Basic ResearchTechnology

Electrochemical Dissolution of Nickel-Titanium Endodontic Files Induces Periodontal Ligament Cell Death
Quinn Mitchell, BDS,* Billie Gail Jeansonne, DDS, PhD,* Diana Stoute, BS, and Thomas E. Lallier, PhD
Abstract
Introduction: Fractured endodontic les present a major problem. A novel method has been proposed to retrieve fractured nickel-titanium (NiTi) endodontic les by using electrochemical dissolution. However, the effect of le dissolution on adjacent soft tissues such as the periodontal ligament (PDL) has not been investigated. The aim of this study was to determine the effects of the dissolution products on PDL broblasts. Methods: Endodontic les were dissolved in sodium uoride (NaF) by passing a 50-mA current through the NiTi les while immersed in the NaF solution. NaF/NiTi solutions were diluted with minimal essential medium-a media containing 10% serum. PDL cells were treated for up to 24 hours, and cell viability was quantied by using calcein AM to label live cells and ethidium homodimer to label dead cells. This was repeated by using articial saliva (AS) as an alternative to NaF. Results: NaF solution reduced PDL cell survival, and the NaF/NiTi solution further reduced PDL cell survival. AS alone did not reduce cell survival, whereas AS/NiTi solution reduced PDL cell survival. Particles that resulted from the electrochemical dissolution of NiTi les were highly cytotoxic. Conclusions: Electrochemically dissolving NiTi les in NaF results in solutions that are cytotoxic to PDL broblasts. AS may be a less toxic alternative for dissolving NiTi les. (J Endod 2013;39:679684)

Key Words
Cytotoxicity, broblasts, nickel-titanium, periodontal ligament

From the *Department of Endodontics, Department of Oral Biology, and Department of Cell Biology and Anatomy, Center of Excellence in Oral and Craniofacial Biology, Louisiana State University Health Science Center, School of Dentistry, New Orleans, Louisiana. Supported by Louisiana State University Health Science Center, School of Dentistry, New Orleans, Louisiana. Address requests for reprints to Dr Thomas E. Lallier, Department of Cell Biology and Anatomy, Department of Oral Biology, Center of Excellence in Oral and Craniofacial Biology, Louisiana State University Health Science Center, School of Dentistry, 1100 Florida Avenue, New Orleans, LA 70119. E-mail address: tlalli@lsuhsc.edu 0099-2399/$ - see front matter Copyright 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.12.013

uring the course of endodontic therapy, fracture of endodontic instruments occurs in approximately 3.3% of cases (1), which may prevent the complete debridement of the root canal system and resulting in endodontic failure (1, 2). Many methods to remove fractured instruments have been proposed. A comparison of 2 current methods of retrieval by using ultrasonic instruments and the instrument removal system revealed no difference between methods, with an overall success rate of 70% (2). Current methods of removal have relied on mechanical means of removal requiring visualization of fractured instrument. This is more attainable in less curved canals with a longer radius of curvature. But because many fractured instruments occur in the apical third of the root beyond a curve (36), obtaining access to the separated instrument is often accompanied by excessive loss of tooth structure, failure to retrieve the fractured instrument, and/or perforation (2, 7, 8). Furthermore, although 85%97% of fractured les can be removed (7, 9), this process can lead to a two-thirds reduction in root strength (10). Thus, better means are needed to eliminate the le remnant from the narrow connes of the root apex. Recently, it has been proposed to remove the le remnant electrochemically (11, 12). These researchers successfully dissolved the majority of a NiTi le fragment by using a sodium uoride (NaF) solution and a current of 50 mA within the course of 1 hour [often the time needed to remove a le remnant manually (2)]. Electrochemical dissolution of NiTi les was performed in vitro and has never been attempted clinically. Currently, there is no evidence regarding the cytotoxic effects of NaF or the resulting NaF/nickel-titanium (NiTi) solution in periapical tissues. Further study is needed to determine whether this procedure is a viable alternative to manual removal of a le remnant. Although metallic NiTi alloys have been shown to be highly biocompatible (1317), little is known about the effects of soluble nickel and titanium in solution on periapical tissues. Specically, what are the effects of a NaF solution with both nickel and titanium on the surrounding soft tissues, specically the cells of the periodontal ligament (PDL)? Furthermore, little research has been done that investigates the toxicity of NaF in solution with nickel and titanium. In this study, we examined cytotoxicity of the solution of NaF/NiTi resulting from the dissolution of rotary NiTi les as previous described (11). We also examined the ability of an alternative saline solution, articial saliva (AS), to electrochemically dissolve rotary NiTi les and the potential cytotoxicity of the resulting solution.

Materials and Methods

PDL Cells PDL cells were isolated from periodontally healthy individuals who required tooth extraction for nonpathologic reasons at the Louisiana State University School of Dentistry. In all cases, tissues were obtained from subjects after informed consent as prescribed in an approved Institutional Review Board protocol (18). Cells were maintained in minimal essential medium-a (MEMa) containing 10% fetal bovine serum, 200 units/mL penicillin, and 200 mg/mL streptomycin (Invitrogen, GIBCO, Grand Island, NY). PDL broblasts between the 5th and 12th passages were used in this study. Isolates were deemed to be of PDL origin if they maintain spindle-shaped morphology in culture and express collagen-1, bronectin, vimentin, and bromodulin. These cells are currently being grown and maintained in the Lallier Lab.

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Creation of NiTi/NaF and NiTi/AS Solutions It has been reported that NiTi les can be dissolved in a NaF solution (120 mmol/L NaF, 17 mmol/L NaCl) by using a 5-mA current for 1 hour in vitro (11). An estimated volume of an average canal system (including the pulp chamber) was determined to be approximately 100 mL. A 3-mm le remnant (typical of one broken off in an endodontic procedure) was estimated to represent 6.75% of the mass of the uted (grooved) portion of an endodontic le. The uted portion of a typical le weighs approximately 10 mg, and NiTi is typically 56% nickel and 44% titanium. The uted portions of 34 NiTi endodontic hand les (50/02, Lexicon Dentsply 02 tapered hand les; DENTSPLY Tulsa Dental Specialties, York, PA) were dissolved in 50 mL NaF, resulting in a solution of 67.5 mmol/L Ti, 67.5 mmol/L Ni, 120 mmol/L NaF, and 17 mmol/L NaCl. This was accomplished by passing a 50-mA current through the NiTi les by using a platinum wire as an anode, while immersed in a NaF solution for more than 60 minutes or until the uted portion of the le dissolved completely to create stock solution NaF/NiTi. Alternatively, the uted portions of 34 endodontic les were dissolved in 50 mL AS (155 mmol/L NaCl, 5.6 mmol/L KCl, 2.4 mmol/L CaCl2, 2.4 mmol/L NaHCO3, pH 7.4) as described above to create a stock solution of AS/NiTi. Treatment of Cells with NiTi Solutions PDL cells were plated into 48-well plates at a density of 10,000 cells/mL in a volume of 200 mL and allowed to adhere for 24 hours before the addition of NiTi-containing solutions (either NaF/NiTi or AS/NiTi). NiTi solutions were diluted with MEMa media containing 10% serum. Cells were exposed to serial dilutions of NiTi solutions for 24 hours (8 samples of each dilution in addition to control). Cells treated with MEMa alone were controls. After treatment the cells were rinsed 3 times with MEMa and then immersed with MEMa. The NiTi solution was used in 4 different ways to determine the effect on PDL cell survival, with the fourth being the stock solution: control, stock solution dilutions of either NaF or AS; complete, solution after electrochemical dissolution containing both soluble and insoluble elements from NiTi les in the solution; soluble, soluble fraction of NiTi-containing solutions, separated by centrifugal force to remove insoluble particles; and insoluble, insoluble particulate fraction of NiTi-containing solutions, separated by centrifugal force to remove soluble ions. Quantication of Cell Viability Cell viability was monitored by using calcein AM (Invitrogen, Carlsbad, CA) to periodically label the live cells. This dye is a nonuorescent ester that is internalized by cells and cleaved by endogenous esterases, producing a uorescent carboxylic acid product that is trapped with the cells (excitation/emission maxima 495 nm/515 nm). Only live cells take up and retain this dye. Quantication was performed by using a BioTek Synergy 2 uorescent plate reader (BioTek, Winooski, VT). Cells were plated in 48-well plates and used for quantication via the microplate reader, with 8 samples per condition (n = 8). Examination of Cell Viability by Using Live/Dead Staining Cell viability was monitored by using calcein AM to periodically label the live cells. At the completion of the assay, dead cells were labeled with ethidium homodimer (excitation/emission maxima 495 nm/635 nm). This dye labels the nuclei of cells with broken plasma membranes (dead cells). Cell morphology was observed and recorded on a TE2000 inverted microscope (Nikon, Tokyo, Japan) equipped with a CoolSnap CF camera (Photometrics, Tucson, AZ).
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Statistical Analysis Comparison of cell survival was performed by using analysis of variance, with P < .001 considered to be signicant.

Results
Dissolving NiTi Files Electrochemically NiTi endodontic (50/02 les) les were electrochemically dissolved in either NaF or AS (Fig. 1A). In NaF solutions, NiTi les completely dissolved in 80 minutes, whereas in AS, les were completely dissolved in 60 minutes. After only 20 minutes, les in both solutions appeared to be noticeably smaller and corroded. After 40 minutes, les in NaF took on a nely tapered appearance, whereas those in AS appeared shorter and wider. In both cases, the les were visibly reduced in mass. A particulate, insoluble precipitate formed in both cases, beginning around 10 minutes after the application of current (data not shown). In NaF, the precipitate had a pale green appearance (likely a combination of NiO2 and TiO2), whereas the precipitate in the articial solution had a dark gray appearance (likely a combination of Ni2O3 and TiO2). NaF Solutions but Not AS Solutions Reduced PDL Cell Survival PDL cell survival after 24-hour exposure to NaF and AS solutions was examined (Fig. 1B). PDL cell survival was signicantly reduced (P < .001) in NaF solutions of 1.25% or greater. In contrast, PDL cells displayed little reduction in cell survival in AS solutions as high as 20%. Therefore, NaF solutions appear to be more cytotoxic than AS solutions. NiTi/NaF Solutions Reduce PDL Cell Survival Low concentrations of NaF/NiTi solutions reduced PDL cell survival. In 1.25% solutions of NaF/NiTi, PDL cell survival was signicantly reduced (P < .001) compared with NaF solutions alone (Fig. 2A). The insoluble particulate component (median lethal dose = 0.1%) of this solution was signicantly more cytotoxic (P < .001) than the soluble component (median lethal dose = 1.5%, Fig. 2B and Table 1). An examination of cells treated with 1% solutions of the NaF solutions revealed that cells assumed a rounded morphology (Fig. 3C) when compared with the elongated spindle morphology typical of untreated PDL broblasts (Fig. 3A). Furthermore, the use of ethidium homodimer stains revealed that roughly 50% of the cells were dead, with disrupted plasma membranes (Fig. 3C). Similarly, cells exposed to 1% soluble NaF/NiT solutions attained a similar rounded morphology and a similar percentage of permanent dead cells (Fig. 3E). Treatment of PDL cells with 1% suspensions of the NaF/ NiTi particles resulted in total cell death, with only cell fragments and debris loosely adhered to the substratum (data not shown). Therefore, NaF solutions (both with and without soluble NiTi) were highly cytotoxic to PDL cells. Only the Insoluble Particulates from NiTi/AS Solutions Reduce PDL Cell Survival Particulate precipitates from AS/NiTi solutions reduced PDL cell survival, whereas the soluble components did not. In 1.25% solutions of AS/NiTi containing insoluble particles, PDL cell survival was signicantly reduced (P < .05) compared with AS solutions containing soluble NiTi ions (Fig. 2C). Soluble NiTi ion containing AS did not display a signicant reduction in cell survival except at concentrations above 10%. The insoluble particulate component (median lethal dose = 0.1%) of this solution was signicantly more cytotoxic (P < .001) than the soluble component (median lethal dose > 20%,
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disrupted plasma membranes (Fig. 3D). Similarly, cells exposed to 1% AS/NiTi solutions maintained their elongated, spindle-shaped morphology and a small percentage of permanent dead cells (Fig. 3F). Treatment of PDL cells with 1% suspensions of the NiTi/ AS particles resulted in total cell death, with only cell fragments and debris loosely adhered to the substratum (data not shown). Therefore, AS solutions with soluble NiTi were slightly more cytotoxic to PDL cells at concentrations above 10%.

120%
100% 80% 60%

40%
20% 0% NaF

AS
Control 1.25% 2.5% 5% 10% 20%

Concentration
120% 100%

NiTi/NaF Solutions Induce Less Cell Death after 2-Hour Exposures Because of the cytotoxicity noted in the 24-hour exposure time of NaF/NiTi solutions, a shorter exposure time of 2 hours was examined (Fig. 1C). Two hours was chosen to be closer to the typical retreatment time needed to remove a fractured le in a clinical setting. Even at these shorter exposure times, the insoluble particulates present in NaF/NiTi solutions caused considerable cell death. In contrast, a 2-hour exposure to NaF induced no detectable reduction in cell survival. Furthermore, the additional soluble NaF/NiTi solutions up to 5% induced no detectable cell death. NaF/NiTi solutions greater than 10% induced signicantly more cell death (P < .001) at 2 hours but exhibited less cell death than after 24-hour exposure (P < .001). Thus, a reduction of exposure time signicantly reduced the cytotoxicity of both NaF solutions as well as soluble NiTi ions and raised the median lethal dose from 2% to 10% (Table 1).

% Cell Survival

Discussion
Quantitative analysis of cell viability of the solutions resulting from the dissolution of NiTi endodontic les in NaF or AS showed differences in cytotoxicity. NaF, the solution recommended for the dissolution of NiTi endodontic les in a previous study (11), was found to be cytotoxic even in very dilute solutions (below 2%). The insoluble dissolution byproducts were found to be highly cytotoxic when either NaF or AS was used for dissolution. In contrast, the soluble elements of these solutions had little effect on the cytotoxicity when compared with the stock solutions before dissolution. Through the course of this study, AS was seen to be a reasonable alternative to NaF. It is able to dissolve a NiTi endodontic le as efciently as NaF (Fig. 1) and is signicantly less cytotoxic to PDL cells (Figs. 2 and 3). After quantifying cell survival at various dilutions, approximate median lethal doses were determined for the various components of the solutions (Table 1). Little difference was seen between the median lethal doses of NaF and NaF/NiTi (soluble) after 24 hours of exposure. AS and AS/NiTi solutions also showed little increase in cytotoxicity when compared with control. The insoluble particles in either NaF/NiTi or AS/ NiTi were equally cytotoxic and 10 more cytotoxic than the NaF/NiTi (soluble) solutions. Thus, the dissolution products of NiTi les are cytotoxic to PDL cells, with a cytotoxicity ranking of insoluble particles > soluble NaF/NiTi > soluble AS/NiTi. Although NiTi alloys have been examined extensively in the oral cavity and have been shown to be safe (13, 14, 16, 17), less is understood about the effects of soluble Ni or Ti ions or the insoluble particulates created by the process of electrochemical dissolution of NiTi les in either NaF or AS solutions. We have calculated that the dissolution of a 3-mm le tip in a 100-mL root canal would result in the release of approximately 67.5 mmol/L Ni and Ti ions. Much of this would form insoluble particles of NiO2 (green), Ni2O3 (black), or TiO2 (white), as seen in this study. However, an unknown amount of Ni may remain in solution, because NiCl2 is soluble well above 1 mol/L. Ni ions are known to have detrimental effects on cells. Ni ions in concentrations as low as 10 mmol/L induce apoptosis in T cells (19,
PDL Cytotoxicity of Dissolved NiTi

80%
% Cell Survival
60% 40% 20%
NaF NaF NiTi-C

NaF NiTi-SF NaF NiTi-ISF

0%

Control

1.25%

2.5%

5%

10%

20%

Concentration

Figure 1. PDL cell survival in diluted NaF and AS solutions. (A) NiTi les were electrochemically dissolved in either NaF or AS solutions. Untreated les represent the les before dissolution (0). Files were completely dissolved in NaF after 80 minutes, whereas those in AS were completely dissolved in 60 minutes. Files dissolved for intermediate times of 40 and 20 minutes are also presented. (B) PDL cells were exposed for 24 hours to various concentrations of NaF or AS solutions. Cell survival was assayed by using calcein AM and quantied uorometrically. NaF solutions were signicantly more cytotoxic than AS solutions. Each point represents the mean and standard deviation of 8 samples. (C) PDL cells were exposed for 2 hours to various dilutions of NaF, NaF containing soluble NiTi and insoluble particles (C), only the soluble fraction (SF), or insoluble particles (ISF). Survival was assayed 24 hours later by using calcein AM and quantied uorometrically. Each point represents the mean and standard deviation of 8 samples. Short-term exposure to NaF and soluble NiTi solutions (up to 5%) did not reduce PDL cell survival.

Fig. 2D and Table 1). An examination of cells treated with 1% solution of AS revealed that cells retained an elongated spindle-shaped morphology (Fig. 3D), comparable with that typical of untreated PDL broblasts (Fig. 3B). Furthermore, the use of ethidium homodimer stains revealed that very few of these cells were dead, with
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120%
NaF

120%
NaF NiTi-C

100%

100%

NaF NiTi-SF

% Cell Survival

60%

% Cell Survival

80%

NaF NiTi-ISF

80%
60%

AS
AS NiTi-C AS NiTi-SF AS NiTi-ISF

40%
20% 0%

40%
20%

Control

1.25%

2.5%

5%

10%

20%

0%

Control

1.25%

Concentration
120% 100% 120%

2.5% 5% Concentration

10%

20%

AS NiTi-C

100% 80%
60% 40% 20%

AS NiTi-ISF

% Cell Survival

60% 40% 20% 0% NaF NaF NiTi-C NaF NiTi-SF NaF NiTi-ISF Control 0.01% 0.1% 0.5% 1% 2%

% Cell Survival

80%

0%

Control

0.01%

Concentration

0.05% 0.1% Concentration

0.5%

1%

Figure 2. PDL cell survival in diluted NaF-NiTi and AS-NiTi solutions. (A and B) PDL cells were exposed for 24 hours to various concentrations of NaF (A and B) or AS (C and D) solutions containing soluble NiTi and insoluble particles (C), only the soluble fraction (SF), or insoluble particles (ISF). Cell survival was assayed by using calcein AM and quantied uorometrically. Each point represents the mean and standard deviation of 8 samples. Concentration ranges from 0%20% (A and C) and 0%2% (B and D) were compared. Solutions containing insoluble NiTi precipitates were signicantly more cytotoxic than those only containing soluble NiTi.

20), as well as kidney cells (21, 22) and neutrophils (23), in part by activation of the AKT/p38 pathway (24). In addition, lower concentrations of Ni ions have been shown to have carcinogenic effects (2527) and can alter the expression of at least 134 transcripts in keratinocytes (28). Furthermore, TiO2 particles greater than 300 nm in diameter are cytotoxic to broblasts and neutrophils (23, 29, 30). Thus, the byproducts of NiTi le dissolution have the potential to cause signicant harm to living tissues, especially if extruded out the apex of the root. Unfortunately, insoluble precipitates that formed as a result of the dissolution process were unavoidable and highly cytotoxic. However, it could be argued that during the course of endodontic therapy a very cytotoxic irrigant, sodium hypochlorite, is used routinely to disinfect the canal system. Only in rare circumstances is this material inadvertently extruded into periapical tissues. A similar rationale could also be made for the insoluble dissolution precipitates. If kept inside the canal system or removed through the ushing action of irrigants, it is reasonable to think that this process could be used clinically without
TABLE 1. Summary of Cytotoxicity Results Test solution
NaF AS NaF/NiTi NaF/NiTi AS/NiTi NaF/NiTi AS/NiTi

State
Soluble Soluble Soluble Soluble Soluble Insoluble Insoluble

Potentially Exposure Median toxic time (hours) lethal dose compounds

24 24 24 2 24 24 24 2% >20% 1% 10% >20% 0.1% 0.1% F NiF2, NiCl2 NiF2, NiCl2 NiCl2 NiO2, TiO2 Ni2O3, TiO2

undue harm to the patient. However, if insoluble precipitates are extruded into periapical tissues, it would be far more cytotoxic than the soluble solution alone. In addition to cytotoxicity, precipitate color may also play a role in determining which solution to use for le dissolution. A light green precipitate was formed during the dissolution of les by using AS, whereas a black precipitate was formed when using NaF. The esthetic effect of these precipitates is currently unknown and should be investigated further. However, the formation of a dark precipitate would also encourage regular irrigation to reduce any negative esthetic effect and reduce any temperature changes encountered during the process. More studies are needed to determine whether there is a clinically applicable delivery method that would be suitable for patient use. Animal studies addressing the effect of a closed circuit current system, heat generated during the dissolution process, and effects of precipitates formed on PDL cells should also be performed. These studies would also be useful in determining the optimal current needed to dissolve a typical fractured le tip in a clinically relevant setting, because it took much more than an hour to dissolve the entire uted portion of a le by using a 50-mA current. These studies would also reveal whether the use of continuous ushing could prevent the extrusion of insoluble particles out of the apex of the tooth during this procedure.

Conclusions
NaF is cytotoxic to PDL broblasts, and insoluble elements of the NaF/NiTi solution were even more cytotoxic. Dissolving les in AS resulted in a solution that was less cytotoxic to PDL broblasts, but both methods produced highly toxic particles. AS completely
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Figure 3. Cell viability of cells treated with NaF and AS solutions containing NiTi. Two stains, calcein AM (green for live cells) and ethidium homodimer (red for dead cells), were used to examine cell viability and morphology after treatment for 24 hours with 1% solution of NaF (C), NaF containing NiTi-soluble (E), 1% solution of AS (D), or AS containing NiTi-soluble (F). Untreated cells served as controls (A and B).

dissolved NiTi les as efciently as NaF and should be considered a good alternative to NaF for these applications.

Acknowledgments
The authors deny any conicts of interest related to this study.

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