“…Furthermore, ROS scavengers decreased their levels induced by 30 mM HEMA, but not by 3 mM HEMA. These results and those from previous studies 11 , 12 support the view that the activation of the Nrf2-ARE/HO-1 transcriptional pathway by HEMA at low concentrations is mainly independent of oxidative stress and is suggestive of direct binding to Keap-1. Figure 6 A schematic diagram showing how 30 mM HEMA increases ATP release in hDPSC-K4DT cells.…”
Section: Discussionsupporting
confidence: 89%
“…ROS production is attributed to HEMA-induced formation of mitochondrial superoxide anions 10 . The expression of antioxidant enzymes, including heme oxygenase (HO-1), following ROS production, protects the cells against HEMA 11 , 12 . HO-1 , a major downstream gene of the Nrf2-ARE (nuclear factor erythroid 2-related factor/antioxidant response element) pathway, is highly upregulated to attenuate cellular oxidative stress, preventing apoptosis and promoting cell survival 13 – 17 .…”
Extracellular ATP (adenosine triphosphate) and transient receptor potential ankyrin 1 (TRPA1) channels are involved in calcium signaling in odontoblasts and dental pain. The resin monomer 2-hydroxyethyl methacrylate (HEMA), used in dental restorative procedures, is related to apoptotic cell death via oxidative stress. Although the TRPA1 channel is highly sensitive to reactive oxygen species (ROS), the effect of HEMA-induced ROS on ATP release to the extracellular space and the TRPA1 channel has not been clarified in human dental pulp. In this study, we investigated the extracellular ATP signaling and TRPA1 activation by HEMA-derived ROS in immortalized human dental pulp cells (hDPSC-K4DT). Among the ROS-sensitive TRP channels, TRPA1 expression was highest in undifferentiated hDPSC-K4DT cells, and its expression levels were further enhanced by osteogenic differentiation. In differentiated hDPSC-K4DT cells, 30 mM HEMA increased intracellular ROS production and ATP release, although 3 mM HEMA had no effect. Pretreatment with the free radical scavenger PBN (N-tert-butyl-α-phenylnitrone) or TRPA1 antagonist HC-030031 suppressed HEMA-induced responses. These results suggest that ROS production induced by a higher dose of HEMA activates the TRPA1 channel in human dental pulp cells, leading to ATP release. These findings may contribute to the understanding of the molecular and cellular pathogenesis of tertiary dentin formation and pain in response to dental biomaterials.
“…Furthermore, ROS scavengers decreased their levels induced by 30 mM HEMA, but not by 3 mM HEMA. These results and those from previous studies 11 , 12 support the view that the activation of the Nrf2-ARE/HO-1 transcriptional pathway by HEMA at low concentrations is mainly independent of oxidative stress and is suggestive of direct binding to Keap-1. Figure 6 A schematic diagram showing how 30 mM HEMA increases ATP release in hDPSC-K4DT cells.…”
Section: Discussionsupporting
confidence: 89%
“…ROS production is attributed to HEMA-induced formation of mitochondrial superoxide anions 10 . The expression of antioxidant enzymes, including heme oxygenase (HO-1), following ROS production, protects the cells against HEMA 11 , 12 . HO-1 , a major downstream gene of the Nrf2-ARE (nuclear factor erythroid 2-related factor/antioxidant response element) pathway, is highly upregulated to attenuate cellular oxidative stress, preventing apoptosis and promoting cell survival 13 – 17 .…”
Extracellular ATP (adenosine triphosphate) and transient receptor potential ankyrin 1 (TRPA1) channels are involved in calcium signaling in odontoblasts and dental pain. The resin monomer 2-hydroxyethyl methacrylate (HEMA), used in dental restorative procedures, is related to apoptotic cell death via oxidative stress. Although the TRPA1 channel is highly sensitive to reactive oxygen species (ROS), the effect of HEMA-induced ROS on ATP release to the extracellular space and the TRPA1 channel has not been clarified in human dental pulp. In this study, we investigated the extracellular ATP signaling and TRPA1 activation by HEMA-derived ROS in immortalized human dental pulp cells (hDPSC-K4DT). Among the ROS-sensitive TRP channels, TRPA1 expression was highest in undifferentiated hDPSC-K4DT cells, and its expression levels were further enhanced by osteogenic differentiation. In differentiated hDPSC-K4DT cells, 30 mM HEMA increased intracellular ROS production and ATP release, although 3 mM HEMA had no effect. Pretreatment with the free radical scavenger PBN (N-tert-butyl-α-phenylnitrone) or TRPA1 antagonist HC-030031 suppressed HEMA-induced responses. These results suggest that ROS production induced by a higher dose of HEMA activates the TRPA1 channel in human dental pulp cells, leading to ATP release. These findings may contribute to the understanding of the molecular and cellular pathogenesis of tertiary dentin formation and pain in response to dental biomaterials.
“…It was reported that HEMA induces dissociation of Keap1 from Nrf2 through oxidative stress and/or direct binding with cysteine residues on Keap1, thus preventing Nrf2 degradation (Becher et al, 2019;Gallorini et al, 2015). We also confirmed increases in Nrf2 protein levels in HEMA-stimulated cells.…”
Section: Discussionsupporting
confidence: 83%
“…Nrf2 is a transcription factor that induces the expression of antioxidant genes, such as heme oxygenase-1 (Hmox1), by binding to the antioxidant response element, whereas Keap1 directly binds to Nrf2 and promotes proteasomal degradation of Nrf2 (Itoh et al, 2004). HEMA induces dissociation of Keap1 from Nrf2 through oxidative stress and/or direct binding with cysteine residues on Keap1 because of its electrophilic property, preventing subsequent Nrf2 degradation (Becher et al, 2019;Gallorini et al, 2015).…”
The hydrophilic compound 2-hydroxyethyl methacrylate (HEMA) is a major component of dental bonding materials, and it enhances the binding of resin-composites to biomolecules. However, HEMA is a well-known contact sensitizer. We reported previously that intradermal injection of HEMA induces the production of IL-1 locally in the skin. Keratinocytes are the first barrier against chemical insults and constitutively express IL-1α. In this study, we analyzed whether HEMA induces the production of inflammatory cytokines from murine keratinocyte cell line Pam212 cells. We demonstrated that HEMA induced the release of 17-kDa mature IL-1α and caused cytotoxicity. The activity of calpain, an IL-1α processing enzyme, was significantly higher in HEMA-treated cells. The thiol-containing antioxidant N-acetyl cysteine (NAC) inhibited HEMA-induced IL-1α release but not cytotoxicity. NAC inhibited intracellular calpain activity and reactive oxygen species (ROS) production induced by HEMA. NAC post-treatment also inhibited IL-1α release and intracellular ROS production induced by HEMA. Furthermore, HEMA-induced in vivo inflammation also inhibited by NAC. NAC inhibited polymerization of HEMA through adduct formation via sulfide bonds between the thiol group of NAC and the reactive double bond of HEMA. HEMA-induced IL-1α release and cytotoxicity were also inhibited if HEMA and NAC were pre-incubated before adding to the cells. These results suggested that NAC inhibited IL-1α release through decreases in intracellular ROS and the adduct formation with HEMA. We concluded that HEMA induces IL-1α release from skin keratinocytes, and NAC may be a promising candidate as a therapeutic agent against inflammation induced by HEMA.
“…The membranes were blocked for 30 min with 3% bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T). The selection of investigated proteins was mainly based on previous in vitro findings [ 26 ]. Primary antibodies targeting each of the proteins Glutamate cysteine ligase catalytic subunit (GCLC), p62, HO-1, Pirin, and NAD(P)H Quinone Dehydrogenase 1 (NQO1) were each diluted in TBS-T containing.…”
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.
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