Objectives Material chemistry and workflow variables associated with the fabrication of dental devices may affect the biocompatibility of the dental devices. The purpose of this study was to compare digital and conventional workflow procedures in the manufacturing of acrylic-based occlusal devices by assessing the cytotoxic potential of leakage products.Methods Specimens were manufactured by 3D printing (stereolithography and digital light processing), milling, and autopolymerization. Print specimens were also subjected to different post-curing methods. To assess biocompatibility, a human tongue epithelial cell line was exposed to material-based extracts. Cell viability was measured by MTT assay while Western blot assessed the expression level of selected cytoprotective proteins.Results Extracts from the Splint 2.0 material printed with DLP technology and post-cured with the Asiga Flash showed the clearest loss of cell viability. The milled and autopolymerized materials also showed a significant reduction in cell viability. However, by storing the autopolymerized material in dH2O for 12 h, no significant viability loss was observed. Increased levels of cytoprotective proteins were seen in cells exposed to extracts from the print materials and the autopolymerized material. Similarly to the effect on viability loss, storing the autopolymerized material in dH2O for 12 h reduced this effect.Conclusions/Clinical relevance Based on the biocompatibility assessments, clinical outcomes of acrylic-based occlusal device materials may be affected by the choice of manufacturing technique and workflow procedures.

The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and l-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and l-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.

Biological evaluation of resin-based dental composites has traditionally been based on in vitro endpoint tests with different methods to determine loss of cell viability and cell morphology changes after exposure to the material or monomer constituents. The data reveals a potential for biological effects, but clinical relevance of such data is limited. Positive allergy tests and allergic clinical reactions to dental monomers are observed in dental personnel and patients. The aim of this review is to address newer research on molecular events caused by exposure to resin-based composites to have a better understanding of the potential for clinical adverse effects. A more accurate understanding of the biological aspects of dental composite materials has been found after studying parameters like glutathione depletion, oxidative stress, genotoxicity, and immunomodulatory key effects in various cell culture models. Using omics-based approaches allow for a broader and non-specified search of changes caused by methacrylate exposure. Defense mechanisms and adaption are observed in cells exposed to monomer concentrations relevant to clinical exposure. The above-mentioned methods are the foundations for modified testing strategies. The clinical relevance of most available in vitro endpoint tests is of limited relevance for the patient. Research focusing on molecular mechanisms has given new insight into methacrylate toxicity in exposed cells. Using this knowledge from mechanistic studies to develop standardized in vitro biocompatibility tests will likely improve their clinical relevance.

AbstractSeveral in vitro studies utilizing 2-dimensional (2D) cell culture systems have linked 2-hydroxyethyl methacrylate (HEMA) with cytotoxic effects in oral mucosa and dental pulp cells. Although such studies are invaluable in dissecting the cellular and molecular effects of HEMA, there is a growing interest in the utilization of appropriate 3-dimensional (3D) models that mimic the structure of oral mucosa. Using a previously characterized 3D-organotypic co-culture model, this study aimed to investigate the cellular and molecular effects of HEMA on a 3D-co-culture model consisting of primary normal oral keratinocyte (NOK) grown directly on top of collagen I gel containing primary oral fibroblasts (NOF). The second aim was to examine the suitability of a 3D-co-culture system consisting of oral squamous cell carcinoma (OSCC) cells as a model system to investigate the biological effects of HEMA. We demonstrated that HEMA treatment led to reduced viability of NOK, NOF and OSCC-cell lines in 2D-culture. The keratinocytes in 3D-co-cultures of NOK and OSCC-cells reacted similarly with respect to cell proliferation and activation of autophagy flux, to HEMA treatment. Nevertheless, NOK was found to be more susceptible to apoptosis following HEMA treatment than OSCC in 3D-co-cultures. These results indicate that 3D-organotypic co-cultures of NOK might represent an appropriate model system for the investigation of the biological effects of HEMA and other dental biomaterials. Given the challenges in obtaining primary cultures of NOK and issues associated with their rapid differentiation in culture, the possible use of OSCC cells as an alternative to NOK for 3D models represents an area for future research.

HovedbudskapForståelse for retentionscementernes sammensætning og egenskaber er afgørende for valget af den bedst egnede cement i enhver given klinisk situation.Plastcementer kan anvendes til alle restaureringstyper, men bør anvendes til cementering af lavstyrke keramiskrestaureringer, under forudsætning af tilstrækkelig tørlægning.Glasionomercementer er et godt alternativ til cementering ved utilstrækkelig tørlægning, dog ikke egnet til lavstyrke keramiske restaureringer samt ej-retentive restaureringer.Zinkfosfatcement må gerne anvendes til cementering af metalliske restaureringer, især ved subgingivale præparationer.

Objective The aims of this in vitro study were to assess if dynamic loading increases the metal ion release of selected dental alloys and to evaluate the cytotoxicity of the released metal ions. Materials and methods One Pd–Ag alloy (Aurolite 2B) and two Co–Cr alloys (Wirobond 280 and d.Sign 30) were investigated. Two different corrosion immersion tests were used: a standardized static test (ISO 22674: 2016) and an experimental dynamic test. Both tests involved immersion of the specimens in a lactic acidic solution (pH = 2.3). Inductively coupled plasma mass spectrometry was used to identify and quantify released elements. A human monocyte cell-line (THP-1) was exposed to serially diluted solutions containing the selected metal ions. Cell viability was measured using the methyl-thiazolyl-tetrazolium assay. Results According to the threshold defined in ISO 22674, only low concentrations of released elements were observed for both corrosion tests. No increase in metal ion release from the dynamic test compared with the static test was observed. Of the released elements, only Zn(II) and Co(II) showed a cytotoxic effect on THP-1 cells at 250 µM and higher concentrations. No increased viability loss was observed when adding other released elements to the exposure mixture. Conclusions The tested alloys showed low levels of metal ion release from both static and dynamic corrosion testing. Dynamic loading did not increase the metal ion release compared to the static corrosion test. Concentrations of 250 µM and above of Zn(II) and Co(II) showed a cytotoxic effect on THP-1 cells.

Methacrylate monomers are major components of resin-based biomaterials. The polymerization of these materials is never complete, and methacrylates leaking from cured materials cause exposure of patients. Only some selected methacrylates have thoroughly been tested for possible interaction with living cells. In the current study, we compared the effects of 2-hydroxyethyl-methacrylate (HEMA; a carefully studied methacrylate) and hydroxypropyl-methacrylate (HPMA; a scarcely investigated methacrylate). Five cell lines differing in both source and cell type were used. The cells were exposed to methacrylates (1-8 mM). Cell viability, cell death, glutathione levels, reactive oxygen species (ROS), and cell growth pattern were measured. Both methacrylates reduced cell viability, and glutathione depletion was observed in all cell lines. The cell death pattern varied among the cell lines. The ROS levels and cell growth pattern also differed between the cell lines after exposure to methacrylate monomers. No difference between HEMA and HPMA exposures were observed in any of the cell lines. The variation between cell lines shows that the measured methacrylate toxicity depends heavily on the test system chosen. Further, the conformity between HEMA and HPMA effects suggests that the two methacrylates similarly affect living cells.

Background. Leakage of unpolymerized methacrylate monomers after placement of methacrylate-containing polymeric dental materials leads to human exposure. Based on studies using murine macrophages and LPS from Escherichia coli (E. coli), dental monomers like 2-hydroxyethyl methacrylate (HEMA) are known to inhibit lipopolysaccharide (LPS) induced cytokine release. The aim of this study was to establish a model system with relevance for human oral monomer exposure using exposure to live gram-positive bacteria, and to confirm the HEMA-induced effects on cytokine release in this model. Methods. The human THP-1 monocyte cell line was differentiated to macrophages using phorbol 12-myristate 13-acetate (PMA), before exposure to 0.5-2 mM HEMA and live Staphylococcus aureus (S. aureus) in various multiplicity of infections (MOI). Cytokine release and cytotoxicity were determined after (i) 2-24 h pre-exposure to HEMA followed by 2-4 h S. aureus exposure and (ii) 2-4 h simultaneous exposure. The 24 h pre-exposure regime was also tested in primary human airway macrophages and for phagocytosis of S. aureus in THP-1 macrophages. Results. HEMA attenuated the cytokine release more strongly in the pre-exposure than combined exposure regime, with a maximal reduction of 95% in the S. aureus-induced cytokine release. A MOI of 0.1 (corresponding to a bacteria-macrophage ratio of 1:10) was determined to be optimal in the THP-1 macrophages as it induced sufficient cytokine release and negligible cytotoxicity. Attenuated release of S. aureus-induced interleukin (IL)-1 beta after HEMA exposure was confirmed in primary airway macrophages, while HEMA increased the phagocytosis of S. aureus in THP-1 cells. Conclusion. The model was successfully established and attenuated bacteria-induced cytokine release after HEMA exposure confirmed. (C) 2019 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

Resin-based biomaterials are widely used in medical and dental treatment, and both clinicians and patients are exposed to the materials. The knowledge of toxicity is mainly based on in vitro studies at exposure concentrations that induce cell death. However, severe cell damage and cell death signaling may overshadow essential cellular events caused by a possible toxicant. For dental resins, the knowledge of interaction with living cells at more clinical relevant exposure doses is sparse. 2-Hydroxyethylmethacrylate (HEMA) is a commonly used monomer in dental resins. Measuring cellular adaptation to HEMA at concentrations that did not reduce cell viability was the main focus of this study. Stable isotope labeling with amino acids in cell culture was used to measure proteome changes in cultured THP-1 cells exposed to HEMA. Western blotting verified the results. Cells exposed to HEMA increased their level of several cytoprotective proteins. The observed adaptation is compatible with increased oxidative burden caused by GSH depletion and the electrophilic characteristic of HEMA. The present approach to analyzing the toxic potential of HEMA yielded information on interactions with living cells is not previously reported. This detailed information is of great value to make better predictions of possible side effects in the clinic. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 851-859, 2019.

BPA has been reported to leach from some resin based dental restorative materials and materials used for orthodontic treatment. To confirm and update previous findings, especially in light of the new temporary lower threshold value for tolerable daily BPA intake, we have investigated the leaching of BPA from 4 composite filling materials, 3 sealants and 2 orthodontic bonding materials. The materials were either uncured and dissolved in methanol or cured. The cured materials were kept in deionized water for 24 hours or 2 weeks. Samples were subsequently analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS-MS). The composite filling material Tetric EvoFlow® and the fissure sealant DELTON® showed significantly higher levels of BPA leaching compared to control samples for all test conditions (uncured, 24 h leaching and 2 weeks leaching). There were no significant differences in amount of leached BPA for any of the tested materials after 24 hours compared to 2 weeks. These results show that BPA is still released from some dental materials despite the general concern about potential adverse effects of BPA. However, the amounts of BPA were relatively low and most likely represent a very small contribution to the total BPA exposure.

Objectives. Cellular responses including cell death are induced by in vitro exposure to the un-polymerized dental monomer 2-hydroxyethyl methacrylate (HEMA). Activation of the Nrf2/ARE signaling pathway has been suggested to mediate the cellular responses. Activation of this pathway may occur either indirectly through generation of increased oxidative stress or through direct binding to cysteine thiols due to the electrophilic properties of HEMA. The objective of this study was to elucidate the potential mechanism of Nrf2/ARE pathway activation after HEMA exposure. Methods. Global gene expression was investigated after exposure of the human bronchial epithelial cell line BEAS-2B to 2 mM HEMA for 4h. After exposure to 0.5, 1 or 2 mM HEMA for up to 24h, western analysis was performed for selected proteins. Finally, the levels of the same proteins were determined after treatment with either the antioxidants Vitamin C, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or BSO (L-buthioninesulfoximine), an inhibitor of GSH formation. Results. Several of the 25 genes with the highest increase in gene transcription are related to oxidative stress responses. Increased levels of 5 corresponding proteins (HO-1, GCLC, GCLM, NQO1 and SQSTM1) were observed. Antioxidant treatment as well as inhibition of GSH did not affect upregulation of these proteins. Thus, increased ROS or reduced GSH levels appear to be of limited importance in the observed HEMA-induced changes. Significance. Knowledge of the cellular responses to HEMA is important to evaluate the safety of HEMA-containing biomaterials. The results support that HEMA activates the Nrf2-ARE transcriptional pathway directly through its electrophilic properties. (C) 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

The present study examined the effects of di-n-butyl phthalate (DBP) on phorbol myristate acetate (PMA)-induced macrophage differentiation of THP-1 monocytes, determined by morphological classification and flow cytometry. Focusing on the expression of the surface marker CD36, the potential role of peroxisome proliferator-activated receptor gamma (PPARγ) was examined using various PPARγ agonists and antagonists. As the PPARγ ligand-binding domain contains multiple ligand-binding sites (LBS), agonist and antagonists targeting the different sites were used. DBP accelerated PMA-induced morphological changes and increased expression of CD36, although to a lesser degree than the PPARγ agonists rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). A proteomics screening revealed that DBP enhanced the expression of PPARγ-regulated proteins. During combined exposures, DBP partly attenuated the effect of rosiglitazone, an agonist binding reversibly to PPARγ's canonical LBS. In contrast, DBP increased expression of CD36 in combination with 15d-PGJ2 which binds irreversibly to the canonical LBS. Thus, DBP appears to interact with both the canonical and alternative LBS. Accordingly, the antagonist GW9662, which binds to the canonical LBS, only partly reduced the DBP-induced CD36 expression, while the dual-site antagonist SR16832 completely blocked the effects of DBP. Overall, the results show that DBP modifies PMA-induced differentiation of THP-1 cells through interaction with PPARγ.

Arbejdet på en tandklinik indebærer kontakt med en række kemiske stoffer, der kan medføre udvikling af temporære eller permanente skader hos personalet, oftest i form af allergiske reaktioner, men også som irritative, cancerogene eller organtoksiske effekter. Allergi kan optræde som straksallergi eller kontaktallergi, og tandlæger og klinikassistenter hører til de faggrupper, der hyppigst får arbejdsbetinget eksem, der også kan være irritativt betinget. De kemiske stoffer kan stamme fra tandfyldningsmaterialer, fx kviksølv fra amalgam og monomer fra resinbaserede materialer. Stofferne kan frigives ved både fremstilling, pudsning og fjernelse af fyldninger. Derudover anvendes forskellige biocider/kemiske desinfektionsmidler til både kliniske og infektionshygiejniske formål som hånd-, overflade- og henstandsdesinfektion. Endelig kan handsker indeholde allergifremkaldende latexprotein og gummikemikalier samt give anledning til irritativt eksem efter langvarig brug.

Methacrylate monomers, like 2-hydroxyethyl methacrylate (HEMA), are common components of resin based dental materials. Leakage of unpolymerized monomers after placement and curing leads to human exposure. HEMA is known to inhibit lipopolysaccharide (LPS) induced cytokine release. In this study we explore a possible role of the antioxidant glutathione (GSH) in this effect. In the RAW 264.7 murine macrophage cell line, HEMA (< 2 mM) did not induce cell death, but reduced cellular GSH levels, increased cellular ROS and decreased the IL-1 beta release from LPS-stimulated cells. Moreover, the IL-1 beta mRNA levels were reduced after 3-6 h exposure, suggesting transcriptional effects of HEMA. The GSH modulators butylsulfoximine (BSO; inhibitor of GSH synthesis) and 2-oxothiazolidine-4-carboxylate (OTC; Cysteine precursor) caused a decrease and increase in the LPS-induced IL-1 beta release, respectively, suggesting a role for GSH in negative regulation of LPS-induced IL-l beta release. However, the magnitude and dynamics of the effects of HEMA and BSO on LPS-induced IL-1 beta release and GSH depletion differed considerably. Thus, GSH depletion alone could not explain the strong attenuation of LPS-induced IL-1 beta release caused by HEMA. Formation of HEMA-protein conjugates due to the thiol reactivity of HEMA emerges as a likely candidate for the molecular mechanism accounting for this effect. 0 2017 Elsevier B.V. All rights reserved.

Tandrestaureringsmaterialer indeholder en mængde forskellige stoffer med ulige biologiske egenskaber. I mundhulen udsættes materialerne for både mekaniske, kemiske, mikrobielle og enzymatiske påvirkninger, og substanser kan frigives fra materialerne. Frigivne substanser kan medføre biologiske effekter i mundslimhinden, eller de kan absorberes og distribueres systemisk og give generelle reaktioner. I artiklen diskuteres biologiske mekanismer, som kan have betydning for frigivelse af stoffer fra polymerbaserede tandrestaureringsmaterialer, samt de biologiske konsekvenser på celleniveau. Endvidere diskuteres mekanismer for mulige kliniske konsekvenser af eksponering for frigivne stoffer med fokus på overfølsomhed, allergi og toksicitet.

2-Hydroxyethyl methacrylate (HEMA) is a methacrylate monomer used in polymer-based dental-restorative materials. In this study, the viability of human lung epithelial cells, BEAS-2B, was investigated after exposure to this monomer. Exposure to HEMA reduced the viability of the BEAS-2B cells as a result of increased apoptosis, interruption of the cell cycle, and decreased cell proliferation. Depletion of cellular glutathione and increased levels of reactive oxygen species (ROS) were seen after exposure of BEAS-2B cells to HEMA. The glutathione synthase inhibitor, L-buthioninesulfoximine (BSO), was used to study whether the reduced viability was caused by glutathione depletion and increased levels of ROS. Similarly to incubation with HEMA, incubation with BSO resulted in glutathione depletion and increased ROS levels, without increasing cell death or inhibiting cell growth. The results indicate that HEMA-induced cell damage is not caused exclusively by these mechanisms. Mechanisms other than glutathione depletion and ROS formation seem to be of importance for the toxic effect of HEMA on lung epithelial cells.

Due to incomplete curing and material degradation, cells in the oral cavity may be exposed to monomers and filler particles from dental composite fillings. The objective of the present study was to investigate if combined exposures to particles and a methacrylate monomer from composite fillings resulted in additive effects on the macrophage immune response.

Polymer-based dental restorative materials are designed to polymerize in situ. However, the conversion of methacrylate monomer to polymer is never complete, and leakage of the monomer occurs. It has been shown that these monomers are toxic in vitro; hence concerns regarding exposure of patients and dental personnel have been raised. Different monomer methacrylates are thought to cause toxicity through similar mechanisms, and the sequestration of cellular glutathione (GSH) may be a key event. In this study we examined the commonly used monomer methacrylates, 2-hydroxyethylmethacrylate (HEMA), triethylenglycol-dimethacrylate (TEGDMA), bisphenol-A-glycidyl-dimethacrylate (BisGMA), glycerol-dimethacrylate (GDMA) and methyl-methacrylate (MMA). The study aimed to establish monomers' ability to complex with GSH, and relate this to cellular toxicity endpoints. Except for BisGMA, all the monomer methacrylates decreased the GSH levels both in cells and in a cell-free system. The spontaneous formation of methacrylate-GSH adducts were observed for all methacrylate monomers except BisGMA. However, we were not able to correlate GSH depletion and toxic response measured as SDH activity and changes in cell growth pattern. Together, the current study indicates mechanisms other than GSH-binding to be involved in the toxicity of methacrylate monomers.

Methacrylate monomers have been identified in aqueous extracts of freshly cured dental fillings. The hypothesis tested presently was that low concentrations of triethyleneglycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) alone or in combination interfere with the LPS-induced release of cytokines from the macrophage cell line RAW264.7. The cells were exposed to 5-200 μM of monomers for 24 h followed by a 24 h combined exposure to monomers and LPS. TEGDMA reduced LPS-induced release of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), whereas HEMA only reduced IL-1β release. Co-exposure to the two monomers indicated an additive effect. Moreover, the reduced cytokine release persisted for 24 h after termination of the monomer exposure. The LPS-induced activation of proteins in pre-transcriptional signaling pathways (CD14, p-ERK1/2, p-p38, p-JNK, p-IκB-α and p-NFκB-p65) was not altered by monomer exposure, neither were the levels of IL-1β and TNF-α mRNA. However, the LPS-induced level of pro-IL-1β was decreased by the monomer treatment. Thus, HEMA and TEGDMA may interfere with post-transcriptional regulation of synthesis and release of these cytokines. Overall, the results suggest that low concentrations of monomers may cause impaired macrophage responses, and that these effects can persist for up to 24 h after exposure.