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Aim To investigate the effects of 2-hydroxy-ethyl methacrylate (HEMA) on cytotoxicity and cyclooxygenase-2 (COX-2) protein expression in human osteoblasts. Methodology Cytotoxicity was judged using an Alamar Blue reduction assay on human osteoblast cell line U2OS. Western blot was used to evaluate the expression of COX-2 protein by HEMA. To determine whether glutathione (GSH) levels were important in cytotoxicity and COX-2 expression of HEMA, cells were pre-treated with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Paired Student's t-tests were applied for the statistical analysis of the results. Results HEMA demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P

Kuan Y-H, Li Y-C, Huang F-M, Chang Y-C. The upregulation of tumour necrosis factor-α and surface antigens expression on macrophages by bisphenol A-glycidyl-methacrylate. International Endodontic Journal, 45, 619–626, 2012. Abstract Aim To evaluate the expression of tumour necrosis factor-α and surface antigens by bisphenol A-glycidyl-methacrylate (BisGMA) on murine macrophage cell line RAW264.7. Methodology Cytotoxicity was measured by tetrazolium bromide reduction assay. Tumour necrosis factor (TNF)-α was analysed by enzyme-linked immunosorbent assay. Cell surface antigens were investigated by flowcytometry. Statistical analyses were performed using anova followed by the Bonferroni’s t-test for multigroup comparisons. Results BisGMA exhibited cytotoxicity to RAW264.7 in a dose-dependent manner (P 0.05). Conclusions Taken together, the ability of macrophages to induce an appropriate immune response when exposed to BisGMA has the potential to upregulate TNF-α production and expression of surface antigens.

Li Y-C, Kuan Y-H, Huang F-M, Chang Y-C. The role of DNA damage and caspase activation in cytotoxicity and genotoxicity of macrophages induced by bisphenol-A-glycidyldimethacrylate. International Endodontic Journal, 45, 499–507, 2012. Abstract Aim To evaluate the potential toxicological implications of BisGMA on murine macrophage cell line RAW264.7. Methodology Lactate dehydrogenase release, flow cytometry, Western blot and fluorometric assays were used to detect cell viability, mode of cell death and caspase activities, respectively. In addition, alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assays were applied to detect genotoxicity. Statistical analyses were performed using anova followed by the Bonferroni’s t-test for multi-group comparisons test. Results BisGMA demonstrated a cytotoxic effect on RAW264.7 cells in a dose-dependent and a time-dependent manner (P

Huang F-M, Li Y-C, Lee S-S, Chang Y-C. Cytotoxicity of dentine bonding agents on human pulp cells is related to intracellular glutathione levels. International Endodontic Journal. Abstract Aim To evaluate ex vivo the mechanisms of cytotoxicity of dentine bonding agents in human pulp cells in vitro. Methodology Human pulp cells were obtained from impacted third molars with informed consent and then cultured using an explant technique. Set specimens from Clearfil SE Bond (CB), Prime & Bond 2.1 (PB), and Single Bond (SB) were eluted with culture medium. Cytotoxicity was judged using an assay of tetrazolium bromide reduction. To determine whether glutathione (GSH) levels were important in the cytotoxicity of dentine bonding agents, cells were pretreated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. Three replicates of each dentine bonding agents were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was by one-way analysis of variance (anova). Tests of differences of the treatments were analysed by Duncan’s test. Results Clearfil SE Bond, PB, and SB were cytotoxic to pulp cells in a concentration-dependent manner (P SB > CB. Addition of OTZ extracellularly protected the pulp cells from dentine bonding agents–induced cytotoxicity (P

Lee T-H, Hu C-C, Lee S-S, Chou M-Y, Chang Y-C. Cytotoxicity of chlorhexidine on human osteoblastic cells is related to intracellular glutathione levels. International Endodontic Journal, 43, 430–435, 2010. Abstract Aim To evaluate the mechanisms of cytotoxicity of chlorhexidine (CHX) in human osteoblastic cells in vitro. Methodology Cytotoxicity, cell proliferation and collagen synthesis assays were performed to elucidate the toxic effects of CHX on the human osteoblastic cell line U2OS. To determine whether glutathione (GSH) levels were important in the cytotoxicity of CHX, cells were pre-treated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. Results CHX demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P

Aim To investigate the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. Methodology The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1α and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student’s t-test. Results IL-1α was found to upregulate RANKL production in U2OS cells (P

Aim To report two cases of palatal root fracture in maxillary molars that were successfully managed in the short term by root canal treatment and root amputation. Summary In the first case, a 48-year-old woman with bony destruction and a deep periodontal pocket on the palatal root of tooth 26 (FDI) underwent root canal treatment. Bleeding into the palatal canal and radiolucent lines over the root suggested a fracture. Further evidence was provided by an electronic apex locator. Subsequent surgery confirmed the presence of a horizontal root fracture and the fractured root was removed. In the second case, a 75-year-old woman presented with pain from the left posterior teeth. Clinical examination revealed an oblique root fracture of tooth 27 palatal roots with abscess formation and a deep periodontal pocket. Palatal root amputation and odontoplasty were performed. This was followed by root canal treatment. Both teeth were preserved in the short term and early healing of these two cases was uneventful. Key learning points • Horizontal/oblique root fracture of the palatal root in molars is rare. • A combination of periodontal and root canal treatment and palatal root amputation may allow short-term preservation of functional teeth.

To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002.The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity.The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P0.05).Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

Aim To compare tissue-type plasminogen activator (t-PA) expression in normal human pulp and inflamed human pulp tissue specimens. Methodology Thirty pulpal tissue specimens (13 normal and 17 inflamed pulps) were obtained from extracted third molars. The levels of t-PA between normal pulp and inflamed pulp tissues were compared using the quantitative reverse-transcriptase polymerase chain reaction analysis. In addition, immunohistochemistry was used to identify the in situ localization of t-PA expression in pulp specimens. Wilcoxon–Mann–Whitney rank sum test was applied for the statistical analysis of the results. Results t-PA mRNA gene was found more in inflamed pulps when compared with normal pulp tissue (P

Aim To investigate the effect of black-pigmented Bacteroides on the expression of interleukin (IL)-8 gene in human pulp fibroblasts and osteoblasts. Methodology The supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-8 gene expression in human pulp fibroblasts and osteoblasts. The levels of mRNAs were measured by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Results Investigations of the time-dependence of IL-8 mRNA expression in black-pigmented Bacteroides-treated pulp fibroblasts and osteoblasts revealed a rapid accumulation of the transcript after 2 h of exposure, and remained elevated throughout the 24-h incubation period. In addition, IL-8 mRNA gene expression was also found in human osteoblasts stimulated with black-pigmented Bacteroides. However, black-pigmented Bacteroides was found to be more effective in the induction of IL-8 mRNA gene expression in osteoblasts than in pulp fibroblasts (P

Aim To investigate the effect of pro-inflammatory cytokines and black-pigmented Bacteroides on the expression of IL-6 gene in human pulp fibroblasts. Methodology IL-1α, tumour necrosis factor-α (TNF-α) and the supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-6 gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. Results Investigations of the time dependence of IL-6 mRNA expression in pro-inflammatory cytokines-treated cells revealed a rapid accumulation of the transcript after 2 h of exposure and remained elevated throughout the 24-h incubation period. In addition, black-pigmented Bacteroides also induced IL-6 gene expression in human pulp fibroblasts. Conclusions Pro-inflammatory cytokines and black-pigmented Bacteroides may be involved in developing pulpal inflammation through the stimulation of IL-6 production.

To investigate the cytotoxicity of five different dentine-bonding agents on human pulp cells in vitro.Set specimens from Clearfil SE Bond (CB), Heliobond (HB), PrimeBond NT (PB), Single Bond (SB), and Syntac Single Component (SC) were eluted with culture medium for 2 and 5 days. Cytotoxicity was judged using tetrazolium bromide reduction assay on human primary pulp cells.Elutes from five dentine-bonding agents were cytotoxic to primary human pulp cells (P0.05). CB was the least toxic sealer amongst the chemicals tested. The cytotoxic response decreased in an order of SBPBSCHBCB.The influence of the cytotoxicity depended on the materials tested. Dentine-bonding agents have significant potential for pulpal toxicity.

Aim The purpose of this study was to determine the cytotoxicity of three different types of root canal sealer on human periodontal ligament (PDL) cells and a permanent hamster cell line (V79 cells). Methodology Set specimens from two resin based sealers (AH26 and AHPlus), three zinc oxide–eugenol-based sealers (Canals, Endomethansone and N2) and one calcium hydroxide-based sealer (Sealapex) were eluted with culture medium for 1, 2, 3 and 7 days. Cytotoxicity was judged using tetrazolium bromide reduction assay on human primary PDL cells and V79 cells derived from a Chinese hamster. Results The results showed that elutes from resin-based, zinc oxide–eugenol-based, and calcium hydroxide-based sealers were cytotoxic to primary human PDL cultures and V79 cells. Calcium hydroxide-based sealer was the least toxic sealer amongst the chemicals tested in both cultures. The cytotoxic response decreased in an order of N2 > Endomethansone > AH26 > AHplus > Canals > Sealapex. Conclusions The sensitivity of toxicity depended on the materials tested and the cell culture system used. Thus, the use of both permanent and primary cells is recommended for screening of the cytotoxic effects of root canal sealers. In addition, the results confirmed that root canal sealers constantly dissolve when exposed to an aqueous environment for extended periods, possibly causing moderate or severe cytotoxic reactions. Use of calcium hydroxide-based material as a root canal sealer initially may result in a more favourable response to periradicular tissues.

To evaluate the expression of tumour necrosis factor-α and surface antigens by bisphenol A-glycidyl-methacrylate (BisGMA) on murine macrophage cell line RAW264.7.Cytotoxicity was measured by tetrazolium bromide reduction assay. Tumour necrosis factor (TNF)-α was analysed by enzyme-linked immunosorbent assay. Cell surface antigens were investigated by flowcytometry. Statistical analyses were performed using anova followed by the Bonferroni's t-test for multigroup comparisons.BisGMA exhibited cytotoxicity to RAW264.7 in a dose-dependent manner (P 0.05) during 2-h incubation period. BisGMA was found to increase TNF-α secretion in a dose-dependent manner (P 0.05). In addition, CD11, CD14, CD45, CD54, CD40, CD80, and MHC II were significantly stimulated by BisGMA in a dose-dependent manner (P 0.05). However, MHC I expression was not affected by BisGMA (P 0.05).Taken together, the ability of macrophages to induce an appropriate immune response when exposed to BisGMA has the potential to upregulate TNF-α production and expression of surface antigens.

To evaluate the potential toxicological implications of BisGMA on murine macrophage cell line RAW264.7.Lactate dehydrogenase release, flow cytometry, Western blot and fluorometric assays were used to detect cell viability, mode of cell death and caspase activities, respectively. In addition, alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assays were applied to detect genotoxicity. Statistical analyses were performed using anova followed by the Bonferroni's t-test for multi-group comparisons test.BisGMA demonstrated a cytotoxic effect on RAW264.7 cells in a dose-dependent and a time-dependent manner (P 0.05). BisGMA was found to induce two modes of cell death. The mode of cell death changed from apoptosis to necrosis as the concentrations of BisGMA elevated. Caspase-3, caspase-8 and caspase-9 activities were significantly induced by BisGMA in a dose-dependent manner (P 0.05). Moreover, BisGMA exhibited genotoxicity via a dose-related increase in the numbers of micronucleus and DNA strand breaks (P 0.05).Cytotoxicity and genotoxicity induced by BisGMA are mediated by DNA damage and caspase activation.

Aim To evaluate whether the initial healing of apical radiolucencies 1 year after root canal treatment could be quantitatively identified by the change in fractal dimension (FD) values for the eventually completely healed cases. Methodology Twenty-six patients with successful root canal treatment were recruited. All teeth were associated with complete healing either before or at 1 year following treatment (six of 26) or still undergoing healing at 1 year after treatment but completely healed thereafter (20 of 26). Two radiographs were selected for the same patient, one taken before treatment and the other taken 1 year after treatment. Eight regions of interests (ROIs) were selected from each radiograph, two as the experimental group located close to the infected root apex, two as the control group in the healthy bone and the other four in the healthy bone ensuring the image quality. Results Based on the FD values of the four ROIs in the healthy bone, the two radiographs were confirmed to have been taken with similar projection angles and exposure. The FD values were shown to significantly increase (P = 0.006) and decrease (P = 0.000) around the root apex and the neighbouring region of the apical lesion, respectively. Conclusion Changes in fractal dimension values may serve as a necessary condition to quantitatively indicate the initial healing status 1 year after root canal treatment.

To evaluate ex vivo the mechanisms of cytotoxicity of dentine bonding agents in human pulp cells in vitro.Human pulp cells were obtained from impacted third molars with informed consent and then cultured using an explant technique. Set specimens from Clearfil SE Bond (CB), PrimeBond 2.1 (PB), and Single Bond (SB) were eluted with culture medium. Cytotoxicity was judged using an assay of tetrazolium bromide reduction. To determine whether glutathione (GSH) levels were important in the cytotoxicity of dentine bonding agents, cells were pretreated with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or buthionine sulfoximine (BSO) to deplete GSH. Three replicates of each dentine bonding agents were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was by one-way analysis of variance (anova). Tests of differences of the treatments were analysed by Duncan's test.Clearfil SE Bond, PB, and SB were cytotoxic to pulp cells in a concentration-dependent manner (P0.05). The cytotoxicity was upregulated by dentine bonding agents in the following order: PBSBCB. Addition of OTZ extracellularly protected the pulp cells from dentine bonding agents-induced cytotoxicity (P0.05). Addition of BSO enhanced pulp cell death on dentine bonding agents-induced cytotoxicity (P0.05).Dentine bonding agents have significant potential for pulpal toxicity. GSH depletion could be the mechanism for dentine bonding agents-induced cytotoxicity.

Aim To compare oncostatin M (OSM) expression in clinically healthy and inflamed specimened human pulp tissue. Methodology Thirty pulpal tissue specimens (15 clinically healthy and 15 inflamed) were obtained from extracted third molars with informed consent from patients. The levels of OSM were compared between clinically healthy pulp and inflamed pulp tissues using the semi-quantitative reverse-transcriptase polymerase chain reaction. In addition, immunohistochemistry was used to identify the in situ localization of OSM expression in pulp specimens. For testing of differences in the OSM between the clinically healthy and inflamed human dental pulps, the Wilcoxon–Mann–Whitney rank sum test was applied. Differences in OSM expression between tissue with low and high levels of inflammation were subsequently analysed by Fisher’s exact test. Results Inflamed pulps exhibited significantly higher OSM mRNA gene expression than clinically healthy pulps (P

To evaluate the mechanisms of cytotoxicity of eugenol in human osteoblastic cells in vitro.Cytotoxicity and cell proliferation assays were performed to elucidate the toxic effects of eugenol on the human osteoblastic cell line U2OS. Furthermore, the effects of antioxidants catalase (scavenger of H2O2), superoxide dismutase (SOD, an extracellular superoxide free radical scavenger) and N-acetyl-L-cysteine (NAC, a cell-permeable glutathione precursor) were added to discover the possible mechanisms of eugenol-induced cytotoxicity. Paired Student's t-test was applied for the statistical analysis of the results.Eugenol demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P0.05). The 50% inhibition concentration of eugenol was approximately 0.75 mmol L(-1). Eugenol also inhibited cell proliferation during a 4-day culture period (P0.05). Addition of NAC extracellularly protected the cells from eugenol-induced cytotoxicity (P0.05). Neither, SOD nor catalase provided any protective effects on eugenol-induced cytotoxicity (P0.05).The levels of eugenol tested inhibited growth and proliferation of U2OS cells. Eugenol has significant potential for periapical toxicity. These inhibitory effects were associated with glutathione levels.

Aim To investigate the effect of black-pigmented Bacteroides on the expression of vascular endothelial growth factor (VEGF) gene in human pulp fibroblasts. Methodology The supernatants of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia were used to evaluate VEGF gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. Results Black-pigmented Bacteroides induced significantly high levels of VEGF mRNA gene expression in human pulp fibroblasts (P

To investigate the effect of black-pigmented Bacteroides on the expression of interleukin (IL)-8 gene in human pulp fibroblasts and osteoblasts.The supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-8 gene expression in human pulp fibroblasts and osteoblasts. The levels of mRNAs were measured by the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.Investigations of the time-dependence of IL-8 mRNA expression in black-pigmented Bacteroides-treated pulp fibroblasts and osteoblasts revealed a rapid accumulation of the transcript after 2 h of exposure, and remained elevated throughout the 24-h incubation period. In addition, IL-8 mRNA gene expression was also found in human osteoblasts stimulated with black-pigmented Bacteroides. However, black-pigmented Bacteroides was found to be more effective in the induction of IL-8 mRNA gene expression in osteoblasts than in pulp fibroblasts (P0.05).Black-pigmented Bacteroides are capable of amplifying the local immune response and promoting pulpal/periapical tissue inflammation by stimulating pulp fibroblasts and osteoblasts to express IL-8.

To investigate the effect of pro-inflammatory cytokines and black-pigmented Bacteroides on the expression of IL-6 gene in human pulp fibroblasts.IL-1alpha, tumour necrosis factor-alpha (TNF-alpha) and the supernatants of Porphyromonas endodontalis, P. gingivalis and Prevotella intermedia were used to evaluate IL-6 gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis.Investigations of the time dependence of IL-6 mRNA expression in pro-inflammatory cytokines-treated cells revealed a rapid accumulation of the transcript after 2 h of exposure and remained elevated throughout the 24-h incubation period. In addition, black-pigmented Bacteroides also induced IL-6 gene expression in human pulp fibroblasts.Pro-inflammatory cytokines and black-pigmented Bacteroides may be involved in developing pulpal inflammation through the stimulation of IL-6 production.

AIM: The purpose of this study was to investigate the expression of cyclooxygenase-2 (COX-2) in radicular cysts. METHODOLOGY: Thirty biopsy specimens of radicular cysts were examined using immunohistochemistry. A peroxidase-labelled streptavidin-biotin technique was used for identification of the COX-2. Fisher's exact test (two-tail) was used for statistical analysis of the results. RESULTS: The result demonstrated that COX-2 expression was significantly higher in radicular cysts with higher levels of inflammatory infiltrates. COX-2 stain was detected in the lining epithelium, subepithelial fibroblasts, macrophages and endothelial cells in all specimens. CONCLUSIONS: COX-2 expression is significantly higher in radicular cysts. COX-2 may play an important role in the pathogenesis of radicular cysts.