Diagnosis of inflammatory peri-implant diseases using an immunochromatographic assay for calprotectin in peri-implant crevicular fluid.

BACKGROUND: The incidence rate of peri-implant diseases is increasing with implant placement. Early detection of peri-implant diseases is important to prevent and treat these diseases, and a simple and objective diagnostic method is expected. Immunochromatographic (IC) assays are used for rapid diagnostic methods for some diseases. The aim of this clinical study was to determine the amount of calprotectin, an inflammatory marker, in peri-implant crevicular fluid (PICF) using an IC chip, and estimate the possibility of this diagnostic system. METHODS: Forty-six individuals with dental implants participated in a pilot study. PICF samples were collected from the peri-implant sites with or without inflammation after clinical examinations including probing depth (PD), bleeding on probing (BOP) and gingival index (GI). Calprotectin in PICF was determined by an IC chip and enzyme-linked immunosorbent assay (ELISA) for calprotectin. The density of calprotectin line on the IC chip was measured using an IC reader (IC reader value). The relationship between IC reader value and ELISA value or clinical parameters was investigated. A receiver operating characteristic (ROC) curve analysis of IC reader value of calprotectin was performed to predict inflammation in peri-implant diseases. RESULTS: IC reader value of calprotectin was significantly correlated with its ELISA value and PD. IC reader values of calprotectin in PICF samples from periodontal sites with GI-1 and GI-2, and with BOP-positive sites were significantly higher than those of PICF samples from GI-0 sites, and BOP-negative sites, respectively. The IC reader value for calprotectin in PICF samples from inflammatory diseased sites was significantly higher than that of non-diseased sites. ROC analysis suggested that the IC reader value of PICF calprotectin was useful for predicting inflammatory peri-implant diseases. CONCLUSION: IC assay for PICF calprotectin may be a possible system for diagnosing the inflammatory peri-implant diseases.

Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01-1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis.

Advanced glycation end-products increase lipocalin 2 expression in human oral epithelial cells.

BACKGROUND AND OBJECTIVES: Diabetes mellitus (DM), a risk factor of periodontal diseases, exacerbates the pathological condition of periodontitis. A major factor for DM complications is advanced glycation end-products (AGEs) that accumulate in periodontal tissues and cause inflammatory events. Lipocalin 2 (LCN2) is an antimicrobial peptide and inflammation-related factor, and LCN2 levels increase in DM. In this study, the effects of AGEs and lipopolysaccharide of Porphyromonas gingivalis (P g-LPS) on LCN2 expression in human oral epithelial cells (TR146 cells) and the role of secreted LCN2 in periodontitis with DM were investigated. MATERIAL AND METHODS: TR146 cells were cultured with AGEs (AGE2) and control BSA and cell viability was estimated, or with P g-LPS. Conditioned medium and cell lysates were prepared from cultures of epithelial cells and used for Western blotting and ELISA to analyze LCN2, RAGE, IL-6, MAPK, and NF-κB. RNA was isolated from AGE-treated TR146 cells and differentiated HL-60 (D-HL-60) cells and used for quantitative real-time PCR to examine the expression of LCN2 and interleukin-6 (IL-6) mRNAs. RAGE- and LCN2-siRNAs (siRAGE, siLCN2) were transfected into epithelial cells, and AGE-induced LCN2 expression was investigated. D-HL-60 cells were co-cultured with TR146 cells that were transfected with siLCN2 and treated with AGEs, and IL-6 mRNA expression in D-HL-60 cells and cell migration was investigated. RESULTS: AGEs increased the expression levels of LCN2 and IL-6 in oral epithelial cells. siRAGE and a neutralizing antibody for RAGE inhibited AGE-induced LCN2 expression. AGEs stimulated the phosphorylation of ERK, p38, and NF-κB in epithelial cells, and their inhibitors suppressed AGE-induced LCN2 expression. In contrast, P g-LPS did not show a significant increase in LCN2 level in TR146 cells that expressed Toll-like receptor 2. In co-culture experiments, AGE-induced LCN2 inhibited IL-6 mRNA expression in D-HL-60 cells, and LCN2 knockdown in epithelial cells suppressed HL-60 cell migration. CONCLUSION: These results suggested that AGEs increase LCN2 expression via RAGE, MAPK, and NF-κB signaling pathways in oral epithelial cells, and secreted LCN2 may influence the pathological condition of periodontitis with DM.

S100A9 Increases IL-6 and RANKL Expressions through MAPKs and STAT3 Signaling Pathways in Osteocyte-Like Cells.

OBJECTIVE: Calprotectin is a heterocomplex of S100A8 and S100A9 and is mainly secreted from neutrophils, monocytes, and chondrocytes in inflammatory condition. Calprotectin binds to RAGE and TLR4 and induces the expression of proinflammatory chemokines and cytokines in various cells. Periodontitis is a chronic inflammatory disease that leads to gingival inflammation and alveolar bone resorption. Calprotectin levels in gingival crevicular fluid of periodontitis patients are higher than healthy patients. In the present study, the effects of S100A8 and S100A9 on the expressions of proinflammatory cytokines and bone metabolism-related factors in mouse osteocyte-like cells (MLO-Y4-A2) were investigated. DESIGN: MLO-Y4-A2 cells were treated with S100A8 and S100A9, and the expressions of RAGE, TLR4, RANKL, and several inflammatory cytokines were analyzed by PCR and Western blotting or ELISA methods. To investigate the intracellular signaling pathways, phosphorylation of MAPK and STAT3 was determined by Western blotting, and chemical specific inhibitors and siRNAs were used. RESULTS: Expressions of IL-6 and RANKL were increased by treatment with S100A9 but not S100A8. However, both S100A8 and S100A9 did not change expression of IL-1ß, IL-8, and TNF-α. Although RAGE and TLR4 expressions were not upregulated by S100A9 treatment, transfection of siRNA for RAGE and TLR4 significantly decreased IL-6 and RANKL expressions. In addition, S100A9 activated p38, ERK, and STAT3 signaling pathways, and inhibitors for these factors significantly decreased S100A9-induced IL-6 and RANKL expressions. CONCLUSIONS: These results indicated that S100A9 induces IL-6 and RANKL production via engagement with RAGE and TLR4 signalings in osteocytes and suggested that S100A9 may play important roles in the periodontal alveolar bone destruction.

Assessing the oral health of in-patients with diabetes using a clinical version of the Diabetes Oral Health Assessment Tool© and its association with dental examinations.

Aims : To compare assessment of the oral health conditions and behaviors of in-patients with diabetes using a clinical version of the Diabetes Oral Health Assessment Tool (C-DiOHAT©) with dental examinations. Methods : A cross-sectional design was used. A nurse assessed 60 in-patients using the C-DiOHAT© (a formatted questionnaire to assess four factors of patients' oral health conditions and behaviors : oral health conditions, oral hygiene behaviors, sharing health information among patients and dental/medical professionals, and perception and knowledge of oral health) while a dentist examined their oral health conditions. Results : "Use of supplementary tools (e.g., interdental brush, dental floss)" in the item of C-DiOHAT© was significantly associated with dental examination of "the number of present teeth" and "no recommendation of further dental visit". "Symptoms of gingival swelling" in the item of C-DiOHAT© was also significantly associated with "recommendation of dental visit". "Knowledge of a relationship between periodontal disease and systemic disease including diabetes" was significantly associated with Community Periodontal Index. Conclusions : These results suggest that nurses should prioritize these assessment items to most quickly acquire useful information about patients' oral health. It is important to encourage nurses to be interested in patients' oral health by such small pile of clue. J. Med. Invest. 66 : 328-336 August, 2019.

6-Shogaol Inhibits Advanced Glycation End-Products-Induced IL-6 and ICAM-1 Expression by Regulating Oxidative Responses in Human Gingival Fibroblasts.

Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM.

Advanced glycation end-product 2 and Porphyromonas gingivalis lipopolysaccharide increase sclerostin expression in mouse osteocyte-like cells.

Sclerostin is a secreted glycoprotein that is mainly expressed in osteocytes, exerts negative effects on bone formation, and is present at elevated levels in diabetes mellitus (DM). Periodontitis is an infectious disease caused by periodontopathic bacteria, a complication of DM, and sometimes associated with severe inflammation and alveolar bone resorption. Advanced glycation end-products (AGEs) are a major pathogen in DM complications and adversely influence periodontitis in DM patients. In the present study, the effects of AGE2 and Porphyromonas gingivalis lipopolysaccharide (P-LPS) on the expression of sclerostin in mouse osteocyte-like cells (MLO-Y4-A2 cells) and its function in osteoblast differentiation were investigated. AGE2 and P-LPS up-regulated the expressions of receptor of AGE (RAGE) and Toll-like receptor 2 (TLR2), respectively, and significantly up-regulated that of sclerostin and interleukin 6 (IL-6) in osteocytes. Sclerostin, RAGE and TLR2 levels were synergistically increased by AGE2 and P-LPS. The siRNAs of RAGE and TLR2 significantly inhibited AGE2- and P-LPS-induced sclerostin expression. AGE2 up-regulated sclerostin expression in osteocyte-like cells via the RAGE, ERK and JNK, and NF-κB signal pathways. On the other hand, P-LPS elevated sclerostin levels via the TLR2, JNK and p38, and NF-κB signal pathways. When osteocytes pre-treated with AGE2 and P-LPS and osteoblastic cells (MC3T3-E1) were co-cultured in the medium with a sclerostin-neutralizing antibody, AGE2- and P-LPS-induced decreases in alkaline phosphatase activity and Runx2 expression in osteoblastic cells were significantly inhibited by the sclerostin-neutralizing antibody. These results suggest that AGE2 and P-LPS influence bone metabolism and inflammation through the regulation of sclerostin expression, and may aggravate periodontitis with DM.

Calprotectin and cross-linked N-telopeptides of type I collagen levels in crevicular fluid from implant sites with peri-implant diseases: a pilot study.

BACKGROUND: Peri-implant crevicular fluid (PICF) contains calprotectin and NTx, which are markers for inflammation and bone resorption, respectively. The aims of this pilot study were to compare calprotectin and NTx levels in PICF from implant sites with or without peri-implant diseases and to evaluate the usefulness of calprotectin and NTx as diagnostic markers for peri-implant diseases. METHODS: Thirty-five patients with dental implants participated in this pilot study. PICF samples were collected from peri-implant disease sites (n = 40) and non-diseased (healthy) sites (n = 34) after clinical indicators including probing depth (PD), bleeding on probing (BOP), gingival index (GI), and bone loss (BL) rate were investigated. Calprotectin and NTx amounts in PICF were measured using their respective ELISA kits and then compared between diseased and healthy samples. The relationship between PICF calprotectin or NTx levels and clinical indicator levels was investigated. A receiver operating characteristic (ROC) curve analysis of calprotectin and NTx was performed to predict peri-implant diseases. RESULTS: Calprotectin and NTx levels in PICF were significantly higher from peri-implant disease sites than from healthy sites. PICF calprotectin amounts correlated with PD, and its levels were significantly higher in the GI-1 and GI-2 groups than in the GI-0 group. PICF NTx amounts correlated with PD and the BL rate. ROC curves indicated that PICF calprotectin and NTx are useful biomarkers for peri-implant diseases. CONCLUSIONS: Calprotectin and NTx in PICF have potential as biomarkers for the diagnosis of peri-implant diseases.

Advanced glycation end-products and Porphyromonas gingivalis lipopolysaccharide increase calprotectin expression in human gingival epithelial cells.

Accumulation of advanced glycation end-products (AGEs) in periodontal tissues of patients with diabetes mellitus aggravates periodontitis, but the mechanisms are unknown. Calprotectin, a heterocomplex of S100A8 and S100A9 proteins, is a constitutive cytoplasmic component of healthy gingival epithelial cells. This study aimed at investigating the effects of AGE and Porphyromonas gingivalis lipopolysaccharide (PgLPS) on calprotectin expression in the human gingival epithelial cell line OBA-9. AGE and PgLPS increased the expression of S100A8 and S100A9 mRNAs, and AGE+PgLPS co-stimulation amplified their expression in OBA-9 cells. A higher concentration of calprotectin in cell lysates was also induced by stimulation with AGE and/or PgLPS. S100A8 was mainly translocated from the nucleus to the cytoplasm by AGE stimulation, while cytoplasmic localization of S100A9 was not altered following stimulation with AGE and/or PgLPS. Calprotectin was found in the cytoplasm of BSA-treated cells, but cytoplasmic and nuclear localization was observed following stimulation with AGE and/or PgLPS. AGE-induced S100A8, and S100A9 mRNA expression was partially suppressed by RAGE-specific siRNA. In contrast, PgLPS-induced S100A8 and S100A9 mRNA expression was strongly suppressed by TLR2-specific siRNA. Furthermore, the inhibition of p38, JNK MAPK, and NF-κB attenuated AGE- and PgLPS-induced S100A8 and S100A9 mRNA expression. Taken together, these results demonstrate that AGE acts in synergy with PgLPS to stimulate RAGE and TLR2 expression and activate p38, JNK MAPK, and NF-κB signaling pathways, resulting in increased activation of calprotectin (S100A8/S100A9) in human gingival epithelial cells. Our results suggest that calprotectin may be involved in the pathogenesis of diabetic periodontitis.