2-hydroxyethyl methacrylate-derived reactive oxygen species stimulate ATP release via TRPA1 in human dental pulp cells

doi:10.21203/rs.3.rs-1616583/v1

Download PDF

Article

2-hydroxyethyl methacrylate-derived reactive oxygen species stimulate ATP release via TRPA1 in human dental pulp cells

https://doi.org/10.21203/rs.3.rs-1616583/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

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.

cytotoxicity

reactive oxygen species (ROS)

oxidative stress

osteogenic differentiation

The monomer 2-hydroxyethyl methacrylate (HEMA) is used in dental restorative procedures such as repair or restoration and root canal sealers, and is one of the main components in dentin bonding resins with contents varying from 30–55% ¹. HEMA can easily diffuse from polymer-based restorative materials ². Numerous studies have reported HEMA-induced cytotoxicity in various cell types, including dental pulp cells ³. HEMA-derived reactive oxygen species (ROS) have been suggested as the main cytotoxic factors ⁴. ROS production is attributed to HEMA-induced formation of mitochondrial superoxide anions⁵. The expression of antioxidant enzymes, including heme oxygenase (HO-1), following ROS production, protects the cells against HEMA ^6,7. 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 ^8–12.

Acrylic monomers have been known to cause allergic reactions in dental patients ^13,14. The release of acrylic monomers from bond-resin-based dental restorative materials causes local and systemic allergic reactions ^15,16. As HEMA can leach out through dentin tubules into the pulp tissue, it may cause pulpal inflammation, resulting in the generation of pain impulses ¹⁷.

Transient receptor potential (TRP) channels respond to various physical, thermal, and chemical stimuli ^18,19. TRPA1, TRPV1, and TRPV4 are known as oxidant-sensitive TRP channels, and TRPA1 has the highest oxidation sensitivity among the TRP channels ²⁰. The channel is predominantly expressed in human dental pulp, and its expression is significantly higher in painful pulp than in normal pulp ²¹. Recent studies have reported that TRPA1 expression is upregulated in odontoblasts aligned along tertiary dentin formed under carious lesions ^22,23. Tertiary dentin is produced by odontoblasts in response to external stimuli. It contains entrapped odontoblasts originating from undifferentiated precursors, including dental pulp stem cells (DPSCs), in response to dentinal tubules-derived stimulation ^24,25. Application of dentin bonding systems to seal both dentin and pulp may affect tertiary dentin formation ²⁶. Extracellular ATP (adenosine triphosphate) is thought to act as a neurotransmitter and neuromodulator, and is considered a powerful algogenic substance ^27,28. Recent studies have reported that human odontoblast-like cells can release ATP in response to TRPA1 activation, which promotes the extracellular calcium influx, suggesting that ATP release from odontoblasts following TRP channel–dependent calcium influx directly activates sensory neurons ²⁹.

Odontoblastic/osteoblastic differentiated dental pulp stem cells are more suitable for understanding cellular responses in dental pulp than immature cells. We previously established immortalized dental pulp stem cells (DPSCs) expressing R24C mutant cyclin-dependent kinase 4 (CDK4^R24C), Cyclin D1, and telomerase reverse transcriptase (TERT), which were termed hDPSC-K4DT cells from the last characters of the introduced genes (CDK4, Cyclin D1, TERT) ³⁰. The hDPSC-K4DT cells have the advantage of maintaining their original chromosomal pattern and stemness characteristics, suggesting that they can be a useful tool with relatively intact characteristics that mimic primary human dental pulp stem cells.

In the present study, we investigated the cytotoxicity of ROS generated following treatment of hDPSC-K4DT cells with different HEMA concentrations and examined whether it is involved in TRPA1-dependent ATP release, which would contribute to pain response to stimuli, such as exposure of dental pulp to chemicals.

Cell Culture and Differentiation

HDPSC-K4DT cells and immortalized DPSCs were previously established ³⁰ and cultured in high-glucose Dulbecco’s minimum essential medium (FUJIFILM Wako Chemicals, Miyazaki, Japan) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin solution (FUJIFILM Wako Chemicals) at 37°C in a humidified atmosphere containing 5% CO₂. As a control for hDPSC-K4DT cells, the human cementoblast cells (HCEM), kindly provided by Dr. Takada³¹, were cultured under similar conditions. Osteogenic differentiation of HDPSC-K4DT cells was performed using osteoinduction medium (Osteoblast-Inducer Reagent, Takara, Shiga Japan) for 12–15 days, according to the manufacturer’s instructions. When the cultured cells reached 70% confluence, the normal culture medium was replaced with osteogenic induction medium. Undifferentiated and differentiated hDPSC-K4DT cells after drug treatment were subjected to quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) analysis, ROS detection assay, and ATP release assay.

Drug treatments

HEMA (Kanto Chemical, Tokyo, Japan) was added to each well at 1–30 µM for 1–24 h. To determine the involvement of ROS and TRPA1 in HEMA-induced responses, the cells were pretreated for 1 h with the free radical scavenger PBN (N-tert-butyl-α-phenylnitrone; Sigma-Aldrich) at 6 mM and the TRPA1-selective antagonist HC-030031 (FUJIFILM Wako) at 30 or 100 µM, and then stimulated with HEMA at 3 or 30 mM for an additional 3 or 24 h.

RT-qPCR analysis

The total RNA was extracted from the collected cells using a NucleoSpin RNA kit (Takara Bio, Shiga, Japan), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Tokyo, Japan). cDNA (4 ng) was amplified by qPCR using Fast SYBR™ Green Master Mix (Applied Biosystems, Thermo Fisher Scientific, Tokyo, Japan) and specific primer sets for human genes and QuantStudio 3 PCR system (Applied Biosystems, Thermo Fisher Scientific). The following specific primer sets were used in this study for human genes: transient receptor potential ankyrin 1 (TRPA1), 5′- TGGACACCTTCTTCTTGCATT-3′ and 5′-TCATCCATTTCATGCAGCAC-3′; TRPV1, 5’-CTACAGCAGCAGCGAGACC-3′ and 5′-CCTGCAGGAGTCGGTTCA-3′; TRPV4, 5′-CAACAACGACGGCCTCT-3′ and 5′-GGATGATGTGCTGAAAGATCC-3′; alkaline phosphatase (ALP), 5′-ATGCTGAGTGACACAGACAAGAAG-3′ and 5′-GGTAGTTGTTGTGAGCATAGTCCAC-3′; heme oxygenase (HO-1), 5′-AGAGAATGCTGAGTTCATGAGGA-3′ and CAGCTCTTCTGGGAAGTAGACAG- 3′; collagen type I α 1 chain (COL1A1), 5′-TGAAGGGACACAGAGGTTTCA-3′ and 5′-ACCATCATTTCCACGAGCA- 3’; GAPDH, 5′-AGCTCACTGGCATGGCCTTC-3′ and 5′-TCACACCCATGACGAACATGG-3′. Gene expression values were calculated based on the ΔΔCt method, using GAPDH as an internal control to normalize mRNA levels.

ROS Detection

To determine the levels of intracellular ROS in hDPSC-K4DT cells treated with HEMA, we used the ROS Detection Cell-Based Assay Kit (DCFDA) (Cayman, Ann Arbor, MI, USA), according to the manufacturer’s protocol. In brief, hDPSC-K4DT cells that are immortalized DPSCs previously established³⁰ were seeded in a black tissue culture-treated 96-well plate, cultured to 70% confluence, and incubated at 37°C in osteogenic induction medium for 12 days. For the reactive oxygen species (ROS) detection assay, hDPSC-K4DT cells were pretreated with 6 mM PBN (N-tert-butyl-α-phenylnitrone) for 1h, then stimulated with 2-hydroxyethyl methacrylate (HEMA) (Kanto Chemical, Tokyo, Japan) at 3 or 30 mM for an additional 3 h at 37 ̊C in a humidified 5% CO₂ atmosphere. Then, the medium was aspirated, the cells were rinsed with 150 µl of cell-based assay buffer supplied with the ROS Detection Cell-Based Assay Kit (DCFDA) (Cayman, Ann Arbor, MI, USA), and 100 µl of ROS staining buffer mixed with 10 µM DCFDA was added and incubated for 90 min at 37 ̊C protected from light. After incubation, the ROS staining buffer was aspirated, and after further addition of 100 µl of cell-based assay buffer to each well, fluorescence was read on a TECAN Infinite 200 PRO plate reader (Tecan, Morrisville, NC, USA) at excitation and emission wavelengths of 485 and 535 nm, respectively.

ATP Release Assays

To evaluate ATP release from hDPSC-K4DT cells, ATP levels were measured in the culture supernatant using a luminescence-based ATP assay kit (TOYO B-Net, Tokyo, Japan). In brief, hDPSC-K4DT cells were seeded in 96-well plates, cultured to 70% confluence, and incubated at 37°C in an osteogenic induction medium for 14 days. Following osteogenic differentiation, HDPSC-K4DT cells were treated with HEMA at 0, 3, and 30 mM for 24 h and ATP levels were measured in cell supernatants using luminescence-based commercial kits (TOYO B-Net, Tokyo, Japan). Luminescence was measured using a microplate reader TECAN infinite 200 PRO with i-control 2.0 software (Tecan, Morrisville, NC, USA). To determine the effect of a free radical scavenger or a TRPA1 inhibitor on HEMA-induced ATP release levels, following osteogenic differentiation, the cells were pretreated with PBN (6 mM) or the TRPA1-selective antagonist HC-030031 (30 or 100 µM, FUJIFILM Wako Chemicals, Miyazaki, Japan) for 1 h, then stimulated with 30 mM HEMA for an additional 24 h before measuring ATP levels as above.

Statistical Analysis

Results are presented as mean ± standard deviation (SD). All the experiments were performed with three or more biological replicates per group. A two-tailed unpaired Student’s t-test was used to evaluate statistical differences between two groups. To evaluate the differences among the three groups, one-way analysis of variance (ANOVA) was used followed by Tukey’s test or Dunnett's test. P-values that were less than 0.05 were considered statistically significant.

TRPA1, TRPV1, and TRPV4 expression

RT-qPCR analysis of the mRNA levels of TRPA1, TRPV1, and TRPV4 in undifferentiated hDPSC-K4DT cells, indicated that those of TRPA1 were the highest among the ROS-sensitive TRP channels (P < 0.05, for both TRPV1 and TRPV4, Fig. 1A). In HCEM cells, used as control, TRPA1 mRNA was undetectable and the levels of TRPV1 and TRPV4 mRNAs were significantly lower than those in hDPSC-K4DT cells (P < 0.01 for both; Fig. 1A). Culture of hDPSC-K4DT cells in the osteoinduction medium, for osteogenic differentiation, resulted in increased mRNA levels of the three TRP channels at 8 and 15 days (P < 0.05; Fig. 1B). TRPA1 expression levels days 8 and 15 were significantly higher than that of TRPV1 and TRPV4 (P < 0.05, Fig. 1B). At 15 days, mRNAs levels of the osteoblastic markers collagen type I α 1 chain (COL1A1) and alkaline phosphatase (ALP) were significantly upregulated compared with non-treatment (0 d: P < 0.01 for both genes; Fig. 1C), indicating successful osteoblastic differentiation of hDPSC-K4DT cells. These results indicate that TRPA1 may function as the main ROS sensor in undifferentiated and differentiated hDPSC-K4DT cells.

HEMA toxicity in hDPSC-K4DT

Prior to examining the effects of HEMA on hDPSC-K4DT cells, we tested the cytotoxic doses of HEMA on cell viability using the CCK-8 assay. Incubation of undifferentiated hDPSC-K4DT cells with HEMA at 1–30 mM for 24 h, showed that concentrations higher than 10 mM significantly reduced cell viability (P < 0.05 for 10 and 30 mM; Fig. 2). Therefore, we used 3 and 30 mM as the non-toxic and toxic doses, respectively, in the following experiments.

ROS production by the toxic HEMA dose

To evaluate ROS production following HEMA application, osteodifferentiated hDPSC-K4DT cells were treated with 3 and 30 mM HEMA for 3 h, and ROS levels were determined using the ROS Detection Cell-Based Assay Kit (DCFDA). Compared to the control group (0 day), the intracellular ROS levels increased in hDPSC-K4DT cells treated with toxic 30 mM HEMA (P < 0.05), but not in cells treated with non-toxic 3 mM HEMA (Fig. 3). Although the free radical scavenger PBN slightly increased ROS levels in HEMA-free conditions, it significantly relieved the overproduction of ROS following treatment with the toxic HEMA dose to the control level (P < 0.05; Fig. 3).

ATP release by the toxic HEMA dose and involvement of TRPA1 activation

To address the effect of HEMA on ATP release, we measured ATP concentration in culture supernatants following treatment of osteodifferentiated hDPSC-K4DT cells with HEMA for 24 h. As shown in Fig. 3A, 30 mM HEMA significantly increased the extracellular ATP concentration (P < 0.05), whereas 3 mM HEMA had no effect (Fig. 4). To determine the involvement of ROS-induced TRPA1 activation, we investigated the effects of PBN and TRPA1 antagonist HC-030031 on HEMA-induced ATP release. As shown in Fig. 4B, pretreatment with both PBN and HC-030031 significantly attenuated the increase in extracellular ATP levels upon treatment with 30 mM HEMA (P < 0.01 by PBN and P < 0.05 by HC-030031 at 30 and 100 µM), whereas these drugs did not show any effects on HEMA-free conditions. These results suggest that a toxic HEMA dose stimulates ATP release following TRPA1 activation via ROS.

HO-1 expression after HEMA exposure

The detected ROS levels in Fig. 3 is the final output of the intracellular oxidative/anti-oxidative responses. To examine the intracellular anti-oxidant response during HEMA exposure, we investigated the mRNA expression of HO-1, known as a marker of activation of the anti-oxidative Nrf2-ARE pathway, in osteodifferentiated hDPSC-K4DT cells following treatment with HEMA for 3 h using RT-qPCR analysis. Incubation with HEMA increased HO-1 mRNA expression in a dose-dependent manner, even at non-toxic doses (P < 0.05; Fig. 5A). Pretreatment with PBN reduced 30 mM HEMA-induced HO-1 upregulation (P < 0.05) but did not affect 3 mM HEMA-induced one (Fig. 5B), suggesting oxidative stress-independent activation of the Nrf2-ARE pathway.

PBN-resistant 3 mM HEMA-induced HO-1 upregulation peaked at 3 h and returned to basal levels 24 h after exposure (Fig. 5C). Simultaneously, TRPA1 mRNA expression tended to be reduced 1 h after 3 mM HEMA exposure, although the difference was not statistically significant (Fig. 5D). We examined the possibility of TRPA1 downregulation along with dedifferentiation of osteodifferentiated hDPSC-K4DT cells, but the osteoblastic markers COL1A1 and ALP did not change throughout the time course of the experiment (Fig. 5E, F).

In this study, we demonstrated that the overproduction of ROS caused by high doses of HEMA activates the TRPA1 channel and stimulates ATP release (Fig. 6). High concentrations of HEMA (30 mM) increased the expression of the oxidative stress marker HO-1 and the production of ROS. Furthermore, ROS scavengers decreased their levels induced by 30 mM HEMA, but not by 3 mM HEMA. These results and those from previous studies ^6,7 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.

Activation of the Nrf2-ARE/HO-1 transcriptional pathway in differentiated hDPSC-K4DT cells exposed to low concentrations of HEMA (3 mM) may increase the levels of proteins critical for detoxification, such as HO-1, and eliminate ROS, indicating that HEMA at low concentrations does not cause accumulation of ROS. In contrast, in cells exposed to high concentrations of HEMA (30 mM), induction of HO-1 cannot counteract HEMA-induced severe oxidative stress, which causes ROS accumulation. The increase in extracellular ATP levels in the cells suggested the possibility of TRPA1 activation by ROS stimulation. Indeed, HEMA-induced ATP release, probably via PANX1 in response to TRP channel activation. Secreted ATP has been shown to activate ionotropic P2X3 receptors on nociceptive trigeminal nerve fibers, which mediate sensory transduction to the brain and induce dental pain (Fig. 6), according to a previous study ³². There were differences in the cellular response and defense mechanisms against oxidative stress induced by HEMA at low and high concentrations. From this accumulative side supportive evidence, we explain the different observations that HO-1 and ROS induced by 30 mM HEMA but not by 3 mM HEMA were counteracted by ROS scavengers.

Recent studies in rat incisor models of tooth bleaching have suggested that strong oxidative stress following treatment with high-dose H₂O₂ induced ROS-mediated cytotoxicity in DPSCs, which could affect the hyperalgesia of tooth by regulating TRPA1 expression, intracellular Ca²⁺ concentration, and ATP release into the extracellular space ^33,34. Moreover, oxidative stress in the periodontal tissue and dental pulp following orthodontic force is suggested to activate and/or mechanically sensitize TRPA1 on nociceptive fibers, resulting in orthodontic nociception ³⁵. Our data showed that HEMA-induced oxidative stress with the accumulation of ROS activates TRPA1 channels, consistent with the fact that TRPA1 can be activated by multiple products of oxidative stress ¹⁹. This study provides new insights into cellular responses to HEMA exposure, which may be involved in the pain response through TRPA1 activation ¹⁹.

In this study, the previously established immortalized human DPSCs, hDPSC-K4DT cells ³⁰, exhibited a time-dependent increase in the mRNA expression of COL1A1 and ALP, markers of differentiation in osteogenic cells, suggesting the maintenance of osteogenic differentiation potential. Our previous study and this study demonstrated that hDPSC-K4DT cells retained the original differentiation abilities of human dental pulp cells. Furthermore, consistent with previous reports^23,36, TRPA1 was highly expressed in hDPSC-K4DT cells, and TRPA1 expression was elevated along with their osteogenic differentiation. These results indicate that hDPSC-K4DT cells are a useful in vitro tool for studying the cytotoxicity of HEMA and can be used for the evaluation of the relationship between cytotoxicity and TRPA1 channels. Further elucidation of the functional role of TRPA1 in DPSCs is necessary to understand the transmission of pain sensations.

In summary, the present study suggests that HEMA activates TRPA1 channels through ROS generation in immortalized human DPSC. This study contributes to the understanding of the underlying molecular mechanism of dental pain conduction via TRPA1 activation in response to resin monomers and biomaterials.

Data Availability Statement:

All relevant data are within the paper.

Acknowledgments

We thank Dr. Tohru Kiyono (National Cancer Center, JAPAN) and Dr. Tomokazu Fukuda (Iwate University) for providing critical research materials and K4DT immortalization methods.

We thank Dr. Ayako Washio (Kyushu Dental University) for help with the cell viability analysis and Dr. Seiwa Horie for help with the ATP release assay. We also thank Dr. Shoichiro Kokabu (Kyushu Dental University) for providing research environmental support throughout the study. This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS) to A. Orimoto. All authors declare that they have no conflicts of interest.

Author contributions

AO did the experiments. AO, CK, and KO contributed to the conception, design, and data analysis. AO drafted the manuscript. AO, CK, and KO approved the final version of the manuscript.

Becher, R. et al. Pattern of cell death after in vitro exposure to GDMA, TEGDMA, HEMA and two compomer extracts. Dent. Mater. (2006). doi:10.1016/j.dental.2005.05.013
Van Landuyt, K. L. et al. How much do resin-based dental materials release? A meta-analytical approach. Dental Materials (2011). doi:10.1016/j.dental.2011.05.001
Gallorini, M., Cataldi, A. & di Giacomo, V. HEMA-induced cytotoxicity: Oxidative stress, genotoxicity and apoptosis. International Endodontic Journal (2014). doi:10.1111/iej.12232
Baldion, P. A., Velandia-Romero, M. L. & Castellanos, J. E. Dental resin monomers induce early and potent oxidative damage on human odontoblast-like cells. Chem. Biol. Interact. (2021). doi:10.1016/j.cbi.2020.109336
Schweikl, H. et al. 2-Hydroxyethyl methacrylate-induced apoptosis through the ATM- and p53-dependent intrinsic mitochondrial pathway. Biomaterials (2014). doi:10.1016/j.biomaterials.2013.12.044
Becher, R., Valen, H., Olderbø, B. P., Bølling, A. K. & Samuelsen, J. T. The dental monomer 2-hydroxyethyl methacrylate (HEMA) causes transcriptionally regulated adaptation partially initiated by electrophilic stress. Dent. Mater. (2019). doi:10.1016/j.dental.2018.11.008
Orimoto, A. et al. Effect of 2-Hydroxyethyl Methacrylate on Antioxidant Responsive Element-Mediated Transcription: A Possible Indication of Its Cytotoxicity. PLoS One (2013). doi:10.1371/journal.pone.0058907
Kobayashi, A., Ohta, T. & Yamamoto, M. Unique Function of the Nrf2-Keap1 Pathway in the Inducible Expression of Antioxidant and Detoxifying Enzymes. Methods Enzymol. (2004). doi:10.1016/S0076-6879(04)78021-0
Kaspar, J. W., Niture, S. K. & Jaiswal, A. K. Nrf2:INrf2 (Keap1) signaling in oxidative stress. Free Radical Biology and Medicine (2009). doi:10.1016/j.freeradbiomed.2009.07.035
Zhong, Y. et al. Heme oxygenase-1-mediated reactive oxygen species reduction is involved in the inhibitory effect of curcumin on lipopolysaccharide-induced monocyte chemoattractant protein-1 production in RAW264.7 macrophages. Mol. Med. Rep. (2013). doi:10.3892/mmr.2012.1138
Durante, W. Heme oxygenase-1 in growth control and its clinical application to vascular disease. Journal of Cellular Physiology (2003). doi:10.1002/jcp.10274
Copple, I. M., Goldring, C. E., Kitteringham, N. R. & Park, B. K. The Nrf2-Keap1 defence pathway: Role in protection against drug-induced toxicity. Toxicology (2008). doi:10.1016/j.tox.2007.10.029
Moore, M. M., Burke, F. J. & Felix, D. H. Allergy to a common component of resin-bonding systems: a case report. Dent. Update (2000). doi:10.12968/denu.2000.27.9.432
Aalto-Korte, K., Alanko, K., Kuuliala, O. & Jolanki, R. Methacrylate and acrylate allergy in dental personnel. Contact Dermatitis (2007). doi:10.1111/j.1600-0536.2007.01237.x
Gawkrodger, D. J. Investigation of reactions to dental materials. British Journal of Dermatology (2005). doi:10.1111/j.1365-2133.2005.06821.x
Yamashiro, K. et al. Allergic Reaction to Zirconia Ceramic Bridge Cementation Using a Dental Adhesive Resin Cement: a Case Report. SN Compr. Clin. Med. (2021). doi:10.1007/s42399-020-00671-9
Daou, M. H. Monomers from resin-based materials: Risk assessment in dental medicine. Int. J. Dent. Oral Sci. (2021). doi:10.19070/2377-8075-21000465
Nassini, R., Materazzi, S., Benemei, S. & Geppetti, P. The TRPA1 channel in inflammatory and Neuropathic pain and migraine. Rev. Physiol. Biochem. Pharmacol. (2014). doi:10.1007/112_2014_18
Andersson, D. A., Gentry, C., Moss, S. & Bevan, S. Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress. J. Neurosci. (2008). doi:10.1523/JNEUROSCI.5369-07.2008
Kozai, D., Ogawa, N. & Mori, Y. Redox regulation of transient receptor potential channels. Antioxidants and Redox Signaling (2014). doi:10.1089/ars.2013.5616
Kim, Y. S. et al. Expression of transient receptor potential Ankyrin 1 in human dental pulp. J. Endod. (2012). doi:10.1016/j.joen.2012.04.024
Tazawa, K. et al. Transient Receptor Potential Ankyrin 1 Is Up-Regulated in Response to Lipopolysaccharide via P38/Mitogen-Activated Protein Kinase in Dental Pulp Cells and Promotes Mineralization. Am. J. Pathol. (2020). doi:10.1016/j.ajpath.2020.08.016
Lou, Y. et al. Activation of Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Channels Promotes Odontogenic Differentiation of Human Dental Pulp Cells. J. Endod. (2021). doi:10.1016/j.joen.2021.06.007
Kawashima, N. & Okiji, T. Odontoblasts: Specialized hard-tissue-forming cells in the dentin-pulp complex. Congenital Anomalies (2016). doi:10.1111/cga.12169
Gronthos, S. et al. Stem cell properties of human dental pulp stem cells. J. Dent. Res. (2002). doi:10.1177/154405910208100806
Cobanoglu, N. et al. Evaluation of human pulp tissue response following direct pulp capping with a self-etching adhesive system containing MDPB. Dent. Mater. J. (2021). doi:10.4012/dmj.2020-145
Krishtal, O. A., Marchenko, S. M. & Obukhov, A. G. Cationic channels activated by extracellular atp in rat sensory neurons. Neuroscience (1988). doi:10.1016/0306-4522(88)90203-5
Julius, D. & Basbaum, A. I. Molecular mechanisms of nociception. Nature (2001). doi:10.1038/35093019
Egbuniwe, O. et al. TRPA1 and TRPV4 activation in human odontoblasts stimulates ATP release. J. Dent. Res. (2014). doi:10.1177/0022034514544507
Orimoto, A. et al. Efficient immortalization of human dental pulp stem cells with expression of cell cycle regulators with the intact chromosomal condition. PLoS One 15, 1–18 (2020).
Kitagawa, M. et al. Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo. Bone (2006). doi:10.1016/j.bone.2006.05.022
Shibukawa, Y. et al. Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction. Pflugers Arch. Eur. J. Physiol. (2015). doi:10.1007/s00424-014-1551-x
Chen, C. et al. H2O2 gel bleaching induces cytotoxicity and pain conduction in dental pulp stem cells via intracellular reactive oxygen species on enamel/dentin disc. PLoS One (2021). doi:10.1371/journal.pone.0257221
Chen, C. et al. TRPA1 triggers hyperalgesia and inflammation after tooth bleaching. Sci. Rep. (2021). doi:10.1038/s41598-021-97040-w
Morii, A. et al. Orthodontic force-induced oxidative stress in the periodontal tissue and dental pulp elicits nociception via activation/sensitization of TRPA1 on nociceptive fibers. Free Radic. Biol. Med. (2020). doi:10.1016/j.freeradbiomed.2019.12.016
El Karim, I. A. et al. Human dental pulp fibroblasts express the ‘cold-sensing’ transient receptor potential channels TRPA1 and TRPM8. J. Endod. 37, 473–478 (2011).

No competing interests reported.

Supplementary050222.docx

Download PDF

Editorial decision: Major revision
25 May, 2022
Reviews received at journal
17 May, 2022
Reviewers agreed at journal
12 May, 2022
Reviewers invited by journal
09 May, 2022
Editor assigned by journal
09 May, 2022
Editor invited by journal
09 May, 2022
Submission checks completed at journal
09 May, 2022
First submitted to journal
02 May, 2022

You are reading this latest preprint version

2-hydroxyethyl methacrylate-derived reactive oxygen species stimulate ATP release via TRPA1 in human dental pulp cells

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell Culture and Differentiation

Drug treatments

RT-qPCR analysis

ROS Detection

ATP Release Assays

Statistical Analysis

Results

TRPA1, TRPV1, and TRPV4 expression

HEMA toxicity in hDPSC-K4DT

ROS production by the toxic HEMA dose

ATP release by the toxic HEMA dose and involvement of TRPA1 activation

HO-1 expression after HEMA exposure

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1