Graphical abstract
Highlights
- •
2–20 ppm of eugenol and Zn ions were released from ZOE.
- •
Zn ions had cytotoxicity to dental pulp cells and macrophages.
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Eugenol induced immunomodulatory/anti-inflammatory response to inflamed cells.
Abstract
Objective
The study assessed the cytotoxicity and immunomodulatory/anti-inflammatory effect of extract from zinc oxide–eugenol (ZOE)-based dental materials during setting using immortalized human dental pulp stem cells (IHDPSCs) and mouse bone marrow monocytes (IMBMMs), and identified the responsible extract component.
Methods
In accord with the ISO 10993-12, we extracted a mixture of ZOE cement and sealer after a specified time. The extract was analyzed by two types of mass spectrometry (ICP-MS and GC–MS). Cell viability was evaluated with extract and serial concentrations of ZnCl 2 , ZnSO 4 , and eugenol liquid by WST assay. The immunomodulatory/anti-inflammatory effect of a ZOE component was determined by RT-PCR to detect the downregulatory effect of inflammatory mRNA expression after lipopolysaccharide (LPS)-induced inflammation.
Results
Zn 2+ and eugenol (2–20 ppm) were detected in the ZOE cement and sealer extracts. During the early stage of setting, significant cytotoxicity was observed in IHDPSCs and IMBMMs (p < 0.05). The half maximal effective concentration of Zn 2+ was 5–8 ppm, whereas that of eugenol could not be detected within 80 ppm. After extract treatment, the expression of inflammatory mRNA was significantly lower in inflamed IHDPSCs, but not inflamed IMBMMs, than in the LPS control (p < 0.05). However, eugenol, not Zn 2+ , at 5–20 ppm downregulated inflammatory mRNA expression in the inflamed IMBMMs with and without the exchange of LPS-pretreated medium.
Significance
ZOE was highly cytotoxic, especially during setting, to both cells due to Zn 2+ while the immunomodulatory/anti-inflammatory effect of ZOE was induced by eugenol.
1
Introduction
Dental materials containing zinc oxide–eugenol (ZOE) have been widely used in dentistry for temporary restoration, cementation, and root canal filler because of its easy handability, low cost, excellent cavity-sealing ability, and therapeutic effects including immunomodulatory/anti-inflammatory and a sedative effect on target teeth . However, the use of ZOE has certain disadvantages, such as cytotoxicity, harm to surrounding tissue including dental pulp stem cell and bone marrow derived monocytes, and inhibition of the resin polymerization . Therefore, it has been suggested that the direct contact of ZOE with oral tissue should be prohibited due to irritation to tissue around pulp and oral mucosa .
Indirect contact between ZOE-based dental materials and the involved or surrounding dental tissues was also considered to have adverse potential . Extracts from set ZOE-based materials are cytotoxic to dental pulp stem cell, gingival fibroblasts and keratinocytes in vitro . Furthermore, freshly set ZOE-based materials are more cytotoxic than the final set state because the degree of cytotoxicity decreases substantially as setting takes place. In the clinical setting, freshly set ZOE can come in contact with the dentinal tubes or periapical lesion, so possible adverse effects to the dental pulp are a concern, as well as effects on periodontal ligament and related alveolar bone tissue. However, ZOE extract-induced cytotoxicity to dental pulp cells and bone marrow-derived immunomodulatory cells during or after setting has not been investigated.
Eugenol is a major ingredient of the extract from ZOE-based materials. Eugenol is believed to play a major role in inducing adverse effects . An investigation of the relationship between cytotoxicity and eugenol released from these materials suggested a strong correlation . However, the pattern of eugenol release and cytotoxicity was not always consistent, suggesting the involvement of other factors, such as zinc ion (Zn 2+ ) .
Immunomodulatory/anti-inflammatory properties are expected from ZOE-based materials owing to the presence of eugenol . When the integrity of dental hard tissue is breached, elements of external origin, such as bacteria and noxious environmental stimuli, can invade the pulp or alveolar bone tissue. Acting as antigens, bacterial constituents potentially prompt a variety of immune reactions, and immunomodulatory cells including bone marrow derived monocytes (renamed macrophages) are recruited to the area of the damaged tissue. Therefore, the therapeutic use of ZOE-based dental materials requires regulation of immunomodulatory cells including monocytes (termed as macrophages in tissue) and dental pulp stem cells (DPSCs) which are responsible for pulp or periapical tissues’ innate immunity . When the innate immune response is triggered by pathogen-associated molecules like lipopolysaccharide (LPS), proinflammatory cytokines including interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α) are produced by immunomodulatory cells . It has been believed that an immunomodulatory/anti-inflammatory effect depends on eugenol concentration . However, whether the immunomodulatory/anti-inflammatory properties of eugenol are correlated with its concentration detected in quantitative analyses of extract has not been investigated with monocytes and DPSCs.
Therefore, the aim of this study was to assess the cytotoxicity and immunomodulatory/anti-inflammatory effect of ZOE extract on immortalized human DPSCs dental pulp stem cells (IHDPSCs) and immortalized mouse bone marrow monocytes (IMBMMs) during the setting of ZOE-based material and to identify the component of the extract that induced these effects. The null hypothesis is that cytotoxicity and immunomodulatory/anti-inflammatory effects are induced by both Zn 2+ and eugenol.
2
Materials and methods
2.1
Extract of ZOE cement
Intermediate Restorative Material (IRM; Lot No. 131022; Dentsply, Tulsa, OK, USA) and Tubli-Seal (Lot No. 3-1340; Kerr Corp., Romulus, MI, USA) were chosen from among the various forms of commercially available ZOE-based products because they had been shown to be very cytotoxic in previous studies . Each product was used after being checked for its expiration date and was stored under manufacturers’ recommended conditions throughout the experiment. Extracts from IRM and Tubli-Seal were prepared according to the international standard . Briefly, after powder and liquid were mixed for 2 min on a mixing pad according to the manufacturers’ instructions (23 °C, relative humidity 20%), the mixed specimen was immediately incubated at 37 °C in a model VS-9160C humidified incubator (Vision Scientific, Gyeonggi-Do, Korea). When the desired time from the start of mixing (see Table 1 ) was reached, the hardened specimen was immersed in a sterilized glass bottle containing distilled water (DW). DW was chosen for extraction instead of serum-free cell culture medium to avoid medium-related analytic interference, which could lead to errors between analytic assays and biological tests.
| Code | Contents of additives | Base liquid | Product or chemical |
|---|---|---|---|
| Control | DW | DW | – |
| 3 min | a 3 min from start of mixing | DW | IRM |
| 6 min | a 6 min from start of mixing | DW | IRM |
| 10 min | a 10 min from start of mixing | DW | IRM |
| T3 min | a 3 min from start of mixing | DW | Tubli-Seal |
| T20 min | a 20 min from start of mixing | DW | Tubli-Seal |
| T60 min | a 60 min from start of mixing | DW | Tubli-Seal |
| Z5 | 5 ppm of Zn 2+ | DW | ZnCl 2 |
| Z10 | 10 ppm of Zn 2+ | DW | ZnCl 2 |
| Z20 | 20 ppm of Zn 2+ | DW | ZnCl 2 |
| E5 | 5 ppm of eugenol | DW | Eugenol |
| E10 | 10 ppm of eugenol | DW | Eugenol |
| E20 | 20 ppm of eugenol | DW | Eugenol |
An extraction ratio for the sample was set at 1 mL DW per 0.2 g of mixed cement according to international standards for irregularly shaped specimens . Each sample was extracted at 37 °C for 24 h in a model SI-600R incubator shaker (Jeio Tech, Seoul, Korea) at 80 rpm. After filtration with a sterilized 0.20-μm nitrocellulose filter (HP045AN, Advantec, Tokyo, Japan), the extract was ready for analytical and biological analyses.
Concerning the extraction starting time for IRM, 3 min was set in order to mimic early exposure on dentin when cementation or filling is performed about 3 min after start of mixing. The middle stage and setting stage time was 6 min and 10 min, respectively, based on the manufacturers’ instructions and the relevant ISO standard . For Tubli-Seal, a time of 3 min (T3) was set to mimic early exposure on periapical lesion when filling was performed in root canal, typically about 3 min after start of mixing. A time of 20 min (T20) and 60 min (T60) was chosen to mimic the conditions of the middle stage and setting stage, respectively, based on the manufacturers’ instructions and the ISO standard .
2.2
Analyses of extracts
Gas chromatography–mass spectrometry (GC–MS) was done using a model 7890A–5977A device (Agilent, Santa Clara, CA, USA) and inductively coupled plasma mass spectrometry (ICP-MS) was done using a NexION 300 device (PerkinElmer, Shelton, CT, USA) to analyze the extracts from the ZOE sealer. GC–MS was used to identify and quantify unknown substances (e.g., eugenol, acetic acid, boric acid, and propyl cyclohexane) and ICP-MS was used to detect ionized metals, such as Zn, Ba, Mg, and Fe. Prior to the GC–MS analyses, extracts were freeze-dried for 24 h (Ilshin Biobase, Dongducheon, Gyeonggi-do, Korea) and equal volumes of ethanol (Duksan Pure Chemicals, Gyeonggi-do, Korea) were added. Calibration curves were constructed for the quantitative analysis of these chemicals and ions. All analyses were independently performed in triplicate (n = 3) and the means ± standard deviations were determined.
2.3
Cytotoxicity tests
Cytotoxicity tests were carried out using IHDPSCs and IMBMMs immortalized by transfection with the telomerase catalytic subunit hTERT gene . IHDPSCs and IMBMMs were respectively incubated in Minimum Essential Medium Alpha Medium (α-MEM, LM 008-01; Welgene, Daegu, Korea) and Dulbecco’s modified Eagle’s medium (DMEM, LM 001-17; Welgene) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% antibiotics (penicillin/streptomycin) (Gibco) at 37 °C with a humidified atmosphere containing 5% CO 2 . IHDPSCs and IMBMMs were kindly given by Professor Takashi Takata (Hiroshima University, Japan) and Professor Je-Wook Yu (Yonsei Univerisity, Korea) . IHDPSCs (passages 60–65) and IMBMMs (passages 5–10) were used; these numbers of passages retain biological characteristics compared to primary cells . According to international standards describing in vitro cytotoxicity, cytotoxicity assay was performed . Briefly, 500 mL of 1 × 10 5 cells (2 × 10 5 /mL) was cultured for 24 h in a standard 24-well plate (142471, Nunclon Delta Surface; Thermo Fisher Scientific, Waltham, MA, USA). Five hundred microliters of extractions from mixed IRM cement or serial concentrations of eugenol and ZnCl 2 liquid (Sigma–Aldrich, St. Louis, MO, USA) were placed on each cell cultured in 500 μL of refresh media. After incubation for another 24 h, cell viability was measured using a water-soluble tetrazolium (WST) salt assay (EZ-Cytox, Daeil Lab, Seoul, Korea). In the control group, DW was added. Cell viability results for each test group were expressed as the percentage of the optical density value of each test sample relative to each control sample following the WST assay. All cytotoxicity tests (n = 5) were independently performed in triplicate, and means ± standard deviations were recorded.
2.4
Confocal laser microscopy
Staining to detect living versus dead cells was performed after 24 h of incubation with extract and various concentration of eugenol or ZnCl 2 . Calcein AM and ethidium homodimer-1 (Molecular Probes, Eugene, OR, USA) were added to each well according to the manufacturer’s instructions, and the results were observed using a model LSM 700 confocal laser microscope (Carl Zeiss, Jena, Germany). Viable cells showed intense green fluorescence and dead cells showed bright red fluorescence. Tests were independently performed in triplicate, and representative images were obtained.
2.5
Proinflammatory gene expression by quantitative PCR analysis
The expression of IL-1β, IL-6, IL-8, and TNF-α in normal and inflamed IHDPSCs and IMBMMs was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) to detect anti-inflammatory effects. RT-PCR and qPCR were performed according to the manufacturers’ protocols. Briefly, the total RNA was extracted by TRIzol (Life Technologies, Carlsbad, CA, USA) and 1 μg of total RNA was reverse transcribed to cDNA using the High-Capacity RNA-to-cDNA Kit (Life Technologies) and a model 2720 Thermal Cycler (Life Technologies). The qPCR experiments were performed using the Power SYBR Green PCR Master Mix (Life Technologies) and a real-time PCR system (7300-RT-PCR; Applied Biosystems) according to the manufacturers’ instructions. The primer sequences used are listed in Table 2 . The level of expression of each sample was normalized with beta-actin for IHDPSCs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression for IMBMMs respectively, which showed constant expression among test groups according to the preliminary studies. The results were obtained by the comparative method of relative quantification (n = 4). Analyses were independently performed in triplicate, and representative data are shown as means ± standard deviations.
| No. | Origin | Gene | Target | Forwar primer sequence 5′–3′ | Reverse primer sequence 5′–3′ |
|---|---|---|---|---|---|
| 1 | Human | B-actin | HDPC | AGG ATG CAG AAG GAG ATC ACT G | ATA CTC CTG CTT GCT GAT CCA C |
| 2 | Human | IL-1b | HDPC | GGC AGA AAG GGA ACA GAA AGG | AGT GAG TAG GAG AGG TGA GAG AGG |
| 3 | Human | IL-6 | HDPC | CTG GCA GAA AAC AAC CTG AAC | ATG ATT TTC ACC AGG CAA GTC |
| 4 | Human | IL-8 | HDPC | CTA GGA CAA GAG CCA GGA AG | AGT GTG GTC CAC TCT CAA TC |
| 5 | Mouse | GAPDH | IMBMM | AAC TTT GGC ATT GTG GAA GG | ACA CAT TGG GGG TAG GAA CA |
| 6 | Mouse | IL-1b | IMBMM | GCC CAT CCT CTG TGA CTC AT | AGG CCA CAG GTA TTT TGT CG |
| 7 | Mouse | IL-6 | IMBMM | AGT TGC CTT CTT GGG ACT GA | TCC ACG ATT TCC CAG AGA AC |
| 8 | Mouse | TNF-α | IMBMM | CTG AAC TTC GGG GTG ATC GG | GGC TTG TCA CTC GAA TTT TGA GA |
For the analysis of gene expression in normal IHDPSCs, 500 μL of 2 × 10 5 /mL cell suspension was cultured in the FBS-supplemented medium for 24 h in a standard 24-well plate, and 500 μL of extracts from mixed cement described in Section 2.1 or serial concentrations of eugenol, ZnCl 2 , and ZnPO 4 liquid were added to the cell suspensions. After another 4 h of incubation, gene expression was measured to minimize cytotoxicity induced changes of proinflammatory or housekeeping gene expression .
For the analysis of gene expression in LPS-stimulated IHDPSCs, cells cultured for 24 h were pretreated with 8 μg/mL of LPS for 4 h in the FBS-supplemented medium. After washing, 500 μL of fresh supplemented medium and 500 μL of specified extract or ingredients were added. Another 4 h of incubation took place for RNA preparation.
For analysis of the therapeutic effect with inflamed IMBMMs, IMBMMs were treated with same manner as IHDPSCs except for the concentration and exposure time of LPS, because 8 μg/mL concentrations caused cytotoxicity (∼60%). Furthermore, inflamed IMBMMs easily converted into nearly normal state with simple washing following a 4 h treatment in preliminary experiments, 4 μg/mL of LPS for 6 h was chosen for stimulating IMBMMs.
IMBMMs cultured for 24 h were pretreated for 6 h with 4 μg/mL of LPS in FBS-supplemented medium. After washing, 500 μL of fresh supplemented medium and 500 μL of specified extract or ingredients were added. Another 6 h of further incubation was chosen to match treatment time between LPS and extract or ingredients before RNA preparation. To assess the therapeutic effect of ingredients in absence of washing step compared to similarly inflamed IMBMMs with and without washing condition, which was considered to be more clinically relevant inflamed state, 500 μL of extract or specified ingredients were directly added to LPS (4 μg/mL) pretreated cells. 4 h of further incubation before RNA preparation was chosen based on preliminary study, when IMBMMs showed similar inflamed state.
2.6
Statistical analyses
All data are reported as the means ± standard deviations after at least triplicate experiments. Statistical analysis was carried out using one-way ANOVA with the Tukey method as a post hoc test after confirming normal distribution by Kolmogorov–Smirnov test, where p < 0.05 was considered statistically significant. The IBM SPSS Statistics 20 software program (Armonk, NY, USA) was used for all statistical analyses.
2
Materials and methods
2.1
Extract of ZOE cement
Intermediate Restorative Material (IRM; Lot No. 131022; Dentsply, Tulsa, OK, USA) and Tubli-Seal (Lot No. 3-1340; Kerr Corp., Romulus, MI, USA) were chosen from among the various forms of commercially available ZOE-based products because they had been shown to be very cytotoxic in previous studies . Each product was used after being checked for its expiration date and was stored under manufacturers’ recommended conditions throughout the experiment. Extracts from IRM and Tubli-Seal were prepared according to the international standard . Briefly, after powder and liquid were mixed for 2 min on a mixing pad according to the manufacturers’ instructions (23 °C, relative humidity 20%), the mixed specimen was immediately incubated at 37 °C in a model VS-9160C humidified incubator (Vision Scientific, Gyeonggi-Do, Korea). When the desired time from the start of mixing (see Table 1 ) was reached, the hardened specimen was immersed in a sterilized glass bottle containing distilled water (DW). DW was chosen for extraction instead of serum-free cell culture medium to avoid medium-related analytic interference, which could lead to errors between analytic assays and biological tests.
| Code | Contents of additives | Base liquid | Product or chemical |
|---|---|---|---|
| Control | DW | DW | – |
| 3 min | a 3 min from start of mixing | DW | IRM |
| 6 min | a 6 min from start of mixing | DW | IRM |
| 10 min | a 10 min from start of mixing | DW | IRM |
| T3 min | a 3 min from start of mixing | DW | Tubli-Seal |
| T20 min | a 20 min from start of mixing | DW | Tubli-Seal |
| T60 min | a 60 min from start of mixing | DW | Tubli-Seal |
| Z5 | 5 ppm of Zn 2+ | DW | ZnCl 2 |
| Z10 | 10 ppm of Zn 2+ | DW | ZnCl 2 |
| Z20 | 20 ppm of Zn 2+ | DW | ZnCl 2 |
| E5 | 5 ppm of eugenol | DW | Eugenol |
| E10 | 10 ppm of eugenol | DW | Eugenol |
| E20 | 20 ppm of eugenol | DW | Eugenol |
An extraction ratio for the sample was set at 1 mL DW per 0.2 g of mixed cement according to international standards for irregularly shaped specimens . Each sample was extracted at 37 °C for 24 h in a model SI-600R incubator shaker (Jeio Tech, Seoul, Korea) at 80 rpm. After filtration with a sterilized 0.20-μm nitrocellulose filter (HP045AN, Advantec, Tokyo, Japan), the extract was ready for analytical and biological analyses.
Concerning the extraction starting time for IRM, 3 min was set in order to mimic early exposure on dentin when cementation or filling is performed about 3 min after start of mixing. The middle stage and setting stage time was 6 min and 10 min, respectively, based on the manufacturers’ instructions and the relevant ISO standard . For Tubli-Seal, a time of 3 min (T3) was set to mimic early exposure on periapical lesion when filling was performed in root canal, typically about 3 min after start of mixing. A time of 20 min (T20) and 60 min (T60) was chosen to mimic the conditions of the middle stage and setting stage, respectively, based on the manufacturers’ instructions and the ISO standard .
2.2
Analyses of extracts
Gas chromatography–mass spectrometry (GC–MS) was done using a model 7890A–5977A device (Agilent, Santa Clara, CA, USA) and inductively coupled plasma mass spectrometry (ICP-MS) was done using a NexION 300 device (PerkinElmer, Shelton, CT, USA) to analyze the extracts from the ZOE sealer. GC–MS was used to identify and quantify unknown substances (e.g., eugenol, acetic acid, boric acid, and propyl cyclohexane) and ICP-MS was used to detect ionized metals, such as Zn, Ba, Mg, and Fe. Prior to the GC–MS analyses, extracts were freeze-dried for 24 h (Ilshin Biobase, Dongducheon, Gyeonggi-do, Korea) and equal volumes of ethanol (Duksan Pure Chemicals, Gyeonggi-do, Korea) were added. Calibration curves were constructed for the quantitative analysis of these chemicals and ions. All analyses were independently performed in triplicate (n = 3) and the means ± standard deviations were determined.
2.3
Cytotoxicity tests
Cytotoxicity tests were carried out using IHDPSCs and IMBMMs immortalized by transfection with the telomerase catalytic subunit hTERT gene . IHDPSCs and IMBMMs were respectively incubated in Minimum Essential Medium Alpha Medium (α-MEM, LM 008-01; Welgene, Daegu, Korea) and Dulbecco’s modified Eagle’s medium (DMEM, LM 001-17; Welgene) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% antibiotics (penicillin/streptomycin) (Gibco) at 37 °C with a humidified atmosphere containing 5% CO 2 . IHDPSCs and IMBMMs were kindly given by Professor Takashi Takata (Hiroshima University, Japan) and Professor Je-Wook Yu (Yonsei Univerisity, Korea) . IHDPSCs (passages 60–65) and IMBMMs (passages 5–10) were used; these numbers of passages retain biological characteristics compared to primary cells . According to international standards describing in vitro cytotoxicity, cytotoxicity assay was performed . Briefly, 500 mL of 1 × 10 5 cells (2 × 10 5 /mL) was cultured for 24 h in a standard 24-well plate (142471, Nunclon Delta Surface; Thermo Fisher Scientific, Waltham, MA, USA). Five hundred microliters of extractions from mixed IRM cement or serial concentrations of eugenol and ZnCl 2 liquid (Sigma–Aldrich, St. Louis, MO, USA) were placed on each cell cultured in 500 μL of refresh media. After incubation for another 24 h, cell viability was measured using a water-soluble tetrazolium (WST) salt assay (EZ-Cytox, Daeil Lab, Seoul, Korea). In the control group, DW was added. Cell viability results for each test group were expressed as the percentage of the optical density value of each test sample relative to each control sample following the WST assay. All cytotoxicity tests (n = 5) were independently performed in triplicate, and means ± standard deviations were recorded.
2.4
Confocal laser microscopy
Staining to detect living versus dead cells was performed after 24 h of incubation with extract and various concentration of eugenol or ZnCl 2 . Calcein AM and ethidium homodimer-1 (Molecular Probes, Eugene, OR, USA) were added to each well according to the manufacturer’s instructions, and the results were observed using a model LSM 700 confocal laser microscope (Carl Zeiss, Jena, Germany). Viable cells showed intense green fluorescence and dead cells showed bright red fluorescence. Tests were independently performed in triplicate, and representative images were obtained.
2.5
Proinflammatory gene expression by quantitative PCR analysis
The expression of IL-1β, IL-6, IL-8, and TNF-α in normal and inflamed IHDPSCs and IMBMMs was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) to detect anti-inflammatory effects. RT-PCR and qPCR were performed according to the manufacturers’ protocols. Briefly, the total RNA was extracted by TRIzol (Life Technologies, Carlsbad, CA, USA) and 1 μg of total RNA was reverse transcribed to cDNA using the High-Capacity RNA-to-cDNA Kit (Life Technologies) and a model 2720 Thermal Cycler (Life Technologies). The qPCR experiments were performed using the Power SYBR Green PCR Master Mix (Life Technologies) and a real-time PCR system (7300-RT-PCR; Applied Biosystems) according to the manufacturers’ instructions. The primer sequences used are listed in Table 2 . The level of expression of each sample was normalized with beta-actin for IHDPSCs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression for IMBMMs respectively, which showed constant expression among test groups according to the preliminary studies. The results were obtained by the comparative method of relative quantification (n = 4). Analyses were independently performed in triplicate, and representative data are shown as means ± standard deviations.
