I carried out biochemical and physiochemical investigations on the adsorption of human salivary proteins to synthetic hydroxyapatite (HAp), which was fractionated from whole saliva by gel filtration. Four commercial proteins (mucin, s-lgA, albumin and α-amylase) were used as controls. The SDS-PAGE, western blotting, amino acid composition assay and sugar composition analysis showed that the fractions A2, B2, C2 and D2 had the biochemical characterization similar to mucin, s-lgA, albumin and α-amylase, respectively. After HAp was dispersed in salivary and commercial protein solutions buffered with 10 mM Tris-HCl (pH 7.0) for 24 hr, the adsorption volumes of the proteins to HAp were measured by spectroscopic analysis. The zeta potential of HAp in these protein solutions was also determined.
     The adsorption volume to HAp occurred in two stages for all proteins. The first stage was Langmuir and occurred at low protein concentration. The second stage was the multilayer type and occurred at high concentration. However, the zeta potential of HAp was only the Langmuir (monolayer adsorption) type for all the protein solutions. It was suggested that analysis of protein adsorption requires both measurement of protein volume and zeta potential. Both basic and hydrophobic amino acid residues decreased in the salivary protein solutions after adsorption, indicating that adsorption of salivary proteins to HAp is dependent on electrostatic and hydrophobic interactions.

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     In order to understand senescence characteristics in the salivary glands, we investigated age-related changes in the activity of NAGase, its enzyme protein properties and its isozyme pattern using N-acetyl-β-D-glucosaminidase (NAGase) extracted from submandibular and sublingual glands of 7, 17, 27 and 37 week-old senescence accelerated mice (SAM-P8//Odu, P substrain) and control mice (SAM-R1/Odu, R substrain).
     In the substrain mice, NAGase activity increased gradually up to 17 weeks of age in the submandibular gland, and up to 27 weeks in the sublingual gland, and remained at those respective levels thereafter. However, the activities of both salivary glands decreased significantly at 27 weeks of age in the P substrain. Age-related changes in the optimal pH, heat stability and the effect of compounds on the NAGase were different in both salivary glands for the two substrains. The NAGase activities of both salivary glands of the R substrain were separated by isoelectric focusing into four fractions: a and b, which had pI in the acidic region, and c and d, which had basic pI. However, the distribution ratio of the fraction increased markedly together with the disappearance of the b fraction in both salivary glands of the P substrain.
     We found that qualitative and quantitative changes in NAGase were expressed at 27 weeks of age with accelerated senescence in the P substrain mice. We propose that these changes are closely related with and may cause structural alterations of the acidic isozyme of NAGase with senescence.

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     I examined the expression of enkephalin during tooth movement, which is occasionally associated with pain. Several immunohistochemical studies have recently shown the expression of peptides related to neurotransmission in central and peripheral tissues. Enkephalin, which acts as an analgesic, has received much attention.
     Male Wistar rats, weighing 200 to 250 g, were killed at 1, 3, 6, 9, 12, 18 and 24 hours, and 2, 7 and 14 days after insertion of elastic bands between the upper left first and second molars (Waldo's method). The first molars with surrounding structures were decalcified and sectioned at 15 μm in a cryostat. The sections from the right maxillary molars were used as controls. All sections were stained by indirect immunofluorescence and hematoxylin-eosin. The examination concentrated on the buccal root periodontal ligament of the first molar.
     Immunohistochemistry using the indirect immunofluorescence method revealed that enkephalin-immunoreactive cells were found in the periodontal ligament at 6, 9 and 18 hours after tooth movement. Particularly strong enkephalin-immunoreactivity was found at 9 hours. When the in situ hybridization method was used, signals were found in the periodontal ligament at 9 and 18 hours.
     The appearance of enkephalin-containing cells in the periodontal ligament during tooth movement suggests that this chemical is associated with neurotransmission and that it can be produced by periodontal ligament cells during the movement.

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     I examine the production of the hydrolytic enzymes β-lactamase, DNase, hyaluronidase, chondroitin sulfatase, lipase, lecithinase, collagenase, trypsin, chymotrypsin and viscous material in clinical strains isolated from periodontal pockets with and without acute inflammation.
     In pocket bacteria with acute inflammation, the distribution of collagenase, DNase and trypsin producers was 30.4, 22.6 and 17.6%, respectively. Other hydrolytic enzyme-active bacteria were found in less than 5% of all isolates. However, in cases without acute inflammation, the averages for collagenase and DNase producers were 22.0 and 18.4%, respectively. The ratios for other enzyme producers were less than 6.0%, suggesting that collagenase, DNase and trypsin producers may be involved in exacerbation of the disease.
     In cases with acute inflammation, Porphyromonas gingivalis was the predominant collagenase producer (33.9%), DNase producer (35.7%) and trypsin producer (82.9%). However, in cases without acute inflammation, "milleri" group streptococci were identified as the major producer of collagenase (16.0%) and DNase (31.8%). P. gingivalis was the predominant trypsin producer (42.0%), although it was a subordinant producer of collagenase (8.2%) and DNase (7.1%).
     The ratio of the above enzyme producers was highest in periodontal pockets with acute inflammation, lower in cases without acute inflammation, and lowest in acute periapical periodontitis cases. This indicates that pocket bacteria have considerable potential to induce acute inflammation.

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     I investigated the effects of unilateral craniomaxillofacial deformity on mandibular growth in 90 5-day-old male Wistar rats. A spring appliance made of orthodontic wire was inserted in the left infraorbital fissure of the maxilla and occipital bone, and the rats were sacrificed at 2, 4 and 6 weeks of age. The effects on the mandible were assessed by analyzing the cephalometric radiographs of the dry skulls and weighing of the dry mandibles. The results were as follows:
1) There was no significant change in body weight.
2) The left mandible (operative side) was shorter than that of the controls, although there was no significant difference in its height.
3) Although there was no significant change in the length of the right mandible (non-operative side), the height decreased on that side.
4) Although radiographic examination indicated a decrease in the mandibular surface area, there was no difference between the two sides.
5) Although the dry weight of the mandible decreased, there was no significant difference between the left and right sides.
     The results indicate that a continuous unilateral retraction force between the maxilla and occipital bone produces craniomaxillofacial and mandibular asymmetry in growing rats. It is assumed that the mandibular asymmetry was produced not only by inhibition, but also by rotational changes in growth.

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     We investigated the spread of methicillin-resistant staphylococci (MRS) in Osaka Dental University Hospital. We examined 36 locations in the operating room and the nasal carriage of four medical staff, and tested the antimicrobial susceptibility of the isolates. MRS were isolated from 18 locations in the operating room and two medical staff. About half the MRS strains were isolated from locations in contact with the medical staff, and the others from locations not in contact. MRS were classified into 8 types according to antibiotic sensitivity. All strains were susceptible to vancomycin.
     These results indicate that both air-borne contamination and contact-infection were occurring in the operating room.

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We investigated the spread of Methicillin-resistant staphylococci (MRS) in Osaka Dental University Hospital. We examined the nasal carriage of the medical staff and patients, and tested the antimicrobial susceptibility of isolates. MRS were isolated from three inpatients and a location in a hospital room, but not from any of the medical staff or outpatients. MRS were classified into 9 types according to sensitivity to antibiotics. All strains were susceptible to both chloramphenicol and vancomycin. Classification of the MRS samples indicated that the same substrains were found among the inpatients, suggesting that cross infection was occurring in the hospital.

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We investigated the competitive adsorption of fluorosurfactants (FSA) and protein to synthetic hydroxyapatite (HAp) by measuring of the zeta potential. When protein was mixed with 1 × 10^-4vol% FSA, the surface tension decreased with increasing protein concentration much in the same way it did when FSA was not present. However, when the FSA was increased to 1 × 10^-2vol%, increasing the protein concentration did not affect surface tension so dramatically. The zeta potential of HAp in mixed solutions was similar to that for protein alone. It was found that the protein dominated the competitive adsorption to HAp in any solution of FSA and protein.

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We investigated the effect of TiO₂ on the adherence of S. mutans to a dental pit and fissure sealant containing powdered semiconductor TiO₂, which exhibits a photocatalytic reaction. The viability of S. mutans on the sealant containing TiO₂ varied with the bacterial strain. The adherence to the sealant was greatest for S. mutans Ingbritt, followed in order by S. mutans NCTC 10449 and S. mutans GS-5. TiO₂ increased the Brinell hardness number of the sealant, although the flexural strength decreased.

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To clarify the mechanism controlling human mandibular position as a function of lateral inclination of the head and body, I carried out electromyographic investigations of the muscle activities of the inferior head of the lateral pterygoid muscle under different postures with respect to the frontal plane. When the head leaned left or right, the lower jaw was displaced towards the inclination, and muscle activities on the ipsilateral side increased. These results indicate that reflection muscle activities have some control over the displaced condyle.

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We investigated the spread of methicillin-resistant staphylococci (MRS) in Osaka Dental University Hospital. We examined 36 locations in the operating room and the nasal carriage of four medical staff, and tested the antimicrobial susceptibility of isolates. MRS were isolated from 18 locations in the operating room and two medical staff. About half the MRS strains were isolated from locations in contact with the medical staff, and the others from non-contact locations. MRS were classified into 8 types according to sensitivity to antibiotics. All strains were susceptible to vancomycin. These results indicate that both air-borne contamination and contact-infection were occurring in the operating room.

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We attempted to determine normal ranges of asymmetry and activity indices, as proposed by Naeije et al., in masseter and temporal muscles during intercuspation at clenching levels of 10% and 50% of maximum voluntary contraction. Normal ranges were calculated with parametric statistics on an asymmetry index in 88 males and 77 females (mean age 22.7 yrs), and on an activity index on each side in 47 males and 39 females (mean age 22.5 yrs).

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The disappearance of tonic discharge has been observed immediately preceding rapid voluntary movements. This phenomenon, referred to as the premotion silent period, is thought to be a learned, rather than automatic, motor response. Seven healthy male subjects who were maintaining steady, slight voluntary contractions of the masseter were asked to respond to an acoustic stimulus by maximum voluntary clenching as rapidly as possible. The premotion silent period was observed in the masseter muscle in 5 of the 7 subjects.

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While the clenching level was gradually increased, dynamic stability of the intercuspal position (ICP) in 10 normal subjects and 10 patients with temporomandibular disorders (TMD) was evaluated from occlusal contacts, deviation of the incisal point of the mandible, and bilateral anterior temporalis and masseter muscle activity. Increases in the clenching level produced changes in the location of occlusal contacts in ICP in the TMD patients. During clenching in ICP, displacement of the mandible in TMD patients was greater than in normal subjects, and the activity of the masseter muscle had greater effect on occlusal contact stability than did that of the anterior temporalis muscle.

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I investigated changes in masticatory force vectors in the mandibular first molar as mastication progressed. 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 force increased. The angle of the resultant force with vertical increased over time when this force peaked. Directional changes in the vector during a single masticatory stroke decreased as mastication progressed, and the vector of resultant forces was vertical during the closing phase.

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We attempted to determine the ability of the atomic force microscope to observe the surface of human dental enamel and to ascertain the effect of saliva adsorption on the ultramicrostructure of the surface. Three types of enamel surface were considered in this study:1) Enamel treated with 0.1 M HCl solution for 15 sec, 2) Polished enamel, and 3) Polished enamel placed in saliva for two hours. The atomic force microscope (AFM) and X-ray photoelectron spectroscope (XPS) were employed to observe and analyze these sample surfaces. The AFM and XPS were able to detect the molecular structure and structural defects in the enamel crystal surface. Adsorption of salivary protein to the enamel surface masked the molecular structure and structural defects in the enamel crystal surface. The AFM technique may be useful in determining the mechanisms of carious resistance and remineralization of dental enamel.

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The purpose of this study was to determine the electrostatic characterization of streptococci and the relation between this characterization and the surface structure. The streptococci zeta potential was not influenced by pH at the same ionic strength as the solution. Furthermore, changes in zeta potential were independent of the peptidoglycan layer density or the presence of COOH and NHn on the surface of the streptococci. However, the presence of phosphorus ion resulted in significant changes. It was concluded that the amount of lipoteichoic acid containing phosphate groups on the surface of streptococci has a considerable impact on the zeta potential.

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We examined the pathogenic potential of methicillin-resistant, coagulase-negative staphylococci (MRCNS). Significant numbers of all the MRCNS strains tested adhered to epithelial cells. In more than half the MRCNS strains tested, 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. Many strains produced lipase (48.8%), lecithinase (43.8%), DNase (28.8%), β-lactamase (26.3%) or hyaluronidase (13.8%). These results indicate that the pathogenic potential of MRCNS may be equivalent to that of methicillin-resistant Staphylococcus aureus.

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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. Five strains of P. intermedia and one of Porphyromonas gingivalis were used. TEM samples were prepared from bacterial cells in broth, by spreading 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 strains had an outer layer ultrastructure consisting of an inner membrane, periplasmic space, peptidglycan layer, periplasmic space, outer membrance, electron dense layer and fibrous structure From the cytoplasm. 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.
It was found that the outer layer of P. intermedia cells consists of seven layers and many vesicles are formed by the outer membrane. The width of each layer can be accurately measured on electron micrographs fixed by FS.

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I investigated microstructural and microvascular changes in a vascular bundle transplanted into the tibia of a rabbit (Group 1) and during the healing process of a mandibular defect in the same animal packed with the secondary living free bone graft from the tibia (Group 2). In Group 1 the transplanted vascular bundles communicated with the pre-existing medullary vessels via the newly-formed capillaries, three weeks after transplantation. In Group 2 establishment of circulation in the pre-existing vessels of the donor was observed five days after transplantation and the donor material was completely fused with the mandible at four weeks. The secondary living free bone graft seems clinically effective for mandibular defects.

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