The mechanism of osteoclastic bone resorption induced by periodontopathic bacteria Actinobacillus actinomycetemcomitans cell-surface components was investigated in mouse organ cultures and in a mouse bone marrow culture system. Lipopolysaccharide and capsular polysaccharide isolated from A. actinomycetemcomitans Y 4 induced bone resorption in C 3 H/HeN mouse calvarium organ culture. Many multinucleated cells were formed when mouse bone marrow cells were cultured with A. actinomycetemcomitans Y 4 capsular polysaccharide or lipopolysaccharide for 9 days. The multinucleated cells showed several characteristics of osteoclasts, the production of including tartrateresistant acid phosphatase and the ability to resorb calcified dentine. in this study, monospecific antimouse recombinant interleukin-1 a serum and indomethacin completely inhibited the formation of osteoclasts in the presence of A. actinornycetemcomitans Y 4 capsular polysaccharide. An IL-1 receptor antagonist significantly inhibited the osteoclast formation in mouse marrow cultures. Bioactive IL-1 was detected in the culture supernatant of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y 4 capsular polysaccharide. These results indicate that IL-1 a is involved in the mechanism of formation of osteoclast-like cells induced by A. actinomycetemcomitans Y 4 capsular polysaccharide. There was also a good correlation between the number of osteoclasts formed in the mouse marrow culture and the amount of prostaglandin E₂ released into the culture medium. These findings suggest that A. actinomycetemcomitans capsular polysaccharide may play an important role in inflammatory bone resorption by promoting osteoclast formation in periodontal diseases.

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The qualitative composition of the microorganisms in the periodontal pocket changes with the progression of periodontal disease. Actinomyces viscosus is involved in the etiology of human gingivitis but not periodontitis. Porphyromonas gingivalis is considered to be a periodontal pathogen. Two bacterial species may interact with each other at the stage of transition from gingivitis to periodontitis. The purpose of this study was to investigate the antibacterial substance against P. gingivalis produced by A. viscosus. In cross inhibition tests, A. viscosus greatly inhibited the growth of P. gingivalis. An antibacterial substance was then extracted from the medium in which A. viscosus T14AV had been cultured anaerobically. The material was purified by ammonium sulfate precipitation, anion exchange chromatography on DEAE sepharose CL-6 B, hydrophobic interaction chromatography on phenyl sepharose CL-4 B and reverse phase high performance liquid chromatography. The activity of the material was evaluated by the diffusion method with P. gingivalis 381 as the indicator strain. Specific activity increased 378-fold with a 26% yield. The material had a molecular weight of 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and had a bactericidal effect on the indicator strain. The activity was lost by trypsin or heat treatment at 90.. for 5 min, but not by endo-a-N-acetylgalactosaminidase, neuraminidase, O-glycanase and N-glycanase. These findings suggest that A. viscosus produces the antibacterial substance against P. gingivalis.

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The purpose of this experiment was to clarify the influence of antibody and complement on the superoxide production [Chemiluminescence (CL) response] and periodontopathic bacteria phagocytosis and killing by polymorphonuclear leukocytes (PMNs). PMNs were obtained from the peritoneal cavity of ICR mice after caseinate injection. Three species of periodontopathic bacteria were used : Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn). Mice were immunized with live Aa, Pg or Fn vaccine. Serum was obtained from the blood by cutting the carotid artery. Normal serum was used as complement and inactivated immune serum was used as the antibody. The antibody titers were measured by ELISA using sonicated Aa, Pg and Fn antigen. Serum with a high antibody titer was obtained. IgG levels were high when mice were immunized with either Aa or Pg, whereas IgM levels were high when immunized with Fn. The reaction mixture for the CL response consisted of luminol solution, serum, gelatin Hanks' solution, PMNs and the bacteria. After the CL response was complete, a portion of the reaction mixture was smeared on a glass slide, dried and stained with May-Gruenwald-Giemsa stain. Phagocytic activity was assessed using a microscope. Surviving bacteria were counted on solid medium after treatment with serum, antibody and PMNs alone or in combination.
An increase in the CL response to Aa occurred and was dependent on the addition of complement, and the response to Pg was dependent on both complement and antibody whereas the response to Fn dependent on neither complement nor antibody. The CL response to Fn was inhibited by Dgalactose, N- acetyl-D-galactosamin and mannan. Phagocytic rate of PMN and the number of Aa and Fn bacteria in PMNs was enhanced in the presence of complement. The co-operation of both complement and antibody was needed to increase the phagocytic ratio. The bactericidal effect of complement, antibody and PMN were examined in single or combination mixtures. There was no marked effect on killing the bacteria in any combination.
In conclusion, complement plays the role of enhancing the CL response and phagocytic activity in response to Aa. Co-operation of complement was necesary for phagocytosis of Fn. High titer antibody, complement and PMN had no bactericidal effect under the experimental conditions.

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The aim of this study was to examine whether new fibrous attachment accompanying keratinized tissue can be achieved following guided tissue regeneration (GTR) treatment of buccal gingival recession defects created in dogs. The recession defects were created with osseous and gingival resective surgery on the buccal aspect of the mesial root of the mandibular aourth premolar and first molar. To achieve experimental periodontitis, a ligature wire was tied at the cervical area to promote plaque accumulation for 4 months. Gingival health was reestablished prior to GTR membrane application by scaling, root planing, and oral hygiene procedures. All defects were managed with coronally positioned mucogingival surgery. Expanded polytetrafluoroethylene membranes were applied for 4 weeks to each randomly selected defect in the test group. In the control group, without membrane application, flaps were adapted as closely as possible to the tooth surface. The dogs were sacrificed for specimen preparation and histological evaluation 8 months after the beginning of the experiment. The test group demonstrated new fibrous attachment with newly formed cementum, alveolar bone and keratinized tissue, while the healing type of long junctional epithelium without new bone formation was primarily observed in the control group. The results indicate that applying the GTR membrance in the treatment of gingival recession defects can enhance new fibrous attachment formation accompanying with newly formed bone and cementur.

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We previously reported that periodontium-derived cells could form spheroids and that the spheroids could be used for periodontal reconstructive therapy because they contained cells with proliferative activity and which produce extracellular matrix. In this study, we performed an immunohistochemical study on spheroids formed by human periodontal ligament fibroblasts (HPLFs), which play an important role in periodontal reconstruction, using the antibodies against osteocalcin (OC) and fibroblast growth factor (FGF). The results showed that HPLF could produce OC in the spheroid environment. Much more OC expression was observed on day 40 after spheroid formation, than on day 7. FGFs, which maintain tissue construction, and growth factor for mesenchymal cells, were also observed. We investigated the viability of the cells in the spheroids by using a fluorescent material fluorescein diacetate-propidium iodide; FD (A-PI), and found that almost all cells survived and that no dead colonies were observed. The finding that, HPLF in spheroids can express OC in vitro, and that OC expression appears to be affected by the spheroid environment. FGFs were also contained in the spheroids, so spheroids may be capable of being applied to periodontal reconstructive therapy.

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The purpose of this investigation was to determine the effect of Porphyromonas gingivalis extracellular vesicles (ECVs) on membrane fluidity and superoxide production in neutrophils.
The ECVs were extracted from culture supernatant using the ammonium sulfate precipitation procedure. Transmission electron microscopic observations, SDS-PAGE profiles, and chemical composition analysis identified the ECVs prepared from the outer membrane of P. gingivalis ATCC 33277. Neutrophils were isolated from the fresh heparinized venous blood of healthy volunteers by the combined dextran/Ficoll sedimentation method. Membrane fluidity and superoxide production of isolated neutrophils were assayed in the increasing concentrations of the ECVs. The membrane fluidity of neutrophils was measured by spin-labeling neutrophils with stearic acids, consisting of nitroxide-free radicals bound to the 5, 16 position of stearic acids (5-, 16-SAL), to detect reductions of the nitroxide radicals at different positions in the membrane. Superoxide production by neutrophils was evaluated by the cytocrome C reduction procedure.
ECVs significantly depressed membrane fluidity in a shallow portion (5-SAL) of lipid bilayers, but had no effect on membrane fluidity in the deep portion (16-SAL) of lipid bilayers. Superoxide production by neutrophils after stimulation with n -formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate was significantly suppressed by increasing concentrations of ECVs.
These results indicate that the membrane fluidity and cell function of neutrophils are impaired by ECVs from periodontal pathogens in the periodontal pockets or periodontal tissues.

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The purpose of this study was to examine the cell population and bacterial contamination in the expanded polytetrafluoroethylene membrane (ePTFE) used for guided tissue reg eneration (GTR), and to compare the results with histopathologic and clinical findings. Eighteen periodontal defects were treated by a flap procedure which included the use of the ePTFE to allow GTR. The surgical sites were measured clinically. At 6 months, all sites showed an average 3.8±0.4mm pocket reduction with an average attachment gain 1.6±0.3mm. The ePTFEs were retrieved after 4 to 6 weeks of healing and sectioned serially at 3 μm in a coronal-apical plane. The sections were examined by light microscopy for the presence of oral bacteria and cells : monounuclear cells, erythrocytes, fibroblasts, neutrophils and plasma cells. The ePTFEs were divided into 6 portions: cervical, middle, apical, outer, central and inner portions, which provided a total of 9 fields. The number of cells and bacteria gradually decreased toward the apical portion. Both cells and bacteria were present even in the central portion. Mononuclear cells and erythrocytes were most encountered. Fibroblasts, neutrophils and plasma cells were rarely encountered. Most bacteria were gram-positive. The ePTFEs were markedly adherent and invaded by inflammatory mononuclear cells and bacteria during retention. Though clinical attachment gains were significant in all cases of 6 months post operatively, the degree of attachment gain was influenced by the bacteria adhering to the membrane. The results suggest that the efforts to reduce microbial colonization on the membrane should be made to ensure maximal periodontal regeneration.

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It has been reported that oral rinses extracted from green tea, which include catechin as a soluble ingredient for chemical plaque control, was effective in preventing experimental gingivitis in man. The purpose of this study was to investigate the effectiveness of an oral rinse containing 0.25% catechin for prevention of plaque formation, gingivitis and halitosis. The subjects were 42 adult volunteers ranging in age from 22 to 48 years without any relevant medical or pharmacotherapy histories. Seven days before each study period, the volunteers underwent removal of plaque and calculus. The subject then requested to refrain from their normal hygiene methods for 7 days. In the catechin group, the subjects were asked to rinse with 10 ml of mouthrinse containing 1.0% green tea extract (catechin). In the control group, the subjects used mouthrinse for which tea extract components except for catechin were eliminated. Both groups rinsed for 30 s three times a day for 7 days. The subjects were not allowed to use a toothbrush or other cleaning devices during the study periods. This results were assessed using a double-blind, two mouth rinses cross-over design balanced for the 1 st and 2 nd periods.
Clinical parameters (Plaque Index (PlI), Gingival Index (GI), gingival crevicular fluid level (GCF) and halitosis) were determined and microbiological samples were collected at the baseline, at 3 and 7 days after the start of the study. The oral rinse containing Japanese tea extract was found to be useful for prevention of halitosis. PlI and GI scores and GCF value in the catechin group were lower than in the control group during the study period. However, plaque formation and gingivitis were not control groups. significantly different between the catechin and

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Various reports have evaluated the cleaning effects of electric toothbrushes in comparison with those of manual toothbrushes. The present study was designed to determine the efficacies for cleaning at denuded root surface, who are often found in the practice of periodontal therapy. The aim of present study is to evaluate the efficacies of a counter rotational action powered toothbrush (Interplaque ®) for cleaning at denuded root surface on jaw models. The one in which the denuded root portion was exposed up to 1.5mm from the CEJ (1.5mm model) and the other with the root exposed up to 3.0mm from the CEJ- (3.0mm model). Normal gingival models were used as controls. In the experiment, artificial plaque was applied to the tooth surface, which then under a constant pressure of 250g, determined by a simple load. measuring device, for 15 sec per site using an electric brush, bristle tips of which were placed perpendicular to the tooth surface, and of 15 strokes per site using the manual brush according to the scrubbing method. Measurement was conducted at 61 4 4 16in each model. After brushing, teeth were placed on a standard stent, and pictures were taken of 4 different surfaces, mesial, buccal, distal and lingual, using a standard photographic apparatus. After printing, the area was measured using a digitalizer to calculate the rates of plaque removal by the electric and manual toothbrushes. Wilcoxon's test was used for statistical analysis. The following results were obtained. The mean total plaque removal rate was 94.2% for the electric brush and 79.4% for the manual brush in the 1.5 mm model, and 92.7% for the electric brush and 83.1% for the manual brush in the 3.0mm model, while the rate was 96.2% for the electric brush and 90.9% for the manual brush in the normal gingival model. When assessed according to the site of measurement, the plaque removal rate was higher with the electric brush than with the manual brush in all sites except the frontal teeth of the 1.5 and 3.0mm models. The differences in all sites except the frontal teeth of the 1.5 and 3.0mm models were statistically significant. These results indicate that, while plaque removal by the electric and manual toothbrushes differed only slightly in the anterior teeth, irrespective of the extent of gingival recession, the difference was greater in the molar region, where plaque removal was particularly effective on the proximal surface .

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The purpose of this study was to investigate the effect of removal of artificial plaque by ultrasonic cavitation from interproximal tooth surfaces with the interdental gingiva receded. For this purpose, we developed the newly tip capable of generating ultrasonic cavitation around the oscillating tip in water, and prepared two jaw models with different degrees of interdental gingival recession (exposed tooth crown surface and root surface). The tooth models in these jaw models were designated the upper central incisors (11, 21), the upper right canine and first premolar (13, 14), and the upper right first and second molars (16, 17). The areas of artificial plaque removed from the tooth crown and root surfaces were mesial in 11 and 14 and distal in 16, were momentarily measured in the two different methods to use the tip: the use by projection on one side, palatal or facial, and use on both sides. We also investigated the relationship between the rate of plaque removal and the shape of the interproximal space in each type of tooth. The following results were obtained. In the model with exposed tooth surface, the rate of plaque removal was the highest in 11 when the tip was used on the facial and palatal side. The reason was as follows: The distance between tips and the interproximal areas, and degree of the shadow for projection of cavitation at 11 were greater than other sites. In addition, the rate of plaque removal was the highest in 11 in the model with exposed root surface. These results suggested that the rate of plaque removed from all tooth models is affected by the distance between tips and interproximal areas or morphological factors in the interproximal space. When the tip was used on both the facial and palatal side, almost all the plaque was found to have been removed more effectively in a shorter time.

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One of the methods used for subgingival plaque control is a local drug delivery system into the periodontal pocket.
The purpose of this study was to compare the clinical effects of a newly designed Strip type Drug for Periodontitis (SDP) with minocycline hydrochloride dental ointment (Periocline ® : PER). SDP is a new degradable controlled release device containing Minocycline hydrochloride. SDP is formed as a strip type such that it is possible to ascertain whether the strip has reached the bottom of the pocket.
The patients participating in this study had chronic periodontitis with periodontal pockets exceeding 4 mm, but were free of systemic disease and had not been on antibiotic therapy within the preceding three months. SDP was applied in 33 cases and PER in 35. Both drugs were applied at baseline and at weeks 1, 2 and 3. Clinical evaluation was performed for 7 weeks, and microbiological evaluation was also performed at baseline and at weeks 4 using routine culture methods. The clinical variables used were clinical probing depth (PD), bleeding on probing (BOP), suppuration (5), plaque and gingival indices (PH, GI), and gingival crevicular fluid (GCF). Porphyromonas gingivalis (P. g.), Prevotella intermedia (P. i), Prevotella melaninogenica (P. m.), Capnocytophaga spp. (Cap.), Actinobacillus actinomycetemcomitans (A. a.), Eikenella corrodens (E. c.), and Fusobacterium nucleatum (F. n.) were isolated from the pockets using selective media, and Black Pigmented Gram-Negative Anaerobic Rods BPNAR) and total viable bacteria were meas u (red using non-selective media.
The following clinical results were obtained:
Significant reductions from baseline of BOP, S, GI and PD were observed in both groups at 3, 4 and 7 weeks, but a marked GCF reduction was observed only in the PER group. The rate of clinical improvement for each index between baseline and at each examination week was apparent in both groups, and no differences were observed between the two groups.
The following microbiological results were obtained:
Reductions in BPNAR and viable counts were observed between baseline and at 4 weeks, with no difference between two groups. Reductions in 7 pathogenic bacteria (P. g., P.i., P. m., Cap., A. a., E. c. and F. n.) were also significant between baseline and at 4 weeks in both groups.
From these results, it is clear that the newly designed local drug delivery system (SDP) effectively improves periodontal disease, with no major differences being observed between the strip type SDP) and the ointment type (PER).

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The effects of local drug delivery on the acute phase of periodontal disease were clinically and microbiologically studied using a resorbable minocycline-containing strip. The subjects of the study were 43 patients with no systemic disease and no history of antibiotic therapy within three months previously. All 43 patients complained of symptoms such as pain and tenderness from the acute phase area with occasional abscess formation. Minocycline containing strips were gently inserted till they reached the bottom of the pocket. Clinical changes were monitored on 0, 3 and 7 day after insertion of strips, and microbiological changes on 0 and 7 day. Probing depth (PD), pain, tenderness, bleeding on probing (BOP), suppuration (S) and the volume of gingival crevicular fluid (GCF) were recorded. Black Pigmented Gram-Negative Anaerobic Rods (BPNAR) and total anaerobic cultivable counts were made on non-selective blood agar.
Clinical symptoms such as pain, tenderness, BOP and S were significantly reduced on day 3 and 7 compared to the baseline. Significant reduction of PD and GCF were also found on 7 day. The prevalence of BPNAR was significantly decreased on 7 day compared to the baseline.
These results of the present study indicate that the application of resorbable minocycline containing strips to pockets in the acute phase may be effective in improving the acute phase of periodontitis.

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