The present study was designed to compare the healing response in the periodontal ligament following the placement of 4 different types of barrier membranes. Using 7 mongrel dogs, semilunar-type incision were made and buccal mucoperiosteal flaps were reflected over the right and left maxillary canines and P 4 and mandibular canines and M 4. Fenestration wounds in alveolar bone, 2mm. 4mm, were made at the mid-root level of each tooth by removing the cortical plate using low-speed drills and hand chisels. The exposed root surface were curetted to remove cementum and then randomly divided into 4 experimental groups of 12 sites each and one control group of 8 sites. Each group of experimental sites was then covered with one of the followingmembranes; Gore-Tex, PTEE, Lyophiliaed dura mater and chitin membrane. None was placed over the control defects. The flaps were replaced with gutures and animals sacrificed at 1, 2, 4, and 6 weeks post-surgically. Block sections of the fenestrated areas were decalcified, stained and examined histologically to evaluate and compare the use the barrier materials for regeneration of the periodontium.
The Gore-Tex and PTEE membranes appeared to act as barriers by excluding the gingival inflammatory an connective tissue from contacting the root surface until week 6. New bone formation was observed under each barrier at week 2. At week 4 a large amount of osteogenesis was observed adhering to the membrane and in the defects covered with Gore-Tex while in descending order lesser amounts were seen in defects covered with PTEE, chitin and dura mater. At week 4 biodegradation of chitin and dura mater was noted. Defects covered with chitin exhibited a high degree of cementogenesis while minimal amounts were seen with the other barriers. At week 6 dura mater-covered defects appeared more organized with development of perpendicular oriented periodontal ligament fibers than chitin-covered defects while none was observed using Gore-Tex and PTEE.
While each barrier material promoted evidence for early osteogenesis, it is suggested that dura mater and chitin present a better potential in reestablishing the periodontium as evidenced by the grater development of cementogenesis and periodontal ligament formation.

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The effect of lipopolysaccharides (LPS) on the production of fibronectin by cultured fibroblasts was studied. Fibroblasts were obtained from healthy and inflamed gingiva. The Gin-1 cell line was also tested. LPS were extracted from Bacteroides gingivalis (Porphyromonas gingivalis) 381 and Bacteroides intermedius (Prevotella intermedia) ATCC 33563 by the hot phenol-water method. The LPS and commercially available LPS from Escherichia coli (B 4: O 111) were added to the fibroblast cultures. Fibronectin levels on the cell surface and in the culture medium were measured periodically for 48 hours using ELISA. Fibronectin levels were expressed as . μg/μg protein. The following results were obtained: 1) The levels of fibronectin in the culture medium increased linearly with time in all dishes. There were no differences in fibronectin levels in the same cells whether LPS were added to the culture medium or not. 2) The profiles of cell surface fibronectin levels differed from dish to dish as did the time to reach the maximum level. Healthy gingival fibroblasts and Gin-1 produced more cell-associated fibronectins upon stimulation with LPS from B. gingivalis and B. intermedius, whereas inflamed gingival fibroblasts produced more fibronectins in response to LPS from B. gingivalis. 3) The total amounts of fibronectin on the cell surface and in the medium were greator in the inflamed cells_

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Langerhans cells (LCs) are present in the epithelial structures of the oral mucosa and the skin epidermis and are stellate-shaped dendritic cells. LCs are known to participate in the antigen -presenting function to T lymphocytes and to pos -sess Ia antigen, Fc-IgG and C₃ receptors.
The present study was designed to histochemically evaluate the response of the LCs of the hamster cheek pouch to application of an allergen (DNFB) and the injection of cortisone (prednisolone: PSL) and sex hormones (testosterone, estradiol, progesterone). Two experiments were performed . The 1st experiment was conducted on 3 groups of animals: 1) male hamsters whose cheek pouches were treated with DNFB 3 times, 2) hamsters injected with PSL 3 times (i. p.) before application of DNFB 3 times, and 3) hamsters injected with PSL whose cheek pouches were simultaneously treated with DNFB 3 times. The hamsters in these three groups were sacrificed 1, 3, 5, 7 and 14 days after the final treatment. The 2nd experiment was also conducted on 3 groups of male and female hamsters. Individual groups were injected with testosterone, estradiol and progesterone 5 times every other day. The animals in the 2nd group were sacrificed one day after final treatment. The hamster cheek pouches of all of the groups were treated with EDTA to produce an epithelial sheet for staining with ATPase and vimentin.
The number of LCs in the hamster cheek pouch mucosa were higher in allergen-treated animals suggesting enhanced antigen-presenting function. The number of LCs was markedly decreased after PSL injection with combined application of DNFB, and the LCs were round with no dendritic processes. The preinjection of animals with PSL inhibits inflammatory states after allergen applications. No sex differences and no effects of sex hormone injection were detected with respect to the number or shape of LCs.

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In the present study, which was designed to histochemically determine the basal cell kinetics after epithelial re-organization, the periodontal tissue of rats was examined during wound healing .
The gingival epithelia of rats 144, 192 and 312 hrs after flap operation were used as experimental sites, while the untreated epithelia served as controls. The cell kinetics of gingival epithelium was investigated by means of the cell cycle analysis using the percent labeled mitosis (PLM) method 3H -thyminine . The results were as follows:
(1) In junctional epithelium, the cell cycle times at 144, 192 and 312 hrs postoperatively were 15 . 25, 22.50 and 27.00 hrs, respectively, while it was 27.50 hrs in the costrols.
The cell cycle times for oral sulcus epithelium at 144, 192 and 312 hrs postoperatively were 19.00, 20.75 and 34.38 hrs, respectively, and the time in the controls was 32.25 hrs.
The cell cycle times for oral epithelium at 144, 192 and 312 hrs postoperatively were 27. 38, 40.50 and 49.00 hrs, respectively, while the time in the controls was 48 . 75 hrs.
(2) The initial labeling indices (Lis) of junctional, oral sulcus and oral epithelia in the experimental groups were similar to the values obtained from the control group, showing no statistically significant differences for each observation period .
(3) The mitotic indices (MIs) tended to decrease in junctional, oral sulcus and oral epithelia of the experimental groups when compared with the indices in the control group. The MIs for junctional epithelium at 144 hrs and for oral epithelium at 312 hrs, postoperatively, were significantly different from those in the controls. No statistically significant difference, however, was observed in the MI for oral sulcus epithelium between the experimental groups and the controls through the observation period.

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This study describes the distribution of Type I collagen, Type III collagen and fibronectin after periodontal flap operations in rats. Full thickness flaps were raised and the dental roots were surgically exposed. The animals were sacrificed at 1, 3, 4, 5, 7, 14, 28 and 56 days and the wounds were examined using polycronal antibodies. The specimens were fixed in 10% neutral buffered formalin, decalcified with EDTA, made into serial paraffin sections, and also examined. The three following different stainings were done Hematoxylin and eosin. Masson-trichrome and indirect immunofluorescence for the Type I collagen, Type III collagen and fibronectin. After flap operation, fibronectin was detected in the fibrin clot which was negative for Type I collagen and Type III collagen (day 1). Newly formed granulation tissue (3-7 days) stained moderately for Type I collagen, and strongly for fibronectin and Type III collagen. As the number of fibroblasts decreased in the newly synthesized granulation tissue, fibronectin gradually decreased and the amount of Type I collagen increased (14-56 days). On the surgically exposed root dentin surface staining was strongly positive for Type I collagen but negative for Type III collagen. Dense fibrillar networks of Type I collagen and fibronectin were aligned parallel to the exposed root dentin surface, evidence of connective tissue regenerating. Eventually, the regenerated connective tissue fibers stained strongly positive for Type I collagen, negative for fibronectin, and at the site of reattachment of the regenerated collagen bundle was stained positive for Type III collagen (56 days). These results indicate that in the regeneration of connective tissue, the appearance of Type III collagen precedes that of Type I collagen, and functions as an extracellular scaffold for Type I collagen. Fibronectin may be important in theorganization of the collagenous connective tissue. They also suggest that, on the root surface, Type III collagen functions as a scaffold for reattachment.

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The present investigation was designed to evaluate the healing sequence when cells from the periodontal ligament are allowed to migrate coronally and populate the curetted root surface. Buccal mucoperiosteal flaps were elevated in all four premolar regions in 24 dogs. The buccal bone was removed to approximately 4 mm apically from the cemento-enamel junction level of the roots. Before the elevated flaps were replaced and sutured, membranes (polytetrafluoroethylene) were adjusted to cover the exposed root surfaces. No membranes were placed over the root surfaces which served as controls. Thedifferent dentition segments were scheduled to provide observation periods of 1, 2, 3, 4, 6 and 12 weeks. All of the roots were subjected to histological examination. In the experimental teeth, cementoblasts were either attached to or in close proximity to the planed root surface. New cementum with inserting collagen fibers was observed on all test roots. The length of this new attachment formation corresponded to approximately 4/5 of the distance from the apical extension of root planing to the cemento-enamel junction. In the control teeth, fibroblasts were either attached to or in close proximity to the planed root surface. No new attachment hadformed but connective tissue with collagen fibers oriented parallel to the tooth surface was observed adjacent to the exposed root surface. New cementumWas observed toform only on the most apical area of root planing. The findings in the present study suggest that cementoblasts which were differentiated from periodontal ligament cells and alveolar bone cells possess the ability to reestablish connective tissue attachment.

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The Purpose of this study was to determine the immediate effects of different toothbrushing methods and toothbrushing pressures on gingival soft tissue. The following two investigations were undertaken.
Experiment 1) The number of oral exfoliated cells resulting from different brushing methods was measured, and the gingival surface was observed immediately after brushing. There were 14 subjects (13 males, 1 female), each with both clinically normal dentition and gingiva. Rolling, horizontal and scrubbing methods were evaluated with respect to the following items. (1) The numbers of oral exfoliated cells in rinsing solution before and after brushing (including during brushing) were counted under a microscope. (2) SEM investigation of the gingival surface after brushing using a replica technique.
Experiment 2) Toothbrushings at different pressures (routine brushing pressure and high pressure) using a rolling and a scrubbing methods, were examined as in (1) and (2). Ten subiects (9 males. 1 female) participated in this experiment. The results obtained were as follows.
Experiment 1) The rolling method resulted in the highest number of exfoliated cells during brushing (26.0±6.3×10⁴/ml) when compared with the horizontal (20.5±3.2×10⁴/ml) and the scrubbing methods (12.5±2.2×10⁴/ml). Statistical significance was detected among the three brushing methods (p<0.05), and there was a difference in the location of gingival abrasion among the brushing methods.
Experiment 2) The number of exfoliated cells using the scrubbing method at routine pressure (11.7±4.5×10⁴/ml) was slightly lower than at high pressure (12.1±5.2×10⁴/ml). The number of oral exfoliated cells using the rolling method at routine pressure (18.9±3.8. 10⁴/ml) was lower than at high pressure (23.1±3.5×10⁴/ml). Statistical significance was not observed among the numbers of exfoliated cells. There was no difference in gingival abrasion between routine pressure and high pressure.

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The purpose of this study was to investigate the ultrastructural localization of heparan sulfate side chains of heparan sulfate proteoglycan in healthy canine gingiva using the high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) stainig technique and enzymatic digestion. HID -TCH-SP stain deposits, identified as heparan sulfate, were distributed mainly on the surface membrane of the cytoplasmic protrusions of gingival epithelial cells and the basement membranes associated with gingival epithelium (internal basal lamina, external basal lamina, oral and oral sulcus epithelial basement membranes). However, few were observed in the intercellular space or the stratum corneum of the gingival epithelium. In connective tissue, stain deposits were also detected on the surface membrane of cells suches fibroblasts and the basement membranes associated with capillaries and nerves, but not found in the collagen fibrils. Four different heparan sulfate distribution patterns were identified. In the internal basal lamina, the stain deposits were largely located in the lamina lucida region. In some basement membranes associated with the external basal lamina, the oral and sulcus epithelium, and endothelial cells of capillaries, the stain deposits were randomly localized in the lamina densa. In part of the external basal lamina, the stain deposits were regularly detected on both sides of the lamina densa. Finally, in the basement membranes of the pericytes of capillaries and nerves, the stain deposits were distributed on the external side of the lamina densa adjacent to the connective tissue. Thus these findings suggest architectural heterogeneity among the various basement membranes.

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5'-Methylthioadenosine (MTA) is formed during the biosynthesis of polyamines. This nucleoside is cleaved to adenine and 5'-methylthioribose-l-phosphate in mammalian cells by methylthioadenosine phosphorylase (MTAPase). 5'-Difluoromethylthioadenosine (DFMTA), a synthetic analog of MTA is known to be an inhibitor of MTA Pase.
In this study, we examined the effects of DFMTA on the cell proliferation and alkaline phosphatase (ALPase) activity of enzyme-releasedr at calvaria cells (RCcells). RC cells were isolated by collagenase digestion of 21-day fetal rat calvariae and cultured in the medium containing 15% NU serum, which was used because its MTA Pase activity is very low. Under the culture conditions employed, the ALPase activity of RC cells was low in the exponential growth phase, but markedly increased when the cells reached confluency (day10). DFMTA strongly inhibited MTA Pase extracted from RC cells (Ki= 0.60μM). DFMTA at low concentrations (5, 10μM) decreased the ALPase activity of RC cells measured on day 7 and 14, without affecting cell proliferation. At higher concentrations (≥25μM), this analog suppressed both ALPase activity and cell proliferation. These results suggested that the metabolism of MTA is involved in regulating cell proliferation and differentiation.

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The purpose of this study was to investigate the role of the neuropeptide Substance P (SP) in periodontitis. We examined the distribution of SP in human gingiva from periodontitis-affected sites, and ELISPOT assay was used to study the effects of SP on immunoglobulin synthesis.
SP-immunoreactive fibers close to the epithelium were observed in periodontitis-affected gingival tissue. SP enhanced immunoglobulin synthesis by mononuclear cells from peripheral blood and periodontitis-affected gingival tissue. The effects of SP were maximal at 10^-8 M. The results suggest that SP might play a role in periodontitis.

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We have examined the effect of the fractions from cytosol and membrane of Actinobacillus actinomycetemcomitans Y 4, Porphyromonas gingivalis 381 and Escherichia coli HB 101 on the proliferation of human gingival fibroblasts. Only the cytosol fraction from A. actinomycetemcomitans Y 4 could inhibit cell growth in a dose dependent fashion and no other fractions gave any effect on cell growth. The inhibitory activity of the cytosol fraction was found in the precipitate by ammonium sulfate addition (100% final concentration). This activity was lost by heating at 70.. for 30 min. These results suggest that the activity found in the cytosol fraction is protein. We further purified that inhibitory activity on Cellurofine GC 700m gel chromatography and found its apparent molecular weight was 50k.

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This study was performed to examine whether decalcified and freeze-dried bone allografts (DFBA) in two-walled bony defects would promote new cementum formation on roots as well as in three-walled defects. Four adult monkeys were used. Maxillary lateral incisors were extracted to provide room for implantation. After reflection of buccal mucoperiosteal flaps, defects of 3 mm in depth were created in the mesial alveolar bone of maxillary canines, resulting in two-walled defects with a mesial and palatal bony wall. The mesial root surfaces were throughly planed. DFBA were placed into the bony defects but not into those of controls. The left and right teeth were treated in the same way in each monkey. The animals were sacrificed to yield observation periods of 4 and 8 weeks. Each block of canines including the tooth and surrounding bone was processed for light and electron microscopy. Implant particles were resorbed and replaced by the new bone. Progressive cementogenesis occurred on the root surface corresponding to the new bone. The results suggested that DFBA in two-walled bony defects could enhance the new connective tissue attachment including the formation of new cementum.

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Polymorphonuclear leukocytes (PMNs) play an important role in host defence against periodontal tissue invading bacteria. On the other hand, PMNs may enhance tissue destruction through the formation of toxic oxygen radicals and the release of tissue reactive proteins. In previous studies, we have shown that lipopolysaccharide (LPS) enhanced N-formyl-mithionyl-leucyl-phenyl-alanine (FMLP) stimulated PMMs-mediated damage to culture human periodontal ligament fibroblast-like cell (HPLF). The present study was to investigate the effect of antibiotics on PMNs-mediated damage of HPLFs. HPLFs were grown to confluent in culture and exposed antibiotics, and then stimulated PMNs by LPS (1000ng/ml) and FMLP (10^-6M). Cytotoxicity of HPLFs were determined by double staining method using fluorescein diacetate (FDA) and propidium iodide (PI).
Minocycline (20μg/ml) and doxcycline (20μg/ ml) had significant inhibitory effect on the PMNs -mediated HPLF injury over the three hour of assay. However, same concentration of ampicillin, streptomycin, chlortetracycline and oxytetracycline had no significant effect on the same system. These results suggest that, in this system, minocycline and doxcycline may suppress PMNs-associated enzymes activity, degranulation and toxic oxygen radicals synthesis.

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The purpose of this study was to evaluate whether the reflex response of the human masseter muscle was inhibited by posterior tooth occlusion.
The reflex response of the masseter muscle was elicited by the following three kinds of stimulation a) tapping of the upper right centralincisor; b) electrical stimulation of the upper right incisor's labialgingiva; and c) tapping of the menton. These stimulations were delivered while the subjects were clenching their tooth under two different biting conditions, i. e., incisor edge-to-edge and posterior tooth biting position.
Masseteric electromyograms were recorded using bipolar surface electrodes. The amplitude and the latency of the reflex responses were measured.
The results were as follows:
1. The reflex responses in the masseter muscle induced by three kinds of stimulation were clearly inhibited by posterior tooth occlusion.
2. The reflex response elicited by tapping on the menton was not inhibited after local anesthesia of the gingiva around the posterior tooth in the occlusal position.
These results suggest that inhibition of the human masseter muscle activity induced by posterior tooth occlusion may be elicited by afferent impulses from the mechanoreceptors in the periodontal ligament in the area of the posterior tooth.

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A double-blind clinical study was conducted to evaluate the effects of dentifrice containing 1% sodium polyphosphate, tranexamic acid and sodium fluoride in preventing calculus formation and gingivitis in comparison with dentifrice containing 5% sodium pyrophosphate and placebo dentifrice. One hundred thirty-nine adult subjects from one company who had developed dental calculus were selected for this study. Initially all of subjects underwent scaling and for 4 weeks thereafter used a placebo dentifrice during a pre-test period. At the end of this period, the degree of calculus formation was measured to divide subjects randomly into 3 equal quality groups. All subjects who received initial prophylaxis were provided with the assigned dentifrice and toothbrush. Assessments of supragingival calculus were made at 4 and 12 weeks using the calculus scoring procedure decribed by Volpe et al. The periodontal condition of subjects with gingivitis was determined at 4 and 12 weeks based on a modified gingival index.
The followig results were obtained.
1. The experimental dentifrice had reduced supragingival calculus significantly more than the dentifrice containing 5% sodium pyrophosphate or the placebo dentifrice at 12 weeks.
2. Both the experimental dentifrice and the dentifrice containing 5% sodium pyrophosphate had reduced gingival inflammation significantly more than the placebo dentifrice at 12 weeks.

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The purpose of these studies was to assess the relationship between factors in the immune network concerned with immunoglobulin synthesis. We examined immunoglobulin production, lymphocyte subset ratios and lymphocyte function using peripheral blood mononuclear cells from 50 subjects consisting of 32 patients with periodontal disease and 18 periodontally healthy subjects. On the basis of serum antibody titers to periodontal bacteria, patients were classified into two groups characterized by either low or high titers. Patients had lower proportional counts of suppressor/cytotoxic T cells and higher immunoglobulin productivity than healthy subjects. Subjects with an elevated or decreased ratio of lymphocyte subsets and lymphocyte functions were detected more frequently among patients, irrespective of antibody titers.
However, a wide distribution of values was observed among subjects in all of the examination. Based on the results obtained, it appears that lymphocyte functions concerned with humoral immune response vary widely among subjects, and each feature must be clarified individually.

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The aim of this experimental analysis was to elucidate the relationship between polyamines and clinical periodontal parameters. Fifteen adult patients with periodontal disease and 45 junior and senior high school students who had a CPITN value of more than 13 were served as subjects of this study. Gingival crevicular fluid was collected, and the amounts and the kinds of polyamines were analysed. Significant positive correlation was found between each polyamine level and age and pocket depth, especially in the case of spermine and putrescine. The individual polyamines were not significantly correlated with BI or GI. A slight correlation was detected between the CPITN value and the amount of cadaverine. A significant positive correlation was detected between the alveolar bone resorption ratio and each of the polyamines other than spermidine . Spermine had the highest correlation coefficient with bone resorption. The results suggest that the severity of periodontal disease depends more on spermine and putrescine levels than those of cadaverine or spermidine.

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This study was designed to investigate multisided scaling of exposed cementum (group S), scaling followed by application of citric acid to exposed cementum (group S+C), root planing of exposed cementum (group R) and root planing followed by application to citric acid of exposed cementum (group R+C).
The results were as follows:
1. The X-ray microanalyzer values for Ca, P and Ca/P were highest in group S and lowest in group R+S.
2. Scanning electron microscope examination revealed distinct striae with the curette-type scaler in group S, and that the striae tended to be weak in group R+C.
3. The relative concentrations of the target elements Ca, P and O/P were lower in group S+C than in group S and lower in group R+C than in group R, but Ca/P valves were similar in each of the groups based on X-ray photoelectron spectroscopy. Ca, P and F binding energies of the cementum were similar in each group.
4. The contact angles in group S and in group R were similar in intact cementum, but in groups treated with citric acid it was impossible to measure because of the extremely hydrophilic sol.

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Clinical investigations were conducted on the effect on periodontal disease of LA-1 gargle, prescribed the same as Acess, a paste used to treat gingivitis and marginal periodontitis, containing as principal components three kinds of medicinal herbs. The subjects consisted of 96 patients diagnosed with gingivitis or marginal periodontitis on the basis of clinical findings and other examinations. Each patient brushed his teeth without dentifrice the same way as before, and after that was given LA-1 gargle every morning and evening for 4 weeks. The results were as follows.
1. Inflammation in ingivitis was improved, and the efficacy rate of this agent was 92.3%.
2. Inflammation in marginal periodontitis was improved, and the effdcacy rate of this agent was 55.7% as of 2 weeks and 77.1% as of 4 weeks.
3. No side effects which seemed to be attributable LA-1 occurred during the period of the experiment.
4. An effect of LA-1 on periodontal disease was demonstrated. LA-1 is considered to be an effective agent as a proprietary drug.

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