change after reconstruction of the JE.

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We did light and electron microscopic observations on interproximal gingival tissue to evaluate the effect of initial preparation in periodontal treatment. The material included biopsies from adults who had undergone initial preparation. A certain degree of inflammatory infiltration remained under the junctional epithelium even after the initial preparation. On the other hand, infiltration was localized and formation of new collagen was seen in some areas. The results suggested that periodontal surgery may not always be required after initial preparation.

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In order to evaluate functional aspects of complete dentures, we compared young and old adults having healthy dentition with complete denture wearers. Recordings were made for mean mandibular displacement and standard deviation in the frontal and sagittal planes for each 1mm of vertical jaw opening. The normal young adults had a greater maximum opening than the other two groups. Complete denture wearers had the highest standard deviation of antero-posterior displacement. Normal young adults had a greater mean lateral displacement than the older adults. Normal young adults also had the lowest standard deviation of lateral displacement. Therefore, it was suggested that aging and wearing dentures influences chewing path.

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