Effect of Porphyromonas gingivalis lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1beta on calprotectin release in human monocytes

Calprotectin is a major cytosolic protein of monocytes, granulocytes, and epithelial cells. It is known that calprotectin is released in inflammatory tissues and detected in gingival crevicular fluid (GCF) of periodontitis patients at high levels. The origin of calprotectin in GCF and its regulation in periodontal disease are unknown. In this study, we investigated the distribution of calprotectin in gingival tissue with inflammation and the induction of calprotectin release from human monocytes by lipopolysaccharide of Porphyromonas gingivalis (P-LPS), tumor necrosis factor-alpha (TNF-alpha), or interleukin-1beta (IL-1beta). Gingival tissues were obtained from a healthy donor and a periodontitis patient and calprotectin in gingival tissues was examined by immunohistochemical staining. Monocytes were isolated from the peripheral blood of healthy donors and cultured with P-LPS, TNF-alpha or IL-1beta for 30 minutes to 4 hours. The content of calprotectin in the cell and medium fractions was determined by enzyme-linked immunosorbent assay (ELISA). Calprotectin was markedly detected at the epithelial and adjacent connective tissue with many inflammatory cells in the gingival tissue from the periodontitis patient. P-LPS increased calprotectin release from monocytes to the maximum level after 30 minutes of treatment and its level was elevated to about 2- to 3-fold of the control level in a dose-dependent manner (1 to 1,000 ng/ml). When the effect of TNF-alpha and IL-1beta on calprotectin release was investigated, calprotectin release significantly increased to about 2.2- and 1.5-fold that of the control level, respectively. These results demonstrate that calprotectin release from monocytes is induced by P-LPS, TNF-alpha, and IL-1beta, which in turn, cause and aggravate periodontal disease.


Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14-Toll-like receptor-nuclear factor kB pathway

Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor kappaB (NF-kappaB). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14-TLR-NF-kappaB pathway and the cellular mechanism of calprotectin release in human neutrophils. Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-kappaB, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-kappaB binding activity using electrophoretic mobility shift assays. P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-kappaB inhibitors suppressed P-LPS-induced NF-kappaB binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. These results suggest that calprotectin release is induced by P-LPS via the CD14-TLR2-NF-kappaB signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems.


Decreased expression of α2 integrin in fibroblasts isolated from cyclosporin A- induced gingival overgrowth in rats

Cyclosporin A (CsA), an immunosuppressive agent, induces fibrous gingival overgrowth through reduction of collagen phagocytosis by fibroblasts. Distinct receptors are involved in the binding of collagen to fibroblasts in collagen phagocytosis, and alpha2beta1 integrin serves as a specific receptor for type I collagen on fibroblasts. To elucidate the role of alpha2beta1 integrin in CsA-induced gingival overgrowth, we investigated collagen phagocytosis and alpha2beta1 integrin expression in rat gingival fibroblasts. Fibroblats were isolated from gingiva of rats fed a powdered diet containing or lacking CsA for 30 d. Flow cytometric analysis were performed to measure the collagen phagocytosis and the alpha2 integrin expression in fibroblasts. Furthermore, total RNAs were isolated from fibroblasts, and the reverse transcriptase-polymerase chain reaction was employed to investigate the mRNA levels of alpha2 integrin. In vitro collagen phagocytosis assay revealed that CsA-treated and control fibroblasts contained a mean of 13.5% and 36.1% phagocytic cells, respectively. CsA-treated fibroblasts had 28% lower expression of alpha2 integrin than that of control. and mRNA expression of alpha2 integrin in CsA-treated fibroblasts was apparently lower than in the controls, but the mRNA expression of beta1 integrin was not affected. These findings suggest that one etiological factor of gingival overgrowth may be inhibition of collagen phagocytosis by reducing alpha2 integrin expression in gingival fibroblasts.


Case Report of an Early-onset Periodontitis Patient Showing Self-Arrest of Alveolar Bone Loss after Puberty

思春期後に歯槽骨吸収が自然停止した早期発症型歯周炎患者の1症例

Journal of the Japanese Association of Periodontology


Okadaic acid stimulates the expression of receptor activator of nuclear factor-kappa B ligand RANKL in mouse osteoblastic cells


MMP 阻害剤の歯槽骨吸収抑制作用

MMP 阻害剤の歯槽骨吸収抑制作用


Nifedipine induces gingival epithelial hyperplasia in rats through inhibition of apoptosis

Nifedipine is used as a long-acting vasodilator; one of its side effects is gingival overgrowth, characterized by an accumulation of collagenous components within the gingival connective tissue and epithelial hyperplasia with elongated, branched rete pegs penetrating into the connective tissue. We investigated the effect of nifedipine on apoptosis of gingival keratinocytes of rats to elucidate the mechanism of nifedipine-induced gingival epithelial hyperplasia. Twenty-day-old rats were fed a powdered diet containing or lacking nifedipine for 8 to 30 days. The mandibular gingiva and palatal mucosa were removed on days 8, 15, or 30, and epithelial thickness was examined by light microscopy. In situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to examine apoptosis of keratinocytes in the epithelium. In addition, we examined the effects of nifedipine on proliferation of keratinocytes and epithelial cell life on day 8 by 5-bromo-2'-deoxyuridine (BrdU) staining. Microscopic examination showed gingival epithelial hyperplasia in nifedipine-treated rats after day 15. Apoptosis of gingival keratinocytes was seen to be inhibited in nifedipine-treated rats on day 8 and 15. Also, nifedipine did not induce an increase of keratinocyte proliferation activity in terms of the number of cells showing positive staining with BrdU. Prolongation of cell life by nifedipine was observed on day 8 in gingival epithelium through a delay of upward cell movement compared to controls. However, epithelial hyperplasia was not detected in palatal mucosa, and there were no significant differences in apoptotic rates of keratinocytes and cell life between nifedipine-treated rats and control rats. These results suggest that nifedipine induces epithelial hyperplasia in gingival overgrowth not by an increase in keratinocyte proliferation, but by prolongation of cell life through reduction of apoptosis before epithelial hyperplasia is detectable.


Effects of TGF-beta1 on Extracellular Matrix Formation in Rat Clonal Dental Pulp Cells


Application of Enamel Matrix Derivative in Autotransplantation of an Impacted Maxillary Premolar

The success of tooth transplantation or replantation depends on the viability of periodontal ligament in the planted tooth. Mechanical injury to periodontal tissues frequently results in dental root resorption and dental ankylosis, which leads to the failure of transplantation or replantation. Enamel matrix derivative (EMD) has been recently used to induce periodontal regeneration. In this report, we show a clinical case of EMD application in the transplantation of an inversely impacted and immature tooth. An impacted second premolar was found in the right maxilla of a 16 year-old girl. The tooth was inversely impacted and the dental root was incomplete. When transplantation was carried out, EMD was applied to the periodontal tissues of the extracted premolar. The tooth was fixed at the correct position and the clinical condition was followed for evaluation for 6 months. Radiographs after 3 months exhibited new bone formation surrounding the transplanted tooth. After 6 months, considerable growth of dental root was evident, periodontal ligament-like radiolucency appeared, the vital reaction of the planted tooth was detected, and there were no signs of root resorption or ankylosis. Short-term results from this case indicate that EMD application was effective in the transplantation of an inversely impacted and immature tooth and that EMD might contribute to the growth of dental root and to the prevention of ankylosis.


Quantitative Analyses of Propeptide of Type Procollagen and Osteocalcin in Gingival Crevicular Fluid of Periodontal Disease Patients

歯周炎患者の歯肉溝滲出液中に含まれるI型プロコラーゲンペプチドおよびオステオカルシンの定量分析

Biochemical markers in gingival crevicular fluid (GCF) have been studied to precisely and objectively diagnose periodontal disease. Carboxy terminal propeptide of type I procollagen (P I CP) and osteocalcin (OCN) are bone-related proteins produced by osteoblasts that are useful clinical indicators of metabolic bone diseases such as osteoporosis and hyperparathyroidism. To determine bone-related markers in GCF, we studied the presence of P I CP and OCN in GCF from periodontitis patients using immunoen-zymatic kits and the relationship between P I CP/OCN amounts and clinical parameters involving gingival index (GD, probing depth (PD) , and bleeding on probing (BOP). GCF samples were collected with paperstrips from 70 diseased sites (PD>4 mm) in 70 periodontitis patients and from 30 healthy sites (PD< 3 mm) in 30 healthy volunteers. A mean of 1.04 ng/site (0.91 ng/μl) P I CP was found at diseased sites and a mean of O . 17 ng/site (0.38 nglpl) at healthy sites, showing a 6 . 1-fold amount of P 1 CP at diseased sites. A mean of 56 .3 pg/site (47.6 pg/μl) OCN was found at diseased sites and a mean of 38.8 pg/site (1203.9 pg/μl) at healthy sites, showing a 1 .5-fold amount of OCN at diseased, sites. Both P 1 CP and OCN in GCF correlated positively with GI and PD, especially P 1 CP, P 1 CP greatly increased at BOP-positive sites (5.5-fold) , suggesting that P I CP may reflect disease activity in alveolar bone metabolism. These results indicate that P 1 C,P and OCN are present in GCF and that these molecules may be useful as a bone-related marker in periodontitis. P 1 CP may prove to be an especially potent marker for bone turnover in periodontitis. J Jpn Soc Periodontol, 44 : 73-81, 2002.

歯周病を正確かつ客観的に診断するために,歯肉溝滲出液(GCF)中の生化学的な指標を見出そうという研究が長年行われている．I型プロコラーゲンC末端ペプチド(P1CP)およぴオステオカルシン(OCN)は,骨芽細胞によって産生される骨代謝関連蛋白であり,骨粗髷症や副甲状腺機能亢進症のような代謝性骨疾患において有用な臨床指標として用いられている．本研究では,GCF中の骨代謝関連指標を探索するために,PICPとOCNがGCF中に存在するかどうかを酵素抗体法キットを用いて検証するとともに,P1CP童およびOCN量を定量し,それらの値と臨床パラメーターである歯肉炎指数(GI),プロービング値(PD),プロービング時の出血(BOP)の有無との関連をそれぞれ調べた．GCF試料はぺ一パーストリップスを用いて,70名の歯周炎患者から歯周ポケット70部位(PD>4mm),およぴ30名の健常者から健常歯肉溝30部位(PD<3mm)を選んで採取された．GCF中のP1CP量はポケット部位で平均1.04ng/site(0.91ng/μl),健常部位で0.17ng/site(0/38ng/μl)であり,ポケット部位で6.1倍のP1CP量が探知された．OCN量はポケット部位で平均56.3pg/site(47.6pg/μl),健常部位で38.8pg/site(123.9pg/μl)であり,ポケット部位で1.5倍のOCN量が探知された．PICP量およぴOCN量は,いずれもGIおよぴPDと正の相関関係を示した．とくに,P1CPは両方の指標と強く相関していた．さらに,P1CP量はBOPの認められる部位で高く(5.5倍値),歯槽骨代謝における疾患活動性を反映している可能性が示唆された．以上の結果は,GCF中にはP1CPおよびOCNが存在し,これらの分子は歯周炎の骨代謝関連指標になりうる可能性があることを示すものであり,とくにPICPは歯周炎における骨代謝回転を反映する指標として有カであることが示唆された．


Autotransplantation of Impacted Teeth and Wisdom Teeth


Visiting service of oral health care for patients suffering from systemic diseases

歯科訪問診療における口腔衛生指導への取り組み


Osteopontin in gingival crevicular fluid

Osteopontin (OPN) is a major glycosylated phosphoprotein in bone matrix and is produced by several cells including osteoblasts, osteoclasts and macrophages. OPN levels increase in active sites of bone metabolism. Recently, several bone-related proteins were identified in gingival crevicular fluid (GCF) to seek markers of alveolar bone resorption in periodontal disease. In this study, we investigated the existence of OPN in GCF and the correlation between OPN level in GCF and probing depth (PD) of sampling sites in 98 periodontitis patients and 35 healthy subjects. An immunoblotting analysis using 10% polyacrylamide gel showed that two forms of OPN with molecular masses of 54 and 66 kDa and several degraded fragments were detected in most GCF samples from diseased sites (PD > 4 mm). In GCF samples from healthy sites (PD < or = 3 mm), only one form (54 kDa) was observed, but any degraded fragments were not detected. When OPN amounts in GCF samples were determined by ELISA, a weak. but significant correlation was observed between OPN amount in GCF and PD (r=0.32, p=0.0013). These results demonstrate that OPN exists in GCF and that OPN level in GCF increases with the progression of periodontal disease.


Nifedipine induces gingival overgrowth in rats through a reduction in collagen phagocytosis by gingival fibroblasts

Nifedipine is used as a long-acting vasodilator, and a primary side effect is the induction of gingival overgrowth, which is characterized by an accumulation of collagenous components within the gingival connective tissue. To elucidate the mechanisms of nifedipine-induced gingival overgrowth, we investigated the effect of nifedipine on Type I collagen metabolism in the gingiva of rats. Twenty-day-old rats were fed a powdered diet containing or lacking nifedipine for 3 to 55 days. Immunohistochemical analysis with anti-Type I collagen antibody was employed to examine the density of Type I collagen in the gingival connective tissue. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 15, 30, and 55, and reverse transcription polymerase chain reaction was performed to investigate the mRNA levels of Type I collagen. In addition, we performed a flow cytometric analysis with collagen-coated latex beads and cultured fibroblasts derived from rat gingiva to measure collagen phagocytosis. Immunohistochemical analysis revealed that Type I collagen was more prevalent in the connective tissue of nifedipine-treated gingiva than in controls on day 55. In the nifedipine-treated group, the expression of Type I collagen mRNA gradually decreased to 1.5% on day 55 compared to day 0. In the control group, Type I collagen mRNA also decreased to 32%; however, mRNA expression was significantly lower in the nifedipine-treated group than in the controls. When the rate of phagocytic cells derived from nifedipine-treated gingiva and controls was represented as the mean +/- SE of the percentage from 3 different experiments, the values were as follows: on day 15, 13.5 +/- 2.1% and 15.0 +/- 1.5%; on day 30, 12.2 +/- 4.3% and 34.5 +/- 6.7% in the nifedipine-treated and the control group, respectively, indicating that phagocytic cells were considerably fewer in the nifedipine-treated gingiva on day 30. This finding demonstrates that the decrease in phagocytosis caused by nifedipine appeared before the detection of severe macroscopic gingival overgrowth. These findings suggest that the decrease in collagen degradation due to lower phagocytosis is closely associated with the increase in Type I collagen accumulation in nifedipine-treated rat gingiva.


Immunohistochemical Localization of Osteopontin in human Pulp Stones.

The organic matrix component of human pulp stones was investigated by immunohistochemistry. Two pulp stones were extracted from the upper molar teeth of two patients suffering from irreversible pulpitis. Both were formed in the center of the pulp cavity and located apart from the dentin walls. After demineralization, serial sections of the stones were prepared and subjected to immunohistochemical procedures using specific antibodies to type I collagen and noncollagenous proteins (osteopontin, osteonectin, and osteocalcin), which are reported to be involved in calcified matrix formation. Type I collagen was localized evenly in the stones, indicating that it is a major matrix component of pulp stones. Strong immunostaining of osteopontin appeared in the peripheral area of the stones, whereas osteonectin and osteocalcin were not detected. We previously reported that dental pulp cells produced osteopontin in vitro. Osteopontin has been commonly found in other pathological calcification, such as urinary stones, atherosclerotic plaques, and dental calculus. Taken together, the present findings suggest that osteopontin produced by dental pulp cells is possibly associated with calcification of the pulp stone matrix.


A Case Report on Gingival Overgrowth in a Patient Receiving Manidipine Hydrochloride

塩酸マニジピン服用患者にみられた歯肉増殖症の1例

Journal of the Japanese Association of Periodontology


Cyclosporin A Decreases the Degradation of Type I Collagen in Rat Gingival Overgrowth

Cyclosporin A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of fibrous gingival overgrowth. The purpose of this study was to investigate the effect of CsA on the type I collagen metabolism in the gingiva of rats fed a powdered diet either containing or lacking CsA. Immunohistochemical analysis revealed that type I collagen was more prevalent in the connective tissue of CsA-treated gingiva than in those of control rats on days 15, 30, and 55 after the start of feeding. Total RNAs were isolated from mandibular molar gingiva on days 0, 3, 8, 15, 30, and 55. Quantitative analysis of mRNA by reverse transcriptase-polymerase chain reaction revealed that the CsA-treated groups showed a gradual decrease in expression of type I collagen and collagenase mRNAs, 0.4% and 18.0% on day 55 compared with those on day 0, respectively. In the control groups, type I collagen and collagenase mRNAs also decreased to 19.7% and 63.0%, respectively, however, both mRNA expressions were significantly lower in the CsA-treated group than in the controls. An electron microscopic analysis of fibroblasts was performed to count the number of cells with collagen fibrils in the cytoplasm, a marker of phagocytosis of collagen by fibroblasts. The collagen fibrils were detected in 4.7% +/- 2.7% and 24.3% +/- 13.7% of fibroblasts in the overgrown gingiva treated with CsA rat for 8 days and 30 days, but in 57.0% +/- 5.3% and 81.3% +/- 9.2% of fibroblasts in the each control group gingiva, respectively. Furthermore, in vitro analysis was performed to measure the phagocytosis of cultured fibroblasts by flow cytometry using collagen-coated latex beads. Fibroblasts isolated from CsA-treated gingiva on day 8 and day 30 contained 5.7% +/- 0.6% and 9.9% +/- 1.5% phagocytic cells, whereas control fibroblasts contained 50.3% +/- 5.5% and 33.3% +/- 4.9% phagocytic cells, respectively. The inhibition rate of phagocytic activity was similar between in vivo and in vitro assays. These findings suggest that the decrease of the collagen degradation due to the lower phagocytosis and the lower collagenase mRNA expression are closely associated with the increase of type I collagen accumulation in CsA-treated rat gingiva.


Mast cells are not involved in the development of cyclosporin A-induced gingival hyperplasia: a study with mast cell-deficient mice

A previous study suggested that mast cells (MC) are involved in the development of cyclosporin A-induced gingival hyperplasia, since an increased number of MC were observed in the tissue sections of enlarged gingiva. To determine the role of MC in gingival hyperplasia, an MC-deficient mouse model was used in the current study. MC-deficient mice (WBB6F1xW/Wv) and their littermates (+/+) were fed sucrose-containing diets supplemented with or without varying concentrations (300, 400, 500, 600 mg) of cyclosporin A/kg of diet. After 30 days, the mice were sacrificed and the degree of gingival hyperplasia was evaluated by the appearance of the gingiva. Tissue MC were stained with toluidine blue to confirm the presence or absence of MC in the enlarged gingiva. Both W/Wv and +/+ mice, when fed with 600 mg cyclosporin A/kg diet for 30 days, exhibited a similar degree of gingival hyperplasia, while other test mice or control mice did not. Toluidine blue staining of the tissue sections confirmed the presence of MC in the enlarged gingiva of the +/+ mice, but not the W/Wv mice. These results indicate that mast cells are not necessary in the development of cyclosporin A-induced gingival hyperplasia, and that the increased number of MC observed in the enlarged gingiva may be a secondary effect of gingival hyperplasia. We also conclude that a study of mice lacking certain molecules or cells would be quite useful in determining the molecules or cell types responsible for the pathogenesis of drug-induced gingival hyperplasia.


Inhibition of Osteoblastic Cell Differentiation by Lipopolysaccharide from Porphyromonas gingivalis and Its Mechanism

Porphyromonas gingivalis 由来リポ多糖による骨芽細胞の分化抑制作用およびその機構解明に関する研究

The Japanese Journal of Conservative Dentistry


Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor-β in rat clonal dental pulp cells

Dental pulps contain sulfated glycosaminoglycans (GAGs), such as chondroitin 4-sulfate (CSA/4CS), dermatan sulfate (CSB/DS), and chondroitin 6-sulfate (CSC/6CS). Sulfated GAGs play important roles in mineralization and collagen fibrillogenesis during primary, secondary, and reparative dentin formations. Transforming growth factor-beta (TGF-beta) is a potent regulator for several extracellular matrix (ECM) components and modulates the proliferation and differentiation. Using rat clonal dental pulp cells (RPC-C2A), we investigated the constituents of GAGs synthesized by the cells and the effect of TGF-beta on their synthesis by measuring the radioactivity of [35S]sulfate incorporated into GAG fractions. Cellulose acetate electrophoresis analysis revealed that RPC-C2A cells synthesized CSA and CSB but not CSC and that 10 ng/ml of TGF-beta increased the production of CSA and CSB in the cell/ECM fraction. Measurement of [35S]sulfate incorporation showed a significant increase in the amount of GAGs by TGF-beta, 1.3-fold CSA, and 1.2-fold CSB in the cell/ECM fraction. In the medium fraction the most secreted GAG was CSA, whereas CSB was stored in the cell/ECM fraction. Secreted CSA in the medium was markedly increased by 10 ng/ml of TGF-beta (1.7-fold). These findings indicate that CSA and CSB are major sulfated GAGs synthesized by RPC-C2A cells and that TGF-beta acts as a stimulator of sulfated GAG synthesis in dental pulp cells.


The association of calprotectin level in gingival crevicular fluid with gingival index and the activities of collagenase and aspartate aminotransferase in adult periodontitis patients

Calprotectin, a major cytosol protein of leukocytes, exists in plasma and other body fluids of healthy human subjects. Since the calprotectin concentration rises markedly in some inflammatory diseases including rheumatoid arthritis, this protein has been thought to be a marker of inflammatory disease. Recently, we identified calprotectin in human dental calculus and gingival crevicular fluid (GCF), and found that the calprotectin concentration in GCF from patients with periodontitis was significantly higher than that in GCF from healthy subjects. In the present study, the association of GCF calprotectin level with GCF volume, gingival index (GI), and levels of biochemical markers including collagenase and aspartate aminotransferase (AST) in GCF was investigated to clarify the relationship between GCF calprotectin level and periodontal inflammation. Ninety GCF samples collected from periodontal pockets with a probing depth of more than 4 mm in 54 patients with adult periodontitis were used for these assays. The GCF volume was measured, and GI in each site was recorded. The calprotectin content in GCF samples was determined by ELISA using a specific antibody. The activity of collagenase or AST was measured by a respective assay kit. The total amount of calprotectin and GCF volume showed a highly significant correlation (r = 0.64, P <0.0001), whereas the calprotectin concentration had no correlation with the GCF volume (r = 0.01, P= 0.924). The mean calprotectin concentration in GCF increased with the degree of GI, and the concentration in individual samples was significantly correlated with the GI score (r = 0.56, P<0.0001). Significant positive correlations were observed in GCF calprotectin versus collagenase (r = 0.57, P <0.0001) and GCF calprotectin versus AST levels (r = 0.40, P <0.005). From the present results and our previous findings, it is shown that the GCF calprotectin level significantly correlates not only with clinical indicators but also with current biochemical marker levels and that calprotectin may be a useful marker for periodontal inflammation.


ハムスター実験的歯周炎におけるプロスタグランディンE2の歯槽骨吸収抑制効果

ハムスター実験的歯周炎におけるプロスタグランディンE2の歯槽骨吸収抑制効果

The Japanese Society of Conservative Dentistry


Epidemiologic Study of Periodontal Conditions in Patients Undergoing Renal Dialysis

人工透析患者の歯周病罹患度に関する疫学的研究


Stimulatory Effects of Phenytoin on Osteoblastic Differentiation in Cultured MC3 T3-E1 Cells

培養骨芽細胞MC3T3-E1におけるフェニトインの分化促進作用


Stimulatory effects of phenytoin on osteoblastic differentiation of fetal rat calvaria cells in culture.

Phenytoin (diphenylhydantoin, DPH), an anticonvulsant drug for epileptic patients, has several adverse effects, including calvarial thickening and coarsening of the facial features, which occur with chronic DPH therapy. While previous studies have demonstrated that DPH has an anabolic action on bone cells in vivo and in vitro, the basis of these effects is not fully understood. In this study, the effect of DPH on osteoblastic differentiation of fetal rat calvaria (RC) cells in culture was investigated by measuring bone nodule (BN) formation, cell growth, alkaline phosphatase (ALPase) activity, collagen synthesis, and expression of osteocalcin (OC) and osteopontin (OP) mRNAs. Continuous treatment of RC cells with DPH for 18 days dose-dependently increased the mineralized BN number by 1.2-1.7-fold at concentrations of 12.5-200 micromol/L DPH. Cell growth was not affected at the same concentrations of DPH. ALPase activity was stimulated by DPH (1.1-1.9-fold) dose-dependently and was maintained at higher levels in DPH-treated cells throughout the experimental period. DPH increased mineralized and unmineralized BN formations both in the presence and the absence of 10(-8) mol/L dexamethasone (Dex). Expression of OC and OP mRNAs was markedly augmented by DPH on days 12-24 and on days 12-18, respectively. While control mRNA levels of OC and OP increased with time, the increases in DPH-treated cells were greater than those of the controls and the stimulatory effects were dose-dependent. Type I collagen was also influenced by DPH; mRNA level was enhanced and the percentage of collagen synthesized was increased significantly, by 200 micromol/L DPH. When DPH was added in three different culture stages, days 1-6 (growth), days 7-12 (matrix development), and days 13-18 (mineralization), BN formation was influenced primarily on days 1-6 and secondarily on days 7-12, but not on days 13-18, suggesting that DPH increased BN formation by enhancing not only the proportion of osteoprogenitor cells in the early stage but also the proportion of functional osteoblasts in the middle stage within mixed-cell populations. Moreover, such increases were detected in conditions of both Dex(+) and Dex(-). These findings demonstrate that DPH stimulates osteoblast-associated markers such as BNs, ALPase, OC, OP, and type I collagen by continuously affecting the stages of growth and matrix development in RC cells, and suggests that the stimulatory effects by DPH may possibly be induced independent of those by Dex.


Calprotectin in gingival crevicular fluid correlates with clinical and biochemical markers of periodontal disease.

Clinical and biochemical markers of periodontal disease have been used for precise objective diagnosis of periodontal inflammation. Interleukin 1beta (IL-1beta) and prostaglandin E2 (PGE2), inflammatory factors, levels in gingival crevicular fluid (GCF) of patients with periodontal disease are elevated and have been studied as biochemical markers. The levels of calprotectin, a leukocyte protein, in body fluids of patients with some inflammatory diseases are raised. Recently, we detected calprotectin in GCF and its concentrations in periodontal pockets were higher than those in healthy gingival crevices. In this study, we investigated the correlations between GCF calprotectin levels and clinical indicators (probing depth and bleeding on probing, BOP), and the IL-1beta or PGE2 levels in GCE Probing depth and BOP at 130 sites of 110 subjects with periodontal or other oral diseases were examined, then GCF samples were collected and their calprotectin, IL-1beta and PGE2 were determined by ELISA. The calprotectin level correlated positively with the probing depth and was significantly higher at BOP-positive than BOP-negative sites. There were significant, positive correlations between the calprotectin and IL-1beta or PGE2 concentrations. These results indicate that the calprotectin level in GCF correlates well with clinical and biochemical markers of periodontal disease and suggest that calprotectin may be useful for evaluating the extent of periodontal inflammation.


Examination of the roots of paramolar tuberoles with computed tomography: Report of 3 cases

Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology


Effect of retinoic acid on osteopontin expression in rat clonal dental pulp cells


Pronounced palatal and mandibular tori observed in a patient with chronic Phenytoin therapy, a case report

Phenytoin, an anticonvulsant drug for epileptic patients, has many adverse effects, including calvarial thickening and coarsening of the facial features. Previous studies have demonstrated that phenytoin has an anabolic action on bone cells. This report describes pronounced palatal and mandibular tori found in a 45-year-old Japanese man undergoing chronic phenytoin therapy. The tori were extremely large, lobular, and symmetrical. A palatal torus appeared along the middle of the hard palate and mandibular tori consisted of 2 pairs of nodular masses extensively filling the lingual floor of the oral cavity. Pronounced osseous outgrowth occurred for the duration of a dose-increase of phenytoin from 1985 to 1997. His parents did not have any palatal or mandibular tori. These facts suggest that these unusual tori may have been the result of chronic phenytoin therapy, rather than association with the familial background.


Stimulatory Effects of Phenytoin on Osteoblastic Differentiation of Fetal Rat Calvaria Cells in Culture


Calprotectin, a leukocyte protein related to inflammation, in gingival crevicular fluid.


Prostaglandin E2 inhibits alveolar bone resorption in experimantal periodontitis in hamster


cDNA cloning of bovine thrombospondin 1 and its expression in odontoblasts and predentin

The extracellular matrix protein thrombospondin 1 (TSP1) was cloned from odontoblasts of bovine mandibular teeth which participate in dentinogenesis. The 5289 bp cDNA contains a complete open reading frame of 1170 amino acids. Bovine TSP1 has high homologies to its human and mouse counterparts. In immunohistochemical analyses of bovine anterior teeth with anti-TSP1 monoclonal antibody, TSP1 was only detectable at the position of predentin, located between dentin and unmineralized dental pulp. Northern blot analysis showed high levels of two sizes of TSP1 mRNAs in odontoblasts but not dental pulp and gingiva. Previously we found that osteotropic factors such as calcitriol and TGF-beta induce TSP1 at the transcriptional level in clonal rat dental pulp cells. These results suggest a role of TSP1 in dentinogenesis and/or maintenance of dentin and dental pulp.

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology


前立腺癌細胞培養上清の骨芽細胞分化抑制作用とその性状

前立腺癌細胞培養上清の骨芽細胞分化抑制作用とその性状


Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro.

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5-30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma.


Identification of calprotein , a calcium binding leukocyte protein, In human dental calculus matrix

Calprotectin is a calcium binding protein produced by leukocytes, macrophages and epithelial cells, and its levels in several tissues increase during infections and in many inflamed areas, suggesting that it may be an indicator of inflammatory activity. Osteopontin is a prominent phosphorylated glycoprotein in bone matrix, having calcium binding capacity. Recently, it has been reported that calprotectin and osteopontin are present in urinary stones (pathological mineralized masses in the body), and that these proteins may be involved in their formation. Dental calculus formed by mineralization of dental plaque is an inflammatory factor which may contribute to periodontal disease. It contains many organic components involved in mineralization. We recently found osteopontin molecules in human dental calculus and suggested that the components of its matrix may be similar to those of urinary stones. In this study, we investigated the presence of calprotectin in human dental calculus by immunohistochemical and immunoblotting analyses using a specific antibody for calprotectin. After fixation and demineralization of dental calculi adhered to tooth roots, sections embedded in paraffin were immunoreacted with the antibody for calprotectin and positive immunostaining for calprotectin was observed. Dental calculus proteins were then extracted with EDTA and separated by electrophoresis on 15% polyacrylamide gels. By immunoblotting analysis, 3 or 4 bands were observed at 11, 14.5, 22-25, 28 or 36.5 kDa and these patterns corresponded to those of calprotectin subunits. When non-immune rabbit serum was used instead of calprotectin-specific antibody as a negative control, no immunoreactivity was observed. These findings indicate that calprotectin is associated not only with antibacterial action but also with calcium binding capacity during dental calculus formation.


Relationship between the expression of parathyroid hormone receptors and hormonal effect during rat osteoprogenitor cell differentiation


ラット胎児頭蓋冠由来細胞培養系におけるプロスタグランディンE2の石灰化促進作用

ラット胎児頭蓋冠由来細胞培養系におけるプロスタグランディンE2の石灰化促進作用

Journal of the Japanese Association of Periodontology


ラット後肢の指骨切断後の治癒過程に及ぼすethane-1-hydroxy-1, 1-diphosphonateの影響—光学顕微鏡所見を中心に—


Identification of osteopontin in human dental calculus matrix.

Osteopontin is a prominent non-collagenous component of bone matrix, although it is expressed in several other tissues. Recently, osteopontin was reported to be involved in urinary stone formation and atherosclerotic lesions of the aorta, suggesting that it may be a key protein associated with these types of pathological mineralization. In this study, whether or not human dental calculus contains osteopontin was investigated by immunoblotting and immunohistochemical analyses. After extraction of calculus proteins with EDTA and separation of the proteins by electrophoresis, immunoblotting analysis revealed the presence of osteopontin. Two forms of osteopontin appeared at 61 and 68 kDa on 10% polyacrylamide gel and the proteins were digested with thrombin, a highly specific protease. Moreover, immunohistochemical analysis revealed that osteopontin was localized in dental calculus adherent to tooth roots. These findings indicate that osteopontin is, in fact, present in human dental calculus and may be involved in calculus formation as the stone matrix.


Identification of osteopontin in human dental calculus matrix


Physiological concentrations of retinoic acid suppress the osteoblastic differentiation of fetal rat calvaria cells in vitro.

The effects of retinoic acid (RA) on osteoblastic differentiation and activity were studied in fetal rat calvaria cells cultured for up to 24 days. Fetal bovine serum used for the experiments was treated with an anion-exchange resin to remove endogenous RA. The depletion of RA in the treated serum was confirmed by high-performance liquid chromatography and tritiated RA tracing. Under the culture conditions employed, the continuous presence of RA for 14 days at 10(-9) mol/l or higher decreased both alkaline phosphatase (ALP) activity on day 12 and the number of bone nodules on day 14 in a dose-dependent manner. Short-term (24 h) exposure to RA at 10(-8) mol/l, which is a physiological concentration, decreased and increased the levels of ALP and osteopontin mRNA on day 6, respectively. Retinoic acid at 10(-8) mol/l also increased the level of osteocalcin mRNA on day 12. However, these effects were not obvious at later stages (days 18 and 24). At a high concentration (10(-6) mol/l), RA increased the level of osteopontin mRNA on day 6 and decreased the levels of ALP and osteocalcin mRNA irrespective of culture period. These results suggest that, at physiological concentrations, RA suppresses the differentiation of osteoprogenitor cells and regulates osteoblastic functions.


Osteopontin expression in clonal dental pulp cells

Annals of the New York Academy of Sciences


株化ラット歯髄細胞培養上清がラット頭蓋冠由来骨原性細胞の石灰化骨様結節形成に及ぼす効果

The Japanese Journal of Conservative Dentistry


Thyroid hormone suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances functional activities of mature osteoblasts in cultured rat calvaria cells.

The effects of thyroid hormone on osteoblastic differentiation and activity were studied in fetal rat calvaria (RC) cells cultured for up to 30 days in medium supplemented with thyroid hormone-depleted serum. In this condition, the cells proliferated and differentiated to form mineralized bone nodules (BN) and expressed osteoblastic markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). The continuous presence of triiodothyronine (T3) at 10(-9)-10(-8) M in the medium inhibited the osteoblastic differentiation: 34% decrease in ALP activity on day 12 and 60% decrease in BN formation on day 15 at 10(-8) M. T3 at these doses had no effect on the DNA content of RC cells at confluence (day 6). Short-term (48-h) exposure of T3 at 10(-9) M or higher decreased ALP activity when RC cells were differentiating (days 7-11). However, when BN formation by the cells had already reached a plateau (day 28), the activity was increased by treatment with T3 at 10(-7)-10(-6) M. OCN production was increased dose dependently by this treatment with T3 (2.1-fold and 1.3-fold of control at 10(-8) M on days 11 and 28, respectively). Similar increases were observed in the levels of OCN mRNA. In addition, increases in phosphorylated OPN in the medium (day 11) and mineralized matrix (day 28) were observed (1.5-fold at 10(-8)-10(-6) M), while OPN synthesis and the level of its mRNA were depressed by T3 (60-70% of control at 10(-8) M). These results suggest that T3 regulates osteoblastic differentiation and activity depending on the state of cell differentiation: T3 suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances the functional activity of mature osteoblasts.


Biochemical study on the tissue compartmentalization of phosphophoryn, osteopontin and bone sialoprotein (BSP) in rat incisor dentin


1α,25-Dihydroxyvitamin D3 stimulation osteopontin expression in rat clonal dental pulp cells


Expression and characterization of parathyroid hormone receptors on fetal rat calvaria cells in culture


Relationship between Calcium Binding Proteins Associated with Mineralized Bone Nodule and 5'-Methylthioadenosine Metabolism in Cultured Rat Calvaria Cells

ラット頭蓋冠由来細胞が合成分泌するカルシウム結合性蛋白とメチルチオアデノシン代謝との関連

5'-Methylthioadenosine (MTA) is formed during the biosynthesis of polyamines. We previously reported that 5'-difluoromethylthioadenosine (DFMTA), a synthetic inhibitor of MTA phosphorylase, caused intracellular adenine depletion and inhibited both alkaline phosphatase activity and bone nodule formation in cultured rat calvaria cells. In addition, exogenous adenine enhanced these two markers and partially reversed the DFMTA-induced inhibition in this culture system. Osteopontin, a non-collagenous protein associated with matrix mineralization, has been reported to be predominantly incorporated into bone nodules in concert with the progresism of in vitro mineralization on rat calvaria cells. In this study, we examined the effects of adenine and DFMTA on osteopontin contents incorporated into mineralized nodules by using polyacrylamide gel electrophoresis and "Stains-All" staining. Adenine increased the accumulation of osteopontin into mineralized nodule whereas DFMTA inhibited it. Furthermore the osteopontin contents which had been decreased by DFMTA recovered with the addition of adenine. These results suggest that adenine formed from MTA is important in the mineralization process of rat calvaria cells.

メチルチオアデノシン(MTA)代謝は種々の細胞の増殖や分化に関与しており,ジフルオロメチルチオアデノシン(DFMTA)はMTAホスホリラーゼの阻害剤,アデニンはMTAの代謝産物である．本研究では,ラット頭蓋冠由来細胞培養系における石灰化骨様コロニー中のオステオポンティン量に及ぼすDFMTAとアデニンの効果を,ポリアクリルアミドゲル電気泳動法とステインズオール染色法を用いることによって検索した．その結果,DFMTAは石灰化骨様コロニー中のオステオポンティン量を減少させたが,アデニンはそれを増加させた．さらに,DFMTAによって減少したオステオポンティン量は,アデニンの添加によって対照群に近いレベルにまで回復した．これらの事実から,MTA由来のアデニンはラット頭蓋冠由来細胞の石灰化過程に重要な役割を担っている可能性があることが示唆された．

Journal of the Japanese Association of Periodontology


Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar)

Biochemical and Biophysical Research Communications


ヒト歯髄細胞培養系におけるインスリンとEGFの作用

ヒト歯髄細胞培養系におけるインスリンとEGFの作用


The role of 5'-methylthioadenosine on rat calvaria cell differentiation.

The role of 5'-methylthioadenosine (MTA), formed during the process of polyamine biosynthesis, on differentiation of osteoprogenitor cells was assessed by its effects on alkaline phosphatase (ALP) activity, bone nodule formation and osteopontin contents of cultured rat calvaria (RC) cells. These three markers were stimulated by exogenous MTA and were depressed by 5'-difluoromethylthioadenosine (DFMTA), a synthetic inhibitor of MTA phosphorylase, which cleaves MTA to adenine and 5-methylthioribose-1-phosphate. 5-Methylthioribose and 2-keto-4-methylthiobutyrate, metabolites of 5-methylthioribose-1-phosphate, had no effects on ALP activity and bone nodule formation in the presence or absence of DFMTA. On the other hand, adenine enhanced ALP activity, bone nodule formation and osteopontin contents in mineralized nodules and also partially reversed DFMTA-induced inhibition of these three markers. MTA, its metabolites and DFMTA did not affect the growth of RC cells under these culture conditions. These results suggest that adenine formed from MTA is important in the differentiation of RC cells.


Role of the Metabolism of 5'-Methylthioadenosine in the Proliferation and Differentiation of Osteoprogenitor Cells : Effects of an Inhibitor of Methylthioadenosine Phosphorylase on the Proliferation and Alkaline Phosphatase Activity of Fetal Rat Calvaria Cells

骨原性細胞の増殖と分化に果たすメチルチオアデノシン代謝の役割 : ラット頭蓋骨由来細胞の増殖とアルカリホスファターゼ活性に及ぼすメチルチオアデノシンホスホリラーゼ阻害剤の影響

5'-Methylthioadenosine (MTA) is formed during the biosynthesis of polyamines. This nucleoside is cleaved to adenine and 5'-methylthioribose-1-phosphate in mammalian cells by methylthioadenosine phosphorylase (MTA Pase). 5'-Difluoromethyl-thioadenosine (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-released rat calvaria cells (RC cells). 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 (day 10). 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.

ラット頭蓋骨由来細胞の増殖,分化にメチルチオアデノシン(MTA)の代謝がどのような役割を果たしているかを調べるために,MTA分解酵素で,かつMTA代謝の律速酵素でもあるMTAホスホリラーゼ(MTA Pase)の阻害剤,ジフルオロメチルチオアデノシン(DFMTA)を用いることによって,本細胞への生理作用をin vitroで追求した．骨原性細胞の増殖および分化の指標としては,それぞれ細胞数およびアルカリホスファターゼ(ALPase)活性を用いた．低濃度(5,10μM)のDFMTAは増殖に影響を与えることなく,培養7日目および14日目のALPase活性を有意に低下させた．さらに,高濃度(50μM)のDFMTAは細胞増殖とALPase活性のいずれも,より著明に抑制した．以上の結果は,MTAの代謝がラット頭蓋骨由来細胞の増殖と分化に重要な役割を果たしていることを示唆している．


ラット歯髄細胞が合成するタンパク質と石灰化の関連

ラット歯髄細胞が合成するタンパク質と石灰化の関連


Inhibitory Effects of Prostaglandin E2 on Alveolar Bone Resorption in Hamster Experimental Periodontitis

ハムスター実験的歯周炎におけるプロスタグランディンE2の歯槽骨吸収抑制効果


Diagnosis of Periodontal Diseases using Biomarkers in Gingival Crevicular Fluid

歯肉溝滲出液中のバイオマーカーを用いた歯周病診断

Periodontal diseases cause an inflammation and degradation of periodontal tissues and missing of teeth. The incidence rate of periodontal diseases is high in middle-aged and elderly people.A reasonable diagnosis of periodontal diseases is very important to keep teeth, however, conventional examinations of periodontal diseases is not necessarily exact and objective. Gingival crevicular fluid (GCF) is an exudate secreted from periodontal tissues and contains many components including proteolytic enzymes, inflammatory cytokines, blood-associated proteins, cellular and bacterial fragments. Because some proteins in GCF are related to inflammation, tissue degradation and bone metabolism, those proteins have been studying as a diagnostic marker of periodontal diseases. GCF is noninvasively collected using a sterile paper strip and biomarkers are determined using enzyme-linked immunosorbent assay (ELISA) and enzyme activity assay. We identified calprotectin, an inflammationrelated protein, in GCF and calprotectin level in GCF from periodontitis sites was significantly higher than that of healthy control. Calprotectin level in GCF was positively correlated to gingival index and other biomarkers and decreased by periodontal treatments. Resistin is an adipocytokine and its level increases in some inflammatory diseases. Resistin level in GCF from periodontitis sites was high compared to the level of healthy control samples. Procollagen type I C-terminal peptide (PICP) is a biomarker for bone metabolism and its level was high in GCF collected from periodontitis sites. These results suggested that calprotectin, resistin and PICP are useful biomarkers for periodontal diseases. On the other hand, we showed that glycated albumin (GA), a marker of diabetes mellitus (DM), was contained in GCF and GA level in GCF from DM patients was significantly higher than that of non-DM individuals. Components in GCF may be biomarkers of systemic diseases as well as periodontal diseases and their determination will be useful diagnostic examination of some diseases. Recently, we have been studying the determining system of GCF calprotectin, including microchip ELISA, surface plasmon resonance assay and immuno-chromatography assay. When GCF biomarkers are determined using the determining systems, we will simply, exactly and objectively diagnose periodontal diseases at our dental offices.


The close relationship between periodontitis and diabetes and the diagnosis of diabetes-associated periodontitis

Proceeding of 2013-Asian-Pacific Society of Periodontology Meeting


FGF-2による歯周組織再生療法

FGF-2による歯周組織再生療法

歯の長期保存の臨床 私はこうして歯を守る

歯の長期保存の臨床 私はこうして歯を守る


Hypercementosis with the progression of chronic apical periodontitis -A case report

慢性根尖性歯周炎の進行に伴って形成されたセメント質過形成症


上顎側切歯にみられた歯内歯(Dens invaginatus/Dens in dente)における歯内治療の一例


Cut-off values of Functional Independence Measure scores for discharge destination.


ラット実験的歯周炎における副甲状腺ホルモンの局所投与による歯槽骨形成作用

ラット実験的歯周炎における副甲状腺ホルモンの局所投与による歯槽骨形成作用

第6回カルシトニン/副甲状腺ホルモン研究会記録集

第6回カルシトニン/副甲状腺ホルモン研究会記録集