Material and methods

To determine the cytotoxicity of orthodontic metals on fibroblasts, we used a human fibroblast cell line 142BR (90011806; European Collection of Cell Cultures, Salisbury, Germany). The cells were cultured in petri dishes (49 cm ² , Sarstedt, Numbrecht, Germany) with minimal essential medium (MEM) (no. M2279) supplemented with 15% fetal bovine serum, 1% antibiotic solution (10,000 μ/mL of penicillin, 10 mg/mL streptomycin, 25 μg/mL amphotericin B), 200 mM of L-glutamine, and 1% nonessential amino acids. Cell cultures were kept in an incubator (Heracel 150; Heraeus, Hanau, Germany) at 37°C, 95% humidity, and 5% carbon dioxide in the air. All products, except those indicated, were purchased from Sigma-Aldrich (St Louis, Mo).

Metals were extracted from 10 samples of 3 different metallic materials used in standard orthodontic treatments. Each sample consisted of 4 buccal tubes and 20 brackets (slot, 0.22 in) all manufactured with the same materials, submerged for 30 days in 30 mL of MEM without phenol red (no. M3024), supplemented with 0.5% fetal bovine serum, and kept in a cell incubator. The control samples contained 30 mL of supplemented MEM without metallic elements and with the same conditions as the other groups. From these 30 mL, 15 mL was used for the determination of metal ions, and the other 15 mL to perform the tetrazolium reduction assay (MTT assay). (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) and the Comet assays (single cell gel electrophoresis assay), a widely-used assay to microscopically detect DNA damage at the level of a single cell.

The experimental groups were as follows.

• 1.

  Control (n = 10): each sample contained 30 mL of MEM without metals.

• 2.

  Stainless steel (n = 10): each sample contained 20 Ultraminitrim brackets and 4 Ortho molar tubes (both, Dentaurum, Ispringen, Germany) immersed in 30 mL of MEM. The alloy is made of 0.10% carbon, 1.0% silcon, 2.0% manganese, 17.0% chromium, 10.0% nickel, 0.50% phosphorus, 69.35% iron, and 0.05% selenium.

• 3.

  Titanium (n = 10): each sample contained 20 Orthos brackets and 4 buccal molar tubes (both, Ormco, Glendora, Calif), immersed in 30 mL of MEM. The alloy is made of 99% titanium and 1% chromium.

• 4.

  Nickel-free (n = 10): each sample contained 20 Minisprint brackets and 4 Flat-line molar tubes (both, Forestadent, Pforzheim, Germany) immersed in 30 mL of MEM. The alloy is made of ≤0.10% carbon, ≤1.0% silicon, 16% to 20% manganese, 16% to 20% chromium, 1.8% to 2.5% molybdenum, ≤0.3% nickel, ≤0.05% phosphorus, and ≤0.05% sulfur.

The determination of metals was performed by using inductively coupled plasma mass spectrometry (Algilent 7500ce; Agilent Technologies, Santa Clara, Calif). For that, the samples (10 mL) were acidified with 0.25 mL of 69% nitric acid (Hiperpur, Panreac, Barcelona, Spain). The metals measured in all samples were those included in the metal composition lists from the companies: Ti ⁴⁷ , Cr ⁵² , Mn ⁵⁵ , Co ⁵⁹ , Ni ⁶⁰ , Mo ⁹² , and Fe ⁵⁶ . In 3 samples of each group, we analyzed also Cu ⁶³ , Zn ⁶⁶ , As ⁷⁵ , Se ⁷⁸ , Cd ¹¹¹ , and Pb ²⁰⁸ . All of these metals were determined in all experimental groups, independently of the alloy compositions provided by the manufacturers. We obtained 3 determinations per sample, so the result of a sample is the average of the 3 values. The metal concentration is in the range of 1 part per billion, equivalent to 1 μg per liter. Standard monoelemental solutions for each metal (Fluka, Madrid, Spain) prepared in the MEM were used for the calibration.

For the MTT assay, cultured fibroblasts were detached from the petri dishes with a 0.25% trypsin-EDTA solution (5 minutes at 37°C) and seeded in 96-well plates at a concentration of 5000 cells per well. Counting procedures were performed with a Neubauer camera (Laboroptik, Bad Homburg, Germany) by using the trypan blue exclusion technique that stains dead cells. The first column contained only MEM (no cells) and served as the blank. The plates were kept in the cell incubator; after approximately 72 hours, they reached 90% to 100% of confluence. Next, the fetal bovine serum concentration in the MEM without phenol red was changed from 15% to 0.5% and maintained for 72 hours. Finally, we started the assay with the following distribution.

• 1.

  Blank column: 200 μL of MEM without cells.

• 2.

  Control column: 200 μL of MEM from the control group obtained in the previous protocol. This was our positive control, or 100% viability.

• 3.

  Toxic column: 160 μL of MEM plus 40 μL of absolute ethanol.

• 4.

  Stainless steel column: 200 μL of MEM from the stainless steel group.

• 5.

  Titanium column: 200 μL of MEM from the titanium group.

• 6.

  Nickel-free column: 200 μL of MEM from the nickel-free group.

In the toxic column, we applied ethanol, a known cellular toxin previously tested in our laboratory to cause dose-dependent toxicity. As in our previous results (data not shown), 25% ethanol also produced great damage to the fibroblasts.

The 96-well plates were maintained in the cell incubator for 7 days with the media containing metals obtained in the previous protocol. After that, the media were removed and replaced by MTT solution (5 mg/mL, in MEM) for 4 hours under the same conditions. Yellow MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is reduced to purple formazan in living cells. Then the excess of the MTT solution (nonreduced MTT) was eliminated from the wells. Next, 100 μL of dimethyl sulfoxide was added to dissolve the purple formazan into a colored solution, and the absorbance was quantified in a spectrophotometer (Labsytems multiskan MCC/340, Helsinki, Finland) at 570 nm with a reference wavelength of 690 nm. The percentage of survival was calculated considering the mean absorbance of the treated cells with respect to the control cells, subtracting before the mean absorbance of the blank column to both of them. Each assay was performed in triplicate.

For the Comet assay, conventional microscope slides (Menzel-Glässer, Braunschweig, Germany) were treated with 2 layers of agarose. The bottom layer was prepared by dipping the slides into 1.0% normal melting agarose (Biorad, Madrid, Spain), allowing it to solidify at 4°C for a minimum of 5 minutes. Then, the top or cell-containing layer consisted of 100 μL of a fibroblast suspension prepared in low melting-point agarose at 0.5%, containing 5000 to 10,000 fibroblasts per milliliter (15 μL of cell suspension + 85 μL agarose). After covering, the slide was kept at 4°C for 5 minutes.

Next, the second coverslip was removed, and the fibroblasts were incubated (24 hours at 37°C) with the MEM containing the different metallic compounds obtained in the first protocol. Thus, the experimental groups (n = 5) were control, toxic, stainless steel, titanium, and nickel-free. In the toxic group, the slides were additionally treated with 1000 μmol/L of hydrogen peroxide (30 minutes at 37°C). Then, all slides were rinsed with a 0.4 mol/L tris base solution (pH 7.5). Except when indicated, all incubations were performed directly on the cell layer with 100 μL of the various solutions, and the slides were kept in a moist dark chamber.

After that, the cells were subjected to lysis with 0.25% trypsin in phosphate-buffered saline solution (30 minutes at 37°C), washed with 0.4 mol/L tris base solution, and treated with proteinase K (1 mg/mL; Roche, Madrid, Spain) for 1 hour. After this, the slides were rinsed, immersed in lysis solution (2.5 mol/l sodium chloride, 0.1 mol/L EDTA, 10 mmol/L tris base; 1% Tritón X-100 (Fluka, Madrid, Spain), and 10% dimethyl sulfoxide; pH 10) for another hour at 4°C and washed again with 0.4 mol/L of tris base solution.

The Comet assay was run by using a horizontal gel electrophoresis tank (Biorad) containing freshly prepared cold (4°C) electrophoresis buffer (1 mmol/L Na2 EDTA and 10 mmol/L NaOH, pH 9) with the slides submerged side by side in the gel tray and left for 20 minutes to produce single-stranded DNA (unwinding). Later, the electrophoresis was run at 25 V and 300 mA for 20 minutes, and the slides were rinsed with tris solution.

The cells were stained with 75 μL of a 20 μg per milliliter solution of ethidium bromide (Biorad) and covered, and the comets were visualized in a fluorescence microscope (Leica, Wetzlar, Germany) by using a barrier filter of 590 nm. The images were taken with a time exposure of 2 seconds, a magnification lens of 10 times, and a resolution of 3900 × 3900. Thus, each image was formed of 12,051 megapixels, and each pixel corresponded to a detected light intensity.

We analyzed 50 randomly chosen nuclei from each slide using the free computerized image analysis system Comet Score (Tritek CometScore Freeware, version 1.5; http://autocomet.com/products_cometscore.php ). The main measure of DNA damage was the olive moment automatically obtained by the software and defined as the product of the distance between the centers of gravity of the tail (CGt) and the head (CGh) along the x-axis of the comet, and the fraction of total DNA in the tail (represented by the intensity of DNA in the tail). Olive moment = (CGt − CGh) ^∗ DNA %/100.

Four slides per sample were evaluated from the experimental groups. From the repeated experiments, the averaged olive moment was calculated for each slide, each sample, and each group. Before any measurement, we calibrated geometrically the imaging software system to know the number of pixels per micrometer of our lens.