Background and objectivesDiabetes 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 methodsTR146 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.ResultsAGEs 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.ConclusionThese 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.

BackgroundPeri-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.MethodsThirty-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.ResultsCalprotectin 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.ConclusionsCalprotectin and NTx in PICF have potential as biomarkers for the diagnosis of peri-implant diseases.

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.

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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.