1.Background

Oral pathologies are primarily infectious diseases related to the immune system. Altered immune response due to cell damage in the dental pulp and surrounding tissues lead to a chronic, mild inflammatory state called inflamm- aging¹⁾. During inflammatory aging, the levels of inflam-matory cytokines such as interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-a increase. These cytokines enhance immune activity, resulting in dysfunction of den-dritic cells, neutrophils, macrophages, and fibroblasts²⁾. The accumulation of senescent cells enhances aging by causing chronic inflammation and metabolic disorders. Excessive secretion of inflammatory mediators by senescent cells with an increased senescence-associated secretion phenotype (SASP) further reduces the efficiency of the immune res-ponse³⁾. The cytotoxic cells that accumulate in the oral tis-sues secrete enzymes, such as matrix metalloproteinases, serine proteases, and cathepsins, which are involved the degradation of the extracellular matrix. As a result, more matrix is exposed to these enzymes, resulting in incresed oral tissue destruction⁴⁾.

Composite resins and dental adhesives are commonly used in clinical dentistry to treat tooth damage caused by dental caries, fractures, and erosion. Common dental com-posite resins and adhesives for tooth restoration include monomers such as 2-hydroxyethyl methacrylate (HEMA), tetraethylene glycol dimethacrylate (TEGDMA), and bis-phenol glycidyl methacrylate (Bis-GMA)⁵⁾. In particular, 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) is the main ingredient of multiple resin-based dental mate-rials such as adhesive systems and bonding resin cements, and it is the most widely used acidic monomer⁶⁾. Although various dental materials containing acidic resin monomers are commonly used in dental clinics, research on their toxicity and effects on physiological functions in the oral environment is lacking. Therefore, this study aimed to inves-tigate the inflamm-aging mechanism of the commonly used acidic resin monomer 10-MDP.

2.Objectives

There is a lack of understanding of the cellular aging me-chanisms to involved in tissue destruction caused by inflam-matory processes in oral diseases. Therefore, the objective of the present study was to investigate the cytotoxic effect of 10-MDP, which is a functional monomer widely used in dental materials, on osteoblasts-like cells and to identify the signaling pathways involved in the cellular senescence.

1.Study design

1) Cell culture and cytotoxicity test

MC3T3-E1 osteoblast-like cells were cultured in a-MEM (Gibco, Life Technologies, Grand Island, NY, USA) sup-plemented with 10% inactivated fetal bovine serum (Gibco) at 37°C in 5% CO₂. After overnight incubation, the cells were seeded into a culture dish and incubated with various concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM) of 10-MDP (Watson International Ltd., Kunshan, China). For cell culture experiments, 10-MDP was directly dissolved in the cell culture medium. Cell viability was measured by WST-8 assays (MediFab, Seoul, Korea), according to the manufacturer’s instructions. The fluorescence signal due to the oxidation of CM-H₂DCFDA dye (Molecular Pro-bes, Eugene, OR, USA) was used to measure the level of intracellular free radicals such as reactive oxygen species (ROS). After treatment with the resin monomer, MC3T3- E1 cells were washed with phosphate-buffered saline (PBS) and loaded with 10 mM CM-H₂DCFDA PBS for 15 minutes at 37°C in the dark. The dichlorofluorescein (DCF) fluorescence of the cells in each test group was assessed using a FACSCalibur^TM flow cytometer (BD Biosciences, San Jose, CA, USA), with excitation and emission wave-lengths of 480 and 530 nm, respectively. The data are pre-sented as mean±standard deviation in triplicate experiments.

2) Detection of molecular markers of aging

Cells were cultured with 0.1 mM 10-MDP for 24 hours. Proteins were extracted from harvested cells using the reco-mmended pro-prep protocol (iNtRON Biotechnology, Seon-gnam, Korea). The protein from each sample was sub-jected to sodium dodecyl sulphate (SDS)-polyacrylamide gel, electrophoresis and then transferred to a membrane by electroblotting. The membranes were incubated for four hours at room temperature (RT) with primary antibodies against p53, p16, p21, and b-actin (Cell Signaling Tech-nology, Danvers, MA, USA), followed by secondary antibody for one hour at RT. Immunoreactive bands were visualized using a chemiluminescence system (Bio-Rad, Hercules, CA, USA). Signals were detected using Micro-Chemi (DNR Bio-Imaging, Modi’in-Maccabim-Re’ut, Israel). Senescence-associated beta-galactosidase (SA-b-gal) acti-vity was determined using a SA-b-gal staining kit (Cell Signaling Technology) according to the manufacturer’s ins-tructions. Senescent cells were identified as blue-stained cells by standard light microscopy at 100× magnification. The representative data is representative of three inde-pendent experiments.

3) Gene expression analysis by RT-PCR

To investigate the effects of 0.1 mM 10-MDP on mole-cular markers of aging in MC3T3-E1 cells, mRNA levels were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells were pretreated with the JNK inhibitor SP600125 (5 mM; Calbiochem, La Jolla, CA, USA), ERK1/2 inhibitor U0126 (5 mM; Calbiochem), or p38 inhibitor SB203580 (10 mM; Calbiochem) for 1 hour, and then treated with 10-MDP for 24 hours. Total RNA was extracted using TRIzol^Ⓡ reagent (Welgene Inc., Daegu, Korea), and cDNA was prepared using AccuPower^Ⓡ RT PreMix (Bioneer, Daejeon, Korea). The RT-generated DNA (2∼5 ml) was then amplified using AccuPower^Ⓡ PCR PreMix (Bioneer). The following primers were used: TNF-a, forward (F): 5’-CCCAAGGCTATAAAGCGG-3’, reverse (R): 5’-CCCAAGGGCTATAAGGCGG-3’; IL-1b, F: 5’-TGCCACCTTTTGACAGTGATG-3’, R: 5’-GGAG CCTGTAGTGCAGTTGT-3’; IL-6, F: 5’-GCCTTCTTG GGACTGATGCT-3’, R: 5’-TGTGACTCCAGCTTATCT CTTGG-3’; IL-8, F: 5’-TGCTTTTGGCTTTGCGTTGA- 3’, R: 5’-GTCAGAACGTGGCGGTATCT-3’; glyceralde-hyde-3-phosphate dehydrogenase (GAPDH), F: 5’-GCAT CTTCTTGTGCAGTGCC-3’, R: 5’-TACGGCCAAATC CGTTCACA-3’. The PCR thermocycling conditions were: 94°C for 30 seconds, followed by 30 cycles of denatu-ration at 95°C for 15 seconds and annealing at 62°C for 30 seconds. Representative data were obtained from three independent experiments.

1.Cytotoxicity effects of the resin monomer 10-MDP

To determine whether 10-MDP affected the viability of MC3T3-E1 cells, we assessed its cytotoxicity by WST-8 assay (Fig. 1A). 10-MDP exerted concentration-dependent (0.05∼1 mM) cytotoxicity. 10-MDP exposure resulted in cell viabilities of 95% and 93% at 0.05 and 0.1 mM, res-pectively. Concentrations of 10-MDP up to 0.1 mM did not exert significant cytotoxicity, while 0.2 mM resulted in a relatively high level of cytotoxicity. Because ROS are known to contribute to cellular senescence⁴⁾, we the ROS levels in MC3T3-E1 cells exposed to 10-MDP. As shown in Fig. 1B, as the 10-MDP concentration increased the fluorescence of DCF in MC3T3-E1 cells increased, indi-cating that intracellular ROS levels were higher than those of the control groups. Therefore, based on the results of both experimental methods, the minimal toxic concent-ration of 10-MDP (0.1 mM) was used.

Fig. 1. Effects of 10-MDP on cytotoxicity and reactive oxygen species (ROS) production in MC3T3-E1 osteoblast-like cells. Cells were treated with 10-MDP (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM) for 24 hours. (A) Cell viability was estimated by WST-8. (B) Production of intracellular ROS is presented as the mean fluorescence intensity of DCF determined by flow cytometry. The values are expressed as mean±standard deviation from triplicate experiments. 10-MDP: 10-Methacryloyloxydecyl dihydrogen phosphate.

2.Involvement of mitogen-activated- protein-kinase pathways in 10-MDP-induced inflamm-aging responses

Because cell senescence or apoptosis occur as a result of cell cycle arrest, we investigated the expression of cell cycle-related molecules. Exposure of MC3T3-E1 cells to 100 mM 10-MDP significantly increased the protein expre-ssion levels of the cell cycle-related senescence markers p53, p16, and p21 (Fig. 2A). When SA-b-gal staining was performed to confirm the degree of cellular senescence, there was a clear increase in SA-b-gal positive cells (Fig. 2B). To confirm the role of mitogen-activated-protein- kinase (MAPK) in senescence-associated cytokine produ-ction in 10-MDP-treated MC3T3-E1 cells, the cells were pretreated with JNK, ERK1/2, p38 inhibitors. Treatment with 0.1 mM 10-MDP induced secretion of TNF-a, IL-1b, IL-6 and IL-8, and their expression was suppressed by MAPK (JNK, ERK1/2, and p38) inhibitors (Fig. 2C).

Fig. 2. Effects of 10-MDP on signal transduction pathways. Cells were cultured with or without 0.1 mM 10-MDP for 24 hours. (A) Expression of cell cycle-related protein. (B) Senescence‐associated β‐galactosidase (SA‐b‐gal) staining. Scale bar=50 mm (C) Production of senescence-associated cytokines. Cells were pretreated with MAPK inhibitors (JNK inhibitor SP600125, ERK1/2 inhibitor U0126, or p38 inhibitor SB203580) for 1 hour and then treated with 0.1 mM 10-MDP for 24 hours. Data of representative of three independent experiments are shown. 10-MDP: 10-Methacryloyloxydecyl dihydrogen phosphate, TNF: tumor necrosis factor, IL: interleukin.

1.Interpretation

Dental resin-based materials are widely used in dental resin restorations, adhesion of prosthetics, and denture manu-facturing, and excessive use of composite resin cement plays an important role in the development of gingivitis and periodontitis⁷⁾. Therefore, important to evaluate the cyto-toxicity of resin monomers toward bone cells. In this study, we found that cellular senescence, which is a state of cel-lular damage, was activated in osteoblasts in response to the cytotoxicity induced by dental resin monomers. To our knowledge, this is the first study to demonstrate that 10- MDP-induced cellular senescence activation is associated with the MAPK pathway in osteoblasts.

2.Key results and comparison

Inflammation is a reaction that occurs when an infection or wound occurs in a tissue to protect it from damage to return it and return the tissue to its normal state. Chronic inflammation is a low-grade, persistent inflammatory con-dition that occurs when acute inflammation does not heal well or when inflammation occurs repeatedly in the same area, and the reaction to stimulation or damage progresses slowly¹⁾. Chronic inflammation of the oral cavity due to periodontal disease has been reported to lead to damage and dysfunction of tissues and organs throughout the body (a direct or indirect cause of several age-related diseases such as periodontitis, cardiovascular disease, type 2 dia-betes, cancer, and neurodegenerative disorders)^8,9).

TEGDMA and HEMA monomers can cause cytotoxicity and allergic reaction in gingival cells^10,11). Previous studies have reported that the negative effects of minimally toxic concentrations of TEGDMA and HEMA on the differen-tiation of dental pulp cells into odontoblasts can reduce the mineralization process, which is an important biological response of pulp tissue^6,12). Bis-GMA is an endocrine dis-ruptor, and animal studies have shown that it has repro-ductive, developmental and systemic toxic effects even at low doses^11,13). These monomers have been shown to induce cellular toxicity and apoptosis in human dental pulp cells through intracellular glutathione depletion and ROS pro-duction. In our study, to investigate the effect of 10-MDP on periodontal inflamm-aging, MC3T3-E1 osteoblast-like cells were treated with 0.05∼1 mM 10-MDP. Kim et al.⁶⁾ reported that 10-MDP inhibited human pulp cell prolifera-tion and increased inflammatory response in a concentration- dependent manner in the range of 0.25 to 0.2 mM. Com-pared with previous studies, 10-MDP result in a similar level of cytotoxicity in the MC3T3-E1 osteoblast-like cells used in this study (Fig. 1A).

A previous study reported that periodontal tissue con-tinuously releases ROS under chronic inflammatory con-ditions, which can damage DNA and cell structure in neighboring cells⁴⁾. Damage from free radicals accumu-lates, causing mutations and tissue damage that trigger aging. Chronic inflammation can also lead to the accumulation of senescent cells, which stop dividing but remain active to produce inflammatory substances¹⁴⁾. Senescent cells acce-lerate the aging process by damaging the surrounding tis-sues and by producing signals that impede cell function¹⁵⁾. The level of ROS increased in 10-MDP-treated cells (Fig. 1B), which is consistent with previous evidence that dental resin monomers cause cytotoxicity in periodontal cell¹⁶⁾.

Cellular senescence is a state of permanent cell cycle arrest and contributes to a pro-inflammatory environment, resulting in tissue dysfunction and reduced regenerative capacity. As a result of the cell cycle arrest, the cells be-come senescent, transform into cancer cells, and undergo apoptosis. The aging marker p53 accumulates in damaged and senescent cells¹⁷⁾. p16 is a marker of apoptosis inde-pendent of p53 and is a known aging marker, whereas p21 is a marker of apoptosis dependent on p53 and has been reported to be an aging marker that promotes aging¹⁸⁾. Consistent with previous results, our findings revealed that the expression levels of the well-known senescence marker of p53, p16, and p21 were increased in the 10-MDP group, indicating that the resin monomer caused cellular sene-scence (Fig. 2A). These results are consistent with the microscopy results and further consistent with increased b-gal activity (Fig. 2B).

However, it is unclear how cellular senescence is acti-vated mechanistically by 10-MDP. Recent studies have re-vealed a key role of MAPK-dependent aging in perio-dontitis¹⁹⁾. Our previous findings showed that autophagy plays an important role in cellular senescence²⁰⁾. Therefore, we investigated the role of MAPK signaling in 10-MDP- induced cellular senescence. Senescent fibroblasts have been reported to release several inflammatory factors, inclu-ding IL-1a, IL-1b, IL-6, and IL-17^14,21). By confirming the SASP signature, we found that IL-1b, TNF-a, and IL-6 were significantly increased in 10-MDP-treated senescent cells (Fig. 2C). We also found that osteoblast senescence was rescued by a MAPK inhibitor, suggesting that the sene-scence induced by 10-MDP is regulated by MAPK. Recent evidence that senescent osteocytes accumulate in murine alveolar bone and display p38 MAPK activation in vivo¹⁹⁾ lends support to our findings are plausible. Therefore, the senescence of osteoblasts in periodontal tissue has been suggested to be evidence of inflammatory cascades.

3.Limitations

Our study had several limitations. This study relied on only a limited number of molecular markers to confirm cellu-lar senescence, and the immune network with surrounding cells, which is important in inflamm-aging, was not inves-tigated. Furthermore, in vivo experiments are insufficient to confirm details of the underlying mechanism. Never-theless, our findings provide a scientific basis for novel molecular targeting of inflamm-aging for dental monomer- related cellular damage.

4.Conclusion

Collectively, our findings indicate that exposure to dental resin monomers results in cell damage and oxidative stress, thereby leading to cellular aging. Additionally, our results indicate that 10-MDP-mediated cellular senescence in osteoblasts is regulated by the MAPK signaling pathway.

None.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Ethical Approval

Not applicable.

Author contributions

Conceptualization: Sang-Im Lee. Data acquisition: Ju Yeon Ban and Sang-Im Lee. Formal analysis: Ju Yeon Ban and Sang-Im Lee. Funding: Sang-Im Lee. Supervision: Sang-Im Lee. Writing - original draft: Ju Yeon Ban and Sang-Im Lee. Writing - review & editing: Sang-Im Lee. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2022R1F1A1076563).

Data availability

The data will be available by corresponding authors upon genuine request.