Possible implications of Ni(II) on oral IL-1β-induced inflammatory processes

Abstract

Objectives

Nickel (Ni) is one of the main metal elements in orthodontic and prosthetic devices. Different effects of Ni are described ranging from an induction of local inflammation to allergy and cancerous/mutagenic properties. Inflammatory reactions are frequently observed in the oral cavity, but the interrelationship of Ni with those events is still unknown. Therefore, we focused on the impact of Ni on inflammation in vitro.

Methods

In accordance to previous immersion tests of our lab, human gingival fibroblasts (HGFs) ( n = 6) were exposed to a pro-inflammatory environment using interleukin-1 beta (IL-1β) and additionally stimulated with different Ni(II) concentrations (400 and 4000 ng/ml). At varying time points the expression of pro- and anti-inflammatory as well as matrix degeneration proteins, i.e. MMPs, were analyzed. Furthermore, proliferation assays, wound healing tests and the detection of NF-κB activation were conducted. Unstimulated HGFs served as control.

Results

Our experiments showed that low clinical average Ni(II) levels did not alter pro-inflammatory cytokines significantly compared to control ( p > 0.05). Instead, a 10-fold higher dose up-regulated these mediators significantly in a time-dependent manner ( p < 0.01). This was even more pronounced combining both Ni(II) concentrations with an inflammatory condition ( p < 0.001), MMP expressions were in line with our findings ( p < 0.001). The mRNA data were supported by proliferation and wound closure assays ( p < 0.001). However, the combination of both stimuli induced contradictory results. Analyzing NF-κB activation revealed that our results may be in part attributed to NF-κB.

Significance

Our in vitro study implicated that Ni(II) has various modifying effects on IL-1β-induced inflammatory processes depending on the concentration.

Introduction

Nickel (Ni) is one of the main elements in orthodontic appliances (arches, brackets and bands) and prosthetic devices (crowns, bridges and removable prostheses) varying from 7 wt.% in stainless steel (SS) to more than 50 wt.% in nickel–titanium (NiTi) alloys. Beside other properties, Ni is incorporated into SS and NiTi devices for chemical stabilization as well as to improve corrosion resistance and durability . Therefore, Ni-based containing dental cast alloys represent around 80% of prosthetic restorations . However, there is general agreement that Ni ions (Ni(II)) are released into the oral cavity as a consequence of corrosion processes. Previous in vitro and in vivo studies examined the content of released Ni(II) demonstrating these metal leaching depends on alloy composition, changes in pH, temperature or mechanical stress . Biocompatibility is not necessarily based on the percent content of the alloy, rather than on the release of metal ions.

Different side effects of Ni are described in the literature varying from induction of local inflammation as well as allergic reaction to cancerous/mutagenic features . Thereby, biocompatibility is related to the duration and route of exposure, form and concentration of this metal. Several studies investigated the impact of Ni(II) on cell metabolism using immune cells like monocytes and dendritic cells (DCs) as well as human dermal or gingival epithelial cells . The authors detected dose- and time-depended cytotoxic and pro-inflammatory reactions. Nontoxic concentrations seem to induce DNA alterations based on strand scission or base damage . Moreover, mutagenic activity of Ni(II) might derive from enzyme inhibitions which are responsible for DNA repair . However, Ni allergy is the most common side effect and contact allergen ranging between 3% in men, up to over 30% in women . Inflammation seems to play a pivotal role in the initiation of Ni(II)-induced hypersensitivity promoting the activation and recruitment of immune cells to the exposure area .

Some of these effects are mediated by a pattern recognition receptor (PRR) of the innate immunity, the Toll-like receptor 4 (TLR4) . TLR4 activation induces the translocation of the transcription factor nuclear factor-κB (NF-κB) leading to an increased expression of pro-inflammatory cytokines like interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) . IL-1β is a potent inducer of other cytokines and inflammatory proteins leading to an inflammatory environment. In general, TLRs share significant cytoplasmic region homology with the interleukin-1 receptors (IL-1R) and both signaling pathways induce similar cellular immune responses .

The balance between pro- and anti-inflammatory mediators is crucial. Pro-inflammatory cytokines and chemokines stimulate the recruitment and activation of professional inflammatory immune cells like macrophages, leukocytes and T-cells leading to an excess of an inflammatory response. This may cause the onset of Ni contact hypersensitivity (CHS) or an irreversible periodontal destruction via matrix metalloproteinases (MMPs). Anti-inflammatory mediators, such as IL-10, try to antagonize these activities. A disturbance of the balance between matrix synthesis and degradation could result in pronounced periodontal tissue destruction finally favoring tooth loss .

The gingival epithelium is the first barrier which comes in contact with corrosive materials like Ni(II) and bacteria. Human gingival fibroblasts (HGFs) are the major component of the gingival tissue and are strongly affected by the release of IL-1β highly synthesized by gingival keratinocytes and monocytes/macrophages . HGFs express both IL-1Rs as well as TLRs and function as non-professional inflammatory cells , therefore, participating in inflammatory reactions like gingivitis, periodontitis or even the initial phase of CHS against Ni .

The increasing incidence of periodontal diseases and Ni allergy on the one hand and the high orthodontic and prosthetic treatment need on the other hand underline the importance for improving the knowledge of Ni(II)-induced mechanisms. In which way existing inflammatory processes like gingivitis, periodontitis or mechanically induced lesions in the oral cavity are affected by Ni(II) is still unknown. The same is true concerning the interaction of TLR4, IL-1/IL-1R and Ni(II) in these events.

The aim of this study was therefore, to analyze the effect of Ni(II) on these processes using the typical quantity of ions released during orthodontic or prosthetic therapy which has been previously analyzed by our working group ( Fig. 1 A) . We hypothesized that Ni(II) enhances existing inflammatory reactions which could facilitate or aggravate periodontal diseases and allergic reactions. Hence, primary human gingival fibroblasts which constitute the major component of the gingival tissue and are affected by keratinocyte expressed IL-1β, were exposed to various Ni(II) concentrations with and without IL-1β-induced inflammation.

Fig. 1
(A) An example of previous studies investigating the Ni(II) release of orthodontic materials using static and dynamic immersion tests DIN/ISO standard 10271 (DIN-Norm 2002). (B) Effect of IL-1β (1 ng/ml) on the mRNA expression of IL-1β in human gingival fibroblasts (HGFs) at different time points. Statistical significances between the non-stimulated control and IL-1β stimulation are calculated using Student’s t -test and marked with asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001. (C and D) Immunofluorescence staining for NF-κB p65 in gingival fibroblasts after stimulation with 1 ng/ml IL-1β (D) for 2 h. Non-stimulated cells served as control (C). NF-κB p65 was visualized by indirect detection with a Cy3-conjugated secondary antibody (red color). Scale bar: 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Materials and methods

Isolation and culture of human gingival fibroblasts

Written consent and approval of the Ethics Committee of the University of Bonn were obtained prior to the collection of human gingival tissues from six healthy adult patients (18–35 years) who underwent tooth extraction or dentoalveolar surgery. Tissue samples that had been severely traumatized or inflamed were not included in this study. Gingival specimens were washed twice with phosphate buffered saline (PBS; Life Technologies, Darmstadt, Germany) supplemented with 1% antibiotic and antimycotic solution (AB/AM) containing penicillin/streptomycin and amphotericin B (Life Technologies). Afterwards, the gingiva was digested with collagenase 2 solution (PAA Laboratories, Cölbe, Germany) at 37 °C for 2.5 h. The connective tissue was subsequently dissected from the epithelial tissue with sterile forceps. The connective tissue was left to adhere to a 60 mm culture plate for 2 min followed by the administration of cell culture medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Life Technologies), 1% AB/AM, and grown at 37 °C in humified atmosphere of 5% CO 2 in air. Medium was changed every 2–3 days. Following our protocol of explant technique, HGFs were used at passage three for experiments. Cell viability was evaluated using trypan blue solution (Sigma–Aldrich, Munich, Germany) followed by counting vital and dead cells.

Cell stimulation

Previous data of our lab showing the release of Ni(II) from orthodontic and prosthetic materials under different conditions were used as basis for our in vitro study ( Fig. 1 A). Ni(II)-chloride-hexahydrate (NiCl 2 ·6H 2 O) was purchased from Sigma–Aldrich and prepared as 10 mg/ml stock solution in sterile water (Ampuwa, Fresenius). To exclude endotoxin contamination, Limulus amebocyte lysate assay (Thermo Fisher Scientific, Bonn, Germany) was performed as recommended by the manufacturer. NiCl 2 concentrations used for stimulation referring to the Ni(II) release experiments were calculated as follows: 100 ng/ml Ni(II) release corresponds to a concentration of 1.7 μM Ni(II). With a molecular weight of 237.7 g/mol, we used an end-concentration of 400 ng/ml NiCl 2 6H 2 O to simulate the clinical average situation of 100 ng/ml Ni(II) (for simplicity, in the following only referred to as 400 or 4000 ng/ml Ni(II)).

For in vitro experiments HGFs were seeded on 6-well plates at an initial density of 100,000 cells per well and left to grow at 37 °C in humified atmosphere of 5% CO 2 in air. After reaching up to 90% confluence, cells were grown under serum free conditions for 24 h before stimulation. Inflammatory condition was mimicked by adding 1 ng/ml IL-1β (R&D Systems) to 2 ml culture medium according to Nokhbehsaim et al. . To investigate the influence of Ni(II) under control and inflammation, cells were stimulated with 400 ng/ml Ni(II) or a 10x higher concentration (4000 ng/ml) at varying time points (2 h, 6 h, 24 h, 72 h). Experiments without Ni(II) and IL-1β stimulation served as control.

RNA-isolation, first strand cDNA synthesis, qPCR

HGFs of six probands were separately used in our experiments and pooled on RNA level. Experiments were performed in triplicate on two occasions. Total RNA was isolated using the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and quantified using a NanoDropHND-1000 Spectrophotometer (NanoDrop, Technologies, Wilmington, DE, USA). First-strand cDNA synthesis was performed with 1 μg RNA using the iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, Munich, Germany) and oligo(dT)-primers.

The mRNA expression of IL-1β, IL-8, transforming growth factor (TGF)–β1, IL-10, MMP-1 and MMP-8 was detected by real-time polymerase chain reaction (RT-PCR) using the iCycler iQ detection system (Bio-Rad Laboratories), SYBR Green (Bio-Rad Laboratories), and gene-specific primers (Metabion, Martinsried, Germany) ( Table 1 ). Verification of all primers was accomplished by computer analysis for specification with the basic logical alignment search tool (BLAST) and synthesized of high quality (Metabion). In addition, the specific annealing temperatures were assessed by a temperature gradient, corresponding PCR-efficiencies for primers were determined with dilution series of sequenced primer-specific PCR-products. In Table 1 primer sequences, annealing temperatures and efficiencies are listed.

Table 1
Primer sequences Primer sequences with corresponding annealing temperatures, efficiencies, and product length in basepairs (bp) used for real time PCR. PCR efficiencies for every set of primers were determined with dilution series of primer specific cloned PCR-products at the corresponding annealing temperature. Efficiency stands for the performance to amplify a cDNA template. Efficiency values of 2.0 mean an amplification of 100%.
Gene Primer sequences (sense/antisense) Efficiency Annealing temperature (°C)
β-Actin 5′-CATGGATGATGATATCGCCGCG-3′
5′-ACATGATCTGGGTCATCTTCTCG-3′
1.84 69
IL-1β 5′-ATGGCAGAAGTACCTGAGCTCGC-3′
5′-TTAGGAAGACACAAATTGCATGGTG-3′
1.83 68
IL-8 5′-ATGACTTCCAAGCTGGCCGTGG-3′
5′-TGAATTCTCAGCCCTCTTCAAAAAC-3′
2.02 68
IL-10 5′-TTAAGGGTTACCTGGGTTGC-3′
5′-GCCTTGCTCTTGTTTTCACA-3′
1.94 65
MMP-1 5′-ATGCACAGCTTTCCTCCACTGC-3′
5′-CACTGGGCCACTATTTCTCCGC-3′
2.05 69
MMP-8 5′-ATGTTCTCCCTGAAGACGCTTCC-3′
5′-CAACGATCACATTAGTGCCATTC-3′
1.85 67
TGF-β1 5′-GAGCCCTGGACACCAACTAT-3′
5′-GACCTTGCTGTACTGCGTGT-3′
1.94 69

For qPCR-analysis, 50 ng cDNA was added to a mastermix containing primers and iQ™ SYBR Green Supermix (Bio-Rad Laboratories). Cloned PCR-products derived from the specific primers were used as positive control, whereas water served as negative control. Every set of experiment was carried out with cDNA of the same sample to compare the expression of the different genes of interest. PCR conditions were defined as follows: 5 min denaturing step at 95 °C, then 50 cycles of 15 s at 95 °C, 30 s at specific annealing temperatures for the primers, and 30 s at 72 °C for elongation. Target gene expression was normalized to β-actin mRNA expression. Relative differential gene expression was calculated using the method described by Pfaffl .

Proliferation/cytotoxicity assay

Cell proliferation was determined using the PromoKine XTT Cell Proliferation Kit (Promocell, Heidelberg, Germany). In brief, gingival fibroblasts were seeded in 96-well-plates for 24 h with 5000 cells per well. To avoid evaporation effects of the peripheral outer wells, cells were only grown in the middle of 96-well-plates, whereas the outer wells were filled with sterile water. In order to induce cytotoxic effects and in accordance to Schmidt et al. , stimulation was performed with up to 100.000 ng/ml Ni(II) and/or 1 ng/ml IL-1β in 100 μl medium per well at 37 °C in a humified atmosphere of 5% CO 2 in air for 24 and 48 h. After the indicated time points, XTT reaction solution was added to the medium for 4 h followed by the measurement of absorbance at 490 nm with correction wavelength 670 nm in a microplate reader. Experiments were performed with HGFs from three different patients in hexaduplicates.

In vitro wound healing assay

The wound fill rate was analyzed using an established in vitro wound healing model . In brief, gingival fibroblasts from six donors were seeded onto 35 mm culture dishes in 2 ml medium (Greiner Bio-One, Frickenhausen, Germany) and grown until confluence at 37 °C in a humified atmosphere of 5% CO 2 in air. Afterwards, defined cell-free areas were induced by disrupting the monolayers with sterile instruments in a standardized manner. Then, medium was changed and cells were stimulated, as described above. Using an inverted microscope (Axiovent 25 C, AxioCam, Zeiss, Oberkochen, Germany), photographs of the wounded area were taken immediately after wounding and at day 1, 2, 3 and 4. Analysis of wound healing was performed with the freely available image-processing software ImageJ 1.43 ( ). The percentage of wound fill rates at day 1, 2, 3, and 4 were determined by relating recovered wound areas to the baseline ( t 0 ). Experiments were performed twice in duplicates.

Immunofluorescence for NF-κB detection

HGFs from three donors were cultured in triplicates on sterile cover slips in 24-well plates for 24 h at 37 °C in a humified atmosphere of 5% CO 2 in air. After incubation with different Ni(II) concentrations and/or 1 ng/ml IL-1β in 500 μl medium for 2 h, cells were fixed with 4% paraformaldehyde (Sigma–Aldrich) for 15 min, washed in PBS and treated with 0.1% Triton X-100 (Sigma–Aldrich) containing PBS for 15 min. In addition, 1 μg/ml lipopolysaccharide (LPS) ( E. coli 0111:B4, Sigma–Aldrich) which is a known TLR4 agonist, was used as positive control for NF-κB activation.

After washing with PBS, cells were blocked with 5% goat serum (PAA) for 1 h at room temperature (RT) and incubated overnight with rabbit anti-NF-κB (p65) antibody (1:100; Santa Cruz, Heidelberg, Germany) diluted in Tris buffered saline (TBS) containing 1% bovine serum albumin (BSA, Sigma–Aldrich) at 4 °C. After extensive washing with PBS, a Cy3-conjugated goat anti-rabbit IgG secondary antibody (Dianova, Hamburg, Germany) (1:250) was applied for 1 h at RT. Finally, cells were washed three times with PBS and mounted with Mowiol/1,4-Diazabicyclo[2.2.2]octane (DABCO) (Roth, Karlsruhe, Germany) for fluorescence microscopic imaging. Quantification of relative nuclear fluorescence intensity was performed for every cover slip by measuring the fluorescence intensity of each cell nucleus related to corresponding cytoplasm in five fields of vision at a primary magnification of 400× with the freely available image-processing software ImageJ 1.43 ( ). To obtain grayscale pictures, each was processed using the tools “color” and “split channels” in ImageJ which resulted in red, green and blue color fractionated grayscale pictures for each channel (Supplementary Fig. 1A–I). Only red fractionated pictures were used for calculation of the relative nuclear fluorescence intensity.

Supplementary Fig. 1
Interactions of Ni(II) and IL-1β-induced inflammation on NF-κB activation. Effect of lipopolysaccharide (LPS) and different Ni(II) concentrations on the nuclear translocation of NF-κB p65 at 2 h. NF-κB p65 was visualized by indirect immunofluorescence staining with Cy3-conjugated secondary IgG. To obtain grayscale pictures, each was processed using the tools “color” and “split channels” in ImageJ software which resulted in red, green and blue color fractionated grayscale pictures for each channel (A and B). Only red fractionated pictures (B–I) were used for calculation of the relative nuclear fluorescence intensity (see also Fig. 6 ). (A) and (B) Non-stimulated control, (C) Stimulation with 1 μg/ml LPS, (D)–(I) stimulation with varying Ni(II) concentrations ((D) 100 ng/ml, (E) 400 ng/ml, (F) 1000 ng/ml, (G) 4000 ng/ml, (H) 10,000 ng/ml, (I) 100,000 ng/ml). Scale bar: 100 μm.

Statistical analysis

For statistical analysis a specific software program was used (GraphPad Software Version 5, San Diego CA, USA). For mRNA expression data, wound healing, and NF-κB activation analysis pooled mean ± standard deviation (SD) were calculated and one-way analysis of variance (ANOVA) and post hoc Tukey’s multiple comparison tests were applied to determine statistical differences between control and stimulation groups. A further one-way ANOVA was used for the pooled mean XTT data for the untreated control and Ni(II) and/or IL-1β treated HGFs with a Student’s t -test comparison to highlight significant differences between Ni(II) and Ni(II) with IL-1β stimulated cells. p -Values less than 0.05 were considered to be statistically significant. In order to delineate statistical significance in the figures, asterisks above graph bars refer to the comparison between the stimulation groups and the control; whereas asterisks above brackets depict statistical significance among the stimulation groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Materials and methods

Isolation and culture of human gingival fibroblasts

Written consent and approval of the Ethics Committee of the University of Bonn were obtained prior to the collection of human gingival tissues from six healthy adult patients (18–35 years) who underwent tooth extraction or dentoalveolar surgery. Tissue samples that had been severely traumatized or inflamed were not included in this study. Gingival specimens were washed twice with phosphate buffered saline (PBS; Life Technologies, Darmstadt, Germany) supplemented with 1% antibiotic and antimycotic solution (AB/AM) containing penicillin/streptomycin and amphotericin B (Life Technologies). Afterwards, the gingiva was digested with collagenase 2 solution (PAA Laboratories, Cölbe, Germany) at 37 °C for 2.5 h. The connective tissue was subsequently dissected from the epithelial tissue with sterile forceps. The connective tissue was left to adhere to a 60 mm culture plate for 2 min followed by the administration of cell culture medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Life Technologies), 1% AB/AM, and grown at 37 °C in humified atmosphere of 5% CO 2 in air. Medium was changed every 2–3 days. Following our protocol of explant technique, HGFs were used at passage three for experiments. Cell viability was evaluated using trypan blue solution (Sigma–Aldrich, Munich, Germany) followed by counting vital and dead cells.

Cell stimulation

Previous data of our lab showing the release of Ni(II) from orthodontic and prosthetic materials under different conditions were used as basis for our in vitro study ( Fig. 1 A). Ni(II)-chloride-hexahydrate (NiCl 2 ·6H 2 O) was purchased from Sigma–Aldrich and prepared as 10 mg/ml stock solution in sterile water (Ampuwa, Fresenius). To exclude endotoxin contamination, Limulus amebocyte lysate assay (Thermo Fisher Scientific, Bonn, Germany) was performed as recommended by the manufacturer. NiCl 2 concentrations used for stimulation referring to the Ni(II) release experiments were calculated as follows: 100 ng/ml Ni(II) release corresponds to a concentration of 1.7 μM Ni(II). With a molecular weight of 237.7 g/mol, we used an end-concentration of 400 ng/ml NiCl 2 6H 2 O to simulate the clinical average situation of 100 ng/ml Ni(II) (for simplicity, in the following only referred to as 400 or 4000 ng/ml Ni(II)).

For in vitro experiments HGFs were seeded on 6-well plates at an initial density of 100,000 cells per well and left to grow at 37 °C in humified atmosphere of 5% CO 2 in air. After reaching up to 90% confluence, cells were grown under serum free conditions for 24 h before stimulation. Inflammatory condition was mimicked by adding 1 ng/ml IL-1β (R&D Systems) to 2 ml culture medium according to Nokhbehsaim et al. . To investigate the influence of Ni(II) under control and inflammation, cells were stimulated with 400 ng/ml Ni(II) or a 10x higher concentration (4000 ng/ml) at varying time points (2 h, 6 h, 24 h, 72 h). Experiments without Ni(II) and IL-1β stimulation served as control.

RNA-isolation, first strand cDNA synthesis, qPCR

HGFs of six probands were separately used in our experiments and pooled on RNA level. Experiments were performed in triplicate on two occasions. Total RNA was isolated using the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and quantified using a NanoDropHND-1000 Spectrophotometer (NanoDrop, Technologies, Wilmington, DE, USA). First-strand cDNA synthesis was performed with 1 μg RNA using the iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, Munich, Germany) and oligo(dT)-primers.

The mRNA expression of IL-1β, IL-8, transforming growth factor (TGF)–β1, IL-10, MMP-1 and MMP-8 was detected by real-time polymerase chain reaction (RT-PCR) using the iCycler iQ detection system (Bio-Rad Laboratories), SYBR Green (Bio-Rad Laboratories), and gene-specific primers (Metabion, Martinsried, Germany) ( Table 1 ). Verification of all primers was accomplished by computer analysis for specification with the basic logical alignment search tool (BLAST) and synthesized of high quality (Metabion). In addition, the specific annealing temperatures were assessed by a temperature gradient, corresponding PCR-efficiencies for primers were determined with dilution series of sequenced primer-specific PCR-products. In Table 1 primer sequences, annealing temperatures and efficiencies are listed.

Table 1
Primer sequences Primer sequences with corresponding annealing temperatures, efficiencies, and product length in basepairs (bp) used for real time PCR. PCR efficiencies for every set of primers were determined with dilution series of primer specific cloned PCR-products at the corresponding annealing temperature. Efficiency stands for the performance to amplify a cDNA template. Efficiency values of 2.0 mean an amplification of 100%.
Gene Primer sequences (sense/antisense) Efficiency Annealing temperature (°C)
β-Actin 5′-CATGGATGATGATATCGCCGCG-3′
5′-ACATGATCTGGGTCATCTTCTCG-3′
1.84 69
IL-1β 5′-ATGGCAGAAGTACCTGAGCTCGC-3′
5′-TTAGGAAGACACAAATTGCATGGTG-3′
1.83 68
IL-8 5′-ATGACTTCCAAGCTGGCCGTGG-3′
5′-TGAATTCTCAGCCCTCTTCAAAAAC-3′
2.02 68
IL-10 5′-TTAAGGGTTACCTGGGTTGC-3′
5′-GCCTTGCTCTTGTTTTCACA-3′
1.94 65
MMP-1 5′-ATGCACAGCTTTCCTCCACTGC-3′
5′-CACTGGGCCACTATTTCTCCGC-3′
2.05 69
MMP-8 5′-ATGTTCTCCCTGAAGACGCTTCC-3′
5′-CAACGATCACATTAGTGCCATTC-3′
1.85 67
TGF-β1 5′-GAGCCCTGGACACCAACTAT-3′
5′-GACCTTGCTGTACTGCGTGT-3′
1.94 69
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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Possible implications of Ni(II) on oral IL-1β-induced inflammatory processes
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