To evaluate the cytotoxicity effects of two different solder materials used for orthodontic appliances on human periodontal ligament fibroblast (HPLF) cells, and to determine whether the mechanism of toxicity may involve oxidative stress and apoptosis.
The silver solder samples (Leone and Summit) were soldered to orthodontic stainless steel bands and exposed to HPLF cells via cell culture inserts for 48 hours. Cytotoxicity effect of the soldered materials on HPLF cells was measured via tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay (n = 10/sample) and morphological observation. In addition, the mechanism of cytotoxicity of the most toxic silver solder was investigated using both a caspase inhibitor Z-VAL-Ala-Asp-flu-oromethylketone (ZVAD-fmk) and the free radical scavenger Trolox (n = 8/sample). Statistical analysis was performed using one-way analysis of variance with a Bonferroni test. P < .05 was considered statistically significant.
Compared to the control (no treatment, cells only), both silver solders were cytotoxic (P < .001). The bands alone were significantly cytotoxic compared to the control. There was a significant difference in cytotoxicity between the stainless steel bands alone and the Summit silver solder (P < .001), but not the Leone silver solder. The Summit silver solder was more cytotoxic than the Leone silver solder (P < .05). MTT results were supported by the microscopic morphological changes of the HPLF cells. Neither ZVAD-fmk nor Trolox provided significant protection.
The two silver solder materials demonstrated different levels of cytotoxicity, and neither oxidative stress nor apoptosis is involved in the mechanism of cytotoxicity.
The fabrication of orthodontic appliances often requires the use of soldering to allow for their intricate design. The use of orthodontic appliances, whether removable or fixed, is associated with different gingival or soft tissue reactions.1 The most common reaction is gingivitis, while other reactions of unknown etiology are lichenoid reactions, hyperplasia of the gingiva, and discoloration. Cytotoxicity, a response to an alteration in cellular function, especially intracellular processes and changes in metabolic pathways, could be a cause of these irritations.2 The oral environment with saliva fluctuations of pH values, temperature, and mechanical forces, leads to stresses and corrosion.3–5
Nickel and copper metal ions released from stainless steel orthodontic appliances are known to be cytotoxic to cells.6–8 However, the extent to which the released metal ions can produce local and systemic effects on human health is not fully understood.9
It has been shown that soldered appliances undergo some amount of corrosion, which facilitates the release of metal ions that may cause adverse effects.10,11 Also, it has been demonstrated that when silver solder materials were fused to orthodontic bands, higher concentrations of iron, nickel, chromium, copper, silver, zinc, and cadmium ions were detected, with silver and copper ions being the main cause of cytotoxicity.12,13 Jacoby et al.14 showed that under identical experimental and culture conditions, different cell lines exhibited variable levels of cytotoxicity in response to silver solder materials.
Goncalves et al.12 found that orthodontic bands with silver solder joints induced stronger cytotoxicity and genotoxicity effects than bands without silver solder joints. Additionally, they investigated the mechanism of the cytotoxicity and DNA damage and found the production of reactive oxygen species (ROS) responsible for the oxidative stress. Recently, it was found that cells tend to counterbalance the production of ROS by increasing the production of some antioxidant enzymes, eg, peroxiredoxin1 (PRDX1) and superoxide dismutase1(SOD1) but not glutathione peroxidase1 (GPX1).15 Still, there are unanswered questions as to whether cytotoxicity was due to overproduction of antioxidants or inadequate specific antioxidant enzymes for ROS. Also unanswered is whether necrosis or apoptosis is primarily responsible for cell death. The conclusion from their experiments recommended further research, with different cell lines and different silver solders to be tested.
To determine which silver solder materials are less toxic, two different silver solder materials with different alloy compositions on the same cell line cells were tested. Additionally, the effect of Trolox, a free radical scavenger antioxidant, and Z-VAD-fmk (Z-Val-Ala-Asp-fluoromethylketone), a caspase inhibitor anti-apoptotic on the PDL cells, was investigated after exposure to the tested silver solder materials. Both reagents have been used extensively in the detection of survival rates of neural cells. It has been shown that cytotoxicity can be inhibited by either of the compounds or not, depending on the mechanism of cell death.16,17
This study analyzed the cytotoxicity effects of two different solder materials used for orthodontic appliances on human periodontal ligament fibroblast (HPLF) cells, and determined whether the mechanism of toxicity involved oxidative stress and apoptosis.
MATERIALS AND METHODS
Orthodontic Silver Solder Materials and Bands
Two commonly used silver solders for orthodontic purposes with published differences in alloy composition were tested:
Leone (Leone Silver Solder; Firenze, Italy); Ag 49%–51%, Cu 26%–29%, Zn 20-22%, Mn 1.5%–3%, Ni .41%–1%.
Summit (Summit Silver Solder; Munroe Falls, OH, USA); Ag 56%, Cu 22%, Zn 17%, Sn 5%.
Stainless steel bands from 3M Unitek (Monrovia, CA, USA) were used as the material to which the solder was fused.
Lengths of 10 mm of each of the two tested silver solder materials were fused to stainless steel bands; each silver solder plus stainless steel band unit created samples weighing approximately 0.16 g. The samples were then placed in a 70% alcohol solution into an ultrasonic bath for 10 minutes to remove excess flux.
Cell Culture and Exposure to Experimental Conditions
HPLF cells were purchased from ScienCell Research Laboratories (cell lines #2630; Carlsbad, CA). The HPLF cells were cultured in alpha-MEM with 10% fetal bovine serum (FBS), and 1% antibiotic-antimycotic solution (penicillin-streptomycin) under standard conditions (37°C, 100% humidity, 95% air and 5% CO2) for 48 hours, and the culture medium was refreshed every 24 hours. The cells were seeded at a density of 2×105/well in 24-well culture dishes for 48 hours, starved with serum-free medium overnight. The different samples were exposed to the HPLF cells via tissue culture inserts of 0.4 μm pore size suspended above the cultured cells for 48 hours. This ensured that the samples were in contact with the media bathing the cultures, but not in direct contact with the cells, avoiding possible cell damage due to physical contact of the cells.18,19 Cells were exposed to the materials for 48 hours in serum-free medium. A positive control of 100% cell death involved exposure of cells to the calcium ionophore, A23187 (10 mmol/L). The negative control was only cells exposed to serum-free medium. The tested groups (n = 10 each group) were: (B) cells exposed to bands only, (BS) cells exposed to bands fused with Summit silver solder, (BL) cells exposed to bands fused with Leone silver solder, (BS+Trolox) cells exposed to bands fused with Summit silver solder with serum-free medium containing Trolox (100μM), and (BS+ZVAD-fmk) cells exposed to bands fused with Summit silver solder with serum free medium containing ZVAD-fmk (100μM). Trolox and ZVAD-fmk were applied to the group with the highest level of cytotoxicity.
After 48 hours, all the inserts were removed from the wells. Photographs of the HPLF cells were taken for each experimental group using an inverted microscope (VWR Trinocular, China) at 40× magnification. Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays were then completed to assess cytotoxicity.
Analysis of Cytotoxicity
Cellular metabolism was measured by means of the MTT colorimetric assay. MTT was reduced by mitochondrial succinate dehydrogenase and accumulates in the cell. After removal of the culture inserts, 50 μL of 10% (V/V) MTT solution was added to each well and incubated for 30 minutes in a cell culture incubator. The MTT solution was removed and replaced by 250 μL of dimethyl sulfoxide (DMSO) in each well to dissolve the formed formazan crystals and cells. MTT measurements were done on 96 well plates by a spectrophotometer (Molecular Devices, San Diego, CA), with a measured absorbance at 570 nm.
Calculations were performed with a Shapiro-Wilk test for normality, and one-way analysis of variance (ANOVA) with a Bonferroni correction. P values less than .05 were considered statistically significant.
Cell Viability after Exposure to the Tested Samples
The MTT test showed statistically significant differences (P < .05) in cell viability among no treatment (control) and both Leone silver solder (BL) and Summit silver solder (BS) groups. The stainless steel bands (B) were also significantly cytotoxic compared to cells only (control). The results also showed a significant difference in cell viability between the stainless steel bands only (B) and the Summit silver solder (BS). However, there was no significant difference between the stainless steel bands (B) and the Leone silver solder (BL). Comparing cell viability between the two silver solders, the Summit silver solder (BS) was significantly (P < .001) more cytotoxic than the Leone silver solder (BL) (Table 1 and 2).
Different morphological features were seen on the different photos (bar on photos is 20 μm) represented as: (negative control group) (Figure 1a), calcium ionophore (positive control group) (Figure 1b), stainless steel bands only (B) (Figure1c), bands with Leone silver solder (BL) (Figure 1d), and bands with Summit silver solder (BS) (Figure 1e).
Cell Viability After Treatment with ZVAD-fmk and Trolox
The MTT experiments were used to detect the effect on cell viability of adding antioxidant factor Trolox and antiapoptotic factor ZVAD-fmk. These experiments were tested on the Summit silver solder group only since it showed the highest level of cytotoxicity. The results of adding Trolox (BS + Trolox), or adding ZVAD-fmk (BS + ZVAD), still showed significant cell death compared to the control (P < .05) (Tables 3 and 4).
The MTT results showed that stainless steel bands were significantly cytotoxic to the HPLF cells (Tables 1 and 2). This was in agreement with other studies showing that stainless steel bands release chromium, nickel, and iron metal ions in concentrations high enough to induce cytotoxicity and genotoxicity in cells.12 A review article elaborated on the cytotoxic effect of nickel metal ions released from stainless bands.20 Another study demonstrated, in a three-dimensional human-derived oral mucosal model, that Ni-based alloys were cytotoxic and could induce oxidative stress and inflammatory cytokine expression.21 Some studies demonstrated contradictory results showing that stainless steel bands alone were not cytotoxic.14,22
Both silver solders were shown to be significantly cytotoxic compared to the control (Tables 1 and 2). These results were also supported by the different morphological patterns of the cells shown in the microscopic photographs, where more rounded cells were seen (Figure 1d,e). A previous study found that fibroblasts had irregularly shaped nuclei when exposed to hexavalent chromium and nickel, pseudopodia when exposed to beryllium and molybdenum, and lipid-droplet formation when exposed to nickel.23 Other researchers found that mice fibroblasts exposed to silver solder experienced inhibition of proliferation, growth, and development of the examined cells. It was also previously demonstrated that silver solder was cytotoxic and genotoxic to two different human cell lineages.12,24
Summit silver solder demonstrated the greatest level of cytotoxicity as shown by the diminished number of viable cells (Tables 1 and 2). One explanation for the difference in cell viability could have been the higher silver concentration in Summit silver solder (56% Ag) versus Leone silver solder (49%–51% Ag). In previous studies, it was shown that a change in the metal concentrations of an alloy would cause a change in their release into the medium in which they were immersed.25 At low concentrations (<2.0 μM/mL) of silver ion, cellular mitochondrial activity is essentially unchanged from normal. However, as the concentration of silver ion increased in the medium, cellular activity decreased exponentially. When concentrations exceeded 10 μM/mL, cellular activity was practically zero.26
Another explanation for the relatively greater cytotoxicity of Summit silver solder may have been its tin content, as the Leone silver solder contained no tin. It has been shown that mouse fibroblasts exposed to tin metal ions suppressed cell activity by 50%.26 A further study could be conducted to determine which metal ions were released from these silver solder materials into the solution and to isolate each orthodontic material to evaluate its toxicity as different appliance combinations may lead to varying responses.
The mechanism of cytotoxicity was investigated by testing the effects of a caspase inhibitor antiapoptotic agent ZVAD-fmk (Z-Val-Ala-Asp-fluoromethylketone) and a free radical scavenger antioxidant Trolox. ZVAD-fmk has been shown to be a consistent marker of apoptotic cell death.27 Trolox is a strong antioxidant that is commonly used in antioxidant assays and oxidative injury studies.16 Results showed that neither Trolox nor ZVAD-fmk showed significant protection against cytotoxicity, which might also indicate that none of them was involved in the mechanism of cell toxicity.
The two silver solders used in this study were tested for their cytotoxicity to PDL fibroblasts. Cytotoxicity studies, being in vitro assays, are considered an initial or primary level of screening for the biocompatibility of dental materials/devices and appear in standards such as ADA/ANSI Standard No. 41, ISO 10993, and ISO 7405. Determining the clinical implications of these in vitro studies is problematic. Overall, the complexity of the interactions that occur in the mouth cannot be modeled in vitro. For instance, the solders and bands were in a static environment for 48 hours whereas, in the mouth, saliva and beverages would dilute or wash corrosion products away. What these studies do provide is evidence that these orthodontic materials can be toxic and that the level of toxicity can vary depending on the materials used. These studies should not be used to disqualify any of these materials from clinical use. Instead, they should be used to direct future studies. It should be recognized that the biocompatibility of a material would also involve assessment of inflammatory, allergic, and mutagenic reactions.
Limitations of this study included lack of standardization of the silver solder surfaces, eg, polishing the soldered surfaces would potentially lead to less corrosion and different cytotoxic results. Use of testing conditions that mimic the oral environment may also lead to different results. Further studies could test the cytotoxicity of other brands of silver solder and alternate means of joining metals, eg, laser welding.
The two silver solder materials demonstrated different levels of cytotoxicity, and neither oxidative stress nor apoptosis was involved in the mechanism of cytotoxicity.
Antioxidant (Trolox) and antiapoptotic (ZVAD-fmk) reagents did not show significant protection against cytotoxicity.
Additionally, stainless steel bands were significantly cytotoxic compared to the control.
Assistant Professor, Department of Orthodontics, Marquette University School of Dentistry, Milwaukee, Wis., USA.
Private Practice, Rosemount, Minn., USA.
Professor, Department of Biomaterials, Marquette University School of Dentistry, Milwaukee, Wis., USA.
Professor & Program Director, Department of Orthodontics, Marquette University School of Dentistry, Milwaukee, Wis., USA.
Assistant Professor, Department of Orthodontics, Marquette University School of Dentistry, Milwaukee, Wis., USA.
Professor, Department of Biomedical Science, Marquette University, Milwaukee, Wis.