Abstract
Objective
Although cobalt–chromium (Co–Cr) dental alloys are routinely used in prosthodontics, the biocompatibility of Co–Cr alloys is controversial. The aims of the present study were to investigate the effects of Co–Cr alloys on human gingival fibroblasts (HGF) and osteoblasts in an in vitro model as well as their potential molecular mechanisms, focusing on NF-E2-related factor 2 (Nrf2) pathways.
Methods
Cells were directly seeded on prepared Co–Cr alloy discs (15.0 mm diameter, 1.0 mm thickness) or indirectly treated with Co–Cr alloy located at the bottom of an insert well and incubated for 3 days. Cytotoxicity and reactive oxygen species (ROS) production was evaluated by MTS assay and flow cytometry, respectively. Protein and mRNA levels were determined by Western blotting and RT-PCR analysis, respectively.
Results
Cell viability and flow cytometric assay demonstrated that the Co–Cr alloy was cytotoxic to HGFs and osteoblasts, and significantly increased ROS production. In addition, the Co–Cr alloys upregulated pro-inflamamtory cytokines (TNF-α, IL-1β, IL-6, and IL-8) and increased levels of various inflammatory mediators (iNOS derived nitrite oxide, and COX-2-derived PGE2) in both cells. A mechanistic study showed that Co–Cr alloys activates the NRF2 pathway and up-regulate antioxidant enzymes including heme oxygenase-1 (HO-1). Co–Cr alloys activated JAK2/STAT3, p38/ERK/JNK MAPKs and NF-κB signaling pathways. Furthermore, antioxidants (resveratrol and NAC) and HO-1 inhibitor (SnPP) significantly inhibited the production of ROS and inflammatory mediators, as well as the activation of NF-κB signaling in Co–Cr alloy stimulated HGFs and osteoblasts.
Significance
This study is the first to show that Co–Cr alloys exert cytotoxic and inflammatory effects via activation of Nrf2/ARE signaling and up-regulation of downstream HO-1, which could represent candidate targets for the regulation of inflammatory responses to Co–Cr alloys.
1
Introduction
Casting alloys have been an important part of restorative dental treatment for more than a century. Although gold alloys possess biocompatible properties and good corrosion resistance , their dental applications have been restricted due to the increasing cost of gold. Consequently, base metal alloy alternatives, such as nickel–chromium (Ni–Cr) and cobalt–chromium (Co–Cr) alloys have increasingly replaced gold alloys in the fabrication of frameworks of removable partial dentures (RPDs), porcelain fused to metal (PFM) restorations, and dental implant superstructures .
Oxidative stress from reactive oxygen species (ROS) enhances inflammatory responses during tissue injury. To avoid the toxic effects of ROS, cells are equipped with protective or cellular defense systems such as the transcription factor NF-E2-related factor 2 (Nrf2)-mediated antioxidant response . Nrf2 is important for protecting cells and multiple tissues through up-regulation of antioxidant response element (ARE)-related detoxification and antioxidant genes such as heme oxygenase-1 (HO-1), γ-glutamylcysteine lygase (GCL), glutathione reductase (GR), and glutathione- S -transferase (GST) . We previously reported that the Nrf2/ARE pathway and especially HO-1 induction is required for this defensive system in stressful conditions and for recovery from injurious events .
The oral environment is a very complex electrolyte environment with a large number of microorganisms, in which metal prosthesis corrode and releases metal ions . Metal ion concentrations are increased in the saliva and oral soft tissue ( e.g. , gingiva and tongue smear) of patients with alloy prostheses . The metal ions released from Ni–Cr alloys have been reported to stimulate adverse reactions such as nickel allergy, gingival inflammation and discoloration, and DNA damage . It was recently demonstrated in three-dimensional (3D) human-derived oral mucosal model that Ni-based alloys are cytotoxic and induce oxidative stress and inflammatory cytokine expression . Because of these problems, clinicians now prefer to use Co–Cr alloys, for metallic frameworks of RPDs and PFM restorations .
Co–Cr alloys possess better biocompatibility as well as higher resistance to corrosion and tarnish than do Ni–Cr-based alloys . In addition, a Co–Cr-based alloy did not elicit adverse oxidative stress or cellular toxicity responses compared with controls in a 3D human-derived oral mucosal model . In contrast, Ni-Cr and Co–Cr alloys caused increase in prostaglandin E 2 (PGE 2 ) and cyclo-oxygenase-2 (COX-2) levels in human gingival fibroblast (HGF) in vitro . Moreover, an average between 3% (women) and 10% (men) of the general population exhibits a hypersensitive reaction to chromium, although oral mucosa allergies are quite unusual . Elevated levels of Co and Cr ions occur in the peripheral blood and in the hip synovial fluid after Co–Cr alloy metal-on-metal (MoM) hip replacement . High metal ion concentrations may lead to adverse health effects such as osteolysis, inflammation, and pain in the periprosthetic tissues . However, the molecular mechanisms involved in inflammatory response can be triggered by Co–Cr alloys was not completely understood.
The aim of this study was to examine (1) whether a Co–Cr alloy is cytotoxic and induces in vitro inflammatory responses in human gingival fibroblasts and osteoblasts, (2) whether a Co–Cr alloy activates Nrf2 signaling and increases downstream ARE-responsive genes, and (3) whether Nrf2/HO-1 signaling could be a major cellular mechanism mediating the potential in vitro inflammatory effects of a Co–Cr alloy.
2
Materials and methods
2.1
Preparation of Co–Cr alloy discs
A commercially available Co–Cr PFM alloy was used. The main constituents of the alloy (in mass %) by manufacturer were listed in Table 1 . Disc-shaped specimens (15 mm diameter and 1.0 mm thickness) were prepared from Co–Cr alloy bar (S-Tech Coporation, Tainan, Taiwan) which was manufactured by vacuum arc remelting. To make 1,0 mm thickness we used wire electric discharge machining with DWC110H (Mitsubish, Tokyo, Japan) cutting machine using PAPS CUT-S (Pungkuk, Seoul, Korea) wire and then grounded with 1000 grit silicon carbide paper. All discs were sterilized individually in an autoclave at 121 °C for 15 min.
| Item | Co | Cr | Mo | Si | Mn | N | Fe | Ni | C |
|---|---|---|---|---|---|---|---|---|---|
| Results | 65.2 | 27.2 | 5.9 | 0.7 | 0.7 | 0.2 | 0.1 | 0.03 | 0.03 |
2.2
Cell culture
HGF cell line by transfection with the E6/E7 open reading frames of HPV type 16, following a previously described method were used. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C. Human osteoblasts were purchased from ATCC (Manassas, VA, USA) and maintained in 1:1 mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium with 2.5 × 10 −3 mol l −1 l -glutamine and 0.3 mg/ml G418 with 10% of FBS.
2.3
Alloy exposure
2.3.1
Direct methods
The alloy discs were placed at the bottom of 6-well tissue culture plates, washed twice with PBS and dried before the cell test. Cells were directly seeded onto the prepared alloy discs and cultured for 1 or 3 days.
2.3.2
Indirect methods
Cells in media containing 10% FBS were seeded in 6-well tissue culture plates and incubated. After an initial attachment period of 24 h, the alloy disc were placed at the bottom of insert wells (Millicell; Millipore, Bedford, MA) with a membrane pore diameter of 0.4 μm in the culture wells, and the cells were incubated for 1 or 3 days.
2.4
Cytotoxicity assay
Following incubation for 24 or 72 h, the cells were checked for the effect of the Co–Cr alloys on their viability (24- or 72-h readings) by using the MTS test (CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) according to the manufacturer’s instructions. The Cytotoxicity was determined from data obtained by measuring the optical absorbance at a wavelength of 490 nm with a microplate reader (Bio Rad, Hercules, CA).
2.5
RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol reagent (Life Technologies, Gaithersburg, MD). 1 mg of RNA was reverse transcribed using oligo (dT) 15 primers (Roche Diagnostics, Mannheim, Germany) and AccuPower RT PreMix (Bioneer Corporation, Daejeon, South Korea). The cDNA was amplified in a final volume of 20 μl containing 2.5 mM magnesium chloride, 1.25 units Ex Taq polymerase (Bioneer, Daejon, Korea) and 1 μM specific primers. The sequences of the specific primers used in this study are detailed in previous our reports . RT-PCR products were electrophoresed on 1.5% agarose gels. PCR products were detected by UV illumination of ethidium bromide-stained gels.
2.6
Quantification of nitric oxide (NO)
Thawed 50 μl aliquots of culture supernatant were mixed with 50 μl Griess reagent (Sigma–Aldrich, St. Louis MO, USA). Samples were incubated at room temperature for approximately 10 min and then read on an enzyme-linked immunosorbent assay microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm.
2.7
Determination of prostaglandin E 2 (PGE 2 ) levels
The concentrations of PGE 2 in the culture supernatants were determined by an ELISA kit, according to the manufacturer’s recommendations (R & D Systems, Minneapolis, MN, USA). The plates were read at 450 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
2.8
Reactive oxygen species (ROS) production
Intracellular ROS generation was measured using the fluorescent dye 5-(and-6)-chloromethyl 20,70-dichlorodihydro-fluorescein diacetate acetyl ester (CM-H2DCFDA; Molecular Probes, Eugene, OR, USA) with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Cells were incubated with the dye for 30 min at the end of the indicated treatments. The mean CM-H2DCFDA fluorescence was determined at 530 nm (band width of 30 nm) with an excitation wavelength of 488 nm using a 15-mW argon laser.
2.9
Western blot analysis
Cells from each set of experiments were rinsed with ice-cold PBS and harvested with a cell scraper followed by centrifugation. The cell pellets were solubilized in ice-cold 1% Triton X-100 lysis buffer followed by centrifugation at maximal speed. After protein quantification (Micro-BCA Protein Assay, Pierce), equal amounts of protein plus loading dye were added per lane on a 12% SDS-polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked and probed with specific primary antibodies with a secondary antimouse or antirabbit horseradish peroxidase-conjugated IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1/5000). Bands were visualized using enhanced chemiluminescence system (Amersham-Pharmacia, Piscataway, NJ), according to the manufacturer’s instructions.
2.10
Immunocytochemistry
Cells were fixed with freshly prepared 3% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 15 min. After a 1-h incubation with 10% normal goat serum/PBS, cells were incubated with anti-p65 Ab (Santa Cruz Laboratories, Santa Cruz, CA) diluted 1:50 in PBS for 2 h, washed and incubated with FITC-conjugated IgG, diluted 1:100 in PBS, for 1 h. In order to identify nuclei, the FITC-labeled samples were counterstained with 10 μg/ml propidium iodide for 2 min. To acquire immunofluorescence images, a confocal microscope (Cell Voyager, Yokogawa, Japan) was used.
2.11
Statistical analysis
Data are presented as mean ± standard deviation of at least three independent experiments. Differences between groups were analyzed by one-way analysis of variance combined with Duncan’s multiple range test. P values <0.05 were considered statistically significant.
2
Materials and methods
2.1
Preparation of Co–Cr alloy discs
A commercially available Co–Cr PFM alloy was used. The main constituents of the alloy (in mass %) by manufacturer were listed in Table 1 . Disc-shaped specimens (15 mm diameter and 1.0 mm thickness) were prepared from Co–Cr alloy bar (S-Tech Coporation, Tainan, Taiwan) which was manufactured by vacuum arc remelting. To make 1,0 mm thickness we used wire electric discharge machining with DWC110H (Mitsubish, Tokyo, Japan) cutting machine using PAPS CUT-S (Pungkuk, Seoul, Korea) wire and then grounded with 1000 grit silicon carbide paper. All discs were sterilized individually in an autoclave at 121 °C for 15 min.
| Item | Co | Cr | Mo | Si | Mn | N | Fe | Ni | C |
|---|---|---|---|---|---|---|---|---|---|
| Results | 65.2 | 27.2 | 5.9 | 0.7 | 0.7 | 0.2 | 0.1 | 0.03 | 0.03 |
2.2
Cell culture
HGF cell line by transfection with the E6/E7 open reading frames of HPV type 16, following a previously described method were used. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO 2 at 37 °C. Human osteoblasts were purchased from ATCC (Manassas, VA, USA) and maintained in 1:1 mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium with 2.5 × 10 −3 mol l −1 l -glutamine and 0.3 mg/ml G418 with 10% of FBS.
2.3
Alloy exposure
2.3.1
Direct methods
The alloy discs were placed at the bottom of 6-well tissue culture plates, washed twice with PBS and dried before the cell test. Cells were directly seeded onto the prepared alloy discs and cultured for 1 or 3 days.
2.3.2
Indirect methods
Cells in media containing 10% FBS were seeded in 6-well tissue culture plates and incubated. After an initial attachment period of 24 h, the alloy disc were placed at the bottom of insert wells (Millicell; Millipore, Bedford, MA) with a membrane pore diameter of 0.4 μm in the culture wells, and the cells were incubated for 1 or 3 days.
2.4
Cytotoxicity assay
Following incubation for 24 or 72 h, the cells were checked for the effect of the Co–Cr alloys on their viability (24- or 72-h readings) by using the MTS test (CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) according to the manufacturer’s instructions. The Cytotoxicity was determined from data obtained by measuring the optical absorbance at a wavelength of 490 nm with a microplate reader (Bio Rad, Hercules, CA).
2.5
RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted using Trizol reagent (Life Technologies, Gaithersburg, MD). 1 mg of RNA was reverse transcribed using oligo (dT) 15 primers (Roche Diagnostics, Mannheim, Germany) and AccuPower RT PreMix (Bioneer Corporation, Daejeon, South Korea). The cDNA was amplified in a final volume of 20 μl containing 2.5 mM magnesium chloride, 1.25 units Ex Taq polymerase (Bioneer, Daejon, Korea) and 1 μM specific primers. The sequences of the specific primers used in this study are detailed in previous our reports . RT-PCR products were electrophoresed on 1.5% agarose gels. PCR products were detected by UV illumination of ethidium bromide-stained gels.
2.6
Quantification of nitric oxide (NO)
Thawed 50 μl aliquots of culture supernatant were mixed with 50 μl Griess reagent (Sigma–Aldrich, St. Louis MO, USA). Samples were incubated at room temperature for approximately 10 min and then read on an enzyme-linked immunosorbent assay microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm.
2.7
Determination of prostaglandin E 2 (PGE 2 ) levels
The concentrations of PGE 2 in the culture supernatants were determined by an ELISA kit, according to the manufacturer’s recommendations (R & D Systems, Minneapolis, MN, USA). The plates were read at 450 nm on a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
2.8
Reactive oxygen species (ROS) production
Intracellular ROS generation was measured using the fluorescent dye 5-(and-6)-chloromethyl 20,70-dichlorodihydro-fluorescein diacetate acetyl ester (CM-H2DCFDA; Molecular Probes, Eugene, OR, USA) with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Cells were incubated with the dye for 30 min at the end of the indicated treatments. The mean CM-H2DCFDA fluorescence was determined at 530 nm (band width of 30 nm) with an excitation wavelength of 488 nm using a 15-mW argon laser.
2.9
Western blot analysis
Cells from each set of experiments were rinsed with ice-cold PBS and harvested with a cell scraper followed by centrifugation. The cell pellets were solubilized in ice-cold 1% Triton X-100 lysis buffer followed by centrifugation at maximal speed. After protein quantification (Micro-BCA Protein Assay, Pierce), equal amounts of protein plus loading dye were added per lane on a 12% SDS-polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked and probed with specific primary antibodies with a secondary antimouse or antirabbit horseradish peroxidase-conjugated IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, 1/5000). Bands were visualized using enhanced chemiluminescence system (Amersham-Pharmacia, Piscataway, NJ), according to the manufacturer’s instructions.
2.10
Immunocytochemistry
Cells were fixed with freshly prepared 3% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 15 min. After a 1-h incubation with 10% normal goat serum/PBS, cells were incubated with anti-p65 Ab (Santa Cruz Laboratories, Santa Cruz, CA) diluted 1:50 in PBS for 2 h, washed and incubated with FITC-conjugated IgG, diluted 1:100 in PBS, for 1 h. In order to identify nuclei, the FITC-labeled samples were counterstained with 10 μg/ml propidium iodide for 2 min. To acquire immunofluorescence images, a confocal microscope (Cell Voyager, Yokogawa, Japan) was used.
2.11
Statistical analysis
Data are presented as mean ± standard deviation of at least three independent experiments. Differences between groups were analyzed by one-way analysis of variance combined with Duncan’s multiple range test. P values <0.05 were considered statistically significant.
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