Biodentine Reduces Tumor Necrosis Factor Alpha–induced TRPA1 Expression in Odontoblastlike Cells

doi:10.1016/j.joen.2015.12.017

Journal of Endodontics

Volume 42, Issue 4, April 2016, Pages 589-595

https://doi.org/10.1016/j.joen.2015.12.017 Get rights and content

Highlights

•
TRPA1 channel expression was modulated by caries-induced inflammation.
•
TRPA1 gene expression and functionality were induced in cultured odontoblastlike cells by tumor necrosis factor alpha.
•
This tumor necrosis factor alpha–induced expression and functionality of TRPA1 were significantly reduced by Biodentine.

Abstract

Introduction

The transient receptor potential (TRP) ion channels have emerged as important cellular sensors in both neuronal and non-neuronal cells, with TRPA1 playing a central role in nociception and neurogenic inflammation. The functionality of TRP channels has been shown to be modulated by inflammatory cytokines. The aim of this study was to investigate the effect of inflammation on odontoblast TRPA1 expression and to determine the effect of Biodentine (Septodent, Paris, France) on inflammatory-induced TRPA1 expression.

Methods

Immunohistochemistry was used to study TRPA1 expression in pulp tissue from healthy and carious human teeth. Pulp cells were differentiated to odontoblastlike cells in the presence of 2 mmol/L beta-glycerophosphate, and these cells were used in quantitative polymerase chain reaction, Western blotting, calcium imaging, and patch clamp studies.

Results

Immunofluorescent staining revealed TRPA1 expression in odontoblast cell bodies and odontoblast processes, which was more intense in carious versus healthy teeth. TRPA1 gene expression was induced in cultured odontoblastlike cells by tumor necrosis factor alpha, and this expression was significantly reduced in the presence of Biodentine. The functionality of the TRPA1 channel was shown by calcium microfluorimetry and patch clamp recording, and our results showed a significant reduction in tumor necrosis factor alpha–induced TRPA1 responses after Biodentine treatment.

Conclusions

In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses.

Section snippets

Cell Culture and Treatments

Dental pulp cells were derived by explant culture as previously described (23). Immature permanent third molar teeth were obtained in accordance with French ethics legislation, and cells were grown in minimal essential medium with L-glutamine supplemented with 10% fetal bovine serum, 100 UI/mL penicillin, and 100 μg/mL streptomycin. To differentiate pulp cells to odontoblastlike cells, the medium was supplemented with 2 mmol/L beta-glycerophosphate, as previously described (14). The odontoblast

TRPA1 Expression in the Odontoblast Layer of Intact and Carious Teeth

TRPA1 expression in healthy and carious teeth was investigated with fluorescent immunohistochemistry as shown in Figure 1. In hematoxylin-eosin staining of intact teeth, odontoblasts appear perfectly aligned at the pulp periphery (Fig. 1A and C). In carious teeth, bacteria infiltrating the dentin are visible, and the dentin structure is irregular at the carious site (Fig. 1B and D). This seems to be mainly caused by cariogenic bacteria as seen in Brown and Brenn staining (Fig. 1J).

Discussion

In the present investigation, we show that human odontoblast TRPA1 expression is increased as a result of caries-induced pulpal inflammation and that this modulation of TRPA1 was mimicked in cultured odontoblastlike cells exposed to the inflammatory cytokine TNF-α. The effect of the clinical application of restorative material on TRPA1 expression in inflamed pulp was simulated by incubating the TNF-α–treated cells with Biodentine extracts. We show that TNF-α–induced TRPA1 expression was

Conclusion

In conclusion, this study showed TRPA1 to be modulated by caries-induced inflammation and that Biodentine reduced TRPA1 expression and functional responses. This suggests a potential property for Biodentine in modulating nociceptive receptors in odontoblasts, which may have implications in postoperative pain relief.

Acknowledgments

The authors would like to thank Catherine Fulton for technical assistance and Dr Jean-Charles Gardon for providing the teeth.

Supported by a research grant from Septodont, France.

The authors deny any conflicts of interest related to this study.

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Limonene, a citrus monoterpene, non-complexed and complexed with hydroxypropyl-β-cyclodextrin attenuates acute and chronic orofacial nociception in rodents: Evidence for involvement of the PKA and PKC pathway
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This may be related to the attenuation of the activation of the TRPV1 and TRPA1 channels and modulation of the inflammatory process. Studies have shown that several inflammatory molecules, such as cytokines, regulate the TRPA1 and TRPV1 channels (El Karim et al., 2015, 2016; Terenzi et al., 2013), indicating an intricate crosstalk between these two molecules in the development of pain. It has also been demonstrated that other terpenes such as carvacrol (Alvarenga et al., 2016), α-bisabolol (Fontinele et al., 2019), carvone (Kang et al., 2015) and 1,4-cineole (Jiang et al., 2016) present similar effects to LIM, acting on the TRPs channels or the inflammatory cascade.
Chronic orofacial pain is a serious public health problem with a prevalence of 7–11% in the population. This disorder has different etiologies and characteristics that make pharmacological treatment difficult. Natural products have been shown to be a promising source of treatments for the management of chronic pain, as an example the terpenes.
The aim of this study was to evaluate the anti-nociceptive and anti-inflammatory effects of one of these terpenes, d-limonene (LIM - a common monoterpene found in citrus fruits) alone and complexed with hydroxypropyl-β-cyclodextrin (LIM/HPβCD) in preclinical animal models.
Orofacial pain was induced by the administration of hypertonic saline on the corneal surface, the injection of formalin into the temporomandibular joint (TMJ), or chronic constriction injury of the infraorbital nerve (CCI-IoN). The study used male Wistar rats and Swiss mice treated with LIM (50 mg/kg), LIM/HPβCD (50 mg/kg), vehicle (control), gabapentin or morphine, and eyes wiping (induced by hypertonic saline), face rubbing (formalin-induced in TMJ) or mechanical hyperalgesia (provoked by CCI-IoN) were assessed. Additionally, ELISA was used to measure TNF-α, and western blot analysis to assess levels of PKAcα, NFκB, p38MAPK and phosphorylated PKC substrates. Serum levels of aspartate aminotransferase (AST) and alanine transferase (ALT) were also evaluated.
LIM and LIM/HPβCD significantly reduced (p < 0.001) corneal nociception and formalin-induced TMJ nociception. In addition, both substances attenuated (p < 0.001) mechanical hyperalgesia in the CCI-IoN model. The antinociceptive effect induced by LIM and HPβCD/LIM was associated with decreased TNF-α levels, downregulation of the NFκB and p38MAPK signalling pathways and reduced PKC substrate phosphorylation and PKA immunocontent. Moreover, the results demonstrated that complexation with HPβCD was able to decrease the therapeutic dose of LIM.
LIM was found to be a promising molecule for the treatment of orofacial pain due to its capacity to modulate some important mediators essential to the establishment of pain, and HPβCD can be a key tool to improve the profile of LIM.
Activation of Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Channels Promotes Odontogenic Differentiation of Human Dental Pulp Cells
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Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are thermosensitive channels that play an important role in thermal sensation or tooth pain by regulating intracellular Ca²⁺ concentration that is essential for pulp tissue repair. The aim of this study was to evaluate the role of TRPA1 and TRPV1 channels in the odontogenic differentiation of human dental pulp cells (HDPCs).
HDPCs were isolated from healthy human intact third molars and cultured in odontogenic differentiation medium. Gene and protein expression levels of TRPA1 and TRPV1 channels during the odontogenic differentiation of HDPCs were evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. HDPCs were then treated with channel agonists or antagonists, and the expression levels of odontogenic markers dentin sialophosphoprotein (DSPP) and osteopontin (OPN) were examined. Alkaline phosphatase activity and alizarin red staining were also conducted to detect mineralization levels.
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TRPA1 is expressed at higher levels than TRPV4 in carious teeth, and it mediates pain and inflammation in the dental pulp [80]. Biodentin can reduce TRPA1 expression and its response to caries-induced inflammation [81]. Another three remarkable mechanosensitive ion channels in odontoblasts are TRPM3, TRPM7 and TRPM8.
Tooth and bone are independent tissues with a close relationship. Both are composed of a highly calcified outer structure and soft inner tissue, and both are constantly under mechanical stress. In particular, the alveolar bone and tooth constitute an occlusion system and suffer from masticatory and occlusal force. Thus, mechanotransduction is a key process in many developmental, physiological and pathological processes in tooth and bone. Mechanosensitive ion channels such as Piezo1 and Piezo2 are important participants in mechanotransduction, but their functions in tooth and bone are poorly understood. This review summarizes our current understanding of mechanosensitive ion channels and their roles in tooth and bone tissues. Research in these areas may shed new light on the regulation of tooth and bone tissues and potential treatments for diseases affecting these tissues.
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
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Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin—a TRPA1 agonist—up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. In vivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.
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Basic ResearchBiodentine Reduces Tumor Necrosis Factor Alpha–induced TRPA1 Expression in Odontoblastlike Cells

Highlights

Abstract

Introduction

Methods

Results

Conclusions

Section snippets

Cell Culture and Treatments

TRPA1 Expression in the Odontoblast Layer of Intact and Carious Teeth

Discussion

Conclusion

Acknowledgments

Neuron

Cell

J Endod

J Biol Chem

Arch Oral Biol

Pain

J Endod

J Dent

Exp Cell Res

Arch Oral Biol

J Biol Chem

J Endod

J Endod

Am J Pathol

Sensing with TRP channels

Nat Chem Biol

Tumor necrosis factor-alpha (TNF-alpha) enhances functional thermal and chemical responses of TRP cation channels in human synoviocytes

Mol Pain

TRPA1 contributes to cold hypersensitivity

J Neurosci

Basic Research
Biodentine Reduces Tumor Necrosis Factor Alpha–induced TRPA1 Expression in Odontoblastlike Cells