Risk factors of periodontal disease include pathogenesis such as periodontal pathogenic bacterial infection and occlusal trauma, host responses, and environmental factors such as smoking, stress, and diet.
Smoking and stress, in particular, are known to affect adversely bone metabolism. In order to know whether or not stress and smoking directly influence bone resorption caused by periodontal disease, we determined the number of tartrate-resistant acid phosphatase (TRAP) -positive multinucleate cells (MNC). Osteoclasts formed particularly in mouse bone marrow cell cultures treated separately with dexamethasone (Dex), lipopolysaccharide (LPS), nicotine, or cotinine. Also, in order to determine the bone formation effects of the above-mentioned factors on the differentiation of mouse osteoblasts, we measured the level of the alkaline phosphatase (ALP) activity generated in response to these factors. In mouse bone marrow cell cultures, TRAP-positive MNC increased in number when LPS or Dex was added, whereas no changes were observed when only nicotine or cotinine was added. In osteoblast cultures, ALP activity was inhibited after nicotine or cotinine stimulation. When the osteoblast culture was treated with LPS alone, the LPS was also inhibited. However, neither nicotine nor Dex inhibited the ALP activity when the osteoblasts were pretreated with LPS. From the study, it can be concluded that smoking factor affects the inhibition of osteoblasts ALP activity and stress factor causes the increase of numbers in osteoclasts. Therefore, the data suggests that smoking factor and stress factor, with the assumption of periodontal disease, directly affects bone metabolism. J. Jpn. Soc. Periodontol., 42: 151-161, 2000.

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Guided tissue regeneration is a method designed to obtain new connective tissue attachment to a root surface previously exposed in a periodontal pocket. This study was designed to investigate the histological response of the new connective tissue attachment to bruxism. Ten Japanese monkeys were used in the experiment. Bony defects were created in the buccal aspect of the mesial roots of the left maxillary second premolars by removing buccal bone to a level of about 5 mm below the cement-enamel junction. Welladjusted expanded polytetrafluoroethylene membranes were applied to cover the bony defects, and the flaps were closed with interrupted sutures. The membranes were removed 6 weeks later. The right maxillary second premolars were untreated and used as controls. Bruxism was induced by the method of Kitamura (1990). Both experimental and control sites were exposed to the force of bruxism for 4 weeks, the animals were sacrificed after another 4 weeks. Half of the sections were stained with hematoxyline-eosin (H-E), and the other half were examined immunohistochemically by using anti-actin and anti-proliferative cell nuclear antigen (PCNA) antibody. The control sites exhibited loss of connective tissue attachment, but the experimental sites did not show loss of the new connective tissue attachment of the periodontal ligament. The number of cementoblasts and fibroblasts on the cementum and the surrounding areas was greater at the experimental sites than at the control sites. The number of PCNA positive cells was statistically greater at the experimental sites than at the control sites (p<0.001). The number of cells positive for actin filaments was also significantly greater at the experimental sites than at the control sites. The results indicate that bruxism-induced tissue remodelling was more active and extensive at the experimental sites than at the control sites. They also suggested that maintenance of new connective tissue attachments is regulated by cementoblasts and fibroblasts in the periodontal ligament. J. Jpn. Soc. Periodontol., 42 : 162 174, 2000.

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Prevotella intermedia is one of the bacteria related to periodontitis. The aim of this study was to investigate whether P. intermedia can utilize proteins available in the oral cavity and change its enzyme activities according to its nutritional environment. P. intermedia ATCC 25611 was grown anaerobically in basal medium (BM) or BM supplemented with albumin, mucin, or glucose. The presence of albumin, mucin, or glucose increased maximal bacterial growth. The presence of albumin or mucin also increased the proteolytic activity of the bacteria as measured by four synthetic subatrates. Addition of glucose, on the other hand, decreased their proteolytic activities. These results indicate that P. intermedia can utilize albumin, mucin and glucose as sources of energy and cell components and suggest that it can modify its pathogenic factors, such as proteolytic activity, depending on the nutrients in the environment. This nutritional flexibility may explain why P. intermedia resides in both subgingival and supragingival areas. J. Jpn. Soc. Periodontol., 42: 175-181, 2000.

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The purposes of this study were to clinically and histologically observe the healing process of Sakagami' s newly developed minimally invasive gingival connective tissue graft. The alveolar mucosa of the canines and the third premolars of ten adult cats were used as experimental sites, and the new surgical procedure was performed on the labial/buccal mucosal area, from which attached/keratinized gingiva was surgically removed in advance. In this experiment, the epithelial tissue was left in the transplant, because it was more difficult to remove than in Sakagami's experiment. Clinical and histological observations were performed 2 days, and 1, 4, 6, and 8 weeks after surgery, with the following results : surgical stress was deemed minimal because no postoperative weight loss, infection, necrosis, or sloughing was observed ; the width of the attached gingiva had increased by two weeks after surgery and was maintained throughout the experimental period ; histopathologically, the implanted tissue maintained its original dense fibrous texture, which is characteristic of palatal gingiva, and gradually bound firmly to the periosteum of the alveolar bone; the epithelium in the implanted tissue did not disappear in the experimental period, but proliferated and dcsquamated inside the mucosal tissue. In conclusion, the new technique proved to be clinically useful in obtaining attached gingiva. Our results also suggested the necessity of excluding the epithelium from the implanted tissue to avoid epithelial proliferation with in the tissue. J. Jpn. Soc. Periodontol., 42: 182-191, 2000.

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The purpose of this study was to verify the effectiveness of the newly developed minimally invasive gingival connective tissue graft technique by clinical andhistopathological observations in monkeys. The surgical procedure can be briefly explained as follows. First, the tip of a 14-gauge aspirating needle is sharpened so that it can be used as a gingival extraction tool. The aspirating needle is inserted in to the palatal gingiva (donor site) of the subject. After inserting the needle to a depth of about 5 mm, a scalpel is used to section off and extract the gingival tissue. Atthe recipient site, a small incision is made with a scalpel, and an 18-gauge injection needle is inserted intothe submucosa to shape a tunnel-like transplantation site. The previously extracted gingival tissue is placed in the tunnel with a small forceps, and as soon as the gingival transplantation has been completed, the site is sutured. Operations have been conducted on two macaques by using the procedure described above. The extracted gingival tissues were transplanted to 8 upper and lower labial sites as experimental sites and 2 labial sites were not transplantatied as control sites. Clinical and histopathological observations were made, during the next 12 weeks and the following results were obtained:
1. The operation required only a short period of time and was easy to perform. Wound healing was uneventful with minimum bleeding.
2. The clinical observations showed that both the keratinized gingiva and the attached gingiva were increased after 12 weeks, compared to their presurgical status.
3. The histopathological observations showed that the transplanted tissues were surrounded by scattered collagenous fiber tissue after 1 to 2 weeks. After 12 weeks, however, the tissue enclosing the transplanted gingiva had formed connective tissue with tightly meshed fibers that had firmly attached to the periosteum of the alveolar bone. The transplanted gingiva had been transformed into attached gingiva, and the overlying epithelium was keratinized as well.
Based on the clinical and histopathological observations, compared to conventional mucogingival surgery, such as subpedicle connective tissue graft procedure, the new surgical method was minimally invasive and effective in achieving attached and keratinized gingiva. A modification of the new technique that excludes the epithelial tissue is also discussed. J. Jpn. Soc. Periodontol., 42: 192-199, 2000.

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The etiopathogenesis of adult periodontal disease is poorly understood, because it is a complex multifactorial disease, and individual differences in initiation and progression of the disease are dramatic. Some recent reports demonstrated that specific genotypes of the polymorphic interleukin-1 (IL-1) gene were a strong indicater of susceptibility to severe adult periodontitis. In this study, to clarify an association of IL-1 gene polymorphism with periodontal disease in Japanese young adults, we examined the distributions of IL-1 A^-889 and IL-1 B⁺³⁹⁵³ genotypes in subjects with periodontal disease and referent subjects. The frequency of IL-1 B genotypes including allele 2, especially homozygous for allele 2, of IL-1 B⁺³⁹⁵³ restriction fragment length bi-allelic polymorphisms was trended to increase in subjects with periodontal disease compared to referent subjects. Furthermore, a significant increase in the frequency of a composite genotype including both IL-1 A-889 allele 1 and allele 2, especially homozygous for allele 2, of IL-1 B+3953 found in subjects with periodontal disease compared to referent subjects. These findings suggest a possible role of IL-1 α and IL-1 β A gene polymorphisms in the susceptibility to periodontal disease in Japanese young adults. J. Jpn. Soc. Periodontol., 42: 200-207, 2000.

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