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 (CDK4R24C), 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.