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Schulz S.D.,Albert Ludwigs University of Freiburg | Ruppell C.,Albert Ludwigs University of Freiburg | Tomakidi P.,Albert Ludwigs University of Freiburg | Steinberg T.,Albert Ludwigs University of Freiburg | And 4 more authors.
Dental Materials | Year: 2015

Objectives The aim of this study was the detection of putative gene expression-related effects of dental composites in conventional and interactive gingival cell systems. Methods Conventional monoculture (MC) and interactive cell systems (ICS) comprising human gingival fibroblast (HGF) and immortalized human gingival keratinocytes (IHGK) were exposed for 24 h and 7 days according to ISO10993-12:2012 manufactured eluates of different composites (Ceram X®, Filtek™ Supreme XT, Filtek™ Silorane, Fusio™ Liquid Dentin, and Vertise™ Flow). qRT-PCR-based mRNA analysis for biomarkers indicating cell proliferation, differentiation, apoptosis, inflammation, and adhesion was performed. Apoptotic cells were quantified by annexin-V labeling. Results Due to low RNA amounts, qPCR could not be performed for Vertise™ Flow and Fusio™ Liquid Dentin at day 7. At 24 h, flowables yielded increased transcription for biomarkers of inflammation and apoptosis in IHGK, irrespective of the cell system. HGF cultures displayed lower transcription for cell adhesion markers in both cell systems. Filtek™ Supreme XT showed increased differentiation by elevated filaggrin gene expression in both cell systems for IHGK at day 7, while Filtek™ Silorane and Ceram X® yielded elevation of inflammation biomarkers in both cell types. Annexin-V labeling revealed high apoptosis rates for both flowables and Filtek™ Supreme XT for IHGK, while low rates were detected for Filtek™ Silorane and Ceram X®. Significance Among the composites evaluated, exposition of IHGK and HGF in conventional and interactive cell systems demonstrated most pronounced gene expression alterations in response to flowables, coinciding with elevated levels of apoptosis. © 2015 Academy of Dental Materials.

Debiak M.,University of Konstanz | Lex K.,University of Konstanz | Ponath V.,University of Konstanz | Burckhardt-Boer W.,University of Konstanz | And 6 more authors.
Toxicology Letters | Year: 2016

Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy.PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the molecular mechanism of PARP1 activation and its functional consequences after mustard treatment in general. Such a study is presented in an accompanying article (. Mangerich et al., 2016). © 2015.

Durner J.,Walther Straub Institute of Pharmacology and Toxicology | Durner J.,Ludwig Maximilians University of Munich | Obermaier J.,Ludwig Maximilians University of Munich | Draenert M.,Ludwig Maximilians University of Munich | Ilie N.,Ludwig Maximilians University of Munich
Dental Materials | Year: 2012

Objectives: This study's purpose was to measure and compare the degree of conversion (DC) and the amount of elutable substances from modern resin-based composites (RBCs) as function of polymerization time. One nano-hybrid RBC based on tricyclodecane-(TCD)-urethane (Venus ® Diamond) and two conventionally formulated RBCs (TetricEvo Ceram ®, Filtek™ Supreme XTE) were considered. Method: DC (n = 5) was investigated in real time for 5 min by Fourier transform infrared spectroscopy (ATR-FTIR) in a filling depth of 2 mm at varied irradiation times (5, 10, 20, 40 s). After storing the specimens in ethanol/water for 7 d at 37 °C the eluates were analyzed by gas chromatography/mass spectrometry. Results were statistically analyzed using a multivariate analysis (α = 0.05) an independent t-test (p < 0.05) and a Pearson correlation analysis. Results: In all groups increasing curing time resulted in a significant increase in DC. For TetricEvo Ceram ® a high significant inverse correlation was found between DC and the amount of eluted camphorquinone (CQ, Pearson correlation coefficient = -0.88), ethylene glycol dimethacrylate (EGDMA, -0.73), 4-N,N-dimethylaminobenzoic acid ethylester (DMABEE, -0.87), triethylene glycol dimethacrylate (TEGDMA, -0.68), Tinuvin P (-0.71) and bisphenol-A-polyetheylene glycol dimethacrylate (BisEMA, -0.84). Unexpectedly DC and the amount of eluted methyl acrylate (MAA) correlated directly (0.72). In the specimens of Venus ® Diamond a significant inverse correlation was found between DC and the amount of eluted CQ (-0.69) and TEGDMA (-0.50), whereas in the specimen of Filtek™ Supreme XTE DC correlated with CQ (-0.96), EGDMA (-0.70), DMABEE (-0.87), TEGDMA (-0.92) and MAA (-0.92). Significance: This study demonstrated a strong inverse correlation between DC and elutable substances in RBCs. Both evaluation methods emphasis the importance of an adequate polymerization (20, 40 s), since short curing-times (5, 10 s) resulted in lower DC and higher amount of eluted substances with toxic potential. © 2012 Academy of Dental Materials.

Ilie N.,Ludwig Maximilians University of Munich | Obermaier J.,Ludwig Maximilians University of Munich | Durner J.,Ludwig Maximilians University of Munich | Durner J.,Walther Straub Institute of Pharmacology and Toxicology
Clinical Oral Investigations | Year: 2014

Objectives: The aim of our study was to analyse whether the irradiation time and/or the modulation of irradiation time influence the degree of conversion (DC) and the amount of elutable substances from modern nano-hybrid resin-based composites (RBCs). Materials and methods: The DC was recorded in real time for 5 min by means of attenuated total reflectance-Fourier transform infrared spectroscopy (n = 5) on the lower surface of 2-mm-thick samples irradiated with continuous and modulated irradiation times for 20 s and 40 s. The modulated times comprise a short polymerisation (2 s or 5 s) followed by a rest period of 1 min and an additional polymerisation to complete 20 s and 40 s of polymerisation (2 s + 18 s, 5 s + 15 s, 2 s + 38 s and 5 s + 35 s). After storing the specimens in ethanol/water for 7 days at 37 °C, the eluates were analysed by gas chromatography-mass spectrometry. Results were statistically analyzed using a one-way ANOVA analysis (α = 0.05). Results: The effect of irradiation time on DC is similar in all three analyzed materials, showing a significant increase in DC by increasing irradiation time from 20 s to 40 s, while the DC is not influenced within one irradiation time (20 s or 40 s) by the modulation of time. Conclusions: The type and amount of eluates are strongly dependent from the material and the irradiation protocol. Clinical relevance: An interrupt irradiation of RBCs is clinically feasible, reducing in general the amount of elutable substances at similar DC as the corresponding continuous polymerisation. © 2013 Springer-Verlag Berlin Heidelberg.

Ilie N.,Ludwig Maximilians University of Munich | Kessler A.,Ludwig Maximilians University of Munich | Durner J.,Ludwig Maximilians University of Munich | Durner J.,Walther Straub Institute of Pharmacology and Toxicology
Journal of Dentistry | Year: 2013

Objectives To assess the effect of irradiation time and distance of the light tip on the micro-mechanical properties and polymerisation kinetics of two bulk-fill resin-based composites at simulated clinically relevant filling depth. Methods Micro-mechanical properties (Vickers hardness (HV), depth of cure (DOC) and indentation modulus (E)) and polymerisation kinetics (real-time increase of degree of cure (DC)) of two bulk-fill resin-based composites (Tetric EvoCeram® Bulk Fill, Ivoclar Vivadent and x-tra base, Voco) were assessed at varying depth (0.1-6 mm in 100 μm steps for E and HV and 0.1, 2, 4 and 6 mm for DC), irradiation time (10, 20 or 40 s, Elipar Freelight2) and distances from the light tip (0 and 7 mm). Curing unit's irradiance was monitored in 1 mm steps at distances up to 10 mm away from the light tip on a laboratory-grade spectrometer. Results Multivariate analysis (α = 0.05), Student's t-test and Pearson correlation analysis were considered. The influence of material on the measured mechanical properties was significant (η2 = 0.080 for E and 0.256 for HV), while the parameters irradiation time, distance from the light tip and depth emphasise a stronger influence on Tetric EvoCeram® Bulk Fill. The polymerisation kinetics could be described by an exponential sum function, distinguishing between the gel and the glass phase. The above mentioned parameters strongly influenced the start of polymerisation (gel phase), and were of less importance for the glass phase. Conclusions Both materials enable at least 4 mm thick increments to be cured in one step under clinically relevant curing conditions. Clinical significance The susceptibility to variation in irradiance was material dependent, thus properties measured under clinically simulated curing conditions might vary to a different extent from those measured under ideal curing conditions. © 2013 Elsevier Ltd.

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