Cytotoxicity and anti-inflammatory effects of zinc ions and eugenol during setting of ZOE in immortalized human oral keratinocytes grown as three-dimensional spheroids

Graphical abstract

Highlights

  • Eugenol and Zn ions were released from ZOE.

  • Zn ions had cytotoxicity to oral keratinocyte.

  • 2D and 3D culture have different biological effects.

  • Eugenol and Zn ions induced anti-inflammatory response to oral keratinocyte.

Abstract

Objectives

The objective of this study is to assess the cytotoxic and anti-inflammatory effects of ZOE cement during setting in two-dimensional (2D) or three-dimensional (3D) cultures of immortalized human oral keratinocytes (IHOKs) with determining the extract components responsible for these effects.

Methods

Extracts of mixed ZOE at different stages of setting were analyzed by a digital pH meter, ICP-MS, and GC–MS. Serial concentrations of extract and their mixture of ZnCl 2 , ZnSO 4 ·H 2 O, and eugenol liquid were added to the 2D and 3D IHOK cultures to determine the half maximal effective concentration in investigating the cause of cytotoxicity by means of WST assay and to investigate mRNA expression levels of inflammatory cytokines by RT-PCR.

Results

Zn 2+ and eugenol (4–19 ppm) were detected in the extracts. In the early setting stage, significant cytotoxicity was observed in the 2D and 3D IHOK cultures ( P < 0.05). The EC50 of Zn 2+ from ZnCl 2 was 5–44 ppm in both cultures, whereas the EC50 of eugenol was not detectable under 100 ppm. Along with the lower levels of inflammatory cytokine gene expressions in the extract, treatment of the 2D IHOKs with Zn 2+ alone and treatment of the 3D IHOKs with Zn 2+ plus eugenol resulted in significantly lower expression levels of IL-1β, IL-6, and IL-8 ( P < 0.05).

Significance

The cytotoxic effect of ZOE on IHOKs was greater during the setting stage owing to the presence of Zn 2+ . The anti-inflammatory response to ZOE was induced by a combination of Zn 2+ and eugenol. Cytotoxic and anti-inflammatory effects differed between the 2D and 3D IHOK cultures.

Introduction

Zinc oxide–eugenol (ZOE) cement has been widely used in dentistry for temporary restorations or cementations because of its easy handling, low cost, excellent cavity-sealing ability, antibacterial properties, and sedative effects on sensitive teeth . ZOE is a material created by combining zinc oxide (ZnO) powder with eugenol liquid extracted from the oil of cloves. An acid–base reaction occurs when hydrated ZnO is mixed with eugenol, resulting in the formation of a long crystal matrix of zinc eugenolate chelate . However, ZOE is cytotoxic and inhibits the polymerization of resin cement . Zinc oxide–non-eugenol (ZONE) cement has been developed as an alternative to ZOE and is more compatible with resin cement in terms of bonding strength, resin polymerization, and lower cytotoxicity .

A few reports have documented the adverse effects of ZOE, such as increased inflammation in oral mucosal tissue and the recession of gingiva adjacent to the treated tooth , possibly owing to direct or indirect contact between the cement and the mucosa. Direct contact results in soft-tissue irritation and is therefore avoided; indirect contact with dental material extracts has been reported to have adverse effects on the oral mucosa or gingiva . In addition, extracts from set ZOE cement have exhibited soft-tissue cytotoxicity in vitro . In its freshly set state, ZOE is more cytotoxic than in its final set state, and the degree of cytotoxicity decreases substantially as setting takes place. In the clinical situation, the setting time can be accelerated by exposing ZOE to saliva before the final stage of setting; consequently, the cytotoxicity of the ZOE extract is also increasing up. However, no studies have reported data on cytotoxicity during setting to mimic clinical circumstance.

Eugenol, a component of ZOE extracts, has been considered to be a primary factor inducing adverse effects on the oral mucosa . The production or acceleration of erythema as well as ulceration has been associated with eugenol exposure . One study showed a strong correlation between cytotoxicity and the release of eugenol from ZOE extracts ; however, the pattern of eugenol release relative to toxicity was not consistent, suggesting that other factors might be involved (e.g., the presence of zinc ions [Zn 2+ ]). Although the use of ZOE cement has been reported to have adverse inflammatory effects on the oral mucosa , eugenol has also been reported to have anti-inflammatory effects on human dental pulp cells and human skin mucosa . Because the adverse (or, conversely, the therapeutic) effects of eugenol were found to depend on the concentration applied , it is necessary to determine the concentrations of all components of the ZOE extract to evaluate their cytotoxic and anti-inflammatory effects on oral keratinocytes, which make up the outer layer of cells of the oral mucosa .

An evaluation of the biocompatibility of dental materials (e.g., through in vitro cytotoxicity assays) is an essential step in determining whether such materials are suitable for clinical use . However, previous studies of ZOE cements have involved animal-based cell cytotoxicity tests with pre-set cements and thus lack clinical relevance to human oral cells . Cytotoxicity levels are known to depend on the type of cell line involved, and the levels differ between animal and human cells . Because ZOE cement would be exposed to human oral saliva, the ZOE extracts would affect human oral keratinocytes in the mucosa during setting.

The biocompatibility of ZOE cement is commonly evaluated using two-dimensional (2D) cell monolayer structures, in keeping with methods established by the International Organization for Standardization (ISO), using a mouse fibroblast cell line (L929) . However, these structures are not physiologically relevant to complex three-dimensional (3D) tissues. The results of cytotoxicity assays involving 2D structures may overestimate susceptibility, thereby exaggerating the cytotoxic effects of ZOE and limiting the clinical applications of ZOE cements . Recently, studies have shown that tissue-engineered 3D oral mucosal models are biocompatible owing to their physiological similarity to the structure in humans . However, high cost and complex preparation have limited the use of such models in evaluating the cytotoxicity of dental materials . As an alternative, 3D spheroid culture systems have been developed in which small cell aggregates are cultured in suspension. Because of their high degree of clinical and biological relevance to natural tissues, spheroid cultures are being widely used to evaluate the cytotoxicity and effectiveness of drugs in vitro .

Cytokines are potent local mediators of inflammation and are produced by a variety of cells . The cytokines expressed in epidermal epithelial cells (keratinocytes) have been investigated as potential markers of cellular injury that induce soft-tissue damage through inflammation. These cytokines include interleukin (IL)-1β, IL-6, and IL-8 . Owing to the presence of eugenol, ZOE is known to have anti-inflammatory effects . However, its protective effects against inflammatory cytokines in oral keratinocytes have not been studied.

The aim of this study was to assess the cytotoxic and anti-inflammatory effects of ZOE using 2D and 3D cultured immortalized human oral keratinocytes (IHOKs) and to determine the component(s) of the ZOE extract responsible for inducing such effects. According to our first null hypothesis, the cytotoxic and anti-inflammatory effects observed in the spheroidal 3D model do not differ significantly from those observed in the 2D monolayer culture system. Our second null hypothesis states that the eugenol extracts from ZOE significantly induce cytotoxic and anti-inflammatory effects in human oral keratinocytes.

Materials and methods

Extract of ZOE cement

We selected IRM ® (intermediate restorative material) (Lot No. 131022, Dentsply, Tulsa, OK, USA) from among the various commercially available ZOE cements owing to its high cytotoxicity . The IRM ® was stored under conditions recommended by the manufacturer, and IRM ® extracts were prepared in keeping with international standards . Briefly, the IRM ® powder and liquid were mixed for 2 min on a mixing pad according to the manufacturer’s instructions (23 °C, relative humidity 20%). The mixed cement solutions were incubated at 37 °C in a humidified incubator (VS-9160C) (Vision Scientific, Daejeon, Korea) for predetermined times, as listed in Table 1 , after which the mixed cement was added to a sterilized glass bottle containing distilled water (DW). The details of the mixing and incubation times are shown in Table 1 . After cementations or fillings, extract acting could be performed at the early, middle, or final stage of setting, which described extract starting point at 3, 6, or 10 min after start of mixing, respectively in this study, after the extracts were prepared according to the manufacturer’s instructions and to ISO standards . The 1 mL per 0.2 g sample extraction ratio between the mixed cement and DW was set according to international standards . Each cement was then extracted in an incubated shaker (SI-600R) (Jeio Tech, Seoul, Korea) at 37 °C at 80 rotations per minute for 24 h. Afterward, the samples were filtered using sterilized, 0.20-μm nitrocellulose filters (HP045AN) (Advantec, Tokyo, Japan), and same volume of filtered extract or the extract diluted with DW was added to IHOKs cultured media in each well of 24-well plates. The final concentrations of extracts were 50%, 25%, 12.5%, and 6.25%. Details about incubation with IHOKs were described in Section 2.3 .

Table 1
Eugenol and acetic acid concentration in extract.
Clinical condition Mixing/incubating time Start of extract (after start of mixing) Code Eugenol (ppm) Acetic acid (ppm)
On set of cementaion/filling 2 min/1 min 3 min 3 min 14.2 ± 2.3 a ND
Middle of setting 2 min/4 min 6 min 6 min 7.2 ± 1.4 b ND
After setting 2 min/8 min 10 min 10 min 4.4 ± 1.2 c ND
Results are shown as means ± standard deviations. Significant differences exist among different letters ( P < 0.05, n = 3). ND: not detected. Experiments were repeatedly performed in triplicate and representative data are shown.

Analysis of extracts

Gas chromatography–mass spectrometry (GC–MS) (7890A-5977A, Agilent, Atlanta, GA, USA) and inductively coupled plasma-mass spectrometry (ICP-MS) (NexION 300, PerkinElmer, Shelton, CT, USA) were used to analyze the extracts. GC–MS was used to identify and quantify unknown substances (i.e., eugenol and acetic acid), and ICP-MS was used to detect ionized metal ions such as Zn 2+ , Mg 2+ , and Fe 2+ . Prior to the GC–MS analyses, extracts were freeze-dried for 24 h (Ilshin Biobase, Gyeonggi-do, Korea), and equal volumes of ethanol (Duksan Pure Chemicals, Gyeonggi-do, Korea) were added to the freeze-dried powder. For quantitative analysis, 0, 5, 10, 20, and 40 ppm of eugenol in ethanol were used to derive a calibration curve. A digital pH meter (Orion 4-Star) (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the pH of the extract in culture media. All analyses were independently performed in triplicate, and the means ± standard deviations were determined.

Cytotoxicity tests

Cytotoxicity tests were performed based on the ISO standards (ISO 3107, 10993-5, 12). Briefly, 5 × 10 4 cells were cultured in standard 24-well plates (142471, Nunclon Delta Surface) (Thermo Fisher Scientific, Waltham, MA, USA) for the monolayer 2D model and in specialized 24-well plates (174930, Nunclon Sphera) (Thermo Fisher Scientific) for the spheroidal 3D structures in 500 μL of culture medium for 24 h (Fig. S1). The Nunclon Sphera surface is designed to cause minimal cell attachment with minimal extracellular matrix protein binding to the well plate surfaces and, consequently, supports the formation of 3D spheroids.

Mixed cement extracts, their dilutions in DW, eugenol dilutions (Sigma–Aldrich, St. Louis, MO, USA), ZnCl 2 (Sigma–Aldrich), and ZnSO 4 ·H 2 O (Sigma–Aldrich) liquid were added to each well. The ratio of cell culture medium-to-additive (extract, diluted extract, and various concentrations of chemical liquid) was set at 0.5. Therefore, 250 μL of the cement extracts was added to 250 μL of cell culture media, indicating a 50% final concentration, and 250 μL of serially diluted extract by DW was added at 25%, 12.5%, and 6.25% final concentrations. Various concentrations (0, 5, 10, 20, 40 ppm) of eugenol, Zn 2+ from ZnCl 2 , and ZnSO 4 ·H 2 O liquid and their mixture were added to each well. Sodium dodecyl sulfate (SDS) 0.001% (Sigma–Aldrich) was used as the positive control group. After 24-h incubation, cell viabilities were measured using a water-soluble tetrazolium (WST) salt assay (EZ-Cytox) (Daeil Lab, Seoul, Korea). Only 250 μL of DW was added to the control wells. L929 cells (mouse fibroblast CCL-1, ATCC) and immortalized human oral keratinocytes (IHOKs) were cultured in RPMI 1640 (LM011-01, Welgene, Daegu, Korea) and DMEM/F-12 3:1 mixture (LM 030-01, Welgene), respectively, with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco) in a humidified incubator at 37 °C containing 5% CO 2 . To compare the cytotoxicity in IHOKs and L929 cells (the ISO 10993 recommended cell line for cytotoxicity tests), we carried out cytotoxicity testing with L929 . Cell viabilities were expressed as the percentage of the optical density value of each test sample relative to their respective controls. All cytotoxicity tests ( n = 5) were independently performed in triplicate, and the means ± standard deviations were calculated.

Confocal laser microscopy

Fluorescence staining was performed after 24-h incubation with the various cement extracts (50%) and the DW control. Calcein AM and ethidium homodimer-1 (Molecular Probes, Eugene, OR, USA) were added to each well, according to the manufacturer’s instructions, and observed under a confocal laser microscope (LSM700, Carl Zeiss, Thornwood, NY, USA). Intense green fluorescence was observed in live cells, and bright red fluorescence was observed in dead cells. Tests were independently performed in triplicate. To obtain more clearly focused photographic images, the magnification of the confocal laser microscope was adjusted differently for the 2D and 3D cultured cells. The average value between the longest and the shortest diameter of the 3D IHOK spheroids was calculated for determining spheroid size before and after treatment ( n = 5) by Image J software, version 1.50b (National Institutes of Health, Bethesda, MD, USA).

Gene expression of interleukin on quantitative PCR analysis

After a 4-h incubation of IHOKs with 50% extract or various concentrations of liquid (see Section 2.3 ), mRNA expression levels of IL-1β, IL-6, and IL-8, normalized to β-actin, were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) analyses of the IHOKs. Briefly, total RNA was extracted by TRIzol (Life Technologies, Carlsbad, CA, USA), and 1 μg of total RNA was reversely transcribed to cDNA using oligo-dT primer (Qiagen, Venlo, The Netherlands), a premixture for reverse transcription (AccuPower RT PreMix) (Bioneer, Gyeonggi-do, Korea), and a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Real-time qPCR experiments were performed using a Power SYBR Green PCR Master Mix (Applied Biosystems) and real-time PCR equipment (StepOnePlus) (Applied Biosystems), according to the manufacturer’s instructions. The primer sequences used were β-actin forward 5′-AGG ATG CAG AAG GAG ATC ACT G-3′ and β-actin reverse 5′-ATA CTC CTG CTT GCT GAT CCA C-3′, IL-1β forward 5′-GGC AGA AAG GGA ACA GAA AGG-3′ and IL-1β reverse 5′-AGT GAG TAG GAG AGG TGA GAG AGG-3′, IL-6 forward 5′-CTG GCA GAA AAC AAC CTG AAC-3′ and IL-6 reverse 5′-ATG ATT TTC ACC AGG CAA GTC-3′, and IL-8 forward 5′-CTA GGA CAA GAG CCA GGA AG-3′ and IL-8 reverse 5′-AGT GTG GTC CAC TCT CAA TC-3′. After confirming qPCR efficiency using no template controls, positive controls (0.001% SDS treatment), and no amplification controls, according to the suggested guidance , mRNA expression levels of each sample ( n = 4) were normalized to a housekeeping gene (β-actin), and the relative fold change in expression was automatically calculated, with a value of 2 −ΔΔCt with respect to the control group, using StepOne software, version 2.3 (Applied Biosystems). Analyses were independently performed in triplicate, and data are shown as representative means ± standard deviations.

Statistical analysis

All data are reported as the means ± standard deviations of at least triplicate experiments. Statistical analyses were carried out using the SPSS Statistics 20 program (Armonk, NY, USA) using one-way or two-way ANOVA tests, along with Tukey post hoc tests. Comparisons were considered significant at P < 0.05.

Materials and methods

Extract of ZOE cement

We selected IRM ® (intermediate restorative material) (Lot No. 131022, Dentsply, Tulsa, OK, USA) from among the various commercially available ZOE cements owing to its high cytotoxicity . The IRM ® was stored under conditions recommended by the manufacturer, and IRM ® extracts were prepared in keeping with international standards . Briefly, the IRM ® powder and liquid were mixed for 2 min on a mixing pad according to the manufacturer’s instructions (23 °C, relative humidity 20%). The mixed cement solutions were incubated at 37 °C in a humidified incubator (VS-9160C) (Vision Scientific, Daejeon, Korea) for predetermined times, as listed in Table 1 , after which the mixed cement was added to a sterilized glass bottle containing distilled water (DW). The details of the mixing and incubation times are shown in Table 1 . After cementations or fillings, extract acting could be performed at the early, middle, or final stage of setting, which described extract starting point at 3, 6, or 10 min after start of mixing, respectively in this study, after the extracts were prepared according to the manufacturer’s instructions and to ISO standards . The 1 mL per 0.2 g sample extraction ratio between the mixed cement and DW was set according to international standards . Each cement was then extracted in an incubated shaker (SI-600R) (Jeio Tech, Seoul, Korea) at 37 °C at 80 rotations per minute for 24 h. Afterward, the samples were filtered using sterilized, 0.20-μm nitrocellulose filters (HP045AN) (Advantec, Tokyo, Japan), and same volume of filtered extract or the extract diluted with DW was added to IHOKs cultured media in each well of 24-well plates. The final concentrations of extracts were 50%, 25%, 12.5%, and 6.25%. Details about incubation with IHOKs were described in Section 2.3 .

Table 1
Eugenol and acetic acid concentration in extract.
Clinical condition Mixing/incubating time Start of extract (after start of mixing) Code Eugenol (ppm) Acetic acid (ppm)
On set of cementaion/filling 2 min/1 min 3 min 3 min 14.2 ± 2.3 a ND
Middle of setting 2 min/4 min 6 min 6 min 7.2 ± 1.4 b ND
After setting 2 min/8 min 10 min 10 min 4.4 ± 1.2 c ND
Results are shown as means ± standard deviations. Significant differences exist among different letters ( P < 0.05, n = 3). ND: not detected. Experiments were repeatedly performed in triplicate and representative data are shown.

Analysis of extracts

Gas chromatography–mass spectrometry (GC–MS) (7890A-5977A, Agilent, Atlanta, GA, USA) and inductively coupled plasma-mass spectrometry (ICP-MS) (NexION 300, PerkinElmer, Shelton, CT, USA) were used to analyze the extracts. GC–MS was used to identify and quantify unknown substances (i.e., eugenol and acetic acid), and ICP-MS was used to detect ionized metal ions such as Zn 2+ , Mg 2+ , and Fe 2+ . Prior to the GC–MS analyses, extracts were freeze-dried for 24 h (Ilshin Biobase, Gyeonggi-do, Korea), and equal volumes of ethanol (Duksan Pure Chemicals, Gyeonggi-do, Korea) were added to the freeze-dried powder. For quantitative analysis, 0, 5, 10, 20, and 40 ppm of eugenol in ethanol were used to derive a calibration curve. A digital pH meter (Orion 4-Star) (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the pH of the extract in culture media. All analyses were independently performed in triplicate, and the means ± standard deviations were determined.

Cytotoxicity tests

Cytotoxicity tests were performed based on the ISO standards (ISO 3107, 10993-5, 12). Briefly, 5 × 10 4 cells were cultured in standard 24-well plates (142471, Nunclon Delta Surface) (Thermo Fisher Scientific, Waltham, MA, USA) for the monolayer 2D model and in specialized 24-well plates (174930, Nunclon Sphera) (Thermo Fisher Scientific) for the spheroidal 3D structures in 500 μL of culture medium for 24 h (Fig. S1). The Nunclon Sphera surface is designed to cause minimal cell attachment with minimal extracellular matrix protein binding to the well plate surfaces and, consequently, supports the formation of 3D spheroids.

Mixed cement extracts, their dilutions in DW, eugenol dilutions (Sigma–Aldrich, St. Louis, MO, USA), ZnCl 2 (Sigma–Aldrich), and ZnSO 4 ·H 2 O (Sigma–Aldrich) liquid were added to each well. The ratio of cell culture medium-to-additive (extract, diluted extract, and various concentrations of chemical liquid) was set at 0.5. Therefore, 250 μL of the cement extracts was added to 250 μL of cell culture media, indicating a 50% final concentration, and 250 μL of serially diluted extract by DW was added at 25%, 12.5%, and 6.25% final concentrations. Various concentrations (0, 5, 10, 20, 40 ppm) of eugenol, Zn 2+ from ZnCl 2 , and ZnSO 4 ·H 2 O liquid and their mixture were added to each well. Sodium dodecyl sulfate (SDS) 0.001% (Sigma–Aldrich) was used as the positive control group. After 24-h incubation, cell viabilities were measured using a water-soluble tetrazolium (WST) salt assay (EZ-Cytox) (Daeil Lab, Seoul, Korea). Only 250 μL of DW was added to the control wells. L929 cells (mouse fibroblast CCL-1, ATCC) and immortalized human oral keratinocytes (IHOKs) were cultured in RPMI 1640 (LM011-01, Welgene, Daegu, Korea) and DMEM/F-12 3:1 mixture (LM 030-01, Welgene), respectively, with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco) in a humidified incubator at 37 °C containing 5% CO 2 . To compare the cytotoxicity in IHOKs and L929 cells (the ISO 10993 recommended cell line for cytotoxicity tests), we carried out cytotoxicity testing with L929 . Cell viabilities were expressed as the percentage of the optical density value of each test sample relative to their respective controls. All cytotoxicity tests ( n = 5) were independently performed in triplicate, and the means ± standard deviations were calculated.

Confocal laser microscopy

Fluorescence staining was performed after 24-h incubation with the various cement extracts (50%) and the DW control. Calcein AM and ethidium homodimer-1 (Molecular Probes, Eugene, OR, USA) were added to each well, according to the manufacturer’s instructions, and observed under a confocal laser microscope (LSM700, Carl Zeiss, Thornwood, NY, USA). Intense green fluorescence was observed in live cells, and bright red fluorescence was observed in dead cells. Tests were independently performed in triplicate. To obtain more clearly focused photographic images, the magnification of the confocal laser microscope was adjusted differently for the 2D and 3D cultured cells. The average value between the longest and the shortest diameter of the 3D IHOK spheroids was calculated for determining spheroid size before and after treatment ( n = 5) by Image J software, version 1.50b (National Institutes of Health, Bethesda, MD, USA).

Gene expression of interleukin on quantitative PCR analysis

After a 4-h incubation of IHOKs with 50% extract or various concentrations of liquid (see Section 2.3 ), mRNA expression levels of IL-1β, IL-6, and IL-8, normalized to β-actin, were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) analyses of the IHOKs. Briefly, total RNA was extracted by TRIzol (Life Technologies, Carlsbad, CA, USA), and 1 μg of total RNA was reversely transcribed to cDNA using oligo-dT primer (Qiagen, Venlo, The Netherlands), a premixture for reverse transcription (AccuPower RT PreMix) (Bioneer, Gyeonggi-do, Korea), and a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Real-time qPCR experiments were performed using a Power SYBR Green PCR Master Mix (Applied Biosystems) and real-time PCR equipment (StepOnePlus) (Applied Biosystems), according to the manufacturer’s instructions. The primer sequences used were β-actin forward 5′-AGG ATG CAG AAG GAG ATC ACT G-3′ and β-actin reverse 5′-ATA CTC CTG CTT GCT GAT CCA C-3′, IL-1β forward 5′-GGC AGA AAG GGA ACA GAA AGG-3′ and IL-1β reverse 5′-AGT GAG TAG GAG AGG TGA GAG AGG-3′, IL-6 forward 5′-CTG GCA GAA AAC AAC CTG AAC-3′ and IL-6 reverse 5′-ATG ATT TTC ACC AGG CAA GTC-3′, and IL-8 forward 5′-CTA GGA CAA GAG CCA GGA AG-3′ and IL-8 reverse 5′-AGT GTG GTC CAC TCT CAA TC-3′. After confirming qPCR efficiency using no template controls, positive controls (0.001% SDS treatment), and no amplification controls, according to the suggested guidance , mRNA expression levels of each sample ( n = 4) were normalized to a housekeeping gene (β-actin), and the relative fold change in expression was automatically calculated, with a value of 2 −ΔΔCt with respect to the control group, using StepOne software, version 2.3 (Applied Biosystems). Analyses were independently performed in triplicate, and data are shown as representative means ± standard deviations.

Statistical analysis

All data are reported as the means ± standard deviations of at least triplicate experiments. Statistical analyses were carried out using the SPSS Statistics 20 program (Armonk, NY, USA) using one-way or two-way ANOVA tests, along with Tukey post hoc tests. Comparisons were considered significant at P < 0.05.

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Cytotoxicity and anti-inflammatory effects of zinc ions and eugenol during setting of ZOE in immortalized human oral keratinocytes grown as three-dimensional spheroids
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