Utility of a haemoglobin test of gingival crevicular fluid: A multicentre, observational study.

OBJECTIVE: The purpose of this study was to verify the accuracy and utility of clinical parameters (plaque index, gingival crevicular fluid volume, probing depth, clinical attachment level, bleeding on probing and gingival index) and biochemical parameters (aspartate aminotransferase, protein and haemoglobin) in a longitudinal analysis during the supportive periodontal therapy period. SUBJECTS AND METHODS: A total of 279 test sites of 128 patients were investigated clinically and biochemically. After the first examination of clinical and biochemical parameters, periodontal support treatments were administered immediately and performed once every three months up to the second examination. RESULTS: All of the clinical and biochemical parameters were significantly lower at the second examination than at the first, except for the plaque index and bleeding on probing. Of these parameters, in particular, aspartate aminotransferase and haemoglobin in the gingival crevicular fluid were significantly reduced compared to those of the first examination in both the ≤4 and ≥5 mm probing depth groups, and they clearly suggested that periodontitis tended to recover. CONCLUSION: Adding the haemoglobin test to the bleeding on probing test strongly improves the accuracy of measurement of clinical parameters after periodontal treatment.

Transcriptional regulation of human odontogenic ameloblast-associated protein gene by tumor necrosis factor-α.

OBJECTIVE: Odontogenic ameloblast-associated protein (ODAM) is produced by maturation stage ameloblasts and junctional epithelium (JE). The function of ODAM is thought to be involved in the attachment of teeth and JE. To elucidate transcriptional regulation of human ODAM gene in inflamed gingiva, we have analyzed the effects of TNF-α on the expression of ODAM gene in Ca9-22 and Sa3 gingival epithelial cells. MATERIALS AND METHODS: Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). ODAM mRNA and protein levels were analyzed by qPCR and Western blotting. Luciferase (LUC) analyses were performed using LUC constructs inserted in various lengths of ODAM gene promoter. Gel shift and chromatin immunoprecipitation (ChIP) assays were carried out. RESULTS: TNF-α increased ODAM mRNA and protein levels at 3 to 24 h. TNF-α induced LUC activities of the ODAM gene promoter constructs, and the activities were inhibited by protein kinase A, tyrosine kinase, MEK1/2, PI3-kinase and NF-κB inhibitors. Gel shift and ChIP assays revealed that TNF-α increased CCAAT/enhancer-binding protein (C/EBP) ß and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements. CONCLUSION: These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter.

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10 ng/ml, 12 h). AMTN and inhibitor of kappa-B kinase beta (IKKß) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3'-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3'-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKß mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3'-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKß inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKß mRNAs in the human gingival epithelial cells.

Tumor necrosis factor-α regulates human follicular dendritic cell-secreted protein gene transcription in gingival epithelial cells.

Follicular dendritic cell-secreted protein (FDC-SP) is a secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium. To elucidate the transcriptional regulation of the human FDC-SP gene by tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with chimeric constructs of the FDC-SP gene promoter linked to a luciferase reporter gene, gel mobility shift and chromatin immunoprecipitation assays using Ca9-22 gingival epithelial cells. TNF-α (10 ng/ml) induced FDC-SP mRNA and protein levels at 3 hr and reached maximum at 12 hr. In transient transfection assays, TNF-α (12 hr) increased the LUC activities of constructs between -116FDCSP and -948FDCSP including the human FDC-SP gene promoter. Transcriptional stimulations by TNF-α were partially inhibited in the -345FDCSP constructs that included 3-bp mutations in the YY1, GATA, CCAAT enhancer-binding protein 2 (C/EBP2) and C/EBP3. Transcriptional activities induced by TNF-α were inhibited by tyrosine kinase, MEK1/2 and phosphoinositide 3-kinase inhibitors. The results of ChIP assays showed that YY1, GATA and C/EBPß transcription factors interacted with the YY1, GATA, C/EBP2 and C/EBP3 elements that were increased by TNF-α. These studies show that TNF-α stimulates human FDC-SP gene transcription by targeting YY1, GATA, C/EBP2 and C/EBP3 in the FDC-SP gene promoter.

MiR-200b attenuates IL-6 production through IKKß and ZEB1 in human gingival fibroblasts.

OBJECTIVE: MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF). MATERIALS AND METHODS: Total RNA and protein were extracted from HGF after stimulation by interleukin-1ß (IL-1ß; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1ß, inhibitor of nuclear factor kappa-B kinaseß (IKKß), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot. RESULTS: IL-1ß and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKß and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKß and ZEB1 and increased E-cadherin mRNA and protein levels in HGF. CONCLUSIONS: These results suggest that miR-200b attenuates inflammatory response via IKKß and ZEB1 in periodontal tissue.

Clinical usability of aspartate aminotransferase to evaluate the prognosis of periodontal regeneration therapies: prospective, longitudinal study.

To evaluate the degree of periodontal tissue destruction, aspartate aminotransferase (AST) levels in the gingival crevicular fluid (GCF) are utilized as a predictor of periodontal therapy. We have previously shown that the usefulness of AST activities [periodontal tissue monitor (PTM) values] using a PTM-kit to evaluate the effects of initial periodontal therapy and periodontal regeneration therapy by enamel matrix derivative (EMD). This prospective, longitudinal study was conducted using 38 healthy and 80 periodontitis sites with probing depth (PD) of 5-10 mm for guided tissue regeneration (GTR) and EMD from 36 patients. GCF samples were used to evaluate PTM values at base line (BL) and after 6 months of surgeries (re-evaluation: RE), and periodontal examinations were performed concurrently. PTM values at BL were statistically improved at RE, accompanied by the improvement of periodontal parameters in both groups. PTM values and PD, and the clinical attachment level (CAL) showed high correlations. PD, CAL and bleeding on probing (BOP) were highly correlated with PTM values in both groups, whereas only PD showed a significant correlation with PTM values at RE in the GTR group. Change in the amounts of PD, CAL and BOP between BL and RE in both groups showed no correlation with PTM values. In the negative PTM value sites at BL in EMD group, the mean PD was significantly reduced at RE compared with positive PTM sites at BL. PTM values are able to be utilized as the biochemical predictor of prognosis after periodontal regeneration therapy.

Evaluation of bleeding on probing and gingival crevicular fluid enzyme activity for detection of periodontally active sites during supportive periodontal therapy.

This study aimed to analyze the enzyme activity in gingival crevicular fluid (GCF) and its association with clinical parameters, especially bleeding on probing (BOP), and thus reconsider the significance and accuracy of recording BOP. A total of 184 patients who had entered supportive periodontal therapy were selected and GCF was collected from 401 sites before recording the clinical parameters, probing pocket depth (PPD), BOP, clinical attachment level, gingival index and plaque index. The enzyme activity of neutrophil elastase and aspartate aminotransferase and amount of protein in GCF were also analyzed. In the clinical parameters for biochemical data, amount of GCF showed the most correlation. A cut-off value for BOP and PPD were determined by the ROC curve and Youden index. Analysis was performed with all clinical parameters and biochemical data. Of the 401 sites, 51 were less than the cut-off value and were BOP-negative. On the other hand, 29 sites had values more than the cut-off value, with 14 BOP-negative sites and 15 BOP-positive sites. A conclusion is as follows: twenty-nine sites with values more than the cut-off value were diagnosed as sites requiring periodontal management, however, 14 of these were BOP-negative. These results suggest that combining other biochemical tests with examination of BOP and PPD may improve the validity of periodontal disease diagnosis. In future studies, it will be essential to find a marker that can precisely detect periodontal disease activity, and to develop a diagnostic tool for chair-side use.

TNF-α regulates the composition of the basal lamina and cell-matrix adhesions in gingival epithelial cells.

Laminin 5, type 4 collagen, and α6ß4 integrin contribute to the formation of hemidesmosomes in the epithelia of periodontal tissues, which is critical for the development and maintenance of the dentogingival junction. As it is not known whether TNF-α alters the composition of the epithelial pericellular matrix, human gingival epithelial cells were cultured in the presence or absence of TNF-α. Treatment with TNF-α accelerated epithelial cell migration and closure of in vitro wounds. These data indicate unexpectedly, that TNF-α promotes the formation of the pericellular matrix around epithelial cells and enhances adhesion of epithelial cells to the underlying matrix, properties which are important for cell migration and the integrity of the dentogingival junction.

Impact of adjunctive procedures on recombinant human fibroblast growth factor-2-mediated periodontal regeneration therapy: A retrospective study.

BACKGROUND: Human fibroblast growth factor-2 (rhFGF-2) therapy has been used for periodontal tissue regeneration. However, few studies have reported their adjunctive procedures based on strategy of tissue engineering. The aim of this retrospective study is to assess the adjunctive effects of modified papilla preservation technique (mPPT) and combination with autogenous bone grafts (AG) on the rhFGF-2 therapy. METHODS: Total of 44 sites underwent rhFGF-2 therapies and the evaluations in the survey periods. The primary outcome was set to the radiographic bone fill by radiographic examinations at 6 and 12 months after surgeries. We analyzed the correlation between influencing factors and the primary outcome, and differences of therapeutic effect by combination therapy with mPPT and that with AG. RESULTS: After surgeries, probing depth (PD), clinical attachment level (CAL) and bone defects significantly improved. The improvements of radiographic bone fill were significantly positive correlated with a number of bone walls, combination with mPPT, and AG at 6 months after surgeries, and with combination with mPPT and AG at 12 months after surgeries. The significant differences of improvements of radiographic bone fill were demonstrated between combination with or without mPPT at 12 months after surgeries, and with or without AG at 6 and 12 months after surgeries. Moreover, the multiple linear regression analysis for the radiographic bone fill indicated the significant regression coefficient with conducts of mPPT. CONCLUSIONS: mPPT and AG had powerfully adjunctive effects on rhFGF-2 therapy. Further studies are needed in order to verify by randomized clinical trials.

Transcriptional regulation of bone sialoprotein gene by Porphyromonas gingivalis lipopolysaccharide.

Lipopolysaccharide (LPS) is a major mediator of inflammatory response. Periodontopathic bacterium Porphyromonas gingivalis LPS has quite different character from Escherichia coli LPS. E. coli LPS is agonist for Toll-like receptor 4 (TLR4), whereas P. gingivalis LPS worked as antagonist for TLR4. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of P. gingivalis LPS on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were decreased by 0.1 microg/ml and increased by 0.01 microg/ml P. gingivalis LPS at 12 h. Results of luciferase assays showed that 0.1 microg/ml decreased and 0.01 microg/ml P. gingivalis LPS increased BSP transcription in -116 to +60 BSP construct. The effects of P. gingivalis LPS were abrogated by double mutations in cAMP response element (CRE) and FGF2 response element (FRE). Tyrosine kinase inhibitor herbimycin A, ERK1/2 inhibitor and antioxidant N-acetylcystein inhibited effects of P. gingivalis LPS. Protein kinase A inhibitor and PI3-kinase/Akt inhibitor only abolished the effect of 0.01 microg/ml P. gingivalis LPS. Furthermore, 0.1 microg/ml LPS decreased the CRE- and FRE-protein complexes formation, whereas 0.01 microg/ml P. gingivalis LPS increased the nuclear protein binding to CRE and FRE. ChIP assays revealed increased binding of CREB1, JunD, Fra2, Runx2, Dlx5, and Smad1 to a chromatin fragment containing the CRE and FRE by 0.01 microg/ml P. gingivalis LPS. These studies therefore indicated that 0.1 microg/ml suppressed, and 0.01 microg/ml P. gingivalis LPS increased BSP gene transcription mediated through CRE and FRE elements in the rat BSP gene promoter.

A multicenter prospective cohort study on the effect of smoking cessation on periodontal therapies in Japan.

Few prospective studies have reported the effects of periodontal therapy on patients who attempted to quit smoking. This study aimed to assess how smoking cessation affects periodontal therapy. Twenty-five smokers with periodontitis were investigated by dividing them into two groups, a smoking cessation support group and a continued smoking group. Those in the support group received counseling and nicotine replacement therapy, followed by periodontal treatment conducted by dentists who had completed an e-learning course on smoking cessation. Clinical parameters were measured at baseline, 3, and 6 months. Most clinical parameters improved for those in the smoking cessation support group. There were no significant improvements in bleeding on probing (BOP) or the number of severe periodontal disease sites in the continued smoking group. Probing pocket depth (PPD) and clinical attachment levels (CAL) at sites that received scaling and root planing (SRP) significantly improved in all subjects. BOP did not improve at reevaluation in the smoking relapse subgroup. Patients in the smoking cessation support program led by dental professionals showed more improvement in BOP than those in the continued smoking group.

IL-1ß enhances cell adhesion through laminin 5 and ß4 integrin in gingival epithelial cells.

The junctional epithelium and dental enamel adhere because of hemidesmosomes containing laminin 5 and α6ß4 integrin, which are important adhesion molecules in the internal basal lamina. Interleukin (IL)-1 is important in the pathogenesis of periodontal disease. IL-1ß induces bone resorption by activating osteoclasts; however, its effects on adhesion of epithelial cells remain to be clarified. Laminin ß3, ß4 integrin, and focal adhesion kinase mRNA levels were higher after 1 h and 3 h of stimulation with IL-1ß (1 ng/mL), and IL-1ß, type I α1, and type IV α1 collagen mRNA levels were higher after 1 h and lower after 3 h of stimulation with IL-1ß. After IL-1ß stimulation, colocalization of laminin 5 and ß4 integrin was increased after 1 h, colocalization of ß4 integrin and plectin was increased after 1 h and decreased after 3 h, and colocalization of ß4 integrin and type IV collagen was decreased after 3 h. Wound healing assays showed that IL-1ß treatment (3 h) delayed wound healing. These results suggest that IL-1ß enhances cell adhesion by altering localization of epithelial adhesion molecules.

Transcriptional regulation of human amelotin gene by interleukin-1ß.

One of the major causes of tooth loss is chronic inflammation of the periodontium, the tissues surrounding the tooth. Amelotin (AMTN) is a tooth enamel protein which is expressed in maturation-stage ameloblasts and also in the internal basal lamina of junctional epithelium, a unique epithelial structure attached to the tooth surface which protects against the constant microbiological challenge to the periodontium. Localization of AMTN suggests that its function could be involved in the dentogingival attachment. The purpose of this study was to investigate the effect of interleukin-1ß (IL-1ß) on AMTN gene transcription in human gingival epithelial Ca9-22 cells. IL-1ß increased AMTN mRNA and protein levels at 3 h, and the levels reached maximum at 6 and 12 h. IL-1ß induced luciferase activities of human AMTN gene promoter constructs (-211, -353, -501, -769, and -950AMTN), but these activities were partially inhibited in -353AMTN constructs that included 3-bp mutations in CCAAT/enhancer binding protein 1 (C/EBP1), C/EBP2, and Ying Yang 1 (YY1) elements. Transcriptional activities induced by IL-1ß were abrogated by protein kinase A (PKA), tyrosine kinase, mitogen-activated protein kinase kinase (MEK1/2), and phosphatidylinositol 3-kinase (PI3K) inhibitors. Gel shift and ChIP assays showed that IL-1ß increased C/EBPß binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 elements after 3 h, and that these DNA-protein interactions were inhibited by PKA, tyrosine kinase, MEK1/2, and PI3K inhibitors. These results demonstrated that IL-1ß increases AMTN gene transcription in human gingival epithelial cells mediated through C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

Correlation Between Gingival Crevicular Fluid Hemoglobin Content and Periodontal Clinical Parameters.

BACKGROUND: Probing depth (PD) and bleeding on probing (BOP) are essential clinical parameters used for periodontal diagnosis. This study investigated whether detection of hemoglobin (Hb) in gingival crevicular fluid (GCF), along with PD and BOP, would improve diagnostic accuracy. METHODS: After plaque index (PI) was measured, GCF was collected from the gingival sulci of 401 anterior teeth in the maxilla and mandible from 184 patients who had entered periodontal maintenance therapy. Clinical parameters (gingival index [GI], PD, clinical attachment level [CAL], and BOP) were recorded. Hb values in GCF were assessed by immunochromatography. Moreover, cutoff values for PI, GI, and CAL based on the degree of PD and amount of GCF were created and analyzed. RESULTS: Hb was detected in 64.8% of GCF samples in 105 BOP-negative (-) sites in the periodontally stable group out of 107 sites that were less than all cutoff values. There were 71 BOP(-) sites in the periodontal-management-required group out of 122 sites that were more than all cutoff values, although no improvement in periodontal disease was observed. Hb was detected in 88.7% of GCF samples from these 71 BOP(-) sites. CONCLUSIONS: Hb was observed in more than 60% of GCF samples in BOP(-) gingival sulci in both periodontally stable and periodontal-management-required groups. These results suggest inspection of Hb derived from microbleeding in gingival sulci may serve as an index for preclinical diagnosis.

Unliganded estrogen receptor α stimulates bone sialoprotein gene expression.

Estrogen is one of the steroid hormones essential for skeletal development. The estrogen receptor (ER) is a transcription factor and a member of the steroid receptor superfamily. There are two different forms of the ER, usually referred to as α and ß, each encoded by a separate gene. Hormone-activated ERs form dimers, since the two forms are coexpressed in many cell types. Bone sialoprotein (BSP) is a tissue-specific acidic glycoprotein that is expressed by differentiated osteoblasts, odontoblasts and cementoblasts during the initial formation of mineralized tissue. To determine the molecular basis of the tissue-specific expression of BSP and its regulation by estrogen and the ER, we have analyzed the effects of ß-estradiol and ERα on BSP gene transcription. ERα protein levels were increased after ERα overexpression in ROS17/2.8 cells. While BSP mRNA levels were increased by ERα overexpression, the endogenous and overexpressed BSP mRNA levels were not changed by ß-estradiol (10(-8)M, 24 h). Luciferase activities of different sized BSP promoter constructs (pLUC3~6) were increased by ERα overexpression, whereas basal and induced luciferase activities by ERα overexpression were not influenced by ß-estradiol. Effects of ERα overexpression were abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that ERα overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were disrupted by ERα, CREB and phospho-CREB antibodies. The AP1/GRE-protein complexes were supershifted by the c-Fos antibody. These studies demonstrate that ERα stimulates BSP gene transcription in a ligand-independent manner by targeting the CRE and AP1/GRE elements in the rat BSP gene promoter.