2.1

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.

2.2

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.

2.3

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.

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 .

2.4

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.

2.5

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.

2.6

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.

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.

2.7

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).

2.1

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.

2.2

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.

2.3

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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