The present study examined the migration, orien-tation of cultured human gingival fibroblasts and in vitro formation of a new fibrous attachment to experimentally treated cemental surfaces of impacted and periodontally diseased roots. Trans-versally cut root slices of human teeth, 100μm thick, were prepared from impacted and peri-odontally involved teeth that were planed with hand scalers previously. Root slices were demineralized in citric acid and then attached to sheets of fibrob-lasts in culture dishes. The cell cultures were examined daily by phase contrast- and dark field-microscopy. After 4h of culture, fibroblasts had migrated and attached, were oriented approximate-ly perpendicular to the partially demineralized root surface, and had produced extracellular matrices. Sheets of cells and fibrial materials anchored between the cementun surface and the floor of the dish were formed, and newly secreted collagen fi-brils continuous with the exposed cementum col-lagen were observed after 1 week. There was no significant difference in the percent increase in cell attachment to dental roots between impacted and periodontally involved teeth after planing and par-tial demineralization of root surface cementum. These results suggest that the treatment by planing and partial demineralization of the root cementum of periodontally involved teeth gave a good condi-tion for attachment of fibrous tissue. This culture system is a useful model system for in vitro assess-ment of the treatment of periodontally diseased root surfaces and determing the mechanisms operat-ing in fibrous attachment to root surfaces.

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Lipopolysaccharide (LPS) plays very important roles in periodontal disease and osteoclastic resorp-tion of alveolar bone. Although LPS stimulates osteoclastic bone resorption in vivo and in vitro, its mechanism is not clear. Recently we showed that long-term human umbilical cord blood and bone marrow cell cultures can adapted to form multinu-cleate cells (MNC's) that express an osteoclast phenotype (resorption lacunae on calcified matrices and cross-reactivity with mAb 23 C 6: CD -51, a monoclonal antibody that preferentially binds osteoclasts in bone biopsy specimens), and we also suggested that the CFU-GM (the granulocyte-macrophage progenitor cell) is the progenitor for the osteoclast. In the present experiments, we studied the biological effect of LPS, an extracel-lular product from Porphyromonas gingivalis (P. gingivalis) and Escherihia coli (E. coli), on the mechanism of osteoclast formation from CFU-GM-derived cells and on bone resorption. Addition of LPS (10-100 ng/ml) from either of these bacteria to these cultures significantly increased the forma-tion of 23 C 6-positive MNC's. Addition of anti-human IL-1 antibody to cultures treated with LPS totally inhibited the increase in MNC formation stimulated by either LPS. Both LPS's stimulated bone resorption, but the P. gingivalis LPS caused a 1.4 fold greater increase in the resorption area compared with the E. coli one. The effects of LPS on regulation of tyrosine phosphorylation were also studied in experiments utilizing osteoclast precur-sor cells. The phosphorylation was detectable in LPS-treated CFU-GM cells. When these cells were incubated with LPS from P. gingivalis, a 42-kD protein band containing phoshotyrosine was detected. Addition of 100 nM herbimycin A to cultures treated with either LPS totally inhibited the bone resorption stimulated by these LPS's. However, herbimycin A did not inhibit the MNC formation. These experiments suggest that LPS stimulates osteoclast formation through a primary action on osteoclast precursor cells, which cells are induced by the action of IL-1 produced by the LPS which in turn stimulates osteoclast formation, resulting in osteoclastic bone resorption. These findings also suggest that c-src kinase is involved in the regulation of bone-resorbing activity of osteoclasts by LPS.

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he purpose of this investigation was to deter-mine the effects of extracellular vesicles (ECV), suspected of having the potential to enhance the periodontal disease process, on polymorphonuclear leukocyte (PMN) functions. We determined the effects of ECV derived from Porphyromonas gin-givalis (P. gingivalis), a suspected periodontal pathogen, on superoxide production, phagocytosis and chemotaxis of PMN. Moreover the interac-tion between ECV and PMN was observed by trans-mission electron microscopy (TEM). ECV from P. gingivalis W 50, W 83 and ATCC 33277 were extracted from the culture supernatant by the ammonium sulfate precipitation procedure. PMN were incubated for 60 minutes with ECV from P. gingivalis W 50. TEM observations showed that PMN phagocytosed ECV. Superoxide production by PMN was assayed by the dose response of ECV from three P. gingivalis strains and was evaluated by the cytochrome c reduction procedure. ECV from the three P. gingivalis strains had little effect on the superoxide production by PMN, but dose dependently significantly depressed the superoxide production by PMN stimulated with N-formyl-methionyl-leucyl-phenylalanine or phorbol myris-tate acetate. PMN were incubated for 60 minutes with three P. gingivalis ECV. ECV depressed the PMN phagocytosis and chemotaxis, but not signifi-cantly. These results suggest that superoxide pro-duction, phagocytosis and chemotaxis by PMN are impaired by P. gingivalis ECV in the periodontal pockets or periodontal tissues and that ECV have the potential to enhance the periodontal disease process.

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The ability of tetracyclines (TCs) to induce chromosome aberrations was examined by using cultured human fibroblasts (HFG/MS cells) derived from gingival tissues. Treatment of HFG/MS cells with tetracycline. EHCl (TC), chlortetracycline. EHCl (CTC), demeclocycline. EHCl (DMC) or minocycline. EHCl (MINO) at 100-1, 000. μM for 48 h reduced colony-forming efficiency (CFE) in a dose-dependent manner. The rank order of the inhibitory effect of TCs on CFE was CTC< MINO<TC<DMC. No significantly different increases in the frequency of chromosome aberrations were observed in HFG/MS cells treated with any compound at 100-1, 000. μM for 24 or 48h. In addition, chromosome aberrations were not observed in HFG/MS cells following treatment with TCs at 10-100. μM for 1h in Ca²⁺-and Mg²⁺-free phosphate-buffered saline. No significant increases in the frequency of chromosome aberrations were detected when HFG/MS cells were treated with MINO at 100-1, 000. μM for 2h in the presence of exogenous metabolic activation mediated by rat liver post-mitochondrial supernatant. The results indicate that there is no clastogenic activity of TCs against cultured human fibroblasts from gingival tissues under certain conditions.

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It has been reported that laser irradiation har-dened enamel. If laser irradiation could also harden cementum. then it would be beneficial in the prevention of root caries and periodontal disease. The purpose of this study was to compare the changes in microhardness of cementum after Nd: YAG laser irradiation : to measure Ca/P ratios after laser irradiation using XMA and to observe the cementum surface structure using SEM. Per-iodontally healthy single rooted teeth were used in this experiment. After extraction of the teeth. remaining remnants of soft tissue were removed useing collagenase. Seventy-five flat cementum surface sites (3× 3mm) selected for the experimet-nts. The roots were cut into 5× 5mm segments. Among them. 75 flat segments were selected for the experiment. Twenty-five segments served as the control (10 for microhardness. 10 for Ca/P. 5 for SEM observation). Fifty segments were used for laser irradiation at 30mJ 10 pps for 30secs (10 for microhardness. 10 for Ca/P. 5 for SEM) and 25 segments were lased at 50mJ 10 pps for 30secs (10 for microhardness. 10 for Ca/P. 5 for SEM). After irradiation. the roots were cut into 5× 5mm seg-ments for measurement of microhardness. Knoop hardness was measured with a 25-gram-load 3 times on each segment. The remaining roots were cut into 3× 3mm segments for analysis of Ca/P ratios and SEM observation. The Ca/P ratios of each segment was measured by XMA (JEOL. Tokyo) The average microhardness of cementum (control) was 24.95±6.59Hk. The average of microhard-ness after laser irradiation was 39.58±16.16Hk for the 30mJ group (p<0.01) and 51.42±20.93Hk for the 50mJ group (p<0.01). The average of the Ca/P ratio (control) was 1.76. The average of the Ca/P ratios for the 30mJ and 50mJ group 1.78 and 1.84 for the 50mJ group (p<0.05). There were no topographical differences between the con-trol and the 30mJ segments. Melting and resolidification of the cementum surface were ob-served on the 50mJ segments. These results sug-gested that laser irradiation at 30mJ 10pps made the cementum surface resistant to demineralization without topographical change on the cementum surface.

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Previously, we reported that the electric tooth-brush is as effective as the manual toothbrush in improving the periodontal condition of patients when used for a short period. The purpose of this study was to compare the effect of electric and manual toothbrushes on various clinical parameters of patients receiving initial treatment and mainte-nance therapy for two years. Twenty-three patients received tooth brushing instructions for using INTERPLAK™ (IP, BAUSCH & LOMB, USA) followed by initial treatment and mainte-nance therapy. The patients used IP 2t home dur-ing the intial treatment and 24 months of mantenan-ce therapy. Fourteen patients who were instructed in plaque control using a manual toothbrush and received initial treatment and maintenance therapy served as a control group. They used manual toothbrushes at home during the initial treatment and maintenance therapy. Clinical assessments (plaque control record, probing depth, bleeding on probing) were made before and after the initial treatment and affer 6, 12 and 24 months of the manitenance therapy. Clinical conditions were im-proved significantly by the initial treatment and the good conditions remained during 24 months of maintenance therapy. These results were compa-rable with those for patients using manual tooth-brushes. It is suggested that an electric toothbrush is as effective as a manual toothbrush for self care of the patients receiving initial treatment and maintenance therapy.

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We investigated the survival of teeth that were subjected to a flap operation with implantation of hydroxyapatite (HAP) granules in periodontal os-seous defects. For 70 cases over a five-year period, the survival rates for individual tooth type were determined every year. The influence of the fol-lowing conditions on survival of the teeth was considered: 1) whether the patient recall at the appropriate interval was consistent; 2) whether the tooth was splinted; 3) whether the tooth had furcation involvement; and 4) how deep the prob-ing pocket depth was at the flap operation. The survival rate for all teeth was 87% after five years, and it decreased with the passing years. The rate decreased drastically after 7 to 8 years, and it was 47% after 10 years. The survival rate by individual tooth type was 44% for upper molars, the lowest among all types of teeth; 75% for upper incisors and canine teeth; 91% for lower incisors and canine teeth and 72% for lower premolars. Teeth with furcation involvement tended to be lost. Keeping a appointment recall, splinting, and probing pocket depth at the operation appeared to have little effect upon tooth survival. Thus, despite the conditions of the teeth, HAP implant applied to a single-rooted tooth with a periodontal osseous defect resulted in a good long-term prognosis.

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Periodontal pockets of 38 periodontal patients who had received no antibiotic therapy within 6 months of sampling, were examined for tetracycline-resistance genes (tet M and tet B) and the Actinobacillus actinomycetemcomitans gene by the polymerase chain reaction. Positive rates of tet M and tet B were respectively 60.5% and 34.2% (significant, chi square test, p<0.05). Positive rates of tet M were 31.6% in shallow pockets (=4 mm, n=19) and 89.5% in deep pockets (>4mm, n=19) (significant, chi square test, p<0.01). Pos-itive rates of A. actinomycetemcomitans were 44.7% in all patients. Positive rates of tet in A. actinomycetemcomitans-positive sites were 52.9% (tet M) and 41.1% (tet B). Tetracycline-resistance genes (tet M and tet B) seemed to already exist at high rates in periodontal pockets before antibiotic therapy. Their presence seemed to depend on the tet kind and the circumstances. Periodontal pathogenic microbes such as A actinomycetemcomitans may acquire the tet-racycline-resistance gene from tet-positive microbes.

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Interleukin-1 (IL-1), IL-1 α and IL-1 β are known to play an important role in the progression of periodontal inflammation, and an IL-1 receptor. antagonist (IL-1ra) has been identified as a spe-cific inhibitor of IL-1 α and IL-1 β This study was designed to investigate the relationship between the levels of IL-1 α, IL-1 β and IL-1 ra in gingival crevicular fluid (GCF) and the clinical indices of the patients with adult periodontitis. Eighteen inflamed moderate pockets (MP) (3mm≤probing depth < 6mm, 3mm≤clinical attach-ment loss < 7mm, positive bleeding on probing) and nine shallow pockets (SP) (probing depth < 3mm, clinical attachment loss < 3mm, negative bleeding on probing) were selected from nine patients with adult periodontitis after completion of oral hygiene instructions. Clinical examination and GCF sam-pling were performed before and after scaling/root planing. The levels of IL-1α, IL-1β and IL-1ra in GCF were determined by the sandwich ELISA system. The results showed that the amounts of IL-1α and IL-1β in MP were significantly higher than those in SP (P<0.01). The amount of IL-1 ra in MP was higher and the ratios of IL-1 ra to IL-1α and/or IL-1β in MP were lower than those in SP, however, the differences were not significant. Following scaling/root planing in MP (n=16), the amounts of IL-1α and IL-1β were significanly reduced (P<0.01) and the amount of IL-1 ra was slightly decreased, while the ratios of IL-1 ra to IL -1α and/or IL-1β were significantly increased (P<0.01). Clinical indices showed significant improvement after the scaling/root planing. These results suggest that the ratio of IL-1ra to IL-1α and/or IL-1 β may be related to the change in gingival inflammation in periodontitis.

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The guided tissue regeneration (GTR) procedure has been reported to result frequently in membrane exposure by sloughing. Barrier membrane expo-sure increases the incidence of infection and decreases the amount of regenerated tissue. It is difficult to cover the regenerated tissue after mem-brane removal in the GTR procedure because of the large amount of sloughing. We use a subepithelial connective tissue graft after membrane removal to protect the regenerated tissue. A 42-year-old woman presented with gingival swelling, which was diagnosed as moderate adult periodontitis. Follow-ing initial therapy, the GTR procedure using a non-resorbable membrane (Gore Tex Periodontal material) was performed for a maxillary first premolar with an intrabony defect. Barrier mem-brane exposure due to sloughing of the flap was evident at 1 week. We removed the membrane at 5 weeks because increasing of slouging and mem-brane exposure. There was rubbery hard regener-ated tissue under the barrier membrane. However it is impossible to cover regenerated tissue with a flap. We used a subepithelial connective tissue graft to cover the regenerated tissue. A good shape of the gingiva could be obtained by means of graft. This report suggests that a subepithelial connective tissue graft for membrane exposure in the GTR procedure is one of the useful clinical options that protect the regenerated tissue and achieves an esthetically pleasing result.

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