The purpose of this experiment was to study activities of dipeptidyl peptidase IV (DP-IV) and collagenase-like peptidase (CLP) released from macrophages by stimulation of Bacteroides gingivalis in vitro.
Considerable evidence has been accumulated that cellmediated immune mechanisms may be involved in the initiation and peogression of periodontal diseases. Macrophages are widely distributed in connective tissues, organs and periodontal tissues. Macrophages may play a central role in inducement and appearance of immunity against bacterial plaque.
It is a remarkable fact that macrophages in inflammatory lesions may have the function not only of self-defense by phagocytosis but also of destruction of connective tissue by released hydrolytic enzymes. On the other hand, B. gingivalis has been recently recognized as a pathogenic microorganism in periodontal diseases. It is reported that B. gingivalis is predominantly isolated from periodontal pocket in some advancing lesions of adult periodontitis. It has been also found that a strong correlation exists between the levels of serum antibody against B. gingivalis strain and periodontal diseases.
As an in vitro experiment, peritoneal macrophages were obtained from 250-300g male rats (Std: Donryu) by irrigating the peritoneum with RPMI1640 media. Macrophages in culture wells were purified by removing non-adherent cells by washing with the media after incubation. B. gingivalis cells, opsonized B. gingivalis cells, latex beads (φ0.81μm), and media were added in macrophage (0.5×10⁶cells) culture wells. Incubated culture media-an enzyme solution-were collected at variable stimulated volumes and time intervals.
The activities of DP-IV and CLP were measured using Gly-Pro-MCA, Suc-Gly-Pro-Leu-Gly-Pro-MCA as substrates by measuring liberated AMC from substrates. The digested products of the action of released DP-IV from macrophages on partially digested peptide from collagen type I as substrate were analyzed using high-pressure liquid chromatography.
Activities of DP-IV and CLP increased against stimulated volumes and maximum activities at stimulated concentration were 0.5×10 cells. Those of latex beads groups were approximately the same as the control media. In the opsonized cell groups, maximum activity of DP-IV was observed after 12hrs. and that of CLP 4 hrs. after stimulation. Both of the enzyme activities of opsonized cell groups were 2.5-times against non-treated cell groups. The enzyme properties were as follows: The optium pH for DP-IV activity was 8.6 using tris-maleate buffer and that for CLP activity was 8.2 using tris-HCl buffer. The Km value of DP-IV was 0.067mM using Gly-Pro-MCA as a substrate and that of CLP was 0.011mM using Suc-Gly-Pro-Leu-Gly-Pro-MCA as substrate. It seemed that neither inhibitor nor acti vator were present in this enzyme solution with DPIV and CLP. This enzyme solution hydrolyzed partially digested peptide from collagen type I and liberated Gly-Pro peptide.
The enzymes of DP-IV and CLP were released from macropahges by stimulation of B. gingivalis and the release was encouraged by opsonization.

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Collagen, one of the structural proteins of the gingival connective tissue, contains a number of prolines in its amino acid sequence. Due to the unique structure of proline, peptide bonds involving proline residues are resistant to the actions of peptidases, even to peptidases of broad specificity. In order to complete the degradation of peptides derived from collagen, proline specific peptidases will be needed. The purpose of this study was to determine the activities of two proline specific peptidases, dipeptidyl peptidase IV (DP) and postproline endopeptidase (PE), in gingiva from patients with periodontal diseases and clinically healthy subjects.
Sixty patients with periodontitis (diseased group) and 20 clinically healthy subjects (control group) who visited Nihon University Dental Hospital were selected for this study. For each subject three clinical parameters were assesed: probing depth (PD), Gingival Index (GI) and Bleeding Index (BI). The diseased group was devided into the following sub-groups: PD-1 (mean PD<4mm) and PD-2 (meanPD≥4mm), GI-1 (GI<1.5) and GI-2 (GI≥1.5) and BI-1 (BI<50%) and BI-2 (BI≥50%). Gingival biopsies were obtained by the method of Shiomi. The gingiva was homogenized in 10mM phosphate buffer (pH7.0) containing 1mM dithiothreitol and 1mM ethylendiaminetetraacetic acid, and the supernatant was obtained through centrifugation at 3, 000rpm for 5min. The activities of DP and PE were assayed by the method of Kato et al. and Yoshimoto et al. with modifications, and protein contents were assayed by the method of Bradford. One unit of enzyme activity was defined as the amount of enzyme which hydrolyzes the 1μmol of substrate per min at 37°C.
The optium pH of DP in both control and diseased groups were 8.6-8.8 and the Km values for glycylproline methylcoumarinamide were 1×10^-4M. The optium pH of PE in both control and diseased groups were 7.0-7.2 and the Km values for succinylglycylproline methylcoumarinamide were 2×10^-5M. The activities of DP in diseased group was significantly higher than control group, and the activities in PD-2, GI-2 and BI-2 groups were significantly higher than PD-1, GI-1 and BI-1 groups. No statistically significant difference was evident between the activities of PE in control and diseased groups.

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The purpose of the present study was to evaluate the blastogenic responses of gingival lymphocytes stimulated by phytohemagglutinin (PHA), Concanavalin A (Con A) and pokeweed mitogen (PWM) following experimental periodontitis in dogs. And then, their differences among the gingival lymphocyte peripheral blood lymphocyte (PBL) and submandibular node lymphocyte (SNL) were examined.
After establishment of ligature-induced periodontitis in seven dogs, gingival lymphocyte, PBL and SNL were isolated by collagenase treatment and Ficoll-gradient procedure. The cell populations were examined by Giemsa staining and the lymphoproliferative responses were determined by the uptake of ³H-thymidine.
80.3% lymphocytes and 3.6% macrophages were occupied in the cell population obtained from the gingival tissues. Viability of the gingival lymphocytes was 77%. The mean count per minute (CPM) and standard error of the gingival lymphocytes, PBL and SNL of unstimulated culture were 399±107, 864±127, 758±204, respectively. The responses of PBL and SNL to PHA, Con A and PWM were remarkably high and peak responses were obtained at days 2 or 3 of culture. On the other hand, the response of gingival lymphocytes stimulated with mitogen was low and almost equal to the level of unstimulated lymphocytes. The peak activity of gingival lymphocytes was shown at day one of culture.
It is suggested that the response of gingival lymphocytes is not parallel to PBL or SNL and blastogenic activity of gingival lymphocytes unstimulated and stimulated with mitogens is low.

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Lipopolysaccharide (LPS) exhibits a wide variety of biological activities and is regarded as one of the important pathogens in periodontal diseases. For example, LPS induces osteoclastic bone resorption and activation of the complement system in vitro. In spite of these investigations, the role of endotoxin in human periodontal diseases is not clearly defined. In the present study, the concentration of LPS in whole saliva, including the extraction and purification of LPS from Fusobacterium, the toxic activity of the preparation and the serum antibody titers against LPS were investigated. The histo-pathological changes in the periodontal tissues due to consecutive injections of LPS were also evaluated. The following results were obtained in the present study:
1. The concentration of LPS in human whole saliva ranged from 0.5 to 7.0μg/ml with correlation noted between the concentration of LPS, plaque accumulation and the severity of gingival inflammation.
2. Purified LPS preparation, extracted from Fusobacterium nucleatum, exhibited high toxic activities, as compared with Escherichia coli endotoxin which is commercially available.
3. The LPS preparation showed lower cytotoxic effects on HeLa cells with an LD₅₀ concentration calculated from cellular death rate at 489μg/ml.
4. The LPS preparation induced chemotaxis of polymorphonuclear leucocytes through interactions with serum.
5. Specific serum antibody responses for LPS were demonstrated in IgM fraction with correlations recognized between antibody titers and severity of periodontal diseases.
6. Consecutive injections of LPS into gingival sites of normal rat induced severe inflammation of gingival tissue, degeneration and necrosis of osteoprogenitor cells followed by osteoclastic bone resorption.
7. Consecutive injections of LPS into gingival sites of preimmunized rat with LPS induced a severe Arthus type of hypersensitivity in gingival tissue and early osteoclastic bone resorption as compared with normal rat.

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1. The first experiment was an in vivo study made using weekly observations of the interrelationship between the wear of toothbrush bristles and toothbrushing force.
The results obtained were as follows:
1) The toothbrushing forces employed by the subjects tended to increase slightly as the bristles became worn.
2) The toothbrush bristles spread out very little regardless of toothbrushing forces.
3) Scanning electron microscopic observations showed no wear in the tip of the bristles themselves after 2 weeks when weak toothbrushing forces were used. However, at 4 weeks the tip of the bristles were observed to overspread slightly.
However, when stronger brushing forces were used, wear of the tip of the bristles was clearly observed at 2 weeks, and at 4 weeks the round cut off tip of the bristles was observed to overspread considerably.
4) Hardness of the toothbrush bristles was observed to decrease with the use of the toothbrush.
2. The second experiment was an in vitro study observing the interrelationship between the wear of the toothbrush bristles and their plaque removing effect.
The results obtained were as follows
1) The actual scratching area was found to decrease for used toothbrushes for all values of brushing force.
2) Quantitative measurement showed that less gold coating was removed by used toothbrushes for all values of brushing force.
3) Hardness of the toothbrush bristles was observed to decrease with the number of brushing trokes.

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In teeth extracted for periodontal disease, cleansing, root planing and root planing of the denuded dentin were undertaken for the denuded and undenuded root surfaces. The permeability of ³⁵S (Na₂ ³⁵SO₄) into the root surface was examined by autoradiography in a polished fragment.
The following results were obtained:
1. ³⁵S seemed to adhere to or slightly permeate the root surface of both dentin and cementum.
2. In cases in which the root surface was the cementum, there was no marked difference in the adhesion or permeability of ³⁵S into the root surface between cleansing and root planing.
3. The adhesion and permeability of ³⁵S into the root surface of dentin were markedly less than those into the root surface of cementum.
4. Considering the various treatments of the root surface, there was no marked difference in the adhesion or permeability of ³⁵S between the denuded and undenuded root surfaces.

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The aim of this study was to evaluate the functions of salivary neutrophils in comparison with peripheral blood neutrophils. For this purpose, we investigated the activity of attachment to foreign bodies, and the oxidative metabolic process induced in the neutro phils.
The experimental methods were detection of IgGFc and C3b receptors on the cell surface of neutrophils by rosette formation, and measurement of luminol dependent chemiluminescence (LDCL).
The results obtained were as follows;
1. Rosette formations were not significantly different in Eagle's MEM suspension.
2. LDCL levels were lower for salivary neutrophils than for peripheral blood neutrophils in Eagle's MEM suspension.
3. In mixed salivary suspension, both rosette formations and LDCL levels fell remarkably, particularly with peripheral blood neutrophils.
From these results we conclude that saliva may exhibit a toxic effect on neutrophils that flow from gingival crevices, and thus the functions are greatly diminished. This suggests that salivary neutrophils may not play an important role in the host defensive system.

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It is necessary to investigate the voltage and density dependence of the Wiener spectrum, because these factors are considered to have a direct influence when the bone trabecular pattern after bone graft is analyzed by the Wiener spectrum method.
Changes in the Wiener spectrum based on variations in voltage and optical density were determined with the use of X-ray films of the edentulous mandibular bone obtained by standardized radiography.
The results of the experiment showed that the Wiener spectrum is influenced more greatly by optical density than by voltage. The influence of this factor was particularly greater for the range of spatial frequency above 5LP/mm, representing noise components, than for that under 5LP/mm, representing bone trabecular components.

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Microorganisms from periodontal pockets of patients with advancing periodontitis after flap operation were examined. Mean values of Plaque index, Gingival index and Pocket depth were 0.42, 0.06 and 2.4mm, respectively. There was no difference in the ratio of anaerobes against total isolates (75%), compared with those after deep scaling (77.6%). However, isolation rate of Gram-positive rods were prominently higher in this experiment (anaerobes, 40.9%; facultatives, 20.5%). Actinomyces (20.5%), Eubacterium (19.1%), Bacteroides (15.9%) and Peptococcus (13.0%) were most prevalent and predominant. These results indicate that flora constituents after flap operation are different from those of healthy gingival crevice.

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Trichomonas tenax (T. tenax) is the sole flagellate founed in the human oral cavity and is well known to exhibit a high incidence in staining plaque samples obtained from patients with advanced perio dontal disease. In spite of these evidence, the pathogenicity or biological characteristics of T. tenax are still unknown because of the difficulty in longitudinal cultivation and axenic culture. Previously, a new media (EY medium) for cultivation of T. tenax was developed in our laboratory, and ascertained that EY medium was not only useful for isolation of the flagellate from oral materials but alsomaintained their growth. In the present study, the epidemiological incidence and distribution of T. tenax was determined and compared to known clinical features.
The results obtained disclosed that the incidence of T. tenax found in subgingival plaque samples is closely correlated with the age of the subjects, pocket depth, severity of gingival inflammation and plaque accumulation at collected sites. The clinical symptoms were much improved by periodontal treatment with disappearance of T. tenax from the pocket.

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New connective tissue attachment is the one of attachment types established following flap surgery. The question still remains, however, whether cementum deposition will take place in that area or not. The purpose of this study is to clarify the possibility of cementum deposition in future.
The maxillary lateral incisors and canines in three monkeys were used for experimentation. Following elevation of a mucoperiosteal flap, the buccal and proximal alveolar bone was removed within an area extending from the mid-root level to a level 2mm apical to the marginal bone crest. Following bone removal, the root surfaces were curetted in order to remove cementum. After inscribing notch the flap was sutured back into place, 1, 2 and 4 months biopsies including the tooth, gingiva and bone were centrally fractured in bucco-lingual direction, parallel to the long axis of the tooth. Subsequently, these specimens were processed for electron probe analysis.
At new connective tissue attachment area P and Ca did not increase its concentration until four months afterwards and no significant difference was also found between coronal and apical area. These results indicated that new cementum would not deposit at this area in future.

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Ultrastructural and immunohistochemical investigations were performed on 2 cases of acute necrotizing ulcerative gingivitis (ANUG). Results obtained were as follows:
1. Lesions of necrosis were composed of large numbers of spirochetes and neutrophils in addition to small numbers of fusiform, rod-like and coccal shaped organisms.
2. Spirochetes that contained protoplasmic cylinder, cell envelope and axial filament had a width of 0.3-0.4μ and invaded in rarely intercellular spaces and within epithelial cells that appeared to have relatively normal structures of epithelial arrangement.
3. Spirochetes were often phagocytized by neutrophils and macrophages.
4. Immunoglobulin positive cells in lamina propria of ANUG were predominantly of IgG, followed by IgA and IgM.
5. Increased numbers of IgA positive cells were found in ANUG compared to periodontitis. Nature of IgA was a monomeric form suggestive of hematogenious origin.

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This study was carried out in order to establish a reliable method of radiographical quantitative analy sis, which provided a correct judgement on the prognosis after hydroxyapatite application. Hydroxyapatite was applied to experimental periodontitis in beagle dogs, then chronological radiographs were taken with a grey-scale under a standardized method. After densitometric measurements of images on the standardized dental radiographs were transformed into micrometers of copper equivalents by microphotometer on-lined with microcomputer, changes of the objective sites were expressed in numerical value.
The results obtained were as follows.
1. Alterations in density of bone defects due to hydroxyapatite-filling were quantitatively estimated.
2. Fluctuations of the numerical value measured on control case and hydroxyapatite-filling case were different, though bone repair seemed to finish similarly in both cases. The numerical value at the bottom of the bone defect in control case became lower than in coronal parts. On the other hand, the value of each part of hydroxyapatite-filling case presented approximation. These findings suggested that hydroxyapatite brought more natural feature to the bone repair.
3. Chronological changes of the numerical value after the filling of hydroxyapatite were classified into three patterns, which were related with situations of hydroxyapatite in the bone defects. The first pattern, which was seen at coronal part of the defect was that the numerical value increased gradually. It was speculated that in this part hydroxyapatite was discharged and the bone repair would not be expected. The second pattern was observed at central part. The numerical value which was elevated initially showed decrease in the passage of time, perhaps this change was resulted both from loosening of hydroxyapatite granules and advance of the bone repair. The numerical value at the bottom of the defect presented the third pattern that it was reduced by degrees and this findings suggested that the bone repair had been in progress.

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The purpose of this study was to evaluate clinical responses of periodontal tissue to Porus Hydroxyapatite Ceramics (P.H.A.).
Twenty-four patients (twenty-eight defects) with advanced periodontal destruction were used in the study. After initial preparation, P. H. A. were implanted into periodontal osseous defects.
Various clinical examinations were performed before the surgery, during the surgery, 1 week, 2, 3, 6 and 12 months after the surgery.
The results are as follows:
1. There was minimal pain and no evidence of infection or swelling. P. H. A. appeared to be well adapted by the periodontal tissues.
2. At 12 months after the surgery, the mean value of pocket depth was decreased by 2.5mm and of clinical attachment level was gained by 1.5mm.
3. Tooth mobility was reduced in all teeth evaluated.
4. In order to obtain the best effect of P. H. A., it was necessary to keep a stable condition and to maintain a high standard of oral hygiene.

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To survey the prevalence of periodontal disease in a nursing home for aged patients, the number of remaining teeth were examined, except for completely edentulous and severely disabled patients. Plaque and gingival indices were recorded in 44 patients.
The results obtained were as follows:
1. The average number of remaining teeth was 7.98. It was demonstrated that in older-aged male patients, the number of remaining teeth was lower than those of younger-aged patients. In contrast, this tendency was not demonstrated in the female aged group.
2. Mandibular canines were the most prevalent of remaining teeth.
3. In spite of a generally high plaque index, a correspondingly high gingival index was not demonstrated.

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The aim of this study was to investigate epidemiological characteristics of periodontal status in the late teens in Japan. The survey was conducted on 1469 senior high school students aged 15-18 years living in Osaka, Japan.
Pocket depth was measured in mm at the maxillary and mandibular first molars and central incisors of all subjects. Sixty students were chosen at random out of a total of 1469 students, and Gingival Index (Löe and Silness) was recorded on maxillary right first molar, left central incisor, first premolar, mandibular left first molar, right first premolar and central incisor. A statistical analysis was carried out, and the following conclusions were drawn:
1. The frequency of pockets deeper than 4mm and 5mm were 2.4% and 0.3% respectively.
2. There was no significant difference among the age groups with regard to pocket depth, on the other hand, boys had significantly deeper pockets than girls.
3. Comparison of the pocket depth of interproximal surfaces of central incisors with that of central area revealed that the former was significantly deeper than the latter, and comparison of pocket depth of first molars with that of central incisors also showed that the former was significantly deeper.
4. Gingivitis was most frequently observed on interproximal surfaces. Femals had higher prevalence but less severe gingivitis than males. The incidence of gingivitis increases with increasing age.
5. A total of 45 students (approximately 3% of subjects examined) had pockets deeper than 5mm on at least one tooth, and 44 of them were further examined clinically to singled out of the cases fulfilled following criteria: (1) No significant medical abnormalities were found; (2) The subject had pockets deeper than 5mm at least three teeth; (3) Local irritants, such as subgingival plaque and calculus, must not commensrate with the deepened pockets. Two girls who were fulfilled those criteria were examined radiografically and detectable vertical bone loss was not demonstrated around any of their teeth. So they were not accepted as juvenile periodontitis.

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The purpose of this study was to investigate the efficacy of this WHO CPITN (The World Health Organization, Community Periodontal Index of Treatment Needs) for the epidemiological survey of periodontal diseases in Miyagi Prefecture. The CPITN survey was performed on 1390 persons aged between 12 and 70 using the WHO periodontal examination probe. Records were taken in each sextant (i.e. sixth of the dentition) according to the presence or the absence of the following items: (1) gingival bleeding after probing, (2) supra or subgingival calculus, (3) pocket depth between 4 and 5mm and, (4) pocket depth between 6mm and more. Six groups (12-14, 15-19, 20-29, 30-44, 45-64, and 65-70y) were chosen in this survey.
Thirty percent of the subjects had pocket depth between 4 and 5mm and the mean number of sextants affected was 2.0 in teenagers. Pocket depth increased in accordance with the increase of age. Eighty percent of the subjects aged between 65 and 70 years had pocket depth of 4mm or more and the mean number of sextants affected in this age group was about 3.5.
Subjects who required the need for oral hygiene instruction (TN 1) were about 80 percent of teenagers and almost 100 percent in age groups more than 30 years. An increase of subjects required a combination of professional and personal cleaning of teeth became apparent in age groups more than 20 years and the percentage of subjects went up to approximately 50 in the age group of 65-70y.
The CPITN is useful for the primary screening of periodontal diseases in large groups, therefore, the index must be valuable for international comparison of periodontal condition.

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The development of a new occlusal sound checker has been taken place. This study was to evaluate the duration of occlusal contacts under various conditions using the equipment. The points of improvement were as follows:
1. A new accelerated ceramic microphone was reformed to cut off outside noises.
2. A control system was set up to input the sensitivity.
3. The tape recorder was introduced to record the occlusal tones.
4. The computeti zed data analysis was introduced to measure a duration of occlusal tones.
In consequence, it was became clear the path for the computetized data analysis on the duration of occlusal tones.

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