Filtration efficiency of surgical and FFP3 masks against composite dust.

The aim of this study was to investigate the protection efficiency of two types of face masks against composite dust and to characterize the particles that penetrated through the masks. Composite dust was created by grinding a commercial nano-filled composite in a plexiglass box without using water cooling or high vacuum evacuation, in order to obtain a worst-case exposure. Dust particles were collected using a personal inhalable aerosol sampler (IOM) fixed inside a custom-made phantom head. Surgical and filtering facepiece (FFP3) masks were tested, and the situation without a mask served as control. The IOM sampler contained a cassette with two filters to collect large inhalable (4-100 µm) and respirable dust particles (<4 µm). The amount of particles was determined gravimetrically by weighing filters before and after composite grinding, and further characterized by electron microscopy. Particle collection for both inhalable and respirable dust was the highest when no mask was used, and the lowest with the use of a FFP3 mask. Different sizes and shapes of particles were observed, with the largest particles (>1 µm) being seen when no mask was applied, whereas only nanoparticles could be detected when either type of face mask was applied. Even though FFP3 masks showed a higher filtration efficacy than surgical masks of the inhalable dust fraction, penetration of a small respirable particle fraction was inevitable for both masks.

Cytotoxic effects of composite dust on human bronchial epithelial cells.

INTRODUCTION: Previous research revealed that during routine abrasive procedures like polishing, shaping or removing of composites, high amounts of respirable dust particles (<5µm) including nano-sized particles (<100nm) may be released. OBJECTIVE: To determine the cytotoxic potential of composite dust particles on bronchial epithelium cells. METHODS: Composite dust of five commercial composites (one nano-composite, two nano-hybrid and two hybrid composites) was generated following a clinically relevant protocol. Polymerized composite samples were cut with a rough diamond bur (grain size 100µm, speed 200,000rpm) and all composite dust was collected in a sterile chamber. Human bronchial epithelial cells (16HBE14o-) were exposed to serially diluted suspensions of composite dust in cell culture medium at concentrations between 1.1 and 3.3mg/ml. After 24h-exposure, cell viability and membrane integrity were assessed by the WST-1 and the LDH leakage assay, respectively. The release of IL-1ß and IL-6 was evaluated. The composite dust particles were characterized by transmission electron microscopy and by dynamic and electrophoretic light scattering. RESULTS: Neither membrane damage nor release of IL-1ß was detected over the complete concentration range. However, metabolic activity gradually declined for concentrations higher than 660µg/ml and the release of IL-6 was reduced when cells were exposed to the highest concentrations of dust. SIGNIFICANCE: Composite dust prepared by conventional dental abrasion methods only affected human bronchial epithelial cells in very high concentrations.

Cytotoxicity and induction of DNA double-strand breaks by components leached from dental composites in primary human gingival fibroblasts.

INTRODUCTION: The public interest steadily increases in the biological adverse effects caused by components released from resin-based dental restorations. OBJECTIVE: In this study, the cytotoxicity and the genotoxicity were investigated of following released components from dental resin restorations in human gingival fibroblasts (HGF): tetraethyleneglycol dimethacrylate (TEEGDMA), neopentylglycol dimethacrylate (Neopen), diphenyliodoniumchloride (DPIC), triphenyl-stibane (TPSB) and triphenylphosphane (TPP). METHODS: XTT based cell viability assay was used for cytotoxicity screening of substances. γ-H2AX assay was used for genotoxicity screening. In the γ-H2AX assay, HGFs were exposed to the substances for 6h. Induced foci represent double DNA strand breaks (DSBs), which can induce ATM-dependent phosphorylation of the histone H2AX. Cell death effects (apoptosis and necrosis), induced by the substances were visually tested by the same investigator using the fluorescent microscope. RESULTS: All tested substances induced a dose-dependent loss of viability in HGFs. Following toxicity ranking among the substances at EC50-concentration were found in the XTT assay (mM, mean±SEM; n=5): DPIC>Neopen>TPSB>TPP>TEEGDMA. DSB-foci per HGF-cell were obtained, when HGFs were exposed to the EC50-concentration of each substance in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP>TEEGDMA. Multi-foci cells (cells that contain more than 40 foci each) in 80 HGF-cells at EC50-concentration of each substance were found as follow (mean±SEM; n=3): DPIC>Neopen>TPP>TPSB>TEEGDMA. Cell apoptosis contained in each substance at EC50-concentration in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP >TEEGDMA. Cell necrosis contained in each substance at EC50-concentration in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP>TEEGDMA. CONCLUSION: Leached components from dental resin restorations can induce DNA DSBs and cell death effects in HGFs.

No evidence for DNA double-strand breaks caused by endodontic sealers.

INTRODUCTION: On extrusion, endodontic sealers might come into close contact with the periapical tissues for long periods. The objective of this study was to test possible mutagenicity of resin-based endodontic sealers by evaluating their potential to induce DNA double-strand breaks (DSBs). METHODS: Human gingival fibroblasts were exposed to subtoxic concentrations of eluates from 1 epoxy resin-based endodontic sealer (AH Plus Jet) and 2 methacrylate-based endodontic sealers (EndoRez and Real Seal). As control, Calcicur, a Ca(OH)(2)-based sealer, was used. The γ-H2AX immunofluorescence assay was used to microscopically detect DNA DSBs, and a custom algorithm was developed to quantify them. RESULTS: The cytotoxicity of the 24-hour eluates could be ranked in the following order: AH Plus Jet > Real Seal > EndoRez >> Calcicur. The γ-H2AX assay revealed that 1.3%-4.3% of the cell nucleus was occupied by foci when the cells were exposed to the eluates of the endodontic sealers. This was not significantly different from the negative control group in which the cells had been exposed to medium (2.1%). CONCLUSIONS: No indications for increased risk of genotoxicity of resin-based root canal sealers caused by the induction of DNA DSBs were found in this study.

Development and application of a simplified sample preparation method for determination of TEGDMA and related metabolites.

Analysis of biological samples obtained from in vivo experiments can be often challenging. In general it is not possible to apply the commonly used matrices that are necessary for the experiments to the desired analysis systems without further conditioning or sample purification steps. Besides possible adverse effects for instruments, interference between analytes and matrices can affect the correct measurement of analytes. Different methods of sample preparation can be used to convert biological samples into samples suitable for analysis; SPE and HS-SPME are two well established methods. Research of in vivo metabolism of triethyleneglycoledimethacrylate (TEGDMA), one of the most frequently contained comonomer in dental restorative materials, demands sample preparation methods that offer separation of TEGDMA and its related metabolites from biological matrices. In the presented study two methods for sample preparation were developed in order to analyze TEGDMA as well as its metabolites triethyleneglycole (TEG), 2,3-epoxymethacrylicacid methylester (2,3-EMME), and methacrylacid methylester (MAME) in Krebs-Henseleit buffer samples to facilitate a subsequent analysis via GC-MS. An easy and time-saving separation protocol was developed. Recovery rates of TEGDMA and TEG after SPE were 21 +/- 3% and 105 +/- 12%, respectively, recovery rate after headspace extraction of 2,3-EMME and MAME was higher at 48 degrees C compared with 20 degrees C extraction temperature. The tested range for 2,3-EMME and MAME concentration after HS-SPME extraction was 0.1-100 mg/L and both analytes showed a good linearity.

Volatile methacrylates in dental practices.

PURPOSE: In recent years, an increase of occupational respiratory diseases, such as asthma caused by methacrylates, has been observed in dental personnel. In this study, the exposure of dental personnel to various volatile methacrylates was investigated. MATERIALS AND METHODS: The air levels of methacrylates were measured during filling treatment while bonding agents were used in 4 dental practices in Munich, Germany. Short-term air sampling (15 min) was performed using solid phase microextraction (SPME). The SPME fibers were coated with carbowax/divinyl benzene to enrich the analytes. For analysis, the analytes were thermically desorbed from the fiber and subsequently analyzed directly by gas chromatography/mass spectrometry. RESULTS: The methacrylates methyl methacrylate (MMA), 2-hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EGDMA), and triethylene glycol dimethacrylate (TEG-DMA) were identified in the air of dental practices. The exposure levels of the four methacrylates varied during the filling treatments. The maximum concentrations found were 0.4 mg/m3 for MMA, 45 microg/m3 for HEMA, 13 microg/m3 for EGDMA, and 45 microg/m3 for TEG-DMA. The detection of TEG-DMA correlated with the application of bonding agents during performance of dental fillings. CONCLUSION: Exposure levels of different methacrylates were observed at all investigated dental practices. The maximum levels of MMA measured in this study were at least 200 times lower than the toxicologically relevant maximum allowable concentrations defined in various countries. Nevertheless, the exposure levels of methacrylates should be kept as low as possible due to the allergenic potential of some methacrylates.

The toxicokinetics and distribution of 2-hydroxyethyl methacrylate in mice.

The cytotoxicity of dental composites has been attributed to the release of residual monomers from polymerized resin-based composites due to the degradation processes or the incomplete polymerisation of materials. 2-Hydroxyethyl methacrylate (HEMA) is one of the major components released from dental resin-based composites. It was shown in vitro that HEMA was released into the adjacent biophase from such materials during the first days after placement. In this study uptake, distribution, and excretion of 14C-HEMA applied via gastric tube or subcutaneous administration at dose levels well above those encountered in dental care were examined in mice to test the hypothesis that HEMA can reach cytotoxic levels in mammalian tissues. 14C-HEMA was taken up rapidly from the stomach and intestines after gastric administration and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as (14)CO(2). Two metabolic pathways of 14C-HEMA can be described. The peak HEMA levels in all tissues examined after 24h were lower than known toxic levels. Therefore the study did not support the hypothesis.

Cytotoxicity of the dental composite component TEGDMA and selected metabolic by-products in human pulmonary cells.

OBJECTIVES: The comonomer triethyleneglycoldimethacrylate (TEGDMA) is a commonly used constituent of resin-based dental materials. Upon placement, light-cured dental polymers may release a wide spectrum of residual compounds due to incomplete monomer-conversion during polymerization. Apart from liberating unreacted monomers, additional compound release might occur due to mechanical wear and enzymatic degradation on the salivary surface of resin fillings. Following delivery into the local bio phase, leached compounds may encounter a variety of different enzymes, which might be present in their oral or systemic environment. Metabolic by-products formerly associated with TEGDMA-degradation include triethylene glycol (TEG), methacrylic acid (MA), 2,3-epoxymethacrylic acid (2,3-EMA), and formaldehyde. METHODS: Cytotoxicitiy of TEGDMA-derived intermediates was measured as mitochondrial dehydrogenase activity assessed by colorimetric measurement of formazan formation as a cleavage-product from the tetrazolium salt XTT by metabolically active A549 cells. EC(50)-values were calculated by using curve fitting software (GraphPad Prism). RESULTS: The following EC(50)-values (mmol/L) (95% confidence interval) were obtained: 2,3-EMA 1.65 (1.28-2.13), TEGDMA 1.83 (1.46-2.30), MA 4.91 (4.22-5.71), and paraformaldehyde (PFA) 5.48 (4.56-6.58). For TEG no cytotoxic effects up to a concentration of 10mM could be found. SIGNIFICANCE: The epoxy compound 2,3-EMA induced comparable toxic effects as the raw comonomer TEGDMA. It is therefore concluded that the formation of toxic intermediates might significantly contribute to TEGDMA-induced cytotoxicity in human pulmonary cells.

Identification of 2,3-epoxymethacrylic acid as an intermediate in the metabolism of dental materials in human liver microsomes.

OBJECTIVES: In previous studies it could be demonstrated that methacrylic acid (MA) is an intermediate in the metabolism of unpolymerized dental comonomers, released from dental restorative materials. This study was performed to identify the possible dental material intermediate 2,3-epoxymethacrylic acid (2,3-EMA) from MA in human liver microsomes. Most epoxy compounds are regarded as highly toxic substances. METHODS: The formation and hydrolysis were studied in defined systems containing only MA and human liver microsomes at 37 degrees C. Hydrolysis was inhibited by cyclohexene oxide, a competitive inhibitor of epoxide hydrolase. The reaction product 2,3-EMA was analyzed by the headspace gas chromatography-mass spectrometry. After 5, 30, and 60 min samples were taken and analyzed. RESULTS: For the reaction of MA to 2,3-EMA the average conversion rate was about 5% within 1h. It was found that without cyclohexene oxide the rate constant of enzymatic hydrolysis at pH 7.4 was about 10 times higher than the rate constant of the formation from MA in combination with cyclohexene oxide (k=8.3 versus 0.83 micromol/l min), indicating an instability and thus a high reactivity of 2,3-EMA. The formation of the MA intermediate 2,3-EMA was not observed when heat-inactivated liver microsomes were used (controls). SIGNIFICANCE: It could be clearly demonstrated that 2,3-EMA is a product of dental material metabolisms in biological systems. Therefore, increased toxicity might occur on dental restorative materials which are able to release (co)monomers which can be metabolized to MA.