Wan-Yu Tseng

Cytotoxicity of three dentin bonding agents on human dental pulp cells

OBJECTIVES Dentin bonding agents (DBA) have been widely used in operative restoration to prevent leakage and promote bonding strength in the resin-dentin interface. However, DBA may exert potentially harmful effects to the dental pulp. In the present study, differential cytotoxicity of three DBA (Syntac Sprint, SP; Prime and Bond 2.1, PB; and Single Bond, SB) on the pulp cells was tested. METHODS Three DBA were diluted with the culture medium by a ratio of 1:1000, 1:2000 and 1:4000 (v/v). Pulp cells (5 x 10(4) cells/well) were then exposed to culture medium containing different diluents of three DBA for 12, 24h and 3 days. Cytotoxicity was measured with a modified 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. RESULTS A 12h experiment revealed that SP was the strongest cytotoxic agent, followed sequentially by SB and PB. Exposure of pulp cells to 1:4000 (v/v) dilution of SP, PB and SB for 24h reduced the cell number by 23, 6 and 45%, respectively. A 1:2000 (v/v) of DBA diluents reduced the cell number for 32, 13 and 65%, respectively, by SP, PB and SB. Dilution of DBA by 1000-fold of culture medium further enhanced the cytotoxic response. Cell number decreased by 89, 65 and 72%, respectively, by SP, PB and SB. Similar to the 12h-cytotoxicity data, SB is more toxic at high dilution condition, whereas, at low dilution condition, SP is the most toxic agent to pulp cells. Similar cytotoxicity was noted when pulp cells were exposed to DBA for 3 days. Toxicity of DBA was concomitant with marked retraction and rounding of dental pulp cells. SIGNIFICANCE These results indicate that DBA exerts potential harmful effects to the pulp. Differential toxic effects of DBA on the pulp cells should be considered during selection of a suitable DBA for operative restoration.

The effect of BisGMA on cyclooxygenase-2 expression, PGE2 production and cytotoxicity via reactive oxygen species- and MEK/ERK-dependent and -independent pathways.

After operative restoration, some monomers released from dentin bonding agents or composite resin may induce tissue inflammation and affect the vitality of dental pulp. Whether BisGMA, a major monomer of composite resin, may induce prostaglandin release and cytotoxicity to pulp cells and their mechanisms awaits investigation. We found that BisGMA induced cytotoxicity to human dental pulp cells at concentrations higher than 0.075 mm as analyzed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. BisGMA (0.1 mm) also stimulated ERK phosphorylation, PGE(2) production, COX-2 mRNA and protein expression as well as ROS production (as indicated by an increase in cellular DCF fluorescence) in dental pulp cells. Catalase (500 and 1000 U/ml) and U0126 (10 and 20 microm, a MEK inhibitor) effectively prevented the BisGMA-induced ERK activation, PGE(2) production and COX-2 expression. Moreover, catalase can protect the pulp cells from BisGMA cytotoxicity, whereas aspirin and U0126 lacked of this protective activity. These results suggest that BisGMA released from composite resin may potentially affect the vitality of dental pulp and induce pulpal inflammation via stimulation of ROS production, MEK/ERK1/2 activation and subsequent COX-2 gene expression and PGE(2) production. Cytotoxicity of BisGMA to dental pulp cells is related to ROS production, but not directly mediated by MEK activation and PGE(2) production.

Carboxylesterase expression in human dental pulp cells: Role in regulation of BisGMA-induced prostanoid production and cytotoxicity

Biocompatibility of dentin bonding agents (DBA) and composite resin may affect the treatment outcome (e.g., healthy pulp, pulpal inflammation, pulp necrosis) after operative restoration. Bisphenol-glycidyl methacrylate (BisGMA) is one of the major monomers present in DBA and resin. Prior studies focused on salivary esterase for metabolism and degradation of resin monomers clinically. This study found that human dental pulp cells expressed mainly carboxylesterase-2 (CES2) and smaller amounts of CES1A1 and CES3 isoforms. Exposure to BisGMA stimulated CES isoforms expression of pulp cells, and this event was inhibited by catalase. Exogenous addition of porcine esterase prevented BisGMA- and DBA-induced cytotoxicity. Interestingly, inhibition of CES by bis(p-nitrophenyl) phosphate (BNPP) and CES2 by loperamide enhanced the cytotoxicity of BisGMA and DBA. Addition of porcine esterase or N-acetyl-l-cysteine prevented BisGMA-induced prostaglandin E(2) (PGE(2)) and PGF(2α) production. In contrast, addition of BNPP and loperamide, but not mevastatin, enhanced BisGMA-induced PGE(2) and PGF(2α) production in dental pulp cells. These results suggest that BisGMA may induce the cytotoxicity and prostanoid production of pulp cells, leading to pulpal inflammation or necrosis via reactive oxygen species production. Expression of CES, especially CES2, in dental pulp cells can be an adaptive response to protect dental pulp against BisGMA-induced cytotoxicity and prostanoid release. Resin monomers are the main toxic components in DBA, and the ester group is crucial for monomer toxicity.

Areca Nut-induced Micronuclei and Cytokinesis Failure in Chinese Hamster Ovary Cells is Related to Reactive Oxygen Species Production and Actin Filament Deregulation

Epidemiological studies have shown a strong association between environmental exposure to betel quid (BQ) and oral cancer. Areca nut (AN), an ingredient of BQ, contains genotoxic and mutagenic compounds. In this study, we found that AN extract (ANE) inhibited the growth of Chinese hamster ovary cells (CHO‐K1) in a dose‐ and time‐dependent manner. Intracellular reactive oxygen species (ROS) levels and micronuclei (MN) frequency were significantly increased following ANE treatment in CHO‐K1 cells. Addition of catalase markedly inhibited ANE‐induced MN formation, indicating that ANE‐induced genotoxicity was correlated with intracellular H2O2. Incubation of CHO‐K1 cells with ANE (400–800 μg/ml) for 24 hr caused G2/M arrest, and prolonged exposure to ANE (800 μg/ml) significantly induced cell death. Surprisingly, ANE itself caused cytokinesis failure and subsequent increase in binucleated cell formation. Coexposure to catalase (2,000 U/ml) and ANE (800 μg/ml) reduced the generation of binucleated cells, indicating that ANE‐induced cytokinesis failure was associated with oxidative stress. Following prolonged exposure to ANE, an accumulation of hyperploid/aneuploid cells concomitant with bi‐, micro‐ or multinucleated cells was found. In summary, our results demonstrate that ANE exposure to CHO‐K1 cells caused increased MN frequency, G2/M arrest, cytokinesis failure, and an accumulation of hyperploid/aneuploid cells. These events are associated with an increase in intracellular H2O2 level and actin filament disorganization. Environ. Mol. Mutagen., 2009.

p-Cresol affects reactive oxygen species generation, cell cycle arrest, cytotoxicity and inflammation/atherosclerosis-related modulators production in endothelial cells and mononuclear cells.

Aims Cresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS) and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells. Methods EA.hy926 (EAHY) endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2′,7′-dichlorofluorescein diacetate (DCF) fluorescence flow cytometry. Prostaglandin F2α (PGF2α), plasminogen activator inhibitor-1 (PAI-1), soluble urokinase plasminogen activator receptor (suPAR), and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA). Results Exposure to 100–500 µM p-cresol decreased EAHY cell number by 30–61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM). P-cresol (>50 µM) also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF2α, and uPA production in EAHY cells. Conclusions p-Cresol may contribute to atherosclerosis and thrombosis in patients with uremia and cresol intoxication possibly due to induction of ROS, endothelial/mononuclear cell damage and production of inflammation/atherosclerosis-related molecules.

Glucosamine promotes osteogenic differentiation of dental pulp stem cells through modulating the level of the transforming growth factor-β type I receptor†

Dental pulp stem cells (DPSCs) are clonogenic, self‐renewing, and multi‐potential DPSCs capable of differentiating into osteoblasts. In this study, primary cell cultures were obtained from human dental pulp tissue of developing third molars, and flow cytometry was used to sort the subpopulation of DPSCs with STRO‐1 and CD146 double‐positive expression (denoted “DPSCs”). It was noted that DPSCs exhibited superior clonogenic potential and osteogenic differentiation capability than the dental pulp cell subpopulation with STRO‐1 and CD146 double‐negative expression (denoted DPCs). Furthermore, a low concentration (0.005 mg/ml) of exogenous glucosamine (GlcN) was effective in promoting the early osteogenic differentiation of DPSCs through the transforming growth factor‐β receptor (TGF‐βr) type I and Smads signal pathways, which upregulated the Runt‐related transcription factor 2/core‐binding factor alpha1 (Runx2/Cbfa1) and alkaline phosphatase at both the mRNA and protein levels. In the presence of osteogenic supplements, GlcN‐treated DPSCs produced more mineralized‐matrix deposition than did the untreated groups. Taken together, this study demonstrates the capacity of GlcN to promote the osteogenic differentiation of human DPSCs, and the underlying mechanism involves a TGF‐βr‐dependent Smad signal pathway. J. Cell. Physiol. 225: 140–151, 2010.

Prostaglandin F2α-Induced Interleukin-8 Production in Human Dental Pulp Cells Is Associated With MEK/ERK Signaling

Prostaglandin F(2alpha) (PGF(2alpha)) and interleukin-1beta (IL-1beta) levels are elevated in inflamed dental pulp. The roles of IL-1beta and PGF(2alpha) in the pathogenesis of pulpal inflammation await investigation. We found that IL-1beta stimulated PGF(2alpha) production of human dental pulp cells. IL-1beta and PGF(2alpha) (0.5-10 mumol/L) also induced IL-8 production and mRNA expression in pulp cells. Aspirin inhibited IL-1beta-induced PGF(2alpha), but not IL-8 production. PGF(2alpha)-induced IL-8 production and mRNA expression were inhibited by U0126 (an inhibitor of mitogen-activated protein kinase kinase [MEK1/2]) inhibitor), whereas SQ22536 (an adenylate cyclase inhibitor) enhanced this event. These results indicate that IL-1beta-induced IL-8 production in pulp cells is not mainly via direct activation of cyclooxygenase and PGF(2alpha) generation. PGF(2alpha)-induced IL-8 production is possibly via activation of MEK/extracellular signal-regulated kinase signaling, but not by activation of adenylate cyclase. IL-1beta and PGF(2alpha) might involve the pathogenesis of pulpal inflammation via induction of IL-8 production.

Comparative cytotoxicity of five current dentin bonding agents: Role of cell cycle deregulation

To compare the cytotoxicity of three nano-dentin bonding agents (nano-DBAs) and two non-nano-DBAs using Chinese hamster ovary (CHO-K1) cells. We found that nano fillers were not the major contributing factor in DBA cytotoxicity, as analyzed by colony forming assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Exposure of CHO-K1 cells to all three tested total-etching DBAs led to G(0)/G(1) cell cycle arrest, whereas exposure to higher concentrations of two tested nano-DBAs induced G(2)/M arrest. All five DBAs further induced apoptosis at the highest concentration, as analyzed by propidium iodide staining flow cytometry. The toxicity of all DBAs (1:4000v/v or higher) is related to increased reactive oxygen species (ROS) production, as analyzed by single cell DCF fluorescence flow cytometry. These results indicate that clinical application of DBAs may be potentially toxic to dental pulp tissues. Cytotoxicity of DBAs is associated with ROS production, cell cycle deregulation and apoptosis. Presence of methacrylate monomers such as PENTA and UDMA is possibly the major cytotoxic factor for DBAs. Further studies on other toxicological endpoints of nano-DBAs are necessary to highlight their safe use.

Biocompatibility and cytotoxicity of two novel low-shrinkage dental resin matrices

BACKGROUND/PURPOSE To reduce the polymerization shrinkage of dental composite resin, we used two different ratios of toluene 2,4-diisocyanate (TDI) or 1,6-hexamethylene diisocyanate (HDI) as functional side chains of bisphenol A-glycidyl methacrylate (bis-GMA) to synthesize two series of new dental resin matrices. This study evaluated the biocompatibility and cytotoxicity of these two series of new resin matrices. METHODS Two series of new dental resin matrices with the ratios of TDI or HDI functional side chain to bis-GMA (defined as B group) being 1:4, 1:2, 1:1 and 3:2 (defined as T1/4, T1/2, T1, T3/2, and H1/4, H1/2, H1, H3/2 groups, respectively) were synthesized. Each resin sample was light cured and immersed in the culture medium for 24 hours to make the extract solution. Then, human gingival fibroblasts were cultured in different extract solutions for 72 hours. The cytotoxicities of different resins were evaluated by microtitertetrazolium (MTT) assay, the levels of cell-produced reactive oxygen species (ROS) induced by different extract solutions was measured. RESULTS Resins of the T1/4 and B groups revealed significantly higher cytotoxicity than resins of other groups. However, resins of the T1 and T3/2 groups exhibited less cytotoxicity. In general, resins of the TDI-modified groups showed equal or less cytotoxicity and induced equal or lower levels of ROS than the corresponding resins of the HDI-modified and B groups. CONCLUSION Our results showed that the TDI-modified resin matrices containing more functional side chains were less cytotoxic than the corresponding HDI-modified resin matrices. When the ratio of functional side chain to bis-GMA is increased, the stereo hindrance of resin structure is increased, more toxic resin monomers are trapped in the complicated resin structure, and thus the resin matrix reveals less cytotoxicity. The TDI-modified resin matrices exhibit higher stereo hindrance of resin structure and thus show less cytotoxicity than the corresponding HDI-modified resin matrices.

7-Ketocholesterol induces ATM/ATR, Chk1/Chk2, PI3K/Akt signalings, cytotoxicity and IL-8 production in endothelial cells

Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimers disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 μg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways.