Effect of printing layer orientation and polishing on the fatigue strength of 3D-printed dental zirconia.

OBJECTIVE: The aim of the study was to evaluate the influence of surface polishing and printing layer orientation on the fatigue behaviour of 3 mol% yttria-stabilized zirconia (3Y-TZP) by stereolithography (SLA) in comparison with subtractive manufacturing. MATERIALS AND METHODS: 60 experimental zirconia bar-shaped specimens were 3D-printed (P) via SLA, and 30 specimens were milled (M) from commercial zirconia block (Lava™ Frame, 3 M ESPE AG). All specimens had the same dimensions (1 mm × 1 mm x 12 mm) after sintering. The 3D-printed specimens were randomly divided according to printing orientations: parallel or perpendicular to the tensile surface in the fatigue test. The specimens were subsequently submitted to two surface finishing protocols (n = 15/gr): unpolished or polished. Their phase compositions were analysed by X-ray diffraction. The fatigue behaviour was evaluated by a stepwise approach. RESULTS: The milled and both 3D-printed groups showed similar phase compositions for the as-sintered condition. Considerable amounts of rhombohedral phase were detected after polishing. Milled unpolished samples presented significantly higher fatigue strength than 3D-printed unpolished samples. Polishing did not improve the fatigue strength for milled zirconia but was advantageous for the 3D-printed specimens. 3D-printed specimens with parallel printing-layer orientation were significantly stronger than specimens with perpendicular layers regardless of surface finishing. CONCLUSION: The manufacturing techniques had a significant influence on the fatigue strength of 3Y-TZP, but not on the phase compositions of the surface. The polishing protocol showed different effects on 3Y-TZP fatigue strength and induced phase transition of the 3Y-TZP from Tetragonal to Rhombohedral. The best fatigue strength was achieved through milling using an unpolished surface and SLA-printed layers that were parallel to the tensile surface, followed by polishing.

3D printed zirconia dental implants with integrated directional surface pores combine mechanical strength with favorable osteoblast response.

Dental implants need to combine mechanical strength with promoted osseointegration. Currently used subtractive manufacturing techniques require a multi-step process to obtain a rough surface topography that stimulates osseointegration. Advantageously, additive manufacturing (AM) enables direct implant shaping with unique geometries and surface topographies. In this study, zirconia implants with integrated lamellar surface topography were additively manufactured by nano-particle ink-jetting. The ISO-14801 fracture load of as-sintered implants (516±39 N) resisted fatigue in 5-55 °C water thermo-cycling (631±134 N). Remarkably, simultaneous mechanical fatigue and hydrothermal aging at 90 °C significantly increased the implant strength to 909±280 N due to compressive stress generated at the seamless transition of the 30-40 µm thick, rough and porous surface layer to the dense implant core. This unique surface structure induced an elongated osteoblast morphology with uniform cell orientation and allowed for osteoblast proliferation, long-term attachment and matrix mineralization. In conclusion, the developed AM zirconia implants not only provided high long-term mechanical resistance thanks to the dense core along with compressive stress induced at the transition zone, but also generated a favorable osteoblast response owing to the integrated directional surface pores. STATEMENT OF SIGNIFICANCE: Zirconia ceramics are becoming the material of choice for metal-free dental implants, however significant efforts are required to obtain a rough/porous surface for enhanced osseointegration, along with the risk of surface delamination and/or microstructure variation. In this study, we addressed the challenge by additively manufacturing implants that seamlessly combine dense core with a porous surface layer. For the first time, a unique surface with a directional lamellar pore morphology was additively obtained. This AM implant also provided strength as strong as conventionally manufactured zirconia implants before and after long-term fatigue. Favorable osteoblast response was proved by in-vitro cell investigation. This work demonstrated the opportunity to AM fabricate novel ceramic implants that can simultaneously meet the mechanical and biological functionality requirements.

Mechanical properties-translucency-microstructure relationships in commercial monolayer and multilayer monolithic zirconia ceramics.

OBJECTIVES: To evaluate the phase composition, microstructure, optical properties and mechanical properties of eight commercially available multilayer and monolayer monolithic dental zirconias. METHODS: Five commercial 3Y-TZP (GC ST, GC HT [GC, Tokyo Japan]; Katana ML, Katana HT [Kuraray Noritake] and Lava Plus [3M Oral Care]) and three Y-PSZ (Katana STML, Katana UTML [Kuraray Noritake]; GC UHT [GC, Tokyo Japan]) zirconia ceramic grades were cut in plate-shaped specimens, sintered according to the manufacturer's instructions and mirror polished. The zirconia chemical composition was determined using X-ray fluorescence (XRF), phase composition was characterized using X-ray diffraction (XRD), while the grain size was measured using scanning electron microscopy (SEM). The translucency Parameter (TP) and Contrast Ratio (CR) were measured with a spectrophotometer (n = 10/group). The indentation fracture toughness (n = 10), Vickers hardness (n = 10) and biaxial strength (n = 20) of the sintered ceramics were assessed. The stress distribution during biaxial testing was assessed by Finite element analysis (FEA). Statistical analysis involved one-way ANOVA and post-hoc Tukey's HSD test and Pearson correlation test (α = 0.05). RESULTS: FEA showed that the stress distribution in plate shape specimens was the same as for disks, rationalizing the use of plates for biaxial strength testing. As expected, higher quantities of Y2O3 were related to a higher cubic ZrO2 phase content and lower tetragonality t-ZrO2, which improved translucency but diminished flexural strength and toughness. While there was no significant correlation between grain size and other material properties, addition of pigments to the zirconia grade statistically negatively affected hardness. CONCLUSION: Even though an improvement in strength and translucency could be recorded for the last Y-TZP generation, future research still needs to strive for combined improvement of optical properties and mechanical reliability of zirconia ceramics.

Mechanical properties, aging stability and translucency of speed-sintered zirconia for chairside restorations.

OBJECTIVE: To evaluate the performance of zirconia ceramics sintered in a speed sintering induction furnace by comprehensive understanding of their optical and mechanical properties, microstructure, phase composition and aging stability, in comparison to ceramics sintered in a conventional furnace. METHODS: Speed sintered (SS) Katana STMLSS (Kuraray Noritake) (total thermal cycle/sintering time/dwell temperature: 30min/16min/1560°C) and CEREC Zirconia (CEREC ZrSS) (Dentsply Sirona) (15min/2min/1578°C) were compared to conventionally sintered (CS) Katana STMLCS (6.8h/2h/1550°C) and inCoris TZICS (4h/2h/1510°C). The translucency parameter (TP) and contrast ratio (CR) were measured with a spectrophotometer. The chemical composition of the materials was determined by XRF and phase composition was characterized using XRD. Hydrothermal aging behavior was evaluated by measuring the tetragonal-to-monoclinic ZrO2 phase transformation after accelerated hydrothermal aging in steam at 134°C. The indentation fracture toughness, Vickers hardness and biaxial strength of the sintered ceramics were assessed. RESULTS: Speed and conventionally sintered zirconia revealed similar density, microstructure, average strength and hydrothermal aging stability. Both Katana STMLSS/CS 5Y-PSZ ceramics were characterized with a higher content of cubic phase (≈53wt%), which resulted in a higher amount of Y2O3 in the remaining tetragonal ZrO2 phases compared to the 3Y-TZP CEREC ZrSS and inCoris TZICS (8 and 20wt%, respectively). The sintering program did not affect the hydrothermal aging behavior of Katana STMLSS and CEREC ZrSS. TP of Katana STMLSS (TP≈32) was not affected by speed sintering, while the translucency of CEREC ZrSS (TP=14) was significantly reduced. Hardness, fracture toughness and Weibull characteristic strength of Katana STMLSS and CEREC ZrSS also reached the optimal level, but speed sintering substantially lowered their mechanical reliability. SIGNIFICANCE: Speed sintering of 3Y-TZP and 5Y-PSZ in a speed sintering induction oven appeared suitable for clinical applications. However, further studies should focus on improving of translucency and mechanical reliability of the speed-sintered zirconia ceramics.

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.

Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo.

BACKGROUND: The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo. RESULTS: We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1ß increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates. CONCLUSION: Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

The effect of water spray on the release of composite nano-dust.

OBJECTIVE: To evaluate the collection efficiency of water spray on the release of airborne composite particles during grinding of composite materials. MATERIALS AND METHODS: Composite sticks (L:35 mm × W:5.4 mm × H:1.6 mm) of seven commercial dental composites were ground with a rough diamond bur (grain size 100 µm, speed 200,000 rpm). All experiments were performed in an enclosed 1-m3 chamber with low particulate background (< 1,000 #/cm3), and airborne particles were evaluated based on their electrical mobility. The number size distribution was determined by scanning mobility particle sizer (SMPS). Particles were collected by an electrostatic precipitator (ESP), and were ultramorphologically and chemically analyzed by a transmission electron microscope equipped with energy-dispersive X-ray spectroscopy (TEM-EDS). RESULTS: SMPS measurements confirmed that both dry and wet grinding generated high concentrations of nanoparticles particles with the highest concentration recorded during the last minute of grinding (1.80 × 106 - 3.29 × 106#/cm3), after which a gradual decline in particle concentration took place. Nevertheless, grinding with water spray resulted in a significant reduction of the number of released particles (5.6 × 105 - 1.37 × 106#/cm3). The smallest particle diameter was recorded during the last minute of grinding followed by a continuous growth for every next measurement. TEM of composite dust revealed a high concentration of particles varying in both size and shape. CONCLUSIONS: Regardless of whether the water cooling spray system was used during bur manipulation of composite materials, predominately nanoparticles were released. However, the particle concentrations were significantly decreased with water spray. CLINICAL RELEVANCE: Since water spray might not be sufficient in nanoparticle collection, special care should be taken to prevent inhalation of composite dust.

Cytotoxic and genotoxic potential of respirable fraction of composite dust on human bronchial cells.

OBJECTIVE: To determine the cytotoxic and genotoxic potential of the respirable fraction of composite dust (<4 µm) on human bronchial epithelial cells. METHODS: Composite sticks of three commercial dental composites (Filtek Supreme XTE, Grandio, Transbond XT) were ground in an enclosed plexiglass chamber with a rough dental bur (grain-size 100 µm) and the generated airborne respirable dust was collected in a personal cyclone on a teflon filter (pore size 5 µm). Immediately after particle collection, the dust was quantified gravimetrically and the particles were suspended in cell culturing medium. Next, human bronchial epithelial cells (16HBE14o-) were exposed to the suspensions (3 µg/ml-400 µg/ml). After 24 h, cell viability (WST-1 assay) and membrane integrity (LDH assay) were evaluated. Furthermore, the genotoxic effect of a sub-cytotoxic concentration (50 µg/ml) of composite dust was evaluated by the comet assay after 3 h exposure and cell cycle disturbances were analyzed by flow cytometry. Cellular uptake of particles was evaluated by transmission electronic microscope (TEM). RESULTS: For all three tested composite materials, a decrease in metabolic activity of 10-35% was observed when the cells were exposed to the highest concentrations (100 µg/ml-400 µg/ml). Toxicity was partially linked to membrane disruption especially after 72 h exposure. All tested composites provoked a mild genotoxic effect after short-term exposure compared to the control groups. TEM revealed that respirable particles of all tested composites were taken up by the cells. SIGNIFICANCE: The respirable fraction of composite dust only showed cytotoxic effects at the highest concentrations, whereas mild genotoxicity was observed after exposure to a sub-cytotoxic concentration.

Long-term elution of monomers from resin-based dental composites.

OBJECTIVE: To bridge the gap between the current alarming literature on resin-based dental materials and the limited clinical observations, more precise knowledge on the actual quantity of released compounds should be acquired. The objective of this study was to quantify the long-term elution of various compounds from resin-based dental composites during one year. METHODS: Eight materials were investigated: G-aenial Anterior, G-aenial Posterior, Venus, Venus Pearl, Venus Diamond, Ceram X mono, Dyract and Filtek Supreme XTE. Cylindrical specimens (6 mm diameter, 2 mm thickness) were immersed in 1mL of three different extraction solutions (water, artificial saliva or ethanol) and stored in the dark at 37°C. Every week, the extraction solution was refreshed. The samples were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: BisEMA3, BisEMA6, BisEMA10, BisGMA, CQ, HEMA, TCD-DI-HEA, TEGDMA, and UDMA were quantified in the samples. Depending on the composite and the extraction solution, certain monomers (BisGMA, HEMA and UDMA) were able to continuously elute from the materials, up until 52 weeks after initial immersion. Monomer elution was clearly higher when ethanol was used as extraction solution. It could be demonstrated that the tested composites continued to release small quantities of monomers over longer periods when a continuous refreshing protocol is followed. SIGNIFICANCE: Even if monomer elution may not lead to a risk at short term, the potential long-term toxicity should be further investigated. Long-term elution and subsequent chronic exposure to monomers from resin-based dental materials should not be neglected when assessing the overall human health risks.

Freshly-mixed and setting calcium-silicate cements stimulate human dental pulp cells.

OBJECTIVES: To evaluate the effect of the eluates from 3 freshly-mixed and setting hydraulic calcium-silicate cements (hCSCs) on human dental pulp cells (HDPCs) and to examine the effect of a newly developed hCSC containing phosphopullulan (PPL) on HDPCs. METHODS: Human dental pulp cells, previously characterized as mesenchymal stem cells, were used. To collect the eluates, disks occupying the whole surface of a 12-well plate were prepared using an experimental hCSC containing phosphopullulan (GC), Nex-Cem MTA (GC), Biodentine (Septodont) or a zinc-oxide (ZnO) eugenol cement (material-related negative control). Immediately after preparing the disks (non-set), 3ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) were added. The medium was left in contact with the disks for 24h before being collected. Four different dilutions were prepared (100%, 50%, 25% and 10%) and cell-cytotoxicity, cell-proliferation, cell-migration and odontogenic differentiation were tested. The cell-cytotoxicity and cell-proliferation assays were performed by XTT-colorimetric assay at different time points. The cell-migration ability was tested with the wound-healing assay and the odontogenic differentiation capacity of hCSCs on HDPCs was tested with RT-PCR. RESULTS: Considering all experimental data together, the eluates from 3 freshly-mixed and setting hCSCs appeared not cytotoxic toward HDPCs. Moreover, all three cements stimulated proliferation, migration and odontogenic differentiation of HDPCs. SIGNIFICANCE: The use of freshly-mixed and setting hCSCs is an appropriate approach to test the effect of the materials on human dental pulp cells. The experimental material containing PPL is non-cytotoxic and positively stimulates HDPCs.

Saturation reduces in-vitro leakage of monomers from composites.

OBJECTIVE: Accurate knowledge of the quantity of released monomers from composites is important. To evaluate the elution of monomers, polymerized composites are typically immersed in an extraction solvent. The objective was to determine whether the volume of extraction solvent and the immersion time influences monomer leachability from dental composite materials. METHODS: Composite disks of two commercial composites, (Filtek Supreme XTE, 3M ESPE and G-aenial Universal Flo, GC) were prepared. The disks (n=10) were placed in a glass vial with 1ml, 2ml or 3ml of extraction solvent (100% ethanol with deuterated diethylphalate as internal standard). After either 7 or 30 days at 37°C, the supernatant was collected and the amount of released monomers (BisEMA, BisGMA, UDMA, TEGDMA) and bisphenol A was measured with liquid chromatography mass spectroscopy. RESULTS: For both tested composites, the highest amount of released monomers was measured after sample incubation in 3ml, while the lowest amount was measured in 1ml of extraction solvent. Furthermore, 30 days did not result in much more monomer release compared to 7 days, and for most monomers, there was no statistically significant difference in release between 7 and 30 days. SIGNIFICANCE: Release kinetics in in-vitro experiments are also influenced by saturation of the extraction solvent with the leached monomers. This is important as it is unlikely that saturation can be reached in an in-vivo situation, where saliva (or pulpal fluid) is continuously refreshed. Saturation of the extraction solvent can be avoided in-vitro by refreshing the extraction medium after equal time intervals.

Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells.

BACKGROUND: Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells. RESULTS: Using fluorometric measurements of intracellular Ca2+ concentration ([Ca2+]i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca2+]i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells. CONCLUSIONS: Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.

Cyto-genotoxic and DNA methylation changes induced by different crystal phases of TiO2-np in bronchial epithelial (16-HBE) cells.

With the increase in use of TiO2-np, a better understanding of their safety is important. In the present study the effect of different crystal phases of TiO2-np (anatase, rutile and anatase: rutile mixture; 20-26nm) were studied for cyto-genotoxicity and global DNA methylation and hydroxymethylation. Cytotoxic response was observed at a concentration of 25µg/ml for the particles tested. Results of comet and micronucleus (with and without CytB) assays revealed significant genotoxic effect of these particles. Flow cytometry revealed cell cycle arrest in the S-phase. Based on the results, toxicity of the particles could be correlated with their physico-chemical properties (i.e. smaller size and hydrodynamic diameter and larger surface area), anatase form being the most toxic. From the results of the cyto-genotoxicity assays, concentrations were determined for the epigenetic study. Effect on global DNA methylation and hydroxymethylation levels were studied at cyto-genotoxic (25µg/ml), genotoxic (12.5µg/ml) and sub cyto-genotoxic (3.25µg/ml) concentrations using LC-MS/MS analysis. Though no significant changes were observed for 3h treatment schedule; significant hypomethylation were observed at 24h for anatase (significant at 3.25 and 25µg/ml), rutile (significant at 3.25 and 25µg/ml) and anatase: rutile mixture (significant at 25µg/ml) forms. The results suggest that epigenetic changes could occur at sub cyto-genotoxic concentrations. And hence for complete characterization of nanoparticle toxicity, epigenetic studies should be performed along with conventional toxicity testing methods.

Simultaneous analysis of bisphenol A based compounds and other monomers leaching from resin-based dental materials by UHPLC-MS/MS.

Resin-based dental materials have raised debates concerning their safety and biocompatibility, resulting in a growing necessity of profound knowledge on the quantity of released compounds into the oral cavity. In this context, the aim of this study was to develop a comprehensive and reliable procedure based on liquid chromatography with mass spectrometry for the simultaneous analysis of various leached compounds (including bisphenol A based compounds) in samples from in vitro experiments. Different experiments were performed to determine the optimal analytical parameters, comprising mass spectrometry parameters, chromatographic separation conditions, and sample preparation. Four internal standards were used as follows: deuterated diethyl phthalate and bisphenol A (commercially available), and deuterated analogues of triethylene glycol dimethacrylate and urethane dimethacrylate (custom-made). The optimized method was validated for linearity of the calibration curves and the associated correlation coefficient, lower limit of quantification, higher limit of quantification, and intra- and interassay accuracy and precision. Additionally, the developed liquid chromatography with tandem mass spectrometry method was applied to the analysis of leaching compounds from four resin-based dental materials. The results indicated that this method is suitable for the analysis of different target compounds leaching from dental materials. This method might serve as a valuable basis for quick and accurate quantification of leached compounds from resin-based dental materials in biological samples.

Epigenetic effects of carbon nanotubes in human monocytic cells.

Carbon nanotubes (CNTs) are fibrous carbon-based nanomaterials with a potential to cause carcinogenesis in humans. Alterations in DNA methylation on cytosine-phosphate-guanidine (CpG) sites are potential markers of exposure-induced carcinogenesis. This study examined cytotoxicity, genotoxicity and DNA methylation alterations on human monocytic cells (THP-1) after incubation with single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). Higher cytotoxicity and genotoxicity were observed after incubation with SWCNTs than incubation with MWCNTs. At the selected concentrations (25 and 100 µg/ml), DNA methylation alterations were studied. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to assess global DNA methylation, and Illumina 450K microarrays were used to assess methylation of single CpG sites. Next, we assessed gene promoter-specific methylation levels. We observed no global methylation or hydroxymethylation alterations, but on gene-specific level, distinct clustering of CNT-treated samples were noted. Collectively, CNTs induced gene promoter-specific altered methylation and those 1127 different genes were identified to be hypomethylated. Differentially methylated genes were involved in several signalling cascade pathways, vascular endothelial growth factor and platelet activation pathways. Moreover, possible contribution of the epigenetic alterations to monocyte differentiation and mixed M1/M2 macrophage polarisation were discussed.

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.