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Junction epithelium (JE) is located apical to the bottom of the gingival sulcus and binds enamel to hemidesmosomes to protect the periodontal tissue from bacterial infection. Function of odontogenic ameloblast-associated protein (ODAM) is suggested by its expression sites (JE and maturation stage ameloblasts) to be involved in the adhesion between the JE and enamel, and odontogenesis. To analyze the changes in ODAM gene and protein expressions in inflamed gingiva, Ca9-22 gingival epithelial cells were stimulated with 1 ng/ml interleukin-1β (IL-1β) for 3-24 h, and ODAM mRNA and protein levels were analyzed by real-time PCR and Western blotting. Luciferase (LUC) constructs were made ligating various lengths of human ODAM gene promoters and performed LUC analyses in Ca9-22 cells. Gel shift and chromatin immunoprecipitation (ChIP) assays were performed. IL-1β induced ODAM mRNA and protein levels at 6-24 h. IL-1β increased LUC activities of the ODAM gene promoter constructs from - 85 to - 950. These activities were blocked by protein kinase A, tyrosine kinase, mitogen-activated protein (MAP) kinase kinase and phosphoinositide 3-kinase inhibitors. Gel shift and ChIP assays showed that IL-1β induced CCAAT/enhancer-binding protein (C/EBP) β and Yin Yang1 (YY1) binding to C/EBP1, 2, 3, and YY1 elements. These data indicate that IL-1β stimulates ODAM gene transcription mediated through C/EBP1, C/EBP2, C/EBP3, and YY1 elements in the human ODAM gene promoter.

Nutritional factors reflect the periodontal parameters accompanying periodontal status. In this study, the associations between nutritional factors, blood biochemical items, and clinical parameters were examined in patients with systemic diseases. The study participants were 94 patients with heart disease, dyslipidemia, kidney disease, or diabetes mellitus. Weak negative correlation coefficients were found between nine clinical parameters and ten nutritional factors. Stage, grade, mean probing depth (PD), rate of PD 4–5 mm, rate of PD ≥ 6 mm, mean clinical attachment level (CAL), and the bleeding on probing (BOP) rate were weakly correlated with various nutritional factors. The clinical parameters with coefficients of determinations (R2) > 0.1 were grade, number of teeth, PD, rate of PD 4–5 mm, CAL, and BOP rate. PD was explained by yogurt and cabbage with statistically significant standardized partial regression coefficients (yogurt: −0.2143; cabbage and napa cabbage: −0.2724). The mean CAL was explained by pork, beef, mutton, and dark green vegetables with statistically significant standardized partial regression coefficients (−0.2237 for pork, beef, and mutton; −0.2667 for dark green vegetables). These results raise the possibility that the frequency of intake of various vegetables can be used to evaluate periodontal stabilization in patients with systemic diseases.

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.

To investigate the effect of topical administration of propolis (a honeybee product) or curry leaf (an herbal product) into the periodontal pockets of periodontitis patients, a double-blind controlled clinical trial was conducted with 24 subjects including one drop-out diagnosed with moderate-to-advanced chronic periodontitis who completed initial periodontal therapy. They were randomly allocated to the following treatments: placebo, propolis, curry leaf, and minocycline. Gingival crevicular fluid (GCF) samples collected before and after the intervention were analyzed to quantify the number of total bacteria and number of six major periodontopathic bacteria by real-time PCR. Periodontitis-related clinical parameters were also analyzed. Among the six propolis-treated patients whose GCF samples were P. gingivalis-positive, three patients converted to be P. gingivalis-negative after the intervention. The minocycline-treated group exhibited a decrease in probing pocket depth (PPD) with statistically significant improvement, but not gain of clinical attachment level (CAL). Both PPD and CAL have been improved in the propolis-treated group at a statistically significant level, but not the curry leaf-treated group. In conclusion, treatment with propolis significantly improved both PPD and CAL, together with a tendency towards reduced P. gingivalis burden in GCF. It is likely that a propolis-based therapy becomes an alternative treatment option for chronic periodontitis during supportive periodontal therapy.

Follicular dendritic cell-secreted protein (FDC-SP) is secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium (JE). Its expression could be controlled during inflammatory process of gingiva; however, responsible mechanism for gingival overgrowth and involvement of FDC-SP in clinical condition is still unclear. We hypothesized that JE-specific genes are associated with the initiation of drug-induced gingival enlargement (DIGE) called gingival overgrowth, and investigated the changes of JE-specific gene's expression and their localization in overgrown gingiva from the patients. Immunohistochemical analysis revealed that the FDC-SP localization was spread in overgrown gingival tissues. FDC-SP mRNA levels in GE1 and Ca9-22 cells were increased by time-dependent nifedipine treatments, similar to other JE-specific genes, such as Amelotin (Amtn) and Lamininβ3 subunit (Lamβ3), whereas type 4 collagen (Col4) mRNA levels were decreased. Immunocytochemical analysis showed that FDC-SP, AMTN, and Lamβ3 protein levels were increased in GE1 and Ca9-22 cells. Transient transfection analyses were performed using luciferase constructs including various lengths of human FDC-SP gene promoter, nifedipine increased luciferase activities of -345 and -948FDC-SP constructs. These results raise the possibility that the nifedipine-induced FDC-SP may be related to the mechanism responsible for gingival overgrowth does not occur at edentulous jaw ridges.

Periodontitis is an inflammatory disease involving complex tripartite cross-interactions among bacterial, host and environment factors. Heat shock proteins (Hsps) are a protein family produced in response to stress conditions. Hsps protect cells under adverse circumstances such as infection, inflammation and disease. One of the causes of periodontal disease is thought to be an imbalance in the expression of Hsps and anti-Hsp antibodies. Hsps are classified according to their molecular weight, and one of the major ones is Hsp70. In the present study, enzyme-linked immunosorbent assay was used to measure the levels of anti-Hsp70 antibody in gingival crevicular fluid (GCF) from two gingival sulci in each of nine patients with chronic periodontitis (CP): one healthy control (HC) site with a probing pocket depth (PPD) of ≤3 mm and one CP site with a PPD of >5 mm. Anti-Hsp70 antibody levels in GCF were higher at HC sites than at CP sites. Moreover, the anti-Hsp70 antibody levels were found to increase after initial periodontal therapy at both HC and CP sites. These results suggest an association of anti-Hsp70 antibody with periodontitis.

Periodontitis is an inflammatory condition that causes the destruction of the supporting tissues of teeth and is a major public health problem affecting more than half of the adult population worldwide. Recently, members of the herpes virus family, such as the Epstein–Barr virus (EBV), have been suggested to be involved in the etiology of periodontitis because bacterial activity alone does not adequately explain the clinical characteristics of periodontitis. However, the role of EBV in the etiology of periodontitis is unknown. This study aimed to examine the effect of inactivated EBV on the expression of inflammatory cytokines in human gingival fibroblasts (HGFs) and the induction of osteoclast differentiation. We found that extremely high levels of interleukin (IL)-6 and IL-8 were induced by inactivated EBV in a copy-dependent manner in HGFs. The levels of IL-6 and IL-8 in HGFs were higher when the cells were treated with EBV than when treated with lipopolysaccharide and lipoteichoic acid. EBV induced IκBα degradation, NF-κB transcription, and RAW264.7 cell differentiation into osteoclast-like cells. These findings suggest that even without infecting the cells, EBV contributes to inflammatory cytokine production and osteoclast differentiation by contact with oral cells or macrophage lineage, resulting in periodontitis onset and progression.

The recurrence risk evaluation has been emphasized in periodontal stabilization during supportive periodontal therapy (SPT). However, nutritional factors, e.g., dietary habits such as the frequency of eating vegetables, are rarely included in the evaluation. In this study, the effect of nutritional factors on clinical periodontal parameters was examined in a lifestyle-related investigation and a periodontal examination in patients with periodontitis undergoing SPT. A total of 106 patients were recruited. Tendencies toward a negative correlation were found between rate of a probing depth (PD) of 4–5 mm, rate of PD ≥ 6 mm, the bleeding on probing (BOP) rate, periodontal inflamed surface area (PISA), and various nutritional factors. The number of teeth was a clinical parameter with a significantly high R2 (≥0.10) influenced by environmental factors, whereas PD, PD of 4–5 mm, the BOP rate, and PISA were influenced by nutritional factors. These results suggested that environmental factors reflected clinical parameters showing long-term pathophysiology, such as the PD rate. Nutritional factors tended to affect the current inflammatory pathophysiology, such as the BOP rate, PISA, and PISA/periodontal epithelial surface area. Therefore, environmental and nutritional factors appear to be useful for evaluating the risk of periodontitis during SPT.

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. 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. 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) β and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements. These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter.

Background/aims The most prevalent infectious disease, chronic periodontitis which leads to alveolar bone destruction and subsequent tooth loss, develops due to proinflammatory cytokine production induced by periodontopathic bacteria. Chronic obstructive pulmonary disease (COPD), a non-infectious disease, is the third leading cause of death globally. This condition exacerbates frequently, and which is attributable to proinflammatory cytokine production induced by infection by respiratory microorganisms such as Streptococcus pneumoniae. Although a positive association has recently been revealed between chronic periodontitis and COPD, how periodontitis contributes to the pathogenesis of COPD remains unclear. Therefore, we hypothesized that some periodontopathic bacteria are involved in the exacerbation of COPD through the induction of proinflammatory cytokine production by respiratory epithelial cells. In this connection, COPD develops in the airways; however, because most periodontopathic bacteria are anaerobic, they are unlikely to exhibit stable virulence in the lower respiratory organs in humans. Hence, we aimed to elucidate whether exposure to heat-inactivated periodontopathic bacteria induces proinflammatory cytokine production by several human respiratory epithelial cell lines and in the lower respiratory organs and serum in mice. Methods Real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were used to investigate in vitro induction by heat-inactivated periodontopathic bacteria and S. pneumoniae for mRNA expression and protein production of interleukin (IL)-8 and IL-6 by human respiratory epithelial cell lines. ELISA was also used to determine in vivo induction of cytokine production in the lower respiratory organs and serum of intratracheally heat-inactivated Fusobacterium nucleatum-inoculated mice. Results Some, but not all, periodontopathic bacteria, especially F. nucleatum, strongly induced IL-8 and IL-6 production by BEAS-2B bronchial epithelial cells. In addition, F. nucleatum induced IL-8 production by A549 alveolar epithelial cells as well as IL-8 and IL-6 production by Detroit 562 pharyngeal epithelial cells. Furthermore, F. nucleatum induced considerably higher cytokine production than S. pneumoniae. This was also observed in the entire lower respiratory organs and serum in mice. Conclusion Exposure to increased number of F. nucleatum potentially induces proinflammatory cytokine production by human bronchial and pharyngeal epithelial cells, which may trigger exacerbation of COPD.

Background/aim Human chronic periodontitis is a major health problem. Although some oral bacteria have been reported to be putative pathogens, Epstein-Barr virus (EBV) is reported to be associated with the progression of periodontitis. However, the role of EBV in the aetiology of periodontitis is unknown. Therefore, we investigated periodontal pathogenesis of EBV to confirm whether EBV-encoded latent membrane protein 1 (LMP1) induces Interleukin-8 (IL8) production in human gingival cells. Materials and methods Real-time polymerase chain reaction, luciferase assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were performed for determining IL8 mRNA expression, nuclear factor kappa B (NF-ĸB) transcription, IL8 production, and the phosphorylation of NF-ĸB p65 and Inhibitor of kappa B alpha (IĸBα), respectively, in Ca9-22 human gingival epithelial cells. Two LMP1 mutants lacking C-terminal activating region (CATR) domains responsible for activating NF-ĸB were used. Results Extremely high IL8 production was induced by LMP1 in time- and dose-dependent manner, where simultaneous phosphorylation of NF-κB p65 and IĸBα and transcription of NF-ĸB were observed. On the contrary, IL8 production and NF-ĸB transcription were drastically inhibited by dominant negative mutant of IĸBα. Moreover, the LMP1 mutants failed to induce IL8 production. Conclusion Our findings suggest that due to CATR domains, LMP1 contributes to the progression of periodontitis via IL8 production attributable to NF-ĸB activation.

The junctional epithelium and dental enamel adhere because of hemidesmosomes containing laminin 5 and α6β4 integrin, which are important adhesion molecules in the internal basal lamina. Interleukin (IL)-1 is important in the pathogenesis of periodontal disease. IL-1β induces bone resorption by activating osteoclasts; however, its effects on adhesion of epithelial cells remain to be clarified. Laminin β3, β4 integrin, and focal adhesion kinase mRNA levels were higher after 1 h and 3 h of stimulation with IL-1β (1 ng/mL), and IL-1β, type I α1, and type IV α1 collagen mRNA levels were higher after 1 h and lower after 3 h of stimulation with IL-1β. After IL-1β stimulation, colocalization of laminin 5 and β4 integrin was increased after 1 h, colocalization of β4 integrin and plectin was increased after 1 h and decreased after 3 h, and colocalization of β4 integrin and type IV collagen was decreased after 3 h. Wound healing assays showed that IL-1β treatment (3 h) delayed wound healing. These results suggest that IL-1β enhances cell adhesion by altering localization of epithelial adhesion molecules.

Periodontal disease is a chronic inflammatory disease of the periodontal tissue. The periodontal inflamed surface area (PISA) is a proposed index for quantifying the inflammatory burden resulting from periodontitis lesions. This study aimed to investigate longitudinal changes in the periodontal status as evaluated by the PISA following the active periodontal treatment. To elucidate the prognostic factors of PISA, mixed-effect modeling was performed for clinical parameters, tooth-type, and levels of periodontal pathogens as independent variables. One-hundred-twenty-five patients with chronic periodontitis who completed the active periodontal treatment were followed-up for 24 months, with evaluations conducted at 6-month intervals. Five-times repeated measures of mean PISA values were 130+/−173, 161+/−276, 184+/−320, 175+/−417, and 209+/−469 mm2. Changes in clinical parameters and salivary and subgingival periodontal pathogens were analyzed by mixed-effect modeling. Plaque index, clinical attachment level, and salivary levels of Porphyromonas gingivalis were associated with changes in PISA at the patient- and tooth-level. Subgingival levels of P. gingivalis and Prevotella intermedia were associated with changes in PISA at the sample site. For most patients, changes in PISA were within 10% of baseline during the 24-month follow-up. However, an increase in the number of bleeding sites in a tooth with a deep periodontal pocket increased the PISA value exponentially.

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.

Periodontal examination data have a complex structure. For epidemiological studies, mass screenings, and public health use, a simple index that represents the periodontal condition is necessary. Periodontal indices for partial examination of selected teeth have been developed. However, the selected teeth vary between indices, and a justification for the selection of examination teeth has not been presented. We applied a graded response model based on the item response theory to select optimal examination teeth and sites that represent periodontal conditions. Data were obtained from 254 patients who participated in a multicenter follow-up study. Baseline data were obtained from initial follow-up. Optimal examination sites were selected using item information calculated by graded response modeling. Twelve sites&mdash, maxillary 2nd premolar (palatal-medial), 1st premolar (palatal-distal), canine (palatal-medial), lateral incisor (palatal-central), central incisor (palatal-distal) and mandibular 1st premolar (lingual, medial)&mdash, were selected. Mean values for clinical attachment level, probing pocket depth, and bleeding on probing by full mouth examinations were used for objective variables. Measuring the clinical parameters of these sites can predict the results of full mouth examination. For calculating the periodontal index by partial oral examination, a justification for the selection of examination sites is essential. This study presents an evidence-based partial examination methodology and its modeling.

Aspiration pneumonia is a major health problem owing to its high mortality rate in elderly people. The secretion of proinflammatory cytokines such as interleukin (IL)-8 and IL-6 by respiratory epithelial cells, which is induced by infection of respiratory bacteria such as Streptococcus pneumoniae, contributes to the onset of pneumonia. These cytokines thus play a key role in orchestrating inflammatory responses in the lower respiratory tract. In contrast, chronic periodontitis, a chronic inflammatory disease caused by the infection of periodontopathic bacteria, typically Porphyromonas gingivalis, is one of the most prevalent microbial diseases affecting humans globally. Although emerging evidence has revealed an association between aspiration pneumonia and chronic periodontitis, a causal relationship between periodontopathic bacteria and the onset of aspiration pneumonia has not been established. Most periodontopathic bacteria are anaerobic and are therefore unlikely to survive in the lower respiratory organs of humans. Therefore, in this study, we examined whether simple contact by heat-inactivated P. gingivalis induced proinflammatory cytokine production by several human respiratory epithelial cell lines. We found that P. gingivalis induced strong IL-8 and IL-6 secretion by BEAS-2B bronchial epithelial cells. P. gingivalis also induced strong IL-8 secretion by Detroit 562 pharyngeal epithelial cells but not by A549 alveolar epithelial cells. Additionally, Toll-like receptor (TLR) 2 but not TLR4 was involved in the P. gingivalis-induced proinflammatory cytokine production. Furthermore, P. gingivalis induced considerably higher IL-8 and IL-6 production than heat-inactivated S. pneumoniae. Our results suggest that P. gingivalis is a powerful inflammatory stimulant for human bronchial and pharyngeal epithelial cells and can stimulate TLR2-mediated cytokine production, thereby potentially contributing to the onset of aspiration pneumonia.

Periodontitis is an inflammatory disease of periodontium which is caused by periodontopathic bacteria. Moreover, various cytokines such as interleukin-1&beta, (IL-1&beta, ), tumor necrosis factor-&alpha, (TNF-&alpha, ), and IL-6 are expressed in the inflamed periodontium. Heat shock proteins (HSPs) protect cells from abnormal conditions including inflammation, microbial infection and diseases. The 70-kDa HSPs (HSP70s) are major HSPs that express in the inflamed tissues. In this study, an enzyme-linked immunosorbent assay was applied to measure the levels of HSP70 in gingival crevicular fluid (GCF) from two periodontal pockets in each of 10 patients with Stage III, Grade B periodontitis. Sites with probing pocket depth (PPD) of &le, 3 mm were named the healthy control (HC) sites, and sites with PPD of &ge, 5 mm were named the diseased sites. HSP70 levels in GCF were expressed higher at diseased sites than at HC sites, and decreased after initial periodontal therapy at diseased sites. These results suggest the association of HSP70 with the stage of periodontitis.

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 (IKKβ) 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 IKKβ 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. IKKβ 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 IKKβ mRNAs in the human gingival epithelial cells.

Amelotin (AMTN) is a secreted protein expressed during the late stages of enamel formation and in the junctional epithelium. Among many differentially expressed genes, we found significantly increased AMTN mRNA level in inflamed gingiva by DNA microarray. The inductions of AMTN mRNA expressions in inflamed gingiva and human gingival fibroblasts (HGF) were confirmed by real-time polymerase chain reaction. To determine the molecular basis of the expression of AMTN and its regulation by proinflammatory cytokines, we have isolated and characterized the promoter region of mouse AMTN gene. Transient transfection assays were performed using luciferase constructs including mouse AMTN gene promoter. Interleukin-1β, Interleukin-6 and tumor necrosis factor-α induced AMTN mRNA levels in HGF. These cytokines increased the luciferase activities of the AMTN promoter constructs in HGF. The results suggest that proinflammatory cytokines induce AMTN gene transcription and a role for AMTN in gingival inflammation.

Peri-implant diseases are classified into two categories: peri-implant mucositis and peri-implantitis. Peri-implant mucositis is a reversible inflammation in the mucosa surrounding a dental implant. Peri-implantitis is an inflammatory reaction associated with the loss of supporting bones around a functional dental implant. They are the most frequent long-term complications related with dental implants. This chapter provides a detailed description of prevalence, risk factors, pathogenesis of peri-implant diseases, and characteristics of structure and composition of peri-implant tissues.

Junctional epithelium (JE) develops from reduced enamel epithelium during tooth formation and is critical for the maintenance of healthy periodontal tissue through ensuring appropriate immune responses and the rapid turnover of gingival epithelial cells. We have previously shown a relationship between inflammatory cytokines and expression of JE‐specific genes, such as amelotin (AMTN), in gingival epithelial cells. Here, we elucidated the effects of Porphyromonas gingivalis‐derived lipopolysaccharide (Pg LPS) on Amtn gene transcription and the interaction of transcription factors. To determine the molecular basis of transcriptional regulation of the Amtn gene by Pg LPS, we performed real‐time PCR and carried out luciferase assays using a mouse Amtn gene promoter linked to a luciferase reporter gene in mouse gingival epithelial GE1 cells. Gel mobility shift and chromatin immunoprecipitation assays were performed to identify response elements bound to LPS‐induced transcription factors. Next, we analyzed protein levels of the LPS‐induced transcription factors and the interaction of transcription factors by western blotting and immunoprecipitation. LPS increased Amtn mRNA levels and elevated luciferase activities of constructs containing regions between −116 and −238 of the mouse Amtn gene promoter. CCAAT/enhancer‐binding protein (C/EBP) 1–, C/EBP2– and Ying Yang 1 (YY1)–nuclear protein complexes were increased by LPS treatment. Furthermore, we identified LPS‐modulated interactions with C/EBPβ, YY1 and Smad3. These results demonstrate that Pg LPS regulates Amtn gene transcription via binding of C/EBPβ–Smad3 and YY1–Smad3 complexes to C/EBP1, C/EBP2 and YY1 response elements in the mouse Amtn gene promoter.

Junctional epithelium (JE) demonstrates biological responses with the rapid turnover of gingival epithelial cells. The state occurs in inflammation of gingiva and wound healing after periodontal therapy. To understand the underlying mechanisms and to maintain homeostasis of JE, it is important to investigate roles of JE-specific genes. Amelotin (AMTN) is localized at JE and regulated by inflammatory cytokines and apoptotic factors that represent a critical role of AMTN in stabilizing the dentogingival attachment, which is an entrance of oral bacteria. In this study, we demonstrated that the AMTN gene expression was regulated by SNAI2 and transforming growth factor β1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) that occurs in wound healing and fibrosis during chronic inflammation. SNAI2 downregulated AMTN gene expression via SNAI2 bindings to E-boxes (E2 and E4) in the mouse AMTN gene promoter in EMT of gingival epithelial cells. Meanwhile, TGFβ1-induced AMTN gene expression was attenuated by SNAI2 and TGFβ1-induced SNAI2, without inhibition of the TGFβ1-Smad3 signaling pathway. Moreover, SNAI2 small interfering RNA (siRNA) rescued SNAI2-induced downregulation of AMTN gene expression, and TGFβ1-induced AMTN gene expression was potentiated by SNAI2 siRNA. Taken together, these data demonstrated that AMTN gene expression in the promotion of EMT was downregulated by SNAI2. The inhibitory effect of AMTN gene expression was an independent feedback on the TGFβ1-Smad3 signaling pathway, suggesting that the mechanism can be engaged in maintaining homeostasis of gingival epithelial cells at JE and the wound healing phase.

Periodontal diseases are a major public health problem affecting over half of the adult population worldwide. Lipopolysaccharide (LPS) produced by the periodontopathic bacterium Porphyromonas gingivalis induces the expression of inflammatory cytokines that promote inflammatory bone destruction. Mounting evidence supports that periodontal diseases are involved in the onset and progression of several systemic diseases, such as aspiration pneumonia and diabetes. Although treatment of periodontal diseases by removing the periodontopathic bacteria by brushing is a standard practice, it has limitations and is not effective in all cases. Therefore, a new method to replace or complement brushing is needed for the treatment of periodontal diseases. In this study, we investigated the anti-inflammatory effects of an extract from Cynara scolymus L. and its pharmacologically effective compound cynaropicrin, a sesquiterpene lactone, on human gingival fibroblasts (HGFs) stimulated by LPS and the potential anti-osteoclastogenic effects on RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL). We found that cynaropicrin inhibited IL-8 and IL-6 mRNA and protein synthesis in LPS-stimulated HGFs in a dose-dependent manner. P. gingivalis LPS-induced degradation of IκBα and phosphorylation of NF-κB p65 were also suppressed by cynaropicrin, as was LPS-stimulated NF-κB transactivation. Thus, cynaropicrin's inhibition of P. gingivalis LPS-induced IL-8 and IL-6 expression may be due to the inhibition of the NF-κB pathway. Furthermore, we showed that cynaropicrin dramatically reduced RANKL-induced osteoclast differentiation. These results suggest that cynaropicrin may be useful for preventing periodontal diseases and could prove valuable in the development of more effective preventative approaches for periodontal diseases.

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-1β (IL-1β) on AMTN gene transcription in human gingival epithelial Ca9-22 cells. IL-1β increased AMTN mRNA and protein levels at 3 h, and the levels reached maximum at 6 and 12 h. IL-1β 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-1β 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-1β increased C/EBPβ 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-1β increases AMTN gene transcription in human gingival epithelial cells mediated through C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

Amelotin (AMTN) is induced upon initiation of apoptosis by transforming growth factor beta1 (TGFβ1) and is mediated by Smad3 in gingival epithelial cells (GE1 cells). This upregulation of AMTN gene expression is temporary, and the mechanism responsible is still unclear. The present study investigated the transcriptional downregulation of TGFβ1-induced AMTN gene expression in GE1 cells during the progression of apoptosis. To examine time-dependent changes in the levels of AMTN, Smad3 and Bax mRNA induced by TGFβ1, real-time PCR analyses were performed. Immunocytochemistry was carried out to detect the expression of Smad3 and Bax. Transient transfection analyses were performed using mouse AMTN gene promoter constructs of various lengths including Smad response elements (SBEs), in the presence or absence of TGFβ1. Changes in Smad3 binding to SBEs resulting from overexpression of Bax were examined using ChIP assays. Overexpression of Bax dramatically downregulated the levels of TGFβ1-induced AMTN mRNA and transcription of the AMTN gene. Smad3 binding to SBEs in the mouse AMTN gene promoter was induced by overexpression of Smad3 or TGFβ1, and this was inhibited by Bax overexpression. These results show that the levels of AMTN mRNA induced by TGFβ1 and Smad3 are decreased by robust expression of Bax in gingival epithelial cells.

Follicular dendritic cell-secreted protein (FDC-SP) is expressed in FDCs, human periodontal ligament (HPL) cells, and junctional epithelium. To evaluate the effects of interleukin-1 beta (IL-1β) on FDC-SP gene expression in immortalized HPL cells, FDC-SP mRNA and protein levels in HPL cells following stimulation by IL-1β were measured by real-time polymerase chain reaction and Western blotting. Luciferase (LUC), gel mobility shift, and chromatin immunoprecipitation (ChIP) analyses were performed to study the interaction between transcription factors and promoter regions in the human FDC-SP gene. IL-1β (1 ng/mL) induced the expression of FDC-SP mRNA and protein levels at 3 h, and reached maximum levels at 12 h. IL-1β increased LUC activities of constructs (-116FDCSP - -948FDCSP) including the FDC-SP gene promoter. Transcriptional inductions by IL-1β were partially inhibited by 3-base-pair (3-bp) mutations in the Yin Yang 1 (YY1), GATA, CCAAT-enhancer-binding protein2 (C/EBP2), or C/EBP3 in the -345FDCSP. IL-1β-induced -345FDCSP activities were inhibited by protein kinase A, tyrosine-kinase, mitogen-activated protein kinase (MEK)1/2, and PI3-kinase inhibitors. The results of gel shift and ChIP assays revealed that YY1, GATA, and C/EBP-β interacted with the YY1, GATA, C/EBP2, and C/EBP3 elements that were increased by IL-1β. These studies demonstrate that IL-1β increases FDC-SP gene transcription in HPL cells by targeting YY1, GATA, C/EBP2, and C/EBP3 in the human FDC-SP gene promoter.

Differentiation of mesenchymal stem cells (MSCs) in human periodontal ligaments (HPDLs) is induced by specific transcription factors that support periodontal tissue regeneration. The maintaining characteristics for HPDL cells could be regulated by two kinds of transcription factors, such as Twist family basic helix-loop-helix (bHLH) transcription factor 2 (TWIST2) and Kruppel-like factor 12 (KLF12). Because TWIST2 and KLF12 may be controlled by microRNAs (miRNAs), we investigated their 3′-untranslated regions (3′-UTRs) that contain seed sequences for miR-141 and miR-200a. HPDL cells were co-transfected with luciferase (LUC) reporter plasmids with or without 3′-UTRs for TWIST2 or KLF12, and miR-141 or miR-200a expression vectors. MiR-141 and miR-200a suppressed LUC activities via the TWIST2 or KLF12-3′-UTRs and reduced the mRNA and protein levels of TWIST2 and KLF12. These effects of miR-141 and miR-200a were related to reduced and increased expressions of the collagen Type I (COL1A1) and aggrecan (ACAN), while increasing the mRNAs for the chondrogenic factors SRY-Box transcription factor 5 (SOX5) SOX6, and Tricho-rhino-phalangeal syndrome type 1 (TRPS1) genes. We validated that these effects also reduce SOX5 and SOX6 proteins levels. Expression of miR-141 and miR-200a was significantly higher in HCS2/8 chondrogenic cells than human gingival fibroblasts (HGF), or HPDL and Saos2 cells. Furthermore, transfection of HPDL cells with miR-141 or miR-200a produced alcian blue staining. Thus, miR-141 and miR-200a directly target TWIST2 and KLF12 and induce chondrogenic differentiation of HPDL cells.

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.