Stephanie Krifka

A review of adaptive mechanisms in cell responses towards oxidative stress caused by dental resin monomers

Dental composite resins are biomaterials commonly used to aesthetically restore the structure and function of teeth impaired by caries, erosion, or fracture. Residual monomers released from resin restorations as a result of incomplete polymerization processes interact with living oral tissues. Monomers like triethylene glycol dimethacrylate (TEGDMA) or 2-hydroxylethyl methacrylate (HEMA) are cytotoxic via apoptosis, induce genotoxic effects, and delay the cell cycle. Monomers also influence the response of cells of the innate immune system, inhibit specific odontoblast cell functions, or delay the odontogenic differentiation and mineralization processes in pulp-derived cells including stem cells. These observations indicate that resin monomers act as environmental stressors which inevitably disturb regulatory cellular networks through interference with signal transduction pathways. We hypothesize that an understanding of the cellular mechanisms underlying these phenomena will provide a better estimation of the consequences associated with dental therapy using composite materials, and lead to innovative therapeutic strategies and improved materials being used at tissue interfaces within the oral cavity. Current findings strongly suggest that monomers enhance the formation of reactive oxygen species (ROS), which is most likely the cause of biological reactions activated by dental composites and resin monomers. The aim of the present review manuscript is to discuss adaptive cell responses to oxidative stress caused by monomers. The particular significance of a tightly controlled network of non-enzymatic as well as enzymatic antioxidants for the regulation of cellular redox homeostasis and antioxidant defense in monomer-exposed cells will be addressed. The expression of ROS-metabolizing antioxidant enzymes like superoxide dismutase (SOD1), glutathione peroxidase (GPx1/2), and catalase in cells exposed to monomers will be discussed with particular emphasis on the role of glutathione (GSH), which is the major non-enzymatic antioxidant. The causal relationship between vital cell functions like the regulation of cell survival or cell death in monomer-treated cell cultures and the availability of GSH will be highlighted. We will also consider the influence of monomer-induced oxidative stress on central signal transduction pathways including mitogen-activated protein kinases (MAPK) ERK1/2, p38, and JNK as well as the stress-activated transcription factors downstream Elk-1, ATF-2, ATF-3, and cJun. Finally, we address signaling pathways originating from monomer-induced DNA damage including the activation of ATM (ataxia-telangiectasia mutated), Chk2, p53, p21, and H2AX. The understanding of the mechanisms underlying adaptive cell responses will stimulate a constructive debate on the development of smart dental restorative materials which come into contact with oral tissues and effective strategies in dental therapy.

The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA)

Resin monomers like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation, or induce cell death via apoptosis. These phenomena are associated with oxidative stress and a reduction in the concentration of the antioxidant glutathione (GSH), resulting in imbalanced redox homeostasis. Thus far, the precise mechanism of how resin monomers interfere with cellular redox regulation is unknown. The present study provides insight into the induction of apoptosis and the differential expression of antioxidant enzymes depending on the availability of GSH. Buthionine sulfoximine (BSO) was used to inhibit GSH synthesis, while 2-oxothiazolidine-4-carboxylate (OTC), and N-acetylcysteine (NAC) as prodrugs supported GSH synthesis in RAW264.7 mouse macrophages exposed to HEMA (0-8 mm) for 24 h. The level of GSH was significantly decreased after cells were preincubated with BSO, and the formation of reactive oxygen species (ROS) increased in cultures subsequently exposed to HEMA. Apoptosis was drastically increased by BSO in HEMA-exposed cell cultures as well, but OTC and NAC retracted HEMA-induced cell death. These results show that dental monomer-induced apoptosis is causally related to the availability of GSH. The hydrogen peroxide decomposing enzymes glutathione peroxidase (GPx1/2) and catalase were differentially regulated in HEMA-exposed cultures. Expression of GPx1/2 was inhibited by HEMA and further reduced in the presence of BSO. SOD1 (superoxide dismutase) expression was inhibited in the presence of HEMA, and was decreased to an even greater extent by BSO, possibly due to H(2)O(2)-feedback inhibition. The expression of catalase was considerably up-regulated in HEMA-exposed cultures, implying that H(2)O(2) is the type of ROS that is significantly increased in monomer-exposed cells. OTC and NAC counteracted the effect of HEMA on GPx1/2, SOD1, and catalase expression. HO-1 (heme oxygenase) expression was strongly enhanced by HEMA, suggesting the need for further antioxidants like bilirubin to support enzyme activities that directly regulate H(2)O(2) equilibrium. Expression of the oxidoreductase thioredoxin (TRX1), the second major thiol-dependent antioxidant system in eukaryotic cells, was slightly reduced, while the oxygen-sensing protein HIF-1α was downregulated in HEMA-exposed cell cultures. These results indicate that cells and tissues actively respond to monomer-induced oxidative stress by the differential expression of enzymatic antioxidants.

Oxidative stress and cytotoxicity generated by dental composites in human pulp cells

Dental composites are a source of residual monomers that are released into the oral environment. Since monomers act on cultured cells through reactive oxygen species (ROS), we hypothesized that composites generate ROS associated with cytotoxicity. Human pulp-derived cells were exposed to extracts of methacrylate-based materials including triethylene glycol dimethacrylate and 2-hydroxyethyl methacrylate-free composites (Tetric Ceram, Tetric EvoCeram, els, els flow, Solitaire 2) and a silorane-based composite (Hermes III). The materials were polymerized in the presence and absence of a polyester film and then extracted in culture medium. The generation of ROS was measured by flow cytometry, and cytotoxicity was determined as well. Methacrylate-based composites reduced cell survival but varied in efficiency. Undiluted extracts of Solitaire 2 specimens prepared in the absence of a polyester film reduced cell survival to 26% compared with untreated cultures. Cytotoxicity was reduced when specimens were covered with a polyester film during preparation. Cytotoxicity of the composites was ranked as follows: Solitaire 2 >> els flow > Tetric Ceram = Tetric EvoCeram = els > Hermes III. The generation of ROS followed the same pattern as detected with cytotoxic effects. A positive correlation was found between ROS production and cell survival caused by extracts made from materials not covered with a polyester film. These findings suggest that components released from composites affect cellular signaling networks through ROS formation. Regenerative and reparative capacities of the dentine–pulp complex may be impaired by biologically active resin monomers released from composite restorations.

Resin monomer-induced differential activation of MAP kinases and apoptosis in mouse macrophages and human pulp cells

Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer which is released from polymerized dental composite materials. It induced apoptosis in various target cells or inhibition of LPS-induced cytokine production in cells of the immune system after prolonged exposure. In these tissues, mitogen-activated protein kinases (MAPK) regulate signal transduction pathways that support cell survival and cytokine synthesis. The time-dependent regulation of MAPK as well as their linkage to the induction of apoptosis and cytokine release under the influence of resin monomers is unknown. It was the aim of the present study to investigate the kinetics of the up- or down-regulation of the MAPK p38, JNK, and ERK1/2, the induction of apoptosis and cytokine release in RAW264.7 mouse macrophages and human pulp-derived cells. ERK1/2, p38 and JNK were differentially activated by phosphorylation in the presence of lipopolysaccharide (0.1 microg/ml; LPS), a known inducer of MAPK activity, and TEGDMA (3 mM) as detected by Western blotting. In macrophages, ERK1/2 was activated about 6-fold by LPS, while no activation was observed in the presence of TEGDMA after 15 and 30 min. A slight activation of p38 was detected in cell cultures after short exposure to TEGDMA (30 min), but activated JNK was identified after LPS stimulation only. After a long 24 h exposure period, ERK1/2 and p38 were strongly activated by LPS, a combination of LPS/TEGDMA, and TEGDMA alone (15-20-fold). In human pulp-derived cells, ERK1/2 was phosphorylated after exposure to TEGDMA up to 2 h, and sustained activation of ERK1/2 as well as p38 (12-15-fold) was detected after prolonged exposure for 24 h. The LPS-induced, time-related increase in the secretion of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) as well as the anti-inflammatory IL-10 was instantaneously inhibited by TEGDMA in mouse macrophages. In parallel, the percentage of cells in macrophage cultures in the stage of apoptosis and necrosis increased with exposure period. Yet, in contrast to the inhibition of cytokine release, apoptosis and necrosis caused by LPS and TEGDMA was a late response in both mouse macrophages and human pulp-derived cells. From these data it appears as if MAPK activation, inhibition of cytokine release and the induction of apoptosis and necrosis by TEGDMA are tightly related. The direct causal correlation of these phenomena, however, requires further investigation.

Partial ceramic crowns: influence of ceramic thickness, preparation design and luting material on fracture resistance and marginal integrity in vitro.

This in vitro study tested the effects of two different ceramic thicknesses, two preparation designs and two different luting agents on the marginal integrity and fracture resistance of partial ceramic crowns (PCC). Eighty extracted human molars were prepared according to the following preparation designs: a) Coverage of functional cusps/butt joint (n=40), b) Horizontal reduction of functional cusps (n=40). PCC (Vita Mark II, Cerec3 System) were fabricated and the ceramic thickness of the functional cusps was adjusted to (1): 0.5-1.0 mm and (2): 1.5-2.0 mm. PCC were adhesively luted to the cavities with either Excite/VariolinkII (VL) or RelyX Unicem (RX). The specimens were exposed to thermocycling and central mechanical loading (5000 x 5 degrees C-55 degrees C; 30 second/cycle; 50,0000 x 72.5N, 1.6Hz). Marginal integrity was assessed by evaluating dye penetration (fuchsine) on multiple sections in the bucco-oral direction by relating the actual penetration distance to the maximal length of the corresponding cavity wall (100%). Restoration/luting agent (RL)--and tooth/luting agent (TL) interfaces were evaluated separately. The data were statistically analyzed with the Mann Whitney U-test and the Error Rates Method (ERM), and the fracture rates were analyzed with the chi2-test. Dye penetration data indicated that ceramic thickness and luting agent had a statistically significant influence upon marginal integrity in general, irrespective of all other parameters (ERM): RX showed significantly lower microleakage along the RL interface than VL. VL revealed significantly lower microleakage at the TL interface than RX. Fifteen PCC of group 1 (0.5-1.0 mm) and two PCC of group 2 (1.5-2.0 mm) were fractured after thermocycling and central mechanical loading, with the difference being statistically significant. PCC fabricated from industrially sintered feldspathic ceramic should have at least a thickness of 1.5-2.0 mm in stress-bearing areas.

2-Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway.

Resin monomers of dental composites like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation of dental pulp-derived cells, or induce cell death via apoptosis. The induction of apoptosis is related to the availability of the antioxidant glutathione, although a detailed understanding of the signaling pathways is still unknown. The present study provides insight into the causal relationship between oxidative stress, oxidative DNA damage, and the specific signaling pathway leading to HEMA-induced apoptosis in RAW264.7 mouse macrophages. The differential expression of the antioxidative enzymes superoxide dismutase, glutathione peroxidase, and catalase in HEMA-exposed cells indicated oxidative stress, which was associated with the cleavage of pro-caspase 3 as a critical apoptosis executioner. A 2-fold increase in the amount of mitochondrial superoxide anions after a 24 h exposure to HEMA (6-8 mM) was paralleled by a considerable decrease in the mitochondrial membrane potential (MMP). Additionally, expression of proteins critical for the signaling of apoptosis through the intrinsic mitochondrial pathway was detected. Transcription-dependent and transcription-independent mechanisms of p53-regulated apoptosis were activated, and p53 was translocated from the cytosol to mitochondria. HEMA-induced transcriptional activity of p53 was indicated by increased levels of PUMA localized to mitochondria as a potent inducer of apoptosis. The expression of Bcl-xL and Bax suggested that cells responded to stress caused by HEMA via the activation of a complicated and antagonistic machinery of pro- and anti-apoptotic Bcl-2 family members. A HEMA-induced and oxidative stress-sensitive delay of the cell cycle, indicating a DNA damage response, occurred independent of the influence of KU55399, a potent inhibitor of ATM (ataxia-telangiectasia mutated) activity. However, ATM, a protein kinase which responds to DNA double-strand breaks, and the signaling pathway downstream were activated in HEMA-exposed cells. Likewise, expression and phosphorylation of the ATM targets H2AX and p53 was reduced in the presence of KU55399. Moreover, the percentage of cells undergoing apoptosis drastically decreased in HEMA-exposed cell cultures pre-treated with KU55933. These findings demonstrate that HEMA-induced apoptosis is mediated through the intrinsic mitochondrial pathway as a consequence of p53 activation via ATM signaling upon oxidative DNA damage.

Activation of stress-regulated transcription factors by triethylene glycol dimethacrylate monomer

Triethylene glycol dimethacrylate (TEGDMA) is a resin monomer available for short exposure scenarios of oral tissues due to incomplete polymerization processes of dental composite materials. The generation of reactive oxygen species (ROS) in the presence of resin monomers is discussed as a common mechanism underlying cellular reactions as diverse as disturbed responses of the innate immune system, inhibition of dentin mineralization processes, genotoxicity and a delayed cell cycle. Yet, the signaling pathway through a network of proteins that finally initiates the execution of monomer-induced specific cell responses is unknown so far. The aim of the present study was to extend the knowledge of molecular mechanisms of monomer-induced cell death as a basis for reasonable therapy strategies. Thus, the monomer-induced expression and phosphorylation of stress-related transcription factors was analyzed in various cell lines. The time-related induction of apoptosis was investigated as well. The expression of p53 increased in HeLa cell cultures treated with camptothecin (positive control) for 24h, and the formation of p53Ser15 and p53Ser46 was detected in cell nuclei by Western blotting. TEGDMA (3 mm) appeared to stimulate p53 expression only slightly, but increased p21 expression was found in cell nuclei and cytoplasm. Both camptothecin and TEGDMA increased p53 expression to some extent in the nuclear fraction in human transformed pulp-derived cells (tHPC), and similar effects were detected in RAW264.7 macrophages. No clear induction of c-Jun and phospho-c-Jun by TEGDMA was detected in HeLa cell nuclei, and the expression of ATF-2 and phospho-ATF-2 was inhibited in the presence of the monomer. ATF-3 expression was found only in the nuclear fraction of camptothecin-treated HeLa cultures. TEGDMA seemed to inhibit the formation of phospho-c-Jun and phospho-ATF-2 in tHPC, and the monomer acted negatively on the expression of c-Jun, ATF-2 and ATF-3 in RAW264.7 macrophages. These changes in the expression and activation of stress-related transcription factors were time-related to the induction of apoptosis by TEGDMA in all cell lines. The present study provides experimental evidence that TEGDMA interferes with the regulation of cellular pathways through transcription factors activated as a consequence of DNA damage like p53 or initiated downstream of MAPK (mitogen-activated protein kinases) like c-Jun, ATF-2 and ATF-3. The direct causal correlation between DNA damage, activation or inhibition of MAPKs and transcription factors, and apoptosis is under current investigation. However, the induction of apoptosis in different cell lines in the presence of monomers like TEGDMA may be subject to a higher level of complexity than currently suggested by simple linear models.

Toll-like receptors, LPS, and dental monomers.

Unreacted monomers released from dental resin-based composites at non-cytotoxic concentrations cause a depletion of glutathione and an increase of reactive oxygen species (ROS), leading to, e.g., DNA damage and apoptosis. ROS-sensitive MAP-kinases are activated by HEMA and TEGDMA. MAP-kinases are also involved in the bacteria-triggered cell responses of the innate immune system, e.g., after bacterial lipopolysaccharide (LPS) binding to the Toll-like receptor (TLR) 4. Therefore, both bacteria and monomers imply environmental stress to pulp tissue, and they may influence the target cell reactions in a combined way. In macrophages, cell-surface antigens and cytokines were up-regulated after exposure to LPS, but TEGDMA caused a significant down-regulation. Regulation was dependent on exposure time, indicating that LPS and TEGDMA act differently on MAP-kinases. Furthermore, the cell type played a decisive role. Inhibition of the immune response may result in a decrease in inflammatory symptoms and/or a reduced defense capacity against bacteria.

Influence of TEGDMA on the mammalian cell cycle in comparison with chemotherapeutic agents

OBJECTIVES The dental resin monomer triethylene glycol dimethacrylate (TEGDMA) caused a cell cycle arrest in response to DNA damage. However, the underlying mechanisms are unclear. Therefore, the influence of TEGDMA on the cell cycle was analyzed in comparison with the chemotherapeutic agents adriamycin and mitomycin C (MMC), which arrest the cell cycle through different mechanisms. METHODS RAW264.7 mouse macrophages were exposed to TEGDMA, adriamycin, or MMC, and flow cytometry (FACS) was used for cell cycle analyses. In addition, the number of surviving cells was determined by a crystal violet assay, and viability in treated cultures was determined by FACS after staining of cells with trypan blue. Morphological changes in cells were interpreted using forward and side scatter (FSC/SSC) cell physical criteria. RESULTS The exposure of cells to 1mM TEGDMA resulted in a delay of the cell cycle in G1 phase since 85.3% of the cells were found in G1 compared with 47.4% in untreated controls. Adriamycin also increased the number of cells (72.1%) in G1 compared to controls. Caffeine, an inhibitor of the checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), had no effect on the TEGDMA and adriamycin-induced cell cycle arrest. In contrast, MMC delayed the cell cycle in G2 since cell numbers increased to 22.1% compared to 10.7% in controls. The effect of MMC on G2 was even increased by low caffeine concentrations (100-400muM), but 1000muM caffeine inhibited MMC activity. SIGNIFICANCE Our results suggest that the mechanism of a TEGDMA-induced arrest of the cell cycle is different from the effect of the direct-acting interstrand crosslinking agent MMC. Since TEGDMA produced oxidative stress, it probably acts indirectly on the cell cycle through reactive oxygen species, unless TEGDMA-DNA adducts are shown experimentally.

Ceramic inlays and partial ceramic crowns: influence of remaining cusp wall thickness on the marginal integrity and enamel crack formation in vitro.

No information is currently available about what the critical cavity wall thickness is and its influence upon 1) the marginal integrity of ceramic inlays (CI) and partial ceramic crowns (PCC) and 2) the crack formation of dental tissues. This in vitro study of CI and PCC tested the effects of different remaining cusp wall thicknesses on marginal integrity and enamel crack formation. CI (n = 25) and PCC (n = 26) preparations were performed in extracted human molars. Functional cusps of CI and PCC were adjusted to a 2.5 mm thickness; for PCC, the functional cusps were reduced to a thickness of 2.0 mm. Non-functional cusps were adjusted to wall thicknesses of 1) 1.0 mm and 2) 2.0 mm. Ceramic restorations (Vita Mark II, Cerec3 System) were fabricated and adhesively luted to the cavities with Excite/Variolink II. The specimens were exposed to thermocycling and central mechanical loading (TCML: 5000 x 5 degrees C-55 degrees C; 30 seconds/cycle; 500000 x 72.5N, 1.6Hz). Marginal integrity was assessed by evaluating a) dye penetration (fuchsin) on multiple sections after TCML and by using b) quantitative margin analysis in the scanning electron microscope (SEM) before and after TCML. Ceramic- and tooth-luting agent interfaces (LA) were evaluated separately. Enamel cracks were documented under a reflective light microscope. The data were statistically analyzed with the Mann Whitney U-test (alpha = 0.05) and the Error Rates Method (ERM). Crack formation was analyzed with the Chi-Square-test (alpha = 0.05) and ERM. In general, the remaining cusp wall thickness, interface, cavity design and TCML had no statistically significant influence on marginal integrity for both CI and PCC (ERM). Single pairwise comparisons showed that the CI and PCC of Group 2 had a tendency towards less microleakage along the dentin/LA interface than Group 1. Cavity design and location had no statistically significant influence on crack formation, but the specimens with 1.0 mm of remaining wall thickness had statistically significantly more crack formation after TCML than the group with 2.0 mm of remaining cusp wall thickness for CI. The remaining cusp wall thickness of non-functional cusps of adhesively bonded restorations (especially for CI) should have a thickness of at least 2.0 mm to avoid cracks and marginal deficiency at the dentin/LA interface.