Objectives: Printed splints may be an alternative as a treatment of functional disorders in addition to physical, manual and physiological therapeutics. The objective is to investigate whether different 3D printed splint materials, which are fabricated with different fabrication orientation and post-processing (washing and post polymerisation) exhibit different in vitro cytotoxicity. Material and Methods: 600 discs (n = 25 per group, 5mmx1mm) were printed (P30+ DLP-printer, Straumann, CH; 100 mu m layer) from splint materials (M1: Luxaprint OrthoPlus, DMG, G; M2: V-Print Splint, Voco, G). Printing was performed under 90 degrees (A1), 45 degrees (A2) or 0 degrees (A3) alignment to the building platform. Specimens were either automatically washed (W1) (Straumann P Wash, Straumann, CH) or manually cleaned (W2) (Voco Pre-/Main Clean protocol, Voco, G), and post polymerization was performed (P1: Cure, Straumann, CH; P2: Otoflash N171, Ernst Hinrichs Dental, G). RAW264.7 mouse macrophages were exposed to extracts of the specimens and cytotoxicity was determined as cell survival using a crystal violet assay. Optical density values obtained from exposed cell cultures were normalized to untreated controls (100%), summarized as means and statistically analyzed (ANOVA, alpha=0.05). Results: Cell survival varied between 9.1+/-1.3% (alignment A2/post cure P2/material M2/wash system W2) and 58.5+/-5.9% (alignment A1/post cure P1/material M1/wash system W1). Univariate analysis of variance revealed significant differences between mean values for post cure (p = 0.000), wash system (p = 0.002) and materials (p = 0.000), but not for the alignment (p = 0.406). With standardised washing and adapted post cure, both tested materials provided lowest cytotoxicity even in all three printing alignments. Conclusions: The selection of the material as well as the post-processing (post-polymerization, washing procedure) show influence on the in vitro cytotoxicity. Alignment during manufacturing does not affect toxicity. Clinical Relevance: Materials, washing and post-polymerization should be matched to reduce cytotoxic effects during additive manufacturing.

One major goal of biobanks is to provide the best possible biospecimen quality for research use. This can be achieved, notably in accredited structures, by using standardized procedures for collection, processing, and storage of biosamples and associated data. Since tissue samples of a clinical biobank are commonly collected at surgical theaters in satellite locations or hospitals in remote areas, adequate temporary storage of the biosample is mandatory to maintain optimal sample quality. In cases where immediate snap freezing of the collected material is possible, interim storage of the samples in portable dewars filled with liquid nitrogen (LN2) is a widely used method. Therefore, the ideal dewar size and maximum storage time need to be considered to maintain an optimal biospecimen quality. In addition, the nature of the cryotube material is an important aspect for keeping the biosample safe while storing it in LN2. The objective of this study was to test different dewar vessels with respect to LN2 volume and consumption and to analyze the impact of LN2 contact on cryotube material through scanning electron microscopy.

OBJECTIVE:Dental pulp cells interact with immunogenic components such as LPS (lipopolysaccharide) or LTA (lipoteichoic acid) released from microorganisms in carious lesions. In the present investigation, the formation of the pro-inflammatory cytokines TNFα and IL-6 in LPS- or LTA-stimulated cells from the dental pulp interface and pulp fibroblasts was analyzed in the presence of the resin monomer 2-hydroxyethyl methacrylate (HEMA) under varying cellular redox conditions. METHOD:Human pulp fibroblasts (HPC) or cells from the dental pulp interface expressing an odontoblast phenotype (hOD-1) were exposed to LTA, LPS or HEMA for 1 h or 24 h. Redox homeostasis was modified by the prooxidant BSO (L-buthionine sulfoximine) or the antioxidant NAC (N-acetyl cysteine). Formation of TNFα or IL-6 was analyzed by ELISA, and cell survival was determined by a crystal violet assay. Statistical analyses were performed using the Mann-Whitney-U-test. RESULTS:Secretion of TNFα was not detected in LPS- or LTA-stimulated HPC or hOD-1, and IL-6 was not found after a short exposure (1 h). After a 24 h exposure, LPS induced a 3-fold increase in IL-6 formation in HPC, while LTA stimulated IL-6 release about 20-fold. Likewise, LTA was more effective than LPS in hOD-1 stimulating IL-6 levels about 50-fold. HEMA inhibited the LPS- and LTA-induced IL-6 release, and this effect was enhanced by BSO but counteracted by NAC in both cell types. IL-6 release was independent of cell survival rates. CONCLUSIONS:The protective immune response in odontoblasts and pulp fibroblasts is impaired by monomers such as HEMA through the disturbance of the redox homeostasis.

Odontogenic MSCs are vulnerable to LPS-triggered bacterial infections, and they respond by secreting inflammatory mediators, such as IL-6, and with mineralization. Since both processes might be prone to a disturbance of the redox homeostasis, the oxidative stress influence on vital functions of human dental pulp cells (HPCs) was investigated. With these aims, a model of LPS-stimulated primary HPCs was established, and anti- and pro-oxidant substances were administered up to 21 days to measure inflammation and mineralization parameters. LPS-stimulated HPCs retained mineralization potential, which was decreased with the antioxidants NAC and fisetin and the pro-oxidant BSO. The expression of surface markers related to odontogenic commitment was influenced accordingly but counteracted by the enhanced expression of BMP2 and ALP at the transcriptional level. LPS triggers an early IL-6 production in non-odontogenic conditions, while it can be measured only after 15 days in the presence of the differentiation medium. The present study shows that HPCs functions causally depend on a tightly regulated cellular redox balance. Our data demonstrate a redox control of pulp MSC odontogenic commitment along with a potential association between an IL-6 late secretion and mineralization. These findings lay the groundwork for investigations on the molecular role of IL-6 in dental hard tissue metabolism.

Human dental pulp stem cell (DPSC) differentiation toward the osteoblastic phenotype is enhanced when culture media are supplemented with differentiating factors, i.e. ascorbic acid, β-glycerophosphate and dexamethasone. Liposomes, spherical vesicles formed by a phospholipid bilayer, are frequently used as carriers for drugs, growth factors and hydrophobic molecules. The aim of this work was to speed up DPSC commitment to the osteogenic lineage by embedding differentiating factors within liposomes. Firstly, liposomes were prepared by rehydrating a phospholipidic thin film and characterised in terms of dimensions. Secondly, liposome-exposed DPSCs were characterised by their immunophenotypic profile. Levels of CD90 were significantly decreased in the presence of liposomes filled with ascorbic acid, β-glycerophosphate and dexamethasone (Lipo-Mix) with respect to normal differentiation medium (DM), while CD73 and CD29 expression were enhanced, suggesting osteogenic commitment. Additionally, an appreciable extracellular matrix deposition is detected. Thirdly, the Lipo-Mix formulation better increases alkaline phosphatase activity and levels of Collagen I secretion with respect to DM. In parallel, the new liposome formulation is capable of decreasing the release of H2O2 and of triggering a precocious antioxidant cell response, redressing the redox balance required upon mesenchymal stem cell commitment to osteogenesis. It can be therefore hypothesised that Lipo-Mix could represent a suitable tool for clinical regenerative purposes in the field of tissue engineering by speeding up DPSC osteogenic commitment, mineralised matrix deposition and remodelling.

Low concentrations of carbon monoxide (CO) were reported to exhibit anti-inflammatory effects when administered in cells by suitable chemotypes such as CO releasing molecules (CO-RMs). In addition, the pH-modulating abilities of specific carbonic anhydrase isoforms played a crucial role in different models of inflammation and neuropathic pain. Herein, we report a series of chemical hybrids consisting of a Carbonic Anhydrase (CA) inhibitor linked to a CO-RM tail (CAI/CO-RMs). All compounds and their precursors were first tested in vitro for their inhibition activity against the human CA I, II, IX, and XII isoforms as well their CO releasing properties, aiming at corroborating the data by means of molecular modelling techniques. Then, their impact on metabolic activity modulation of RAW 264.7 mouse macrophages for 24 and 48 h was assessed with or without lipopolysaccharide (LPS) stimulation. The compounds were shown to counteract the inflammatory stimulus as also indicated by the reduced tumor necrosis factor alpha (TNF-α) release after treatment. All the biological results were compared to those of N-acetylcysteine (NAC) as a reference antioxidant compound. Within the series, two CAI/CO-RM hybrids (1 and 2), bearing both the well-known scaffold able to inhibit CAs (acesulfame) and the cobalt-based CO releasing portion, induced a higher anti-inflammatory effect up to 48 h at concentrations lower than NAC.

Inducible nitric oxide synthase (iNOS) is a crucial enzyme involved in monocyte cell response towards inflammation, and it is responsible for the production of sustained amounts of nitric oxide. This free radical molecule is involved in the defense against pathogens; nevertheless, its continuous and dysregulated production contributes to the development of several pathological conditions, including inflammatory and autoimmune diseases. In the present study, we investigated the effects of two new iNOS inhibitors, i.e., 4-(ethanimidoylamino)-N-(4-fluorophenyl)benzamide hydrobromide (FAB1020) and N-{3-[(ethanimidoylamino)methyl]benzyl}-l-prolinamidedihydrochloride (CM554), on human LPS-stimulated monocytes, using the 1400 W compound as a comparison. Our results show that CM544 and FAB1020 are selective and decrease cytotoxicity, IL-6 secretion and LPS-stimulated monocyte migration. Furthermore, the modulation of iNOS, nitrotyrosine and Nrf2 were analyzed at the protein level. Based on the collected preliminary results, the promising therapeutic value of the investigated compounds emerges, as they appear able to modulate the pro-inflammatory LPS-stimulated response in the low micromolar range in human monocytes.

The effect of Actovegin® was investigated on PMA- and LPS-induced human peripheral blood mononuclear cells (PBMCs). PBMCs (1 × 106 cells/ml) from five blood donors (2 f, 3 m; 45–55 years) were grown in medium and exposed to Actovegin® in the presence or absence of PMA or LPS. Supernatants were collected to assess the concentration of cytokines (TNF-α, IL-1beta, IL-6 and IL-10). The reactive oxygen species (ROS) were assessed by a ROS-GloTM H2O2 assay. Stimulation of cells by PMA or LPS (without Actovegin®) significantly increased the secretion of IL-1beta, IL-6, IL-10 and TNF-α from PBMCs, compared to controls. Pre-treatment of cells with Actovegin® (1, 5, 25, 125 µg/ml) plus PMA significantly decreased the secretion of IL-1beta from PBMCs, compared to controls (PMA without Actovegin®). In contrast, addition of Actovegin® (1, 5, 25, 125 and 250 µg/ml) plus LPS did not alter the IL-1beta production, compared to controls (LPS without Actovegin®). TNF-α, IL-6 and IL-10 do not contribute to the reduction of inflammatory reactions with Actovegin®. Actovegin® can reduce the PMA-induced IL-1beta release and the ROS production from PBMCs. These findings may help to explain the clinically known positive effects of Actovegin® on athletic injuries with inflammatory responses (e.g., muscle injuries, tendinopathies).

Background: Dental odontoblasts produce dentin mineralized matrix, trigger immune responses and act as sensory cells. The understanding of the mechanisms of these functions has been particularly restricted due to the lack of odontoblasts being cultivable in vitro. Because of the lack of specific markers to identify cells of the odontoblastic lineage, properties of the cells isolated from the dentin-pulp interface were compared to dental pulp cells, periodontal ligament cells, osteoblasts, skin fibroblasts, epithelial cells (A549) and HeLa in the present study. Methods: After surgical procedures, the pulp tissue was removed from the tooth crown, and cells were scrapped off the dentin-pulp interface. Explants from teeth of three patients were routinely cultivated, and cells were harvested after several weeks. Cell morphology and ultrastructure was studied by light microscopy (LM), scanning (SEM) or transmission electron microscopy (TEM). Expression of dentin sialophosphoprotein (DSPP), dentin matrix protein I (DMP1), TRPV4, and 5100 calcium binding protein A4 (5100A4) were analyzed at the protein level by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using specific antibodies. The differential expression of S100A4 in the various cell lines was further investigated at the gene level by semiquantitative real-time PCR. Mineralization in the various cell types was observed after alizarin red staining after a 28 days incubation period. The immunophenotype of the cells was examined by flow cytometry using monoclonal anti-human antibodies CD9O-FITC, CD73-PE, CD105-PE, CD29-PE, CD140a-FITC, CD144-PE, CD45-FITC or CD34-FITC. Differences between median values were statistically analyzed (Mann-Whitney U-test). Results: Cells from the dentin-pulp interface retain the polarity of odontoblast morphology in culture with an elongated, rounded cell body, and an extended cellular process. Ultrastructural analysis of the cells indicates high secretory activity including the extracellular deposition of fibrillar collagen. An extended rough endoplasmic reticulum is lined by a large number of ribosomes, and a vast number of secretory granules merges with the cell membrane. Protein expression of DSPP, DMP1, and TRPV4 as a transient receptor potential cation was detected in all cell lines. S100A4 was found differentially expressed in cultures of cells from tooth tissues. High expression of S100A4 was observed at the protein and gene level in two fractions of cells isolated from the dentin-pulp interface, but was absent or only weakly expressed in pulp cells. S100A4 expression in cells from the dentin-pulp interface and pulp cells is consistent with the intensity of the formation of mineralized nodules detected by alizarin red staining. Immunophenotyping revealed that a high percentage of CD73 (ecto-5-nucleotidase), an enzyme active on the surface of immune-competent cells, was expressed in cells of the dentin-pulp interface. While 72%-78% of positive cells were detected in dentin-pulp interface fractions, only 28-64% of the cells in pulp cell cultures were stained. Conclusions: The present findings obtained with a variety of cells of different origin provide experimental evidence that cells isolated from the dentin-pulp interface express unique properties different from dental pulp cells in particular. The differential expression of S100A4 is a relevant marker candidate for differentiating between dental pulp cells and cells of the odontoblast lineage. (C) 2020 Elsevier GmbH. All rights reserved.

Objective: The release of inflammatory cytokines from antigen-stimulated cells of the Resin monomer immune system is inhibited by resin monomers such as 2-hydroxyethyl methacrylate HEMA (HEMA). Although the formation of oxidative stress in cells exposed to HEMA is firmly estab- Oxidative stress lished, the mechanism behind the inhibited cytokine secretion is only partly known. The Cytokine present investigation presents evidence regarding the role of HEMA-induced oxidative stress NF-KB in the secretion of the pro-inflammatory cytokine TNFoL from cells exposed to the antigens Nrf2 LTA (lipoteichoic acid) or LPS (lipopolysaccharide) of cariogenic microorganisms using BSO (L-buthionine sulfoximine) or NAC (N-acetyl cysteine) to inhibit or stabilize the amounts of the antioxidant glutathione. Method: RAW264.7 mouse macrophages were treated with LTA, LPS or HEMA in the presence of BSO or NAC for 1h or 24h. Secretion of TNFoL from cell cultures was analyzed by ELISA, and the formation of reactive oxygen (ROS) or nitrogen species (RNS) was determined by flow cytometry. Protein expression was detected by Western blotting. Results: The release of TNF alpha in both LTA- and LPS-exposed cells was decreased by HEMA, and this concentration-dependent inhibitory effect was amplified by BSO or NAC. LTA- and LPS-stimulated expression of the redox-sensitive transcription factor NF-alpha B (p65) in cell nuclei decreased in the presence of HEMA because the translocation of p65 from the cytosol was prevented by oxidative stress specifically increased by the monomer. Conclusions: A disturbance of the cellular redox balance, particularly induced by HEMA, is a crucial factor in the inhibition of LTA- and LPS-stimulated signalling pathways leading to TNF alpha secretion. (C) 2020 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

PURPOSE The effects of four different self-adhesive resin cement materials on cell viability and apoptosis after direct and indirect exposure were evaluated using different cell culture techniques. MATERIALS AND METHODS Self-adhesive cements were applied to NIH/3T3 mouse fibroblasts by the extract test method, cell culture inserts, and dentin barrier test method. After exposure periods of 24 h and 72 h, the cytotoxicity of these self-adhesive materials was evaluated using the MTT assay (viability) and the Annexin-V-FITC/PI staining (apoptosis). RESULTS The lowest cell viability was found in cells exposed to BeautiCem SA for 24 h in the extract test method. Cell viability was reduced to 70.6% compared to negative controls. After the 72 h exposure period, viability rate of cell cultures exposed to BeautiCem SA decreased more than 2- fold (29.5%) while cells exposed to RelyX U200 showed the highest viability rate of 71.4%. In the dentin barrier test method, BeautiCem SA induced the highest number of cells in apoptosis after a 24 h exposure (4.1%). Panavia SA Cement Plus was the material that caused the lowest number of cells in apoptosis (1.5%). CONCLUSION The used self-adhesive cements have showed different cytotoxic effects based on the evaluation method. As exposure time increased, the materials showed more cytotoxic and apoptotic effects. BeautiCem SA caused significantly more severe cytotoxic and apoptotic effects than other cements tested. Moreover, cements other than BeautiCem SA have caused necrotic cell death rather than apoptotic cell death.

Human dentin is not only a composite material of a collagenous matrix and mineral to provide strength and elasticity to teeth, but also a precious reservoir full of bioactive proteins. They are released after demineralization caused by bacterial acids in carious lesions, by decalcifying irrigants or dental materials and they modulate tissue responses in the underlying dental pulp. This work describes a first-time analysis of the proteome of human dentin using a shotgun proteomic approach that combines three different protein fractionation methods. Dentin matrix proteins were extracted by EDTA and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), OFFGEL isoelectric focusing (IEF) or strong cation exchange chromatography (SCX). Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 813 human proteins with high confidence, however, isoelectric focusing turned out to be the most beneficial prefractionation method. All Proteins were categorized based on the PANTHER system and representation analysis revealed 31 classes and subclasses to be overrepresented. The acquired knowledge provides a comprehensive insight into the number of proteins in human dentin as well as their physiological and pathological functions. Thus, the data presented paves the way to the analysis of specific functions of dentin matrix proteins in vivo and their potential in tissue engineering approaches to regenerate dental pulp.

Composites and porous scaffolds produced with biodegradable natural polymers are very promising constructs which show high biocompatibility and suitable mechanical properties, with the possibility to be functionalized with growth factors involved in bone formation. For this purpose, alginate/hydroxyapatite (Alg/HAp) composite scaffolds using a novel production design were successfully developed and tested for their biocompatibility and osteoconductive properties in vitro. Redox homeostasis is crucial for dental pulp stem cell (DPSC) differentiation and mineralized matrix deposition, and interleukin-6 (IL-6) was found to be involved not only in immunomodulation but also in cell proliferation and differentiation. In the present study, we evaluated molecular pathways underlying the intracellular balance between redox homeostasis and extracellular matrix mineralization of DPSCs in the presence of composite scaffolds made of alginate and nano-hydroxyapatite (Alg/HAp). Prostaglandin-2 (PGE2) and IL-6 secretion was monitored by ELISA assays, and protein expression levels were quantified by Western blotting. This work aims to demonstrate a relationship between DPSC capacity to secrete a mineralized matrix in the presence of Alg/HAp scaffolds and their immunomodulatory properties. The variation of the molecular axis Nrf2 (nuclear factor erythroid 2-related factor 2)/PGE2/IL-6 suggests a tight intracellular balance between oxidative stress responses and DPSC differentiation in the presence of Alg/HAp scaffolds.

Tissue engineering is widely recognized as a promising approach for bone repair and reconstruction. Several attempts have been made to achieve materials that must be compatible, osteoconductive, and osteointegrative and have mechanical strength to provide a structural support. Composite scaffolds consisting in biodegradable natural polymers are very promising constructs. Hydroxyapatite (HAp) can support alginate as inorganic reinforcement and osteoconductive component of alginate/HAp composite scaffolds. Therefore, HAp-strengthened polymer biocomposites offer a solid system to engineer synthetic bone substitutes. In the present work, HAp was incorporated into an alginate solution and internal gelling was induced by addition of slowly acid-hydrolyzing D-gluconic acid delta-lactone for the direct release of calcium ions from HAp. It has been previously demonstrated that alginate-based composites efficiently support adhesion of cancer bone cell lines. Human dental pulp stem cells (DPSCs) identified in human dental pulp are clonogenic cells capable of differentiating in multiple lineage. Thus, this study is aimed at verifying the mineralization and differentiation potential of human DPSCs seeded onto scaffolds based on alginate and nano-hydroxyapatite. For this purpose, gene expression profile of early and late mineralization-related markers, extracellular matrix components, viability parameters, and oxidative stress occurrence were evaluated and analyzed. In summary, our data show that DPSCs express osteogenic differentiation-related markers and promote calcium deposition and biomineralization when growing onto Alg/HAp scaffolds. These findings confirm the use of Alg/HAp scaffolds as feasible composite materials in tissue engineering, being capable of promoting a specific and successful tissue regeneration as well as mineralized matrix deposition and sustaining natural bone regeneration.

Objective. Resin monomers like 2-hydroxyethyl methacrylate (HEMA) interfere with effects induced by stressors such as lipopolysaccharide (LPS) released from cariogenic microorganisms. In this study, mechanisms underlying monomer-induced inhibition of the LPS stimulated secretion of inflammatory cytokines from immunocompetent cells were investigated. Methods. Secretion of pro-inflammatory cytokines TNF-alpha, IL-6 and the anti-inflammatory IL-10 from RAW264.7 mouse macrophages exposed to LPS and HEMA (0-6-8 mM) was determined by ELISA. The formation of reactive oxygen (ROS) and nitrogen species (RNS) was determined by flow cytometry (FACS) after staining of cells with specific fluorescent dyes. Cell viability was analyzed by FACS, and protein expression was detected by Western blotting. Results. Secretion of TNF-alpha, IL-6 and IL-10 from LPS-stimulated cells increased after a 24 h exposure. A HEMA-induced decrease in cytokine secretion resulted from the inhibition of LPS-stimulated NF-kappa B activation. Nuclear translocation of NF-kappa B was inhibited possibly as a result of enhanced levels of hydrogen peroxide (H2O2) and nitric oxide (NO center dot) in HEMA-exposed cells. Oxidative stress caused by HEMA-induced formation of H2O2 and LPS-stimulated peroxynitrite (ONOO-) also enhanced nuclear expression of Nrf2 as the major regulator of redox homeostasis, as well as Nrf2-controlled stress protein HO-1 to inhibit NF-kappa B activity. HEMA inhibited the LPS-stimulated expression of NOS (nitric oxide synthase) to produce NO center dot but counteracted the expression of Nox2, which forms superoxide anions that combine with NO center dot to peroxynitrite. Conclusions. Resin monomers like HEMA inhibit LPS-stimulated NF-kappa B activation essential for cytokine release as a crucial response of immunocompetent cells of the dental pulp to invading cariogenic pathogens. (C) 2018 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

Objective. Resin monomers released from unpolymerized dental adhesives or composites and bacterial products like lipopolysaccharide (LPS) or lipoteichoic (LTA) are simultaneously present in specific applications following treatment of deep caries lesions. This review is focused on evidence concerning cell responses as a result of the interactions between adaptive mechanisms activated by resin monomers and signaling pathways of the immune response triggered by LPS or LTA originating from cariogenic microorganisms.Methods. Current understanding of dental caries progression and pathways in eukaryotic cells in response to LPS stimulation in a clinical situation as well as cell reactions to oxidative stress caused by resin monomers is analyzed based on publications available through online databases.Results. LPS and LTA activate the redox-sensitive transcription factor NF-kappa B as a major regulator in immunocompetent dental pulp cells. Cell reactions to LPS/LTA associated with oxidative stress are downregulated by the redox-sensitive transcription factor Nrf2. Thus, activation of Nrf2 through resin monomer-induced oxidative stress due to the increased formation of reactive oxygen species (ROS) could be a molecular mechanism underlying the inhibition of LPS-stimulated responses such as the release of pro- or anti-inflammatory cytokines. Likewise, crosslinking of NF-kappa B and Nrf2-regulated biocompatibility pathways regulates cell death induced by the interaction of LPS and resin monomers.Significance. A multidimensional scenario through independent but linked NF-kappa B- and Nrf2-regulated pathways is activated in the clinical situation of caries treatment. Unfavorable or beneficial consequences strictly depend on a wide range of combinations and concentrations of bacterial products and resin monomers. (C) 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Objective. Oxidative stress induced by compounds of dental composites like 2-hydroxyethyl methacrylate (HEMA) due to excess formation of reactive oxygen species (ROS) disturbs vital cell functions leading to apoptosis. The sources of ROS in cells exposed to resin monomers are unknown. The present study investigates functions of flavin-containing ROS and RNS (reactive nitrogen species) producing enzymes in cells exposed to HEMA.Methods. The formation of oxidative stress in RAW264.7 mouse macrophages exposed to HEMA (0-6-8 mM) was determined by flow cytometry (FRCS) after staining of cells with 2'7'-dichlorodihydrofluorescin diacetate (H2DCF-DA), dihydroethidium (DHE) or dihydrorhodamine 123 (DHR123). Cells in apoptosis or necrosis were identified by annexin-V-FITC/propidium iodide labeling followed by FACS analysis. Expression of ROS/RNS producing enzymes was analyzed by Western blotting.Results. DCF fluorescence increased in cells exposed to HEMA for 1 h suggesting the production of hydroxyl radicals, H2O2, or nitric oxide and superoxide anions which form peroxynitrite (ONOO-). Increased DHR123 fluorescence after 24 h indicated the formation of mostly H2O2. The induction of apoptosis in the presence of HEMA was decreased by low concentrations of diphenylene iodonium (DPI), an inhibitor of flavin-containing enzymes. Expression of p47(Phox), a regulatory subunit of the superoxide producing Nox2, was downregulated, and the expression of NOS which produces nitric oxide (NO) was possibly inhibited by feedback loop mechanisms in HEMA-exposed cultures. Inhibition of HEMA-induced apoptosis by VAS2870 or apocynin further suggested a crucial function of Nox2.Significance. The present findings show the physiological relevance of flavin-containing enzymes in monomer-induced oxidative stress and apoptosis. (C) 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.