The present study was performed to investigate the effects of endotoxins from periodontopathic bacteria on collagen metabolism. Endotoxins were extracted from Bacteroides gingivalis 381 and Bacteroides intermedius ATCC 25611 using the hot-phenol method. A commercially available endotoxin from Escherichia coli 0111: B 4 was also used as a control. Human gingival fibroblasts (Gin-1, ATCC CRL 1292) were maintained with DMEM containing 10% FBS. When the fibroblasts became confluent they were exposed to each of the endotoxins in various concentrations (0, 5, 10, 15, 20μg/m/). The effects of these endotoxins on the collagen metabolism of the fibroblasts were assessed on the basis of collagen synthesis and collagenase activity. The former was assessed by ³Hproline incorporation and bacterial collagenase digestable protein. The latter was assessed by fibril assay .
The results were as follows:
There was no change in glucose consumption, cell viability or morphology under the light microscope when the concentration of endotoxin was 20μglml or less. ³H-proline incorporation into protein and collagen synthesis were inclined to decrease. Endotoxin from B. gingivalis resulted in an acceleration of collagenase activity.
There findings indicate that the endotoxins from these bacteria might affect collagen metabolism early in gingivitis in vivo.

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The purpose of this study was to examine the effect of fibronectin on cell attachment and the growth of cultured cells on the surface of the dentin. In the first experiment, dentin specimens were divided into following three groups: dentin surface polished with carborundum-point, carborundum-point and #150 sandpaper and carborundum-point, #150 sandpaper and #240 sandpaper. The roughness of the surface was measured with a profilometer (Surfcom e-st-sa) for the purpose of comparing differences among the three groups. The foregoing treated samples were added to cultures of periodontal ligament (PDL) cells and following fixation, the number of cells was counted under a light microscope and examination under a scanning electron microscope (SEM) was also performed.
1. Greater numbers of cells attached to the smooth surface (#240) than the rough surface.
2. SEM observations showed increased numbers of attached and proliferated cells on the smooth surface.
In the second experiment, the dentin surfaces were polished with carborundum point, #150 and #240 sand paper. The dentin tube side was treated with fibronectin. MEM-treated samples not immersed in fibronectin were employed as controls. The foregoing treated root samples were cultured with PDL cells. Following fixation, the cells attached to the root surface were examined under the light microscope and using a SEM.
1. A significant increase in the number of the attached cells as observed after treatment with fibronectin.
2. SEM observation suggested PDL cells migrated more and proliferated more on the side treated with fibronectin.
3. Fibronectin application increased cell attachment and proliferation more on the smooth surface of dentin.

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Clinico-histological studies were made to obtain basic data on the application of 2 piece-type hydroxyapatite (hereinafter refered to as HAP) originally designed and prepared so as to be shaped to make its clinical application easy as an anchor (HAP anchor) in the corrective movement of teeth. An immobility identification experiment and intrusive experiment were performed in order to identify absent loss of anchor to the corrective force and to identify actually practicable tooth movement by means of the HAP anchor, respectively, and clinico-histological evaluation was made of its function in both of the above cases. The results revealed no change in the distance of HAP anchor in the former experiment and tooth intrusion of 3.7 mm on average in the latter experiment and, histologically, close surrounding of almost the entire region embedded by the HAP anchor, with newly formed osseous tissues in both experiments. The above results suggest the usefulness of this trial HAP anchor as an anchor to tooth movement in periodontal therapy.

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Granulated artificial bone implant materials such as hydroxyapatite (HAP) and tricalcium phosphate (TCP) have been frequently used in attempts to restore periodontal tissue lost as a result periodontitis. However, these materials are considered insufficient for the maintenance and stability of granules at sites losing bone, for active bone formation, or for periodontal ligament regeneration. We have now developed a complex consisting of HAP or TCP coated with atelocollagen (which has recently received attention as a biomaterial) and have conducted experiments to determine the effects of this material on the reconstruction of periodontal tissue. Implantations were performed using a HAP-atelocollagen complex, TCP-atelocollagen complex, HAP and TCP at three-wall bone defect sites in experimentally-induced periodontitis in dogs. A control group without the implants was included in the study. Histopathological observations were conducted 2, 4 and 12 weeks after surgery. Compared with the control group, the groups implanted with the complex displayed enhanced maintenance and stability of granules, suppression of epithelial downgrowth, and acceleration of new bone and cementum formation. These results indicate that the implant method using an atelocollagen-coated artificial bone implant material would be useful for periodontal surgical treatment, eliminating some of the disadvantages of conventional implant methods.

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Cytotoxicity of povidone-iodine on cultured mammalian cells was studied using cultured Chinese hamster lung cells (V 79 cells). Treatment of V 79 cells with povidone-iodine at 100-500μg/ml for 24 to 72hr induced a growth inhibition of the cells in a dose-dependent manner. Although minimum inhibition of survival of V 79 cells was elicited in cultures treated with povidone-iodine at 100-300μg/ml for 2 to 24hr, treatment at 500μg/ml for 6 to 24 hr resulted in decrease in cell survival in a treatment time-dependent fasion. Exposure of cells to povidone-iodine at 100-500μg/ml for 2hr caused inhibition of DNA, RNA or protein synthesis of cells up to 13.9% of untreated cells. These results indicate that cytotoxicity of povidone-iodine occurs in doseand treatment time-dependent manners and that povidone-iodine induces inhibition of DNA, RNA and protein syntheses of cells, even by the treatment which causes no reduction of cell survival.

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It seems to be generally agreed that periodontal disease is a local manifestation of a systemic immune response. Interleukin-1 (IL-1), which has multiple biologic activities, is detected in the gingival sulcus fluid of periodontitis sites. Recent investigations have revealed that IL-1 and tumor necrosis factor (TNF) are analogous to osteoclast activating factor and promote bone resorption. These findings have suggested the possibility that IL-1 and TNF may play a significant role in the initiation and development of periodontal disease. However, it remains to be determined whether these cytokines influence periodontal tissue breakdown in periodontitis. To elucidate the mechanisms of tissue breakdown in periodontitis, we examined cytokine production by human periodontitis gingival tissue.
Twelve periodontitis patients were included in this study. Control subjects with healthy periodontium consisted of nine individuals. Gingival samples were biopsied from inflamed or healthy gingival tissues. Biopsy specimens were dissected into fragments 3mm in diameter and plated onto 24 well culture plates with RPMI 1640 medium. IL-1 activity was measured by a growth inhibition assay using melanoma cell line A 375. An enzyme-linked immunosorbent assay (ELIZA) was used for measuring levels of human IL-1α, IL-1β. TNFα activity was measured by a growth inhibition assay using cell line LM2D6.
IL-1 activity was detected in significantly (p< 0.001) higher levels in culture supernatants from gingival tissues in periodontitis (48.0 ± 23.3units/ml) than in control tissues (2.3 ± 0.6 units/ml), however, levels of IL-1 activity were not associated with periodontal pocket depth or extent of alveolar bone resorption in periodontitis. IL-1αand IL-1β were also detected in culture supernatants from gingival tissues in periodontitis using ELISA. IL-1β levels were higher than IL-1α levels in the samples. No TNFα activity was detected in any of the samples.
These results suggest that IL-1 may play a critical role in inflammatory destruction, such as alteration of gingival connective tissue and loss of alveolar bone in periodontitis.

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The purpose of the experiment was to study the possible penetration of extrinsic tracers with different molecular weights into exposed cementum in vitro and pathway of penetration.
75 human extracted teeth-55 periodontally diseased teeth and 20 embedded 3rd molars-were used. Each tooth was maintained in 0.005% fluorecein isothiocyanate (FITC) solution (M. W. 400), 0.05% FITC conjugated peroxidase solution (M. W. 40, 000), or 0.01% FITC conjugated human IgG solution (M. W. 160, 000) for 10 days. Morphological observations were made by means of fluorescence microscopy. Other extracted teeth were maintained in 0.05% microperoxidase solution (M. W. 1, 900) for 5 days. Observations were then made using the TEM, and micrographs were taken and analyzed with an image analyzer.
As a result, solutions of higher molecular weight showed lower penetration, and unexposed cementum showed a tendency toward greater penetration. Furthermore, the penetration of tracers on the surface of the cementum was along collagen fibers, but in the inner cementum, was into cementocyte lacunae and canaliculi.

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Bone morphogenetic protein (BMP) is known to be a protein which induces new bone at heterotopic sites. Purification of BMP has not been perfected, and obtaining large amounis of BMP is very difficult, so it seems better to use some carrier or frame material for BMP to work effectively. Various kinds of hydroxyapatite (HAP) have been used to repair periodontal osseous defects, but they do not have osteogenetic or osteoinductive properties. If osteoinductive proteins such as BMP could retain their biologic properties after being implanted into living tissue with HAP, it would be an advantage in repairing periodontal osseous defects. In this experiment, we prepared BMP-HAP complex and investigated its osteoinductivc activity.
BMP was extracted from bovine cortical bones in accordance with the Urist's procedure. The ability of this BMP to stimulate new bone growth was ensured by implantation in the muscle pouch of mice. HAP was synthesized by the wet method. The BMP-HAP complex was implanted in the muscle pouch of mice, and osteoinduction was examined 3, 7, 14, and 21 days after implantation to assess its osteo-inductive ablity.
New bone formation was studied by roentgenographic and histologic observation.
In the BMP-HAP group, new bone formation was seen on the roentgenograms and new cartilage and bone were observed histologically in the tissue surrounding the apatite. In the HAP group, no new cartilage or bone formation was noted.

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This study was designed to investigate the effect on new attachment formation using a biodegradable membrane as a barrier to the regeneration of periodontal tissue. One-wall wide periodontal osseous defects with exposed root surfaces were prepared in three adult mongrel dogs. After surgical debridement of the periodontal defects, 3 types of biodegradable membranes, [Poly (_L-lactic acid) ; (P-_L-LA)] membrane, [Poly (lactic acid-co-glycolic acid) ; (PLGA)] (81: 19 mole%) membrane and PLGA (50: 50mole%) membrane, were arranged to cover the denuded root surfaces. Specimen blocks were removed 8 weeks postoperatively for histological evaluation of their effect on the regeneration of periodontal tissue.
The results were as follows:
1. Gingival regions containing P-L-LA or PLGA (81: 19 mole%) membranes showed delayed wound healing macroscopically.
2. Almost all P-_L-LA membranes were present in the experimental sites failing to be resorbed during the 8-week experimental period. PLGA (81: 19 mole%) membranes were also present but showed with some degree of resorption and fragility, while PLGA (50: 50 mole%) membranes were completely resorbed.
3. This showed that the membranes used in this experiment are possible barriers to the generation of new attachment. It is important to make sure keeping the membrane.
4. There was no relationship between osteogenesis and cementogenesis. It seems that osteogenesis was depressed by membrane shrinkage and the gingival pressure which limited “the regenerative space of bone”.
5. Moderate resorption was observed on the root surface, and new cementum was formed at the resorbed surface.
6. Ankylosis was observed between the new cementum and bone at the experimental sites. Newly formed cementum was seen on coronal sections of this area.

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The purpose of this study was to evaluate the effect of implants of granular hydroxyapatite (HAP). HAP was implanted into twenty-five vertical bone defects of twenty-one patients as bone graft material. Various clinical and radiographic examinations were performed postoperatively over a twelve month period. Redness and swelling of the gingiva, gingival bleeding, postoperative pain and increased tooth mobility developed transiently, but they all recovered in time. Open wounds and out-flow of HAP disappeared within the first month. After twelve months, mean probing depth decrease was 3.7 mm and clinical attachment gain was 2.5 mm. In all cases there was radiographic evidence of alveolar bone repair. These results suggest that HAP is clinically effective as a bone graft in periodontal therapy.

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The purpose of this study was to examine the relationship between clinical findings and subgingival microbial flora in periodontitis.
The results obtained were as follows:
1. In a phase-contrast microscopic study, no correlation was found between the clinical findings, total bacteria or proportional distribution of spirochetes or motile rods in the periodontal pocket.
2. Anaerobic incubation revealed no correlation between clinical findings, total bacteria or proportional distribution of black-pigmented Bacteroides in the periodontal pocket.

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The purpuse of this study was to clinically and microbiologically evaluate the effects of supragingival plaque control, scaling and root planing on periodontal therapy. Six patients affected with moderate or severe periodontitis were selected. The clinical status of each patient was recorded with such clinical parameters as probing pocket depth (PD), probing attachment level (PAL), gingival crevicular fluid flow (GCF), gingival index (GI), gingival bleeding index (GBI), suppurative index (SI), and mobility (Mo). After the patients had learned to perform their own oral prophylaxis, they were treated by scaling and root planing. Microbiological status was assessed by phase contrast microscopy and bacterial forms were classified into following six groups: coccoid cells, rods, filaments, fusifOrms, motile rods, spirochetes. The number and the relative proportion of each group were compared statistically. Clinical and microbiological status from baseline to 5 months after scaling and root planing were re-examined.
The results obtained were as follows.
1. The most significant change in clinical and microbiological status was observed one month after scaling and root planing. In particular, the total number of the microorganisms and the number and relative proportion of motile organisms were reduced.
2. Thereafter clinical and microbiological status was maintained during experimental period by means of supragingival plaque control.

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Scaling and root planing is one of the most fundamental and effective procedures in periodontal therapy. The purpose of this study was to investigate statistically whether clinical or microbiological parameters before treatment are related to changes in probing pocket depth (PD). Two kinds of numerical values represented changes in PD. One was pocket difference (A-B: A; PD before the treatment and B; PD after treatment) and the other was rate of pocket decrease [(A-B) /Ax100]. Twenty four sites in six patients with periodontal pockets deeper than 4 mm were selected for this study. Clinical parameters such as PD, probing attachment level (PAL), gingival crevicular fluid (GCF) volume, gingival bleeding index (GBI) and suppurative index (SI) were recorded at each site. Subginigival plaques sampled at the same site were evaluated by phase contrast microscopy.
The results obtained were as follows:
1. Of all the clinical parameters changes in PD were most positively correlated with PD before treatment.
2. The microbiological approach revealed that total bacterial count divided by PD value was more negatively correlated with changes in PD than total bacterial number. It was further, revealed that numbers of rods and motile bacteria were more closely correlated with changes in PD than the counts of bacterial groups. At sites where coccoid cells were relatively dominant, PD tended to decrease readily. Motile bacteria, on the other hand, exhibited just the opposite relationship. These results suggest that periodontal treatment should be more effective in periodontal pockets harboring both smaller numbers of rods and motile bacteria.

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The purpose of this study was to assess the methods of establishing of clinically (experimental) healthy gingiva and to evaluate the status of healthy gingiva in Macaca irus. Three monkeys (2 males and 1 female) were used. After preliminary feeding with hard food for 6 months, plaque control procedures (scrubbing method, modified Stillman method, and flossing) were started. Frequency of plaque removal was three times per week under KETALAR (®) (SANKYO. Co. Tokyo), dissociative anesthesia. We also monitored clinical data.
Results:
1. Forty-nine days later, clinically healthy gingiva were achieved. During this period, no side effects and no tolerance occurred using 12.5 mg/kg of KETALAR (®), and effective time to perform the procedure was 21.8 minutes. 2. Depending on the plaque control procedure, plaque index (PlI), gingival index (GI), and probing depth (PD) were reduced (especially in the first week). However, the level of marginal gingiva (LMG) did not change. 3. Initially and throughout the experiment, PlI, GI, PD were lower value in lower jaws, but by the end of experiment there were no differences between the two jaws in PlI and PD. However, GI was still lower in the lower jaw at this time. There were no differences between the data for the right and left sides of the jaws at any time throughout the experiment. 4. At the end of experiment, the clinically (experimental) healthy gingiva yielded the following data: PlI, 1.7± 0.61; GI, 0.1± 0.60; PD, 1.3± 0.53. No individual differences were found in any of the monkeys at the end of experiment.

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Examination of gingival morphology was conducted using ultrasonic imaging equipment in a 57-year-old female with epulis. The ultrasonogram showed external gingival morphology including epulis and root surface caries which was difficult to detect because of masking by gingival enlargement.
It appears that ultrasonograms are useful not only in examining gingival morphology but also in detecting changes in root surfaces hidden by the gingiva .

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The purpose of this study was to compare differences in pocket reduction rate between different tooth types and sites after initial treatment. Forty-one adult periodontitis patients (mean age 40.8) whose O'Leary plaque records during inital treatment were maintained at a level of less than 10% (mean level 9.02± 4.93%) were selected for this study. A total of 5983 surfaces were monitored by recording probing pocket depths (P.P.D.).
The results obtained were as follows.
1. Significant probing pocket depth reduction was obtained following initial treatment (P<0.001).
2. Significantly improved teeth were 4 2 1 1 2 4 (p<0.001), 5 5 and 5 3 3 5 (p<0.05), and 3 2 2 3 (p<0.05). Deep residual P.P.D. were observed i n 1 1, 6 6, 7 7, 616 and 7 7 (p<0.001), and 2 2 and 3 3 (p<0.05).
3. Response to treatment by tooth type was favorable in 5 4 4 5 and 4 3 1 1 3 4 and was poor in 7 1 1 7, 7 7.
4. Mandibular residual pocket depths were much deeper on proximal surfaces than on the mid-facial and lingual surfaces. Maxillary pockets on palatal surfaces were significant deeper than on facial ones.
5. Poor response to treatment was treatment was observed on the mesiopalatal surfaces (p<0.001), mid-palatal surfaces (p<0.01) of 616 (p<0.001), the distolingual surfaces of 7 7, and 7 7 (p<0.001), and the mesio-palatal surfaces of 1 1 (p<0.05). Favorable responses were observed on the disto-buccal and the mid-palatal surface of 4 4 (p<0.05), mesiodistal buccal surfaces of 5 5 (p<0.05) and mesiolingual surfaces of 4 4 (p<0.05).
This results clearly indicate which tooth types and root surfaces must be paid careful attention to during treatment.

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The purpose of this study was to evaluate the clinical effect of mace extract and egg-white lysozyme in two brands of chewing gum on gingival condition. Ever since mace extract containing dihydroguaiaretic acid was reported to inhibit the growth of Streptococcus mutans, plans were devised to include it in commercially available chewing gum.
Before starting this study, two different types of experimental chewing gum containing mace extract or egg-white lysozyme were made up. A control was also prepared containing neither agent.
The periodontal condition of 68 patients with gingivitis was determined based on PMA index (PMA), gingival index (GI), gingival bleeding index (GBI) and plaque scoring system (PSS) and randomly classified into three groups. Each group was instructed to use one or the other of the above type chewing gums after every meal.
The results were as follows:
1. No clinical changes were observed in the control group during this study.
2. Gingival inflammation (PMA, GI, GBI) significantly improved as a result of using the experimental gums.
3. Plaque reduction was found only in the meceextract gum group.
4. No clinical side effects were detected during this study.

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A double-blind clinical study was conducted to evaluate the effect of a tartar-control dentifrice containing 1% sodium polyphosphate as tartar control agent compared with dentifrice A containing 5% sodium pyrophosphate and a placebo dentifrice. One hundred forty-eights who continued to have a degree of calculus formation after using a regular dentifrice containing no tartar control agent for 1 month in the pretest, were stratified randomly on the basis of pretest, calculus score, age and sex into 3 homogeneous groups. All subjects who received initial prophylaxis were provided the assigned dentifrice and toothbrush. No instructions regarding frequency or method of toothbrushing were allowed. Assessment of supragingival calculus was made at 4 and 12 weeks using the calculus scoring procedure proposed by Volpe et al. Side effects such as oral irritation, ablation of oral mucosa and discoloration of teeth caused by dentifrices were also diagnosed after 12 weeks of use. The following results were obtained in this study.
1. The test dentifrice had reduced (P<0.01) supra-gingival calculus significantly more than dentifrice A and the placebo at 12 weeks.
2. A significant reduction (P<0.01) was observed when dentifrice A was compared with the placebo dentifrice. 30.1% reduction was obtained using the test dentifrice and 9.0% reduction using dentifrice A when assessed in subjects who had a pretest VMI score of more than 6. 0.
3. No side effects caused by the dentifrice which contained sodium polyphosphate were observed.

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The purpose of the present investigation was to determine bacterial contamination of chlorhexidine coated and uncoated (normal) nylon filaments of toothbrushes.
Ten healthy subjects were employed in this study and the test toothbrush of four lines and thirty-eight tufts were used twice a day. Test periods were 1, 8, and 20 days for each filaments of toothbrush and after they brushed the used toothbrush was kept at the constant condition (20°C, 65% RH). After each test period, those toothbrushes were immediately collected and kept at the same condition for drying. After 0, 3, 6, 9, and 24 hours of drying, two tufts of filaments were pulled out from the toothbrush and cutted into two parts (end- and root-side of tufts) by sterile scissors. Each cutted part of filaments was washed with PBS and the aliquots was incubated on Brucella blood agar plate at 37°C for 48 hours. The number of bacteria attached to filaments was enumerated.
The results were as follows:
1. The number of bacteria attached to both endand root-sides of chlorhexidine coated filaments decreased with the time of drying.
2. At the root-side of the normal filaments, the number of contaminated bacteria increased with the test periods.
3. The isolated bacteria from tested toothbrushes were mainly gram positive at shorter period, however, gram negative rods were also found at longer period.
4. The anti-bacterial activity of the end-side of chlorhexidine coated filaments diminished at eight days of the use, however, their activity at the rootside still remained even at twenty days.
From these results, it was clear that the bacterial contamination of toothbrush was affected by several factors such as the condition of drying, the site of tufts, the using period of toothbrush and so on. To prevent this problem, it is important to keep it at good storage condition and to change it periodically. The developed chlorhexidine coated filaments of the toothbrush also indicates to be one of the useful way for prevention of bacterial contamination of toothbrush.

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