Clinical Investigation of the Inhibitory Effects of Tooth-Coating Materials on Initial Active Root Caries: A Pilot Randomized Controlled Trial.

Background and Objectives: Caredyne ZIF-C is a novel, capsule-mixed zinc-containing prototype glass ionomer cement (GIC). Zinc ions are reported to inhibit root dentin demineralization, dentin collagen degradation, bacterial growth, acid production, and in vitro bacterial biofilm formation. However, the effectiveness of GICs against initial root caries lesions is unclear. Therefore, this study aimed to evaluate the efficacy of GICs, especially the new zinc-containing Caredyne ZIF-C GIC, as tooth-coating materials in patients with initial active root caries. Materials and Methods: A total of 58 lesions in 47 older adults (age > 65 years) were randomly allocated to one of the following three groups: Caredyne ZIF-C, Fuji VII (a conventional GIC), and sodium fluoride (NaF). All the lesions were treated with the assigned materials without removing the infected dentin, and the rates of dental plaque attachment and coating material fall-out were evaluated after 3, 6, and 12 months. The failure rate was defined as the number of teeth that needed restoration due to caries progression. Results: The plaque attachment rates tended to be lower in the material-coated root surfaces than in the healthy exposed root surfaces after 3, 6, and 12 months, although the differences among the three groups were not significant. Moreover, the coating material fall-out rate tended to be lower in the Caredyne ZIF-C group than in the Fuji VII group. There was no significant difference in the failure rate among the three groups at the 12 months mark. Conclusions: Though this pilot study offers a new direction for suppressing the progression of initial active root caries by controlling plaque attachment using GICs including Caredyne ZIF-C, clinical studies with a larger sample size are needed.

Effect of Periodontal Treatment on Reducing Chronic Inflammation in Systemically Healthy Patients With Periodontal Disease.

BACKGROUND: We determined the effects and an accurate marker of periodontal treatment on serum interleukin (IL)-6 and high-sensitivity C-reactive protein (HsCRP) levels in systemically healthy individuals with periodontal disease. METHODS: This multicenter study included systemically healthy individuals with periodontal disease who received initial periodontal treatment and had no periodontal treatment history. Periodontal parameters, including periodontal inflamed surface area, masticatory efficiency, and periodontal disease classification; serum IL-6 and HsCRP levels; and serum immunoglobulin (Ig)G titers against periodontal pathogens were evaluated at baseline and after treatment. Subjects were classified as low or high responders (group) based on periodontal inflamed surface area changes. RESULTS: There were 153 participants. Only periodontal inflamed surface area changes were markedly different between low and high responders. Periodontal treatment (time point) decreased both serum IL-6 and HsCRP levels. The interaction between group and time point was remarkable only for serum IL-6 levels. Changes in serum immunoglobulin (Ig)G titers against periodontal pathogens were not associated with IL-6 changes in high responders. We analyzed the indirect effect of serum anti-Porphyromonas gingivalis type 2 IgG titer changes using mediation analysis and found no significance. However, the direct effect of group (low or high responder) on IL-6 changes was considerable. CONCLUSIONS: Periodontal treatment effectively decreased serum IL-6 levels, independent of periodontal pathogen infection, in systemically healthy individuals with periodontal disease.

Umami taste sensitivity is associated with food intake and oral environment in subjects with diabetes.

OBJECTIVE: Dysgeusia is a serious problem in patients with diabetes because it often leads to overeating, which is associated with disease progression. This study aimed to investigate the association between taste sensitivity, eating habits, and the oral environment. SUBJECTS AND METHODS: In this cross-sectional study of 75 subjects with diabetes, gustatory function was assessed using the whole-mouth method, and lingual taste receptor gene expression was measured by real-time PCR. Food intake was evaluated using a food frequency questionnaire based on food groups. The oral environment was assessed using xerostomia and periodontal comprehensive examination. RESULTS: In total, 45.3%, 28.0%, and 18.7% of subjects showed lower umami taste sensitivity, low sweet taste sensitivity, and low salt taste sensitivity, respectively. Lower umami sensitivity correlated with lower estimated glomerular filtration rate and higher energy-source food intake. Subjects with diabetes with higher plaque control record showed significantly higher T1R3 gene expression than those with lower plaque control record. CONCLUSION: Reduced umami taste sensitivity is associated with decreased renal function and high energy food intake in diabetes. Subjects with diabetes with higher plaque control record showed significantly higher T1R3 gene expression, suggesting that the oral environment affects taste gene expression. J. Med. Invest. 70 : 241-250, February, 2023.

ß-defensin 2 synthesized by a cell-free protein synthesis system and encapsulated in liposomes inhibits adhesion of Porphyromonas gingivalis to oral epithelial cells.

ß-defensin 2 (BD-2), an antimicrobial peptide (AMP), is expressed by oral epithelial cells and plays an important role in innate immunity of the oral cavity. Cell-free protein synthesis (CFPS) systems have been studied for the synthesis of various proteins, however, the synthesis of BD-2 by a CFPS system has not been extensively explored. Liposomes have been developed as tools for drug delivery. A delivery of liposome-encapsulated AMP to oral epithelium may be useful to prevent oral infectious diseases. In the present study, we investigated the antimicrobial activity of the BD-2 protein, artificially synthesized using a CFPS system and encapsulated in liposomes. BD-2 protein was artificially synthesized using template DNA and a reconstituted CFPS system and was identified by western blotting. Bilayer liposomes were prepared using 1,2-dioleoyl-sn-glycero-3-phospho-choline and 3-sn-phosphatidylcholine from egg yolk. The artificially synthesized BD-2 was encapsulated in liposomes, collected by ultrafiltration, and detected by western blotting. Human oral epithelial cells were cultured with the liposome-encapsulated BD-2 and the concentration of BD-2 in the cell lysate of the culture with the synthesized BD-2 was higher than that of the control cultures. The antimicrobial activity of the synthesized BD-2 was investigated by an adhesion assay of Porphyromonas gingivalis to oral epithelial cells. The artificially synthesized BD-2 and its liposome significantly inhibited adhesion of P. gingivalis to oral epithelial cells. These results suggest that artificially synthesized BD-2 and liposome-encapsulated BD-2 show antimicrobial activity and can potentially play a role in oral healthcare for periodontal diseases.

Effects of Candidalysin Derived from Candida albicans on the Expression of Pro-Inflammatory Mediators in Human Gingival Fibroblasts.

Candida albicans (Ca) is frequently detected in the peri-implant sulcus with peri-implantitis, a major postoperative complication after oral implant therapy. However, the involvement of Ca in the pathogenesis of peri-implantitis remains unclear. In this study, we aimed to clarify Ca prevalence in the peri-implant sulcus and investigated the effects of candidalysin (Clys), a toxin produced by Ca, on human gingival fibroblasts (HGFs). Peri-implant crevicular fluid (PICF) was cultured using CHROMagar and Ca colonization rate and colony numbers were calculated. The levels of interleukin (IL)-1ß and soluble IL-6 receptor (sIL-6R) in PICF were quantified by enzyme-linked immunosorbent assay (ELISA). Pro-inflammatory mediator production and intracellular signaling pathway (MAPK) activation in HGFs were measured by ELISA and Western blotting, respectively. The Ca colonization rate and the average number of colonies in the peri-implantitis group tended to be higher than those in the healthy group. IL-1ß and sIL-6R levels in the PICF were significantly higher in the peri-implantitis group than in the healthy group. Clys significantly induced IL-6 and pro-matrix metalloproteinase (MMP)-1 productions in HGFs, and co-stimulation with Clys and sIL-6R increased IL-6, pro-MMP-1, and IL-8 production levels in HGFs compared with Clys stimulation alone. These findings suggest that Clys from Ca plays a role in the pathogenesis of peri-implantitis by inducing pro-inflammatory mediators.

The Effect of Probiotic Lactobacillus acidophilus and Ethanolic Propolis Compound toward Nucleic Acid Deposition in the Extracellular Polymeric Substance of Root Canal Bacteria.

OBJECTIVE: This study aimed to evaluate the effects of Apis trigona ethanolic propolis and probiotic bacteria Lactobacillus acidophilus on the nucleic acid concentration in the extracellular polymeric substances (EPS) derived from biofilm of root canal bacteria. MATERIALS AND METHODS: Clinical bacteria of the root canal were cultured with ethanolic extract of propolis (EEP; 10 or 0.1%) and L. acidophilus. After the formation of biofilm was observed in the monolayer bacterial culture under several conditions, the enzymatic treatment and nucleic acid quantification were sequentially performed. STATISTICAL ANALYSIS: Independent t-test and Mann-Whitney were performed following data normality to analyze the significant differences of the treatment effect on the nucleic acid concentration in EPS from the isolated biofilm. RESULTS: The results showed that the nucleic acid concentration in EPS biofilm were not increased by coculture with L. acidophilus as probiotics. However, the treatment with 10% EEP could significantly increase nucleic acid concentration. CONCLUSION: This study suggested that the biosurfactants from probiotic bacteria L. acidophilus might be a promising candidate for endodontic treatment, arguably better than EEP in inhibiting biofilm maturation and complexity.

Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells.

BACKGROUND AND OBJECTIVE: Lipocalin 2 (LCN2), a glycoprotein expressed in epithelial cells and leukocytes, has an antibacterial effect and plays a role in innate immunity. The delivery of LCN2 encapsulated in liposomes to oral epithelium may be useful to prevent oral infectious diseases. This study aimed to investigate the inhibitory effect of LCN2, artificially synthesized using a cell-free protein synthesis (CFPS) system, on the adhesion of Porphyromonas gingivalis to oral epithelial cells in order to approach oral healthcare using LCN2. METHODS: LCN 2 was synthesized using a CFPS system and assayed by Western blotting, mass spectrometry and enzyme-linked immunosorbent assay (ELISA). The bilayer liposomes were prepared by the spontaneous transfer method using 1,2-dioleoyl-sn-glycero-3 phosphocholine (DOPC), 3-sn-phosphatidylcholine from Egg Yolk (Egg-PC), and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine (DOPE). The cellular and medium fractions derived from the culture of oral epithelial cells with liposome-encapsulated LCN2 were assayed by Western blotting and ELISA. The effect of the synthesized LCN2 on adhesion of the labeled P. gingivalis to oral epithelial cells was investigated as an evaluation of its antibacterial activity. RESULTS: The synthesized LCN2 protein was identified by Western blotting; its amino acid sequence was similar to that of recombinant LCN2 protein. The additions of DOPE and octa-arginine in the outer lipid-layer components of liposome significantly increased the delivery of liposomes to epithelial cells. When oral epithelial cells were cultured with the synthesized and liposome-encapsulated LCN2, LCN2 was identified in the cellular and medium fractions by Western blotting and its concentration in the cellular fraction from the culture with the synthesized LCN2 was significantly higher than that of a template DNA-free protein. The synthesized LCN2 and liposome-encapsulated LCN2 significantly inhibited the adhesion of P. gingivalis to oral epithelial cells compared with template DNA-free protein. CONCLUSION: LCN2 was artificially synthesized by a CFPS system, encapsulated in liposomes, and delivered to oral epithelial cells, and demonstrated an antibacterial action against P. gingivalis. This approach may become a useful model for oral healthcare.

Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways.

Porphyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. Pg-OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that Pg-OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.

Effectiveness of a Diabetes Oral Nursing Program Including a Modified Diabetes Oral Health Assessment Tool for Nurses (M-DiOHAT©) : A 12-Month Follow-Up Intervention Study.

PURPOSE: This study aimed to evaluate the effectiveness of a diabetes oral nursing intervention program for individuals with diabetes. METHODS: Fifty-six participants with diabetes underwent a diabetes oral nursing intervention program. The program's effect was evaluated through questionnaires and small interviews. The modified diabetes oral health assessment tool (M-DiOHAT©) was used to assess and educate four factors;oral conditions, behaviors, perceptions and knowledge about diabetes and periodontal disease, and health information-sharing, among participants at baseline, 3, 6, and 12 months later. Primary outcomes included changes in the M-DiOHAT© total scores. Secondary outcomes included scores on the motivation stage of changes in oral health behaviors' scales, dental visits, number of present teeth, hemoglobin A1c (HbA1c), and participants' comments. RESULTS: The M-DiOHAT© total score and the motivation stage score significantly improved with the narrative comment of "being motivated to practice oral health behaviors" between the baseline and 12 months later. Eight participants visited the dentist, whereas no differences were observed in the number of present teeth or HbA1c. CONCLUSIONS: This program improved participants' M-DiOHAT© total score, motivation stage score, and dental visits. These results suggest the program improved oral health perceptions and behaviors among individuals with diabetes. J. Med. Invest. 69 : 86-96, February, 2022.

Anti-Inflammatory and Protective Effects of Juncus effusus L. Water Extract on Oral Keratinocytes.

Periodontitis is a chronic inflammatory disease caused by periodontopathogenic bacteria that form biofilms in periodontal pockets. The gingival epithelium acts as the first physical barrier in fighting attacks by periodontopathogenic pathogens, such as the primary etiological agent Porphyromonas gingivalis, and various exogenous chemicals, as well as regulates the local innate immune responses. Therefore, the development of novel oral care products to inhibit inflammatory reactions caused by bacterial infection and protect the gingival epithelium is necessary. Juncus effusus L. has generally been used as an indigenous medicine, such as a diuretic, an antipyretic, and an analgesic, in ancient practice. In this study, we examined the effects of a water extract from J. effusus L. on the inhibition of the inflammatory reaction elicited by bacterial infection and protection of the oral epithelium by chemical irritation. Pretreatment of oral epithelial cells with the water extract from J. effusus L. significantly reduced P. gingivalis or its lipopolysaccharide- (LPS-) mediated production of chemokines (interleukin-8 and C-C-chemokine ligand20) in a concentration-dependent manner with comparable to or greater effects than epigallocatechin gallate and protected oral epithelial cells from injury by chemical irritants, cetylpyridinium chloride, and benzethonium chloride. Moreover, the water extract from J. effusus L. in the presence of antimicrobial agents or antifibrinolytics already used as ingredients in mouthwash could significantly reduce the production of chemokines from P. gingivalis LPS-stimulated oral epithelial cells in a concentration-dependent manner. These findings suggest that the water extract from J. effusus L. is potentially useful for oral care to prevent oral infections, such as periodontal infections, and maintain oral epithelial function.

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.

Conversion from epithelial to partial-EMT phenotype by Fusobacterium nucleatum infection promotes invasion of oral cancer cells.

The ability of cancer cells to undergo partial-epithelial mesenchymal transition (p-EMT), rather than complete EMT, poses a higher metastatic risk. Although Fusobacterium nucleatum mainly inhabits in oral cavity, attention has been focused on the F. nucleatum involvement in colorectal cancer development. Here we examined the p-EMT regulation by F. nucleatum in oral squamous cell carcinoma (OSCC) cells. We cultured OSCC cells with epithelial, p-EMT or EMT phenotype with live or heat-inactivated F. nucleatum. Expression of the genes involved in epithelial differentiation, p-EMT and EMT were examined in OSCC cells after co-culture with F. nucleatum by qPCR. Cell growth and invasion of OSCC cells were also examined. Both live and heat-inactivated F. nucleatum upregulated the expression of p-EMT-related genes in OSCC cells with epithelial phenotype, but not with p-EMT or EMT phenotype. Moreover, F. nucleatum promoted invasion of OSCC cells with epithelial phenotype. Co-culture with other strains of bacteria other than Porphyromonas gingivalis did not alter p-EMT-related genes in OSCC cells with epithelial phenotype. F. nucleatum infection may convert epithelial to p-EMT phenotype via altering gene expression in OSCC. Oral hygiene managements against F. nucleatum infection may contribute to reduce the risk for an increase in metastatic ability of OSCC.

Evaluation of Temperature Increase From Joule Heat in Numerical Tooth Model by Applying 500 kHz Current for Apical Periodontitis Treatment-Effect of Applied Voltage and Tooth Conductivity.

For apical periodontitis treatments, a new method with the insertion of an electrode into the root canal of a tooth and application of a current at 500 kHz to sterilize the area by Joule heat has attracted attention. However, few studies have quantified the temperature increase in the root canal. This study aimed to investigate the basic characteristics of the temperature increase in a simple and standard tooth model when energizing a current at 500 kHz to the numerical tooth model with typical electrical and physical properties. We developed a numerical model of a standard tooth (dentin) and periodontal tissues consisting of an alveolar bone, cortical bone, and gingiva, and physiological saline in a root canal and calculated the temperature increase inside the numerical model by a coupled analysis of current and heat when a voltage was applied across the electrodes. The calculated results for the different applied voltages showed a temperature increase at the apical portion of the root canal, which increased with the applied voltage even for the same total supplied energy. The temperature increase occurred at the apical portion of the root canal as the tooth conductivity decreased. When the tooth conductivity was high, a current passed through the dentin, which led to a decrease in the temperature at the apical portion of the root canal. However, a chemical solution with a higher conductivity in the root canal tended to increase the temperature at the apical portion of the root canal, regardless of the tooth conductivity. More efficient approaches for increasing the spatial and temporal temperature for the tooth model target are needed. © 2021 Bioelectromagnetics Society.

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.

A large-scale observational study to investigate the current status of diabetic complications and their prevention in Japan (JDCP study 6): baseline dental and oral findings.

Japan Diabetes Complication and Prevention prospective (JDCP) study was conducted to examine the association between glycemic control and oral conditions in a large database of Japanese patients with diabetes. It included a total of 6099 patients with diabetes (range, 40-75 years) who had been treated as outpatients between 2007 and 2009. The mean number of present teeth at baseline was 19.8 and women with type 2 diabetes had fewer teeth than men with type 2 diabetes. Within the previous year, 17% of all patients had lost teeth. At baseline, 32% had experienced gingival swelling, 69% had brushed more than twice a day, 37% had used interdental cleaning aids, and 43% had undergone regular dental checkups. Multiple logistic regression analysis indicated that type 1 patients with HbA1c ≥ 7.0% were at higher risk of having fewer than 20 teeth (odds ratio [OR] 2.38; 95% confidence interval [CI] 1.25-4.78), and type 2 patients with HbA1c ≥ 8.0% also were at high risk of having fewer than 20 teeth (OR 1.16; 95% CI 1.00-1.34), after adjustment for nine possible confounding factors. In conclusion, patients with diabetes were found to be at high risk of tooth loss, and the poorer the glycemic control, the higher the risk of tooth loss in these patients.

Expression of fibroblast growth factor receptor1, -2c, and -3c transcripts in mouse molars after tooth eruption.

A previous study suggested that fibroblast growth factor (FGF) signaling plays an important role in dentin formation during tooth development. In this study, to examine dentin formation after tooth eruption involving secondary and tertiary dentin, we analyzed the expression patterns and expressing cells of Fgfr1, -2c, and -3c in mouse maxillary first molars (M1). Since it is difficult to recover the mRNAs from mineralized tissues, we tested methods for extraction after fixation and decalcification of teeth. We successfully obtained consistent results with quantitative real-time PCR (qPCR) using ß-actin transcripts for validation. qPCR for Dentin sialo phosphoprotein (Dspp), Fgfr1, -2c, and -3c transcripts was performed on mice at ages of 2-20 weeks. The results showed that the highest expression levels of Dspp and Fgfr2c occurred at 2 weeks old followed by lower expression levels after 4 weeks old. However, the expression levels of Fgfr1 and Fgfr3c were constant throughout the experimental period. By in situ hybridization, Dspp, Fgfr1, and Fgfr3c transcripts were detected in odontoblasts at ages of 2 and 4 weeks. In addition, Dspp and Fgfr1 transcripts were detected in odontoblasts facing reactionary dentin at 8 weeks old. These results suggest that FGF-FGFR signaling might be involved in the regulation of odontoblasts even after tooth eruption, including secondary and tertiary dentin formation. Moreover, our modified method for extracting mRNA from mineralized tissues after fixation and decalcification successfully produced consistent results.

Assessment of the functional efficacy of root canal treatment with high-frequency waves in rats.

The purpose of this study was to develop a high-frequency wave therapy model in rats and to investigate the influence of high-frequency waves on root canal treatment, which may provide a novel strategy for treating apical periodontitis. Root canal treatments with and without high-frequency wave irradiation were performed on the mandibular first molars of 10-week-old male Wistar rats. The mesial roots were evaluated radiologically, bacteriologically, and immunohistochemically. At 3 weeks after root canal treatment, lesion volume had decreased significantly more in the irradiated group than in the non-irradiated group, indicating successful development of the high-frequency therapy model. The use of high-frequency waves provided no additional bactericidal effect after root canal treatment. However, high-frequency wave irradiation was found to promote healing of periapical lesions on the host side through increased expression of fibroblast growth factor 2 and transforming growth factor-ß1 and could therefore be useful as an adjuvant nonsurgical treatment for apical periodontitis.

Suppressive effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of Candida species and MRSA to acrylic denture resin.

OBJECTIVES: The effects of 2-methacryloyloxyethyl phosphorylcholine (MPC)-polymer on the adherence of microorganisms such as non-Candida albicans Candida (NCAC) and methicillin-resistant Staphylococcus aureus (MRSA), frequently detected in oral infections in immunocompromised and/or elderly people, to denture resin material, are still unclear. Here, we report the effects of MPC-polymer on the adherence of C. albicans, NCAC, and MRSA to acrylic denture resin. METHODS: Sixteen strains of C. albicans, seven strains of C. glabrata, two strains of C. tropicalis, one strain of C. parapsilosis, and six strains of MRSA were used. We cultured the fungal/bacterial strains and examined the cell growth and adherence of fungi/bacteria to mucin-coated acrylic denture resin plates (ADRP) with or without MPC-polymer coating, by scanning electron microscopy. The cell surface hydrophobicity of the fungal/bacterial strains was measured by the adsorption to hydrocarbons. RESULTS: MPC-polymer did not affect the growth of all strains of Candida species and MRSA, but significantly suppressed adherence to ADRP in most strains of C. albicans and all strains of NCAC and MRSA. A significant positive correlation was found between cell hydrophobicity and the reduction rates of microbial adherence to ADRP treated with 5% of MPC-polymer. CONCLUSIONS: MPC-polymer treatment for acrylic resin material suppresses the adherence of C. albicans, NCAC and MRSA via their hydrophilicity interaction. CLINICAL SIGNIFICANCE: The application of MPC-polymer for denture hygiene is potent to prevent oral candidiasis, denture stomatitis and opportunistic infection, caused by Candida species and MRSA, via suppressing the adherence of those fungus/bacteria.

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.