Human gingival keratinocytes were cultured in vitro in serum-free medium. The survival of cells treated with tetracyclines (TCs) and the intracellular concentrations of the incorporated TCs were examined. Gingival keratinocytes, grown at clonal densities, were treated with minocycline (MINO), tetracycline (TC), or chlortetracycline (CTC), and cell survival was determined by the colony-forming efficiency of the cells. Treatment of cells with each agent reduced cell survival in a dose-dependent manner. The order of the TCs in relation to their reducing effect on cell survival was: MINO>TC> CTC. In addition, survival of cells treated with MINO was dependent on the concentration of Ca²⁺ in medium used in the experiments. The survival was reduced more in low-Ca²⁺ medium than in high-Ca²⁺ medium.
The concentrations of the TCs incorporated into the cells were determined by a fluorometric method. The intracellular concentrations of the TCs varied with the TCs tested. The highest concentration was observed in the cells treated with MINO, and intermediate and the lowest concentrations were detected in cells treated with TC and CTC, respectively. The order of the TCs in regard to incorporation into cells during the first 24h was as follows: MINO>CT>CTC. MINO persisted in the cells longer than TC. The results of this study indicate that the inhibitory effect of TCs on the survival of cultured human gingival keratinocytes varies with the kind of TCs, and that the effect depends on both the intracellular concentration of the TCs incorporated and the persistence of the TCs in the cells.

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The purpose of this study was to determine the effect of Bisphosphonate on alveolar bone regeneration after guided tissue regeneration. In 10 beagle dogs a two-wall alveolar bone defect was created adjacent to a mandibular premolar tooth after removal of a full thickness flap. The defect was then covered with resorbable membrane (Resolut ®), and the flap was replaced. The experimental group was injected with Bisphosphonate (Aredia ®) (0.6mg/kg) every 3 days and the control group was injected with saline. Peripheral blood was collected every week until 11 weeks after the operation. Standardized X-ray films were taken every week. Serum osteocalcin (OC) levels were evaluated as a marker of alveolar bone formation.
Serum OC levels were higher on day 3 after surgery than on day 0 and then decreased gradually in both groups. However, serum OC levels in the experimental group were higher than in the control group at one and two weeks. Standardized X-ray films in the experimental group showed higher bone density than in the control group from 3 to 5 weeks after the operation. These findings suggest that Bisphosphonate enhances the alveolar bone regeneration in the early period after GTR treatment.

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The purpose of this study was to identify changes in masticatory efficiency after periodontal treatment. The parameters of masticatory efficiency investigated were occlusal area, mean pressure, and biting force. Each of these parameters was measured in every tooth using the Dental Prescale ® (Fuji Photo Film Co., Ltd., Tokyo).
The changes in masticatory efficiency in the intial preparation of 75 teeth in 9 periodontal patients were first used to observe and analyze the changes in masticatory efficiency is this the intended meaning initial preparation. The changes in masticatory efficiency after periodontal surgery were measured in 42 teeth of 20 patients who had undergone flap operations were used to analyze changes over time. The following results were obtained.
Initial preparation resulted in 7.7% and 5.6% increases in occlusal area and the biting force, respectively, and 1.6% decrease was observed in mean pressure. One week after flap operation, occlusal area and the biting force had decreased by 44.1% and 42.1%, respectively, and mean pressure increased 11.1%. Three months after the operation, all parameters had almost completely returned to their preoperative values, and 6 months after the operation, increases of about 10% were observed in occlusal area and biting force.
These results suggested that the masticatory efficiency changed according to the degree of inflamation of the periodontal tissue.

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This study was performed to evaluate the osteogenic characteristics of human periodontal ligament-derived cells, and to compare them with human alveolar bone-derived cells. Both cells were obtained from extracted healthy lower third molars and interradicular septa at the same site. Five volunteers were the subjects of this study.
Mineralized nodule formation, alkaline phosphatase (ALPase) activity, and osteocalcin production were evaluated as parameters of osteoblastic characteristics, every 3 days for 21 days.
Mineralized nodule formation was observed by both types of cells in medium containing betaglycerophosphate and dexamethasone.
The ALPase activity of human periodontal ligament-derived cells and human alveolar bonederived cells was increased by the addition of 1, 25- (OH) ₂D₃ or beta-glycerophosphate, but the changes in activity in the two types of cells during the culture period were different.
Human periodontal ligament-derived cells produced osteocalcin only when the medium was supplemented with 1, 25- (OH) ₂D₃, but they produced less than human alveolar bone-derived cells.
These results suggest that human periodontal ligament-derived cells have osteoblast-like properties.

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Alcian blue has been used to detect glycosaminoglycans after they have been separated by electrophoresis on cellulose acetate membranes.
We have already described a microprocedure that can be used for direct measurement of sulfated glycosaminoglycans by using an Alcian blue dyebinding assay without interferences by other anions such as DNA, hyaluronate, or bovine serum albumin.
The aim of this study was to evaluate interference with direct measurement of sulfated glycosaminoglycans, by other anionic macromolecules in biological fluids.
Biological samples, 2μl each, were blotted onto cellulose acetate strips and stained with 0.2% Alcian blue containing 0.05M MgCl₂, 0.4M guanidine-HC1, 0.02M sulfuric acid, and 0.25% TritonX-100 at pH1.5. After drying at room temperature, the dye-substrate complex was analyzed with a dualwave length chromatoscanner. The amount of dye bound to anionic macromolecules in human saliva, serum, and synovial fluid was minimal. The sulfated glycosaminoglycan levels determined by direct assay were approximately 53% of the levels of purified gingival sulfated glycosaminoglycans measured by the electrophoretic method. This procedure is technically simple, rapid, and sensitive, and is useful even in the presence of other polyanions associated with biological fluids, although the yield of sulfated glycosaminoglycans must be improved.

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Tetracycline-resistance gene Q (tet Q), Porphyromonas gingivalis (P. gingivalis) and Prevotella intermedia (P. intermedia) in the periodontal pockets of 38 periodontal patients who had received no antibiotic therapy within 6 months of sampling were investigated by using polymerase chain reaction (PCR) methods and cultures (on trypticasesoy blood agar plates with tetracycline at 4 μg/ml) PCR-positive rates for tet Q, P. gingivalis, and P. intermedia were 89.5%, 65.8%, and 36.8%, respectively. The PCR-positive rate for tet Q in deep pockets (>4mm, n=19) was higher than that in shallow pockets (=4mm, n=19) (significant, chi Square test, p<o. 05). The PCR-tet Q positive rates in P. gingivalis and P. intermedia positive plaque sample were 96.0% and 92.9%, respectively. Neither P. gingivalis nor P. intermedia with tet Q were isolated by culture, but periodontal pathogenic microbes such as P. gingivalis and P. intermedia may acquire the tetracycline resistance gene from tet Q-positive microbes.

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Since pH in the human gingival crevice ranges from below 7.0 in healthy subjects to over 8.0 in periodontal disease, we investigated the effect of environmental pH on bacterial adhesion to human gingiva cells and on fimbrial protein synthesis by Porphyromonas gingivals 381. P. gingivalis cells were cultured in 30 mM HEPES-buffered culture medium at various pH ranges (6.0-8.5). Human gingival carcinoma cell line Ca 9-22 was cultured on microculture plates, and adherence was detected by using [³H] -labeled P. gingivalis. P. gingivalis growth was dependent on pH in the pH 6.0 to pH 7.0, but was inhibited in the pH 7.5 to 8.5 range. Stable adhesion of P. gingivalis to Ca 9-22 cells was observed at pH levels between 6.5 and 8.0, with maximum adhesion obtained at pH 7.5. This adherence was blocked by anti-P. gingivalis fimbriae monoclonal antibody (EF 3-11). The highest fimbrial protein synthesis was observed at pH 7.5. These results indicate that environmental pH regulates P. gingivalis growth and adhesion to host cells and fimbriae expression. They also suggest that the pH of the periodontal pocket rises during the host inflammatory response and that fimbriae synthesis increase markedly, possibly enabling it to evade host defenses by invasion of P. gingivalis to gingival cells.

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The aim of the present study was to evaluate the effects of root conditioning with citric acid (pH 1, 2, 3, or 4) and tetracycline hydrochloride solution (pH 2, 3, or 4) on root and calculus surfaces. Eighty root tips (5×5×1mm) with subgingival calculus were prepared for this experiment. Forty tooth root tips were carefully planed with hand curets until dentin could be seen, and the planed and the unplaned root tips were separated into 8 groups each. Each group of tip was fixed on slide glasses with twe-side adhesive tape. Root tips treated by curettage and those with calculus were conditioned with citric acid solution (pH 1, 2, 3, or 4), tetracycline hydrochloride solution (pH 2, 3, or 4), and distilled water (control) for 3 minutes by using cotton pellets, that were changed every 30 seconds. After conditioning, the root tips were irrigated with distilled water for 1 minutes. And then freezedried with tert butyl alcohol, and coated with sputtered gold. All specimens were examined by scanning electron microscopy. The higher concentrations of both citric acid solution (pH 1 or 2) and tetracycline hydrochloride solution (pH 2) had the strongest demineralizing effect on the dentinal surfaces. Citric acid solution and tetracycline at pH 3 and 4 produced a milder changes in dentin than higher concentrations, and differences were recognized between those and samples conditioned with distilled water. Mineralized objects and many rods were observed on every unplaned root surfaces, at all concentrations of citric acid and tetracycline hydrochloride, the same as in the control. Higher concentrations of citric acid and tetracycline hydrochloride (pH 1 or 2) shold be used for conditioning to demineralize root surfaces. It was concluded that citric acid causes greater degrees of morphologic change in root dentin than tetracycline hydrochloride. The layers of calculus were so thick, however that could not be removed by root conditioning with either citric acid or tetracycline hydrochloride.

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The purpose of this study was to evaluate the effect of dentifrice containing shellac on the adherence of Streptococcus mutans to the enamel surface of the human tooth.
Each enamel surface was polished with dentifrice containing 0.5% or 1% shellac, dentifrice without shellac, and with water (control). After polishing, each enamel specimen was placed into culture medium with S. mutans for 72 hours. Adherence of S. mutans to the enamel surface was assessed by scanning electron microscopy.
Masses of S. mutans were observed on the enamel surfaces polished with water. When the enamel surfaces were polished with the dentifrice that did not contain shellac, only a few S. mutans were observed on the enamel surface. In contrast, almost no S. mutans were observed on the enamel surfaces polished with dentifrice containing 0.5% or 1% shellac.
These results suggest that dentifrice not containing shellac inhibit the adherence of S. mutans to the enamel surface and that dentifrices containing 0.5% or 1% shellac strongly inhibit the adherence of S. mutans to the enamel surface.

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Recently, the application of laser irradiation has been investigated for periodontal therapy. However, the effects and the biological principle are still unclear. Our research has focused specifically on the application of high power lasers to periodontal therapy. The purpose of this study was to evaluate the changes of lipopolysaccharide (LPS) caused by Er: YAG laser irradiation. We also investigated the irradiated root surfaces by Er: YAG laser using scanning electron microscopy. Five. μl of 50EU/ml LPS (Escherichia coli 0111: B4) solution was dropped on glass pellets, and all of the glass pellets were irradiated with the Er: YAG laser at an energy density of 5J/cm², 10J/cm², 15J/cm² and 20 J/cm². LPS was washed out from the glass pellets by ultrasonic cleaner with pyrogen-free water. The concentration of residual LPS solution was determined by a spectrophotometer at 405nm. Freezed-dried LPS was irradiated with the Er: YAG laser at an energy density of 5J/cm², 10 J/cm², 15J/cm² and 20J/cm². The irradiated. freezed-dried LPS was analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE).
Premolars extracted for orthodontic reasons were planed with hand curettes, cut below the cement-enamel junction and longitudinally sectioned. Then, root tips (5×5×2mm) were prepared. These specimens were irradiated with the Er: YAG laser at an energy density of 5J/cm², 10 J/cm², 15J/cm² and 20J/cm².
The results showed that the removal rates of LPS were found to become higher as the energy density increased. Several proteins having molecular weights of 10 to 40kDa and 97kDa were recognized as quantitative changes on SDS-PAGE after Er: YAG laser irradiation. The larger ablated defect is confirmed with increasing energy density.

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The purpose of this study was to evaluate the inhibitory effect of the application of crystal violet solution (0.8mg/l: CV) and yellow He-Ne laser irradiation (4.3 mW, 594 nm : laser) on dental plaque formation on posterior tooth surfaces in rats. Twenty-four 22-23-day-old female Donryu rats were used, and divided into 4 groups. The right and left maxillary 1st molars of the rats in each group were assigned to one of the following 4 treatment groups: 1) He-Ne + CV group-application of CV plus laser irradiation (25 sec, 3 times daily for 2 weeks period) ; 2) He-Ne group-laser irradiation alone; 3) CV group-application of CV alone, and 4) Control group-no application of CV or laser irradiation. Three rats in each group were sacrificed 1 week and 2 weeks later. Inhibitory effecton plaque formation on the maxillary left 1st molars was assessed by stereomicroscopy and scanning electron microscopy. The upper right 1st molars were evaluated histologically. The thickness of the plaque on the section was measured under a microscope. At 1 week, the inhibitory effect on plaque formation in the He-Ne+CV group was statistically less than in He-Ne, CV, and control, the groups. Howevery there were no statistically significant differencs in inhibitory effect on plaque formation between the groups at 2 weeks.
The results indicate that application of CV and laser irradiation may be a useful method of inhibiting early plaque formation.

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Menopause represents a dramatic physiological change in females. The purpose of this study was to investigate the effect of decreasing hormone secretion on alveolar bone loss and clinical parameters by measuring them in pre-and post-menopausal periodontal disease patients. In a total of 233 women with advanced peinodontal disease without treatment of periodontal disease, juvenile periodontitis or trauma from occlusion were examined, and classified into a premenopausal group (n=125) and a postmenopausal group (n=108). The postmenopausal group was classified into three subgroups: a 1-5-year follow-up group, a 6-10-year follow-up group, and an over 11-year follow-up group. One hundred six adult male periodontal disease patients were examined as a control group. The subjects were also classified according to decade of life, number of years since menopause, and the grade of advanced periodontal disease. Clinical parameters (number of teeth present except third molars, plaque control record, probing pocket depth, Gingival Index, mobility, Gingival Bleeding Index) and the amount of alveolar bone loss (calculated on dental X-ray films) were examined. The pre- and post-menopausal group and control group were compared with regard to the amount of alveolar bone loss and clinical parmeters. The results indicated that the amount of alveolar bone loss in the postmenopausal group was significantly greater than in the premenopausal group. The anterior teeth in the postmenopausal groups showed showed larger amounts of alveolar bone loss than in the premenopausal group. The amount of alveolar bone loss in the postmenopausal group was greater than in the male control groups of the same age (50' s). These suggest that alveolar bone loss increases in the menopausal stage as hormone secretion decreases.

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The purpose of this study was to compare standardized reproducible radiographs densitometrically and evaluate changes alveolar bone quantification before and after periodontal surgery by computer-assisted interpretation with pseudocolor transformation. A mandibular molar right site with bone defects was selected as the subject of this study. Alveolar bone quantification was performed in the furcation and the interdental crest areas of the first and second molars. A total of 7 series radiographic densities were measured before and 1, 3, 5, 9, 16 and 25 months after surgery. Image interpretation was performed in eight different colors for the 8-step wedge thicknesses, indicating alveolar bone values as aluminum equivalent values. The calibration deviation in 7 images adjusted by a nonparametric contrast correction method was only 1. 6±1.11 gray levels (n=30, mean.±SD), and the geographic distortion, measured horizontally and vertically, registered only 1.8% and 0.8%. The change in alveolar bone, ranged from three to five mmAl, of the furcation area at 1 month postsurgery to the baseline was 156.6%, and it decreased gradually to 40.5% by 25 months. The distances from cement-enamel junction to the alveolar bone crest at the root surface points of the interdental area, which contained the vertical bony defect, were measured. The distances from both points were decreased in each color interpretation of four different mmAl. It was diagnosed the leveling of the bone height in the interdental area.
The results showed that the changes in alveolar bone after periodontal surgery can be easily and clearly evaluated by using the pseudocolor image system.

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A test paper strip coated with anti-human hemoglobin monoclonal antibody to appropriate immunologically detect salivary occult blood (immunological method: IM) was used in a preliminary trial as a screening test for periodontal disease, and clinically evaluated in comparison with a commercially available conventional test paper using the peroxidase method (PM). The subjects were 132 healthy adults (106 males and 26 females with age ranges between 35 and 55 years old, average: 45.3 years). Prior to beginning the oral examinations for probing depth (PD) and bleeding on probing (BOP), non-stimulated saliva samples were collected to detect them for occult blood by the IM test and the PM test. The positive rates by the IM and PM tests were 54.5 and 55.3%, respectively, and not significantly different, however, the positive rate by the IM test was strongly correlated with age, one of the risk factors for periodontal disease. The average BOP rates of the IM- and PM-positive subjects were 24.3 and 24.1%, respectively, and BOP rates of 7% and 8.2% were found in the IM- and PM-negative subjects. Seven of the 8 subjects who were IM-positive and had BOP rates of less than 10%, had a PD over 4mm (BOP+) at one or more probing sites. However, 2 of the 13 subjects who were PM-positive with BOP rates less than 10% had a PD over 4mm (BOP+). Sixteen of the 60 IM-negative subjects and 26 of the 59 PM-negative subjects had one or more sites with a PD over 4mm (BOP+). Comparison between the results of the IM test and the PM test showed 80.3% correspondence. Therefore, the immunological method appears to be useful as a novel screening test for periodontal disease.

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The aim of this study was to evaluate whether gain of attachment following guided tissue regeneration (GTR), can be sustained over longer periods of maintenance therapy. Twenty-one teeth in 21 patients (29-65 years old [average : 42.5 years], 7 males and 14 females) with adult periodontitis were treated by the GTR procedure (GTR group), and 21 teeth in 21 patients (34-60 years old [average: 43.7], 6 males and 15 females) with adult periodontitis were treated by the flap operation procedure (FOP group). Clinical assessments, including probing depth (PD), clinical attachment level (CAL), and location of gingival margin (GM) were made at baseline, and at 1-year and 5-year examinations. GTR group : PD reductions from baseline were PD of 5.1±1.8mm (1-year) and 4.7±1.7mm (5-year), and gains in CAL were 3.6±1.3mm (1-year) and 3.1±1.6mm (5-year). FOP group : PD reductions were 3.4±1.1mm (1-year) and 2.6±1.1mm (5-year), gains in CAL were 2.3±1.2mm (1-year) and 1.1±1.4mm (5-year). The PD reductions and the gains in CAL were statistically significantly greater (p<0.01) in the GTR group. The diffrenccs in changes in CAL between the two groups were significant (p<0.01) from baseline to 1-year post surgery. CAL in the GTR group was stable during 5-year maintenance, but the changes in CAL in the FOP group was significant (p<0.01). The results demonstrated that periodontal defects treated by the GTR procedure can be successfully sustained over periods up to 5 years.

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The purpose of this study was to evaluate the inhibitory effect of acid electrolyzed water on plaque formation. Seven healthy subjects participated in this study. Before the experiment, the Gingival Index (GI) of all teeth and Gingival Crevicular Fluid (GCF) units of the upper canines were examined, and all teeth were cleaned with an air polisher and hand scaler. The subjects were asked to stop their own daily oral hygiene procedures for 4 days and to rinse with 150 ml of acid electrolyzed water 4 times daily, one minute each time. Two and 4 days later, supragingival plaque was collected from the upper 1st or 2nd premolars and colony forming unit (CFU) were assayed. After 4 days, GI, GCF units, and Plaque Index (PH) were recorded. Povidone iodine solution and physiological saline were used as positive and negative controls, respectively.
GI was significantly lower in the acid electrolyzed water group than in the physiological saline group. PlI in the acid electrolyzed water group was the same as in the physiological saline group, and significantly higher than in the povidone iodine solution group. There were no significant differences in GCF units among the three groups. After 2 days, CFU in the acid electrolyzed water group was significantly lower than in the physiological saline group, and the same as in the povidone iodine solution group. However, no significant improvements were observed after 4 days. These results suggested that both acid electrolyzed water and povidone iodine solution had bactericidal activity after 2 days, but were no longer effective after 4 days, and that acid electrolyzed water was not as effective as povidone iodine solution for plaque inhibition after 4 days.

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