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Open Access 01.12.2022 | Research

Oxidative stress impairs the calcification ability of human dental pulp cells

verfasst von: Satomi Shirawachi, Katsuhiro Takeda, Tomoya Naruse, Yohei Takahasi, Jun Nakanishi, Satoru Shindo, Hideki Shiba

Erschienen in: BMC Oral Health | Ausgabe 1/2022

Abstract

Background

The relationship between internal root resorption and oxidative stress has not yet been reported. This study aimed to add molecular insight into internal root resorption. The present study was conducted to investigate the effect of hydrogen peroxide (H₂O₂) as an inducer of oxidative stress on the calcification ability of human dental pulp cells (hDPCs) and the involvement of inositol 1, 4, 5-trisphosphate (IP3).

Material and methods

hDPCs (Lonza, Basel, Switzerland) were exposed to H₂O₂. Cell viability and reactive oxygen species (ROS) production were then evaluated. To investigate the effect of H₂O₂ on the calcification ability of hDPCs, real-time PCR for alkaline phosphatase (ALP) mRNA expression, ALP staining, and Alizarin red staining were performed. Data were compared with those of hDPCs pretreated with 2-aminoethyldiphenylborate (2-APB), which is an IP3 receptor inhibitor.

Results

H₂O₂ at concentrations above 250 µM significantly reduced cell viability (P < 0.01). More ROS production occurred in 100 µM H₂O₂-treated hDPCs than in control cells (P < 0.01). 2-APB significantly decreased the production (P < 0.05). H₂O₂-treated hDPCs showed significant reductions in ALP mRNA expression (P < 0.01), ALP activity (P < 0.01), and mineralized nodule deposition compared with negative control cells (P < 0.01). 2-APB significantly inhibited these reductions (P < 0.01, P < 0.05 and P < 0.01, respectively). Data are representative of three independent experiments with three replicates for each treatment and values are expressed as means ± SD.

Conclusion

To the best of our knowledge, this is the first study documenting the involvement of IP₃ signaling in the calcification ability of human dental pulp cells impaired by H₂O₂.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

hDPCs

Human dental pulp cells

H₂O₂

Hydrogen peroxide

ROS

Reactive oxygen species

2-APB

2-Aminoethyldiphenylborate

Introduction

Internal root resorption in permanent teeth is characterized by destruction of intraradicular dentin and dentinal tubules along the root canal wall [1]. It occurs in pathological conditions, including trauma, infections, or unknown causes [1, 2]. Concern and curiosity about resorption of dental structures are not recent. Numerous theories have been proposed as a possible cause of internal root resorption. One study reported that dental pulp cells have an innate ability to attenuate dentin resorption by inhibiting osteoclastogenesis [3]. Therefore, it is assumed that the damaged dental pulp cells are probably related to the onset of internal root resorption. However, the underlying pathology is not fully understood.

Dental pulp cells can differentiate into odontoblasts and generate a mineralizing matrix, particularly during reparative dentin formation associated with injury or disease [4, 5]. Odontoblasts, which are organized in a layer at the dentin-pulp interface, secrete type I collagen, osteocalcin (OCN), alkaline phosphatase (ALP), and other noncollagenous proteins [6]. The impairment of reparative dentin formation in permanent teeth may be a key factor implicated in internal root resorption.

Oxidative stress is a damaging response and refers to excessive intracellular levels of reactive oxygen species (ROS). Hydrogen peroxide (H₂O₂) is one of the major ROS. At low physiological levels in the nanomolar range, H₂O₂ is the major agent signaling through specific protein targets that engage in metabolic regulation and stress responses to support cellular adaptation to a changing environment and stress. Many previous studies reported the effects of oxidative stress on pulp cells [7‐9]. One study showed that the oxidative stress of human dental pulp cells mediated by H₂O₂ promotes reduction of odontoblastic capability [8]. On the other hand, Matsu et al. [9] found an increase in osteopontin (OPN) and osteocalcin (OCN) in H₂O₂-treated-human dental pulp cells. Further investigations seem to be needed to clarify the mechanism. In addition, the relationship between internal root resorption and oxidative stress has not yet been reported. This study focused on H₂O₂ as an inducer of oxidative stress.

2-Aminoethyldiphenylborate (2-APB) was originally described as a membrane-permeant inhibitor of the inositol 1, 4, 5-trisphosphate (IP₃) receptor, and it was also used as one of the store-operated Ca²⁺ entry (SOCE) inhibitors [10]. Yamamura et al. [11] reported that oxidative stress reduced SOCE, and the decrease was recovered by 2-APB in brain capillary endothelial cells.

The present study was conducted to add molecular insight into root canal resorption. In the current study, it was demonstrated for the first time that IP₃ signaling is involved in the calcification ability of H₂O₂-treated human dental pulp cells. A possible mechanism to explain the influence of the oxidative stress caused by H₂O₂ on the calcification ability of human dental pulp cells is presented. The present study may provide a clue to help clarify a mechanism of onset of internal root resorption.

Materials and methods

Reagents

Hydrogen peroxide (H₂O₂) and 2-aminoethoxydiphenylborane (2-APB) were purchased from FUJIFILM Wako Pure Chemicals Corporation (Osaka, Japan) and Bio-Techne Corporation (Minneapolis, MN, USA), respectively.

Cell culture

Human dental pulp cells (hDPCs) were obtained from Lonza (Basel, Switzerland). Cells were cultured in growth medium (GM) (Eagle’s Minimum Essential Medium, ALPHA modification (MEM-α)) supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum; Sigma-Aldrich, MO, USA), 100 IU/mL penicillin (FUJIFILM Wako Pure Chemicals Corp.), and 100 µg/mL streptomycin (FUJIFILM Wako Pure Chemicals Corp.) (medium A). The cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Cell cultures between the sixth and ninth passages were used in this study.

To differentiate into odontoblast-like cells, the cells were cultured in osteo-odontogenic medium (OM), MEM-α supplemented with 2.5% heat-inactivated FBS (Sigma-Aldrich), 100 IU/mL penicillin (FUJIFILM Wako Pure Chemicals Corp.), 100 µg/mL streptomycin (FUJIFILM Wako Pure Chemicals Corp.), 10 mM β-glycerophosphate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and 50 mg/mL ascorbic acid (FUJIFILM Wako Pure Chemicals Corp.).

WST assay

Cell viability was assessed by the WST assay using the Cell Counting Kit-8 according to the manufacturer’s protocol (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, cells were plated at a density of 5.0 × 10³ cells/well in 96-well plates and maintained in 0.1 mL of medium A. After 24-h incubation, culture medium was replaced to serum-free medium with H₂O₂ at concentrations of 0, 50, 100, 250, 500, and 1000 µM (final concentration) and incubated for 24 h in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Then, CCK-8 solution was added, and the cells were incubated for another hour. Absorbance at 450 nm was then measured with the reference wavelength at 620 nm using a microplate reader (Multiskan™ FC; Thermo Fisher Scientific, CA, USA). Data are representative of three independent experiments with three replicates for each treatment.

Intracellular reactive oxygen species measurements

Measurement of intracellular ROS was performed by dichlorodihydrofluorescein diacetate (DCFH-DA) (ROS Assay Kit-Highly Sensitive DCFH-DA-; Dojindo Laboratories). Cells were seeded at a density of 1.0 × 10⁴ cells in 6-well plates and incubated overnight in medium A. The medium was replaced to serum-free medium with 10 µM 2-APB or PBS for control cells. One hour later, the cells were stimulated with 100 µM H₂O₂ for 2 h in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. DCFH-DA solution was added after 3-h incubation, and the cells were then incubated for another 30 min. Fluorescence signals were observed using an all-in-one fluorescence microscope (BZ-X700; Keyence, Tokyo, Japan). Data are representative of three independent experiments with three replicates for each treatment.

ALP staining

To evaluate alkaline phosphatase activity, the cells were stained by BCIP-NBT solution (SIGMA FAST™BCIP®/NBT; Sigma-Aldrich). hDPCs were seeded into 48-well plates at a density of 1.0 × 10⁴ cells/well in medium A. After the cells reached 70–80% confluency, culture medium was replaced to fresh MEM-α supplemented with 2.5% FBS (Sigma-Aldrich), 100 IU/mL penicillin (FUJIFILM Wako Pure Chemicals Corp.), and 100 µg/mL streptomycin (FUJIFILM Wako Pure Chemicals Corp.) (medium B). The cells were treated with 10 µM 2-APB or PBS for control cells. One hour later, cells were stimulated with 100 µM H₂O₂ for 6 h. Then, medium was replaced to OM and cultured for 7 days. The medium was changed on the third day. After incubation, the cells were rinsed with PBS, fixed with 10% neutral buffered formalin, and stained with BCIP-NBT solution. The stained area was quantified by Image J software (National Institutes of Health, Bethesda, MD, USA). To briefly explain the quantification method, images were converted to RGB stack, and “blue slice” images were picked up (Fig. 2A(b)). The threshold of the blue slice images was adjusted, and a limited threshold area was highlighted in red, as shown in Fig. 2A(c). Thereafter, the % stained area was automatically measured by the software. Data are representative of three independent experiments with three replicates for each treatment.

Alizarin red staining

hDPCs were seeded into 6-well plates at a density of 1.0 × 10⁵ cells/well and cultured in medium A. After the cells reached 70–80% confluency, the culture medium was replaced to medium B and treated with 10 µM 2-APB or PBS for control cells. One hour later, cells were stimulated with 100 µM H₂O₂ for 6 h. The medium was then replaced to OM and cultured for 28 days. The medium was changed every 3 days. To evaluate calcium deposition, Alizarin red S Solution (PG research, Tokyo, Japan) was used after fixation with 10% neutral buffered formalin. The images were captured by an all-in-one fluorescence microscope (BZ-X700; Keyence), and the stained area was quantified by Image J software (National Institutes of Health). Data are representative of three independent experiments with three replicates for each treatment.

Isolation of total RNA and reverse transcription

hDPCs were seeded into 6-well plates at a density of 1.0 × 10⁵ cells/well and cultured in medium A until subconfluent. The cells were treated with 10 µM 2-APB or PBS for control cells. One hour later, the cells were stimulated with 100 µM H₂O₂ for 6 h. The cells were replaced to OM and cultured for 7 days. The medium was changed on the third day. Total RNA from each culture was extracted using RNA iso plus (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocol. cDNA was synthesized from 250 ng of total RNA in a final volume of 10 µL using the Rever Tra Ace qPCR RT Master Mix with gDNA remover kit (Toyobo Co., Ltd, Osaka, Japan) with a thermal cycler (Veriti™ 96-Well Thermal Cycler; Life Technologies, CA, USA).

Real-time PCR

Two-step qPCR was performed with the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, CA, USA) using THUNDERBIRD® Next SYBR® qPCR Mix (Toyobo Co., Ltd, Osaka, Japan). The cycling protocol was as follows: DNA polymerase activation at 95 °C for 30 s, followed by denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 10 s, for 40 cycles in fast mode. Gene expression was normalized to that of Gapdh mRNA in the same samples, using the 2^−ΔΔCt method. The sequences of relevant primers were as follows: Gapdh: forward 5′-AACGTGTCAGTGGTGGACCTG-3′; reverse 5′-AGTGGGTGTCGCTGTTGAAGT-3′; ALP: forward 5′-CGCCTACCAGCTCATGCATAAC-3′; reverse 5′-GTCAATTCTGCCTCCTTCCACC-3′. Data are representative of three independent experiments with three replicates for each treatment.

Statistical analysis

Each result is presented as a mean ± standard error of the mean. All experiments were performed in at least triplicate. Comparisons between two groups were evaluated by an unpaired two-tailed Student’s t-test. For comparisons of more than two groups, one-way ANOVA was followed by Tukey’s test for multiple comparisons. Significance was set at p < 0.05.

Results

hDPCs underwent severe cell death after high-dose H₂O₂ (250, 500 and 1000 μM) treatment for 24 h as determined by the WST assay (Fig. 1A). H₂O₂ at 50 and 100 µM did not decrease cell viability. ROS were increased in 100 μM H₂O₂-treated hDPCs (Fig. 1B). Therefore, in the present study, H₂O₂ at 100 μM, which is enough to promote ROS production, was used.

To determine whether H₂O₂ has an effect on the calcification ability of hDPCs in the present experimental system, the effects of H₂O₂ on hDPCs cultured in osteo-inductive medium were tested via ALP activity. After osteogenic differentiation for 7 days, H₂O₂ at 100 μM significantly decreased ALP activity in hDPCs (P < 0.01) (Fig. 2A, B).

Real-time PCR was used to determine the effect of H₂O₂ addition on the mRNA expression of ALP. H₂O₂ significantly decreased the ALP mRNA levels compared with control cells (P < 0.01). 2-APB reversed the decrease significantly (P < 0.05) (Fig. 3A).

The percentages of the BCIP-NBT stained area of the control group, positive control group (cultured in osteo-inductive medium), H₂O₂ group and H₂O₂/2-APB treated group were 24.4 ± 1.3%, 76.9 ± 5.9%, 45.2 ± 0.6%, and 62.6 ± 4.1%, respectively. H₂O₂ down-regulated ALP activity compared with positive control cells (cultured in osteo-inductive medium). 2-APB significantly rescued the decrease of ALP activity in H₂O₂-treated cells (P < 0.01) (Fig. 3B, C).

The percentages of the Alizarin red-stained area of the control group, positive control group (cultured in osteo-inductive medium), H₂O₂ group, and H₂O₂/2-APB treated group were 19.1 ± 3.6%, 67.1 ± 3.2%, 26.9 ± 4.8%, and 57.1 ± 1.5%, respectively. Alizarin red staining showed that 2-APB significantly increased H₂O₂-decreased hDPC calcification (P < 0.01) (Fig. 4A, B).

ROS were increased in 100 μM H₂O₂-treated hDPCs (Fig. 5A(a, b). 10 µM 2-APB inhibited the ROS production induced by 100 μM H₂O₂ (Fig. 5A(c)). 10 µM 2-APB did not affect the ROS production(Fig. 5A(d)). FITC fluorescence intensity of the H₂O₂/2-APB treated group was significantly decreased compared to that of the H₂O₂-treated group (P < 0.05) (Fig. 5B).

Discussion

H₂O₂ promotes ROS production in various cells, such as human periodontal ligament cells, human retinal epithelial cells, and vascular endothelial cells [12‐14]. H₂O₂ can induce oxidative destruction of organs and tissues, associated with other inflammatory responses when overexpressed in cells [15]. The effect of H₂O₂ in a living system is dependent on the type of cell, its concentration, its physiological state, and duration of exposure [16, 17]. Deng et al. [18] reported that, like other ROS molecules, a high concentration of H₂O₂ is cytotoxic to cells. In the present study, high-dose H₂O₂ (250, 500 and 1000 μM) significantly reduced the viability of hDPCs. Loss of cell viability is one characteristic of apoptotic cells. Therefore, in the present study, to avoid the influence of apoptosis, H₂O₂ at 100 μM, which is enough to promote ROS production without inducing apoptosis, was used.

Alkaline phosphatase (ALP), involved with the initial phase of dentin matrix biomineralization, promotes dephosphorylation of extracellular matrix proteins, providing inorganic phosphate [19]. In the present study, H₂O₂ decreased ALP activity in hDPCs cultured in osteo-inductive medium. These data are in agreement with those from previous studies [8, 20]. Correlated with ALP activity, the present study also showed decreased ALP mRNA expression by H₂O₂ in hDPCs.

2-APB has been described as a membrane-permeant inhibitor of the IP₃ receptor, and the inhibition of SOCE by 2-APB was taken as evidence for IP₃ receptor activation of calcium release-activated Ca²⁺ channels [21]. It has been reported that the increase in intercellular Ca²⁺ concentration regulates cell functions, such as proliferation, differentiation, and migration [22]. A previous study reported that oxidative stress reduced SOCE, and the decrease was reversed by 2-APB in brain capillary endothelial cells [11]. In the present study, the H₂O₂-decreased human dental pulp cell calcification ability was reversed by 2-APB. In addition, an important criterion for the characterization of odontoblastic cells is their ability to mineralize the collagenous matrix they secrete. In the present study, 2-APB rescued the H₂O₂-decreased amounts of mineralization of the extracellular matrix, judged by Alizarin red staining. Contrary to the present findings, it has been reported that vascular smooth muscle cell calcification was reduced by 2-APB [23, 24]. 2-APB is also used as an inhibitor of transient receptor potential channels (TRPCs), in which Ca²⁺ entry was inhibited directly, rather than as a result of the inhibition of IP₃ [25]. Further experiments are needed to clarify the detailed mechanism underlying the relationships between IP₃, SOCE, TRPC and the calcification ability of human dental pulp cells.

It has been reported that 2-APB reduced ROS production in neutrophils and prevented ROS-induced cardiomyocyte death [26, 27]. The present study showed for the first time that 2-APB reduces ROS production in hDPCs. Therefore, administration of 2-APB may represent a promising therapeutic strategy for the treatment of ROS-related endodontic disease. Further experiments beyond the scope of this paper are required to determine the relationship between ROS and endodontic disease.

In summary, H₂O₂ decreased the calcification ability of human dental pulp cells, and the reduction was reversed by 2-APB. To the best of our knowledge, this is the first study documenting the involvement of IP₃ signaling in the calcification ability of human dental pulp cells. The results of the present study add molecular insight into internal root resorption and may provide a clue to the development of a new therapeutic agent for such resorption in endodontic therapy.

Acknowledgements

This work was in part supported by a Grant-in-Aid for Scientific Research (C) (JP25463218) of the Japan Society for the Promotion of Science.The authors would like to thank the Analysis Center of Life Science of Hiroshima University for the use of facilities.

Declarations

Not applicable.

We, Katsuhiro Takeda (corresponding author), Satomi Shirawachi, Tomoya Naruse, Yohei Takahasi, Jun Nakanishi, Satoru Shindo, and Hideki Shiba hereby declare that we participated in the study and the development of this manuscript. We have read the final version and give our consent for the article to be published in BMC Oral Health.

Not applicable.

Competing interests

The authors of the paper declare that there are no conflicts of interest.

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Titel: Oxidative stress impairs the calcification ability of human dental pulp cells
verfasst von: Satomi Shirawachi
Katsuhiro Takeda
Tomoya Naruse
Yohei Takahasi
Jun Nakanishi
Satoru Shindo
Hideki Shiba
Publikationsdatum: 01.12.2022
Verlag: BioMed Central
Erschienen in: BMC Oral Health / Ausgabe 1/2022
Elektronische ISSN: 1472-6831
DOI: https://doi.org/10.1186/s12903-022-02467-w

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Oxidative stress impairs the calcification ability of human dental pulp cells