Development and evaluation of two PVD-coated β-titanium orthodontic archwires for fluoride-induced corrosion protection

Acta Biomaterialia 7 (2011) 1913–1927

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Acta Biomaterialia

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Development and evaluation of two PVD-coated b-titanium orthodonticarchwires for fluoride-induced corrosion protection

Vinod Krishnan a,⇑, Anand Krishnan b, R. Remya c, K.K. Ravikumar d, S. Asha Nair b, S.M.A. Shibli c,H.K. Varma e, K. Sukumaran d, K. Jyothindra Kumar f,1

a Department of Orthodontics, Sri Sankara Dental College, Trivandrum, Kerala, Indiab Translational Cancer Research Laboratory, Department of Molecular Medicine, Rajiv Gandhi Centre for Biotechnology, Kerala, Indiac Department of Chemistry, University of Kerala, Trivandrum, Kerala, Indiad Materials Characterization Division, National Institute of Interdisciplinary Science and Technology, CSIR, Trivandrum, Kerala, Indiae Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Science and Technology, Trivandrum, Kerala, Indiaf Government Dental College, Kottayam, Kerala, India

a r t i c l e i n f o

Article history:Received 21 August 2010Received in revised form 19 November 2010Accepted 19 November 2010Available online 24 November 2010

Keywords:Orthodontic archwiresPhysical vapor deposition coatingCathodic arc physical vapor depositionMetal surface treatmentFluoride corrosion

1742-7061/$ - see front matter � 2010 Acta Materialdoi:10.1016/j.actbio.2010.11.026

⇑ Corresponding author. Tel.: +91 9447310025.E-mail address: [email protected] (V. Krishna

1 Principal.

a b s t r a c t

The present research was aimed at developing surface coatings on b titanium orthodontic archwirescapable of protection against fluoride-induced corrosion. Cathodic arc physical vapor deposition PVD(CA-PVD) and magnetron sputtering were utilized to deposit thin films of titanium aluminium nitride(TiAlN) and tungsten carbide/carbon (WC/C) coatings on b titanium orthodontic archwires. Uncoatedand coated specimens were immersed in a high fluoride ion concentration mouth rinse, following a spe-cially designed cycle simulating daily use. All specimens thus obtained were subjected to critical evalu-ation of parameters such as electrochemical corrosion behaviour, surface analysis, mechanical testing,microstructure, element release, and toxicology. The results confirm previous research that b titaniumarchwires undergo a degradation process when in contact with fluoride mouth rinses. The study con-firmed the superior nature of the TiAlN coating, evident as many fewer changes in properties after fluo-ride treatment when compared with the WC/C coating. Thus, coating with TiAlN is recommended in orderto reduce the corrosive effects of fluorides on b titanium orthodontic archwires.

� 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Substituted titanium alloy archwires are utilized in all stages oforthodontic mechanotherapy and have received much attention inthe recent past due to their excellent biocompatibility, corrosionresistance and appropriate mechanical properties [1]. It is wellknown that upon exposure to air a passivating layer of TiO2 isformed on the titanium surface, imparting corrosion resistance.This insoluble titanium oxide surface layer forms within nanosec-onds (10�9 s) and reaches a thickness of 20–100 Å within 1 s [2].Oxidative agents, known for their corrosive nature, can thickenand condense a titanium oxide surface layer, improving resistanceto corrosion. In contrast, reductive agents such as fluoride (F�) mayhave the opposite effect [3], resulting in dissolution of the TiO2 sur-face layer, exposing the parent metal to corrosion and discolour-ation [4–6].

In order to prevent dental caries and decalcification with pla-que accumulation around orthodontic attachments mouthwashes,

ia Inc. Published by Elsevier Ltd. A

n).

gels and toothpastes containing fluoride, at the level of 100–10,000 ppm, are commonly prescribed. In addition, various mecha-nical assaults, such as frictional forces at the bracket–archwireinterface, brushing and masticatory loads, further aggravatedissolution of the passivating layer. This might result in formationof hydrides, leading to hydrogen embrittlement and a reduction inthe mechanical properties of titanium-based alloy archwires [4–6].Coating titanium alloy based orthodontic archwires has been at-tempted by various researchers, in order to improve the aestheticsand frictional characteristics, as well as reduce nickel release. Untilnow no attempts have been made to protect titanium-based alloyarchwires against the corrosive effects of commonly used fluoridegels, toothpastes and mouth rinses.

Using physical vapor deposition (PVD), atoms and ions or, lessfrequently, molecules (activated by plasma, laser or high energyelectron and ion beams) can be condensed on substrates. In PVDthe major process control parameters are substrate temperature,background gas pressure and energy, flux and angle of incidenceof the depositing particles (atoms and/or ions). The microstructureand properties of films greatly depend on the deposition processand the conditions used [7]. There are many variants of thePVD coating process, such as electron beam PVD, evaporative

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deposition, magnetron sputtering, cathodic arc deposition andpulsed laser deposition. PVD coatings in general help to improvehardness, wear resistance and oxidation resistance, at the sametime reducing the frictional characteristics [8,9]. Even though themedical field utilizes this technology to improve the efficiency oftheir surgical tools, the application of this technique in the fieldof dentistry, especially orthodontics, has not been explored. Thepresent study is aimed at developing and evaluating titanium alu-minium nitride (TiAlN) and tungsten carbide/carbon (WC/C) PVDcoated b titanium orthodontic archwires for protection againstfluoride-induced corrosion. The effects of fluoride immersion onthe electrochemical behavior, surface characteristics, microstruc-ture, load deflection properties and element release, as well as tox-icology, were assessed.

2. Materials and methods

2.1. Specimens

b Titanium orthodontic archwires (0.017 � 0.025 inches, 77.55%titanium, 11.5% molybdenum, 6% zirconium, 4.5% tin, 0.35% iron)were obtained from Ormco Corp. (Glendora, CA). The uncoatedand coated wires were divided into six groups: group 1, BTUC (un-coated b titanium); group 2, BTFL (uncoated b titanium after fluo-ride immersion cycle); group 3, BTCT1 (b titanium coated withtitanium aluminium nitride, TiAlN); group 4, BTCT1FL (b titaniumcoated with TiAlN and subjected to a fluoride immersion cycle);group 5, BTCT2 (b titanium coated with tungsten carbide/carbon,WC/C); group 6, BTCT2FL (b titanium coated with WC/C and sub-jected to a fluoride immersion cycle).

2.2. Physical vapor deposition

Cathodic arc physical vapor deposition (CA-PVD) and magnetronsputtering were used to deposit thin films of TiAlN and WC/C,respectively, on b titanium orthodontic arch wires (groups 3 and5). In CA-PVD a high current, low voltage arc is connected to thecathode, which acts as a highly energetic emitting area known as acathode spot. This extremely high temperature results in vaporizedcathodic material (titanium and aluminium) travelling at highvelocity (10 km s–1) through a cloud of ions (plasma). Nitrogen isintroduced and reaction between titanium, aluminium and nitrogengenerates TiAl nitrides (TiAlN). This TiAlN mixture will travel to-wards the negatively charged substrates and will condense on itssurface. Magnetron sputtering is the removal of atomized materialfrom a solid (tungsten) due to bombardment of its surface layersby ionized or neutral particles at high impact velocities. Sputteringis performed in a near vacuum. During particle bombardment a con-trolled flow of inert gas (carbon) is introduced in order to raise thepressure. A highly negative voltage source is applied to the substratematerial which attracts positive ions at high speed [10]. Coating wasperformed under 250 �C for 4–5 h.

2.3. Fluoride immersion cycle

Three groups of wires (groups 2, 4 and 6) were immersed in ahigh fluoride ion concentration mouth rinse (Phos-Flur mouthrinse containing sodium fluoride in acidulated phosphate solution,0.44% sodium fluoride (0.02% fluoride ion) with 0.1 M phosphate,pH 4, Colgate Pharmaceuticals, Canton, MA) for 12 weeks. Thisimmersion mimicks the use of a fluoridated mouth rinse. Animmersion cycle of 5 min three times a day was followed. The restof the time all three groups of wires were immersed in a commer-cially available artificial saliva (pH 6.75 ± 0.15 with 0.40 mg l–1

NaCl, 0.40 mg l–1 KCl, 0.80 mg l–1 CaCl2bH2O) and 1.0 mg l–1 car-

bamide (Co(NH2)2) in distilled water (Cirurgicas Ltd., Juiz de Fora,Brazil).

2.4. Linear scratch test

Six specimens from each of groups 3 and 5 were mounted in ac-rylic resin. Progressive loading linear scratch tests were performedusing a micro-combi tester (MCT S/N 06-0213, CSEM, Switzerland)with a Rockwell diamond indenter with a tip radius of 100 lm, at aspeed of 6 mm min–1, loading rate of 9.97 N min–1, load range of0.03–10 N and scratch length of 6 mm. The equipment is equippedwith an integrated optical microscope, an acoustic emission (AE)detection system and a tangential friction force sensor, having asensitivity of approximately 10 mN. Three scratches were madeand the critical load (Lc), the point at which a sharp peak in theAE curve occurred, determined.

2.5. Electrochemical characterization

2.5.1. Substrate preparation and electrolyteGroup 1, 3 and 5 wires were physically cleaned with hard and

fine emery papers (grade 120–400) successively, followed bychemical cleaning along with etching with a solution of HF(08.00%) and HNO3 (12.00%) for about 1 min. All treated wires wererinsed with distilled water and then dried at 37 �C for 10 min. Thephysiological solution used as the electrolyte for electrochemicalcharacterization was Fusayama–Mayer artificial saliva [11,12],maintained at a temperature of 37 ± 1 �C and in the pH range5.2–7.8. The solution was stirred at 200 rpm by means of a mag-netic stirrer. 2% NaF was added to the artificial saliva to producethe test solution to evaluate the corrosive effects of fluoride on btitanium archwires.

2.5.2. Surface potential changesThe changes in surface potential of the three groups of arch-

wires were monitored by measuring the open circuit potential(OCP) with reference to a saturated calomel electrode (SCE) at reg-ular time intervals. The OCP was continuously measured using adigital multi-meter (Systronics, India) from the time of immersionin the respective test solution. The specimen electrical contactswere externally masked using epoxy resin before immersion andthe exposed length and area of the specimens were maintainedconstant during the tests. The specimens were cleaned with sol-vent and rinsed with distilled water prior to exposure. The testspecimens were placed in a conventional electrochemical cellassembly containing the reference electrode and the test solution,which was kept at 37 ± 1 �C. Three batches each having three testspecimens were used for the study and the resultant potential val-ues were compared to ensure reproducibility. Only those caseswhich gave reproducible results are presented here. Corrosion ofthe three groups of archwires was also assessed from the surfacepotential changes in artificial saliva solution containing 2% NaFas electrolyte. There were no significant changes when the corro-sion potential of the specimens was determined by extrapolatingthe tafel slopes after polarization and, hence, this is not discussedhere.

2.6. Surface characterization

2.6.1. ProfilometryThe line profile, which measures the height of a surface along a

straight line, was performed in order to assess the surface rough-ness. The line profile was assessed using a Talysurf CLI 1000. A scanlength of 10 mm with a 1 lm spacing at the measurement speed of50 lm s–1 was used to asses Ra (average roughness) and Rp (peakroughness). Six specimens from each group were utilized for line

V. Krishnan et al. / Acta Biomaterialia 7 (2011) 1913–1927 1915

scans and the mean roughness parameters obtained. The thicknessof the coating over the archwires was also determined by manualadjustment of the step height measurement in the software with aGaussian filter of 25 lm from four different fields for each sample.

2.6.2. Surface morphology and elemental analysisThe surface morphology of all groups of archwire specimens

were studied by environmental scanning electron microscopy(ESEM) (FET Quanta 200, FEI, Eindhovel, The Netherlands). Speci-mens were observed under low (300�) and higher magnification(1000�) and representative micrographs obtained. In order to as-sess the stability of the PVD coatings on the substrate b titaniumarchwires right-angled bending of the wires from three groups(groups 1, 3 and 5) were also observed under ESEM at low(250�) as well as higher (500�) magnification. The elements pres-ent in the archwires of all groups were determined by energy-dis-persive spectroscopy (EDS) (Oxford X-ray Microanalysis Software,Oxford Instruments, Abingdon, UK).

2.7. Microstructural characterization

The microstructures of all six groups of archwires were evalu-ated with the help of an optical microscope (Leica DMRX) follow-ing American Society for Testing and Materials (ASTM) standards.The straight portions of the specimens were cut to suitable lengthand vertically mounted on acrylic blocks. The mounted specimenswere then subjected to polishing with different grades (120–400)of silicon carbide paper and final polishing with a fine diamondsuspension on a polishing buff. The highly polished reflective sur-faces thus obtained were washed thoroughly in running water,dried and then examined under a reflected light microscope. Rep-resentative micrographs were obtained to evaluate the microstruc-ture of the uncoated and coated archwires before and after fluorideattack. The surface coating thickness was also measured with thehelp of the Qwin image analysis programme, integrated into themicroscope.

2.8. Element release determination by inductively coupled plasmaatomic emission spectrometry (ICP-AES)

Measurement of element release (Ti and Mo) by the archwiresimmersed in fluoride solution was performed with the help ofinductively coupled plasma–atomic emission spectroscopy (ICP-AES) (Optima 5300 dual view, Perkin-Elmer, Waltham, MA) onthe fluoride solution in which groups 1, 3 and 5 wires had been im-mersed. The samples were analysed after constructing a calibration

Fig. 1. Representative stress–strain curve

plot for a standard solution (Phos-Flur mouth rinse without arch-wire immersion) as supplied by the manufacturer. Plasma wasviewed axially in the entire analysis.

2.9. Load deflection characteristics

Mechanical testing of the archwire specimens was carried outaccording to American National Standard/American Dental Associ-ation Specification No. 32 for orthodontic wires. The load deflec-tion characteristics of five specimens from each group wereevaluated by three point bending in an Instron universal testingmachine (model No. 1195-5500R, Instron corporation, Canton,MA) as described by Miura et al. [13]. The distance between thetwo supports was fixed at 14 mm. The test wire was secured onbrackets fixed to specially designed areas on the support polesusing 0.3 mm elastomeric ligatures. Each specimen was loaded to1.5 mm and then unloaded to zero deflection at a cross-head speedof 1 mm min–1 and a full scale load of 10 N. Load, in Newtons, wasrecorded at deflections of 0.5 mm loading (0.5 L), 1 mm loading(1 L), 1.5 mm loading (1.5 L), 1 mm unloading (1 UL) and 0.5 mmunloading (0.5 UL). The load value and corresponding extensionduring the initial stages of loading and unloading were noted foreach specimen. The loading and unloading flexural moduli of elas-ticity (E) were calculated from the formula:

E ¼ L3m=4bd3;

where L is the length of the span, b is the width of the loaded spec-imen, d is the depth of the loaded specimen and m is the gradient/slope of the initial straight line part of the load deflection curve. Thevalue for m is calculated by dividing the load by the correspondingextension obtained from the initial straight line part of the loaddeflection curve. The values thus obtained for the loading andunloading elastic moduli were tabulated and analysed statistically.The load–deflection curve obtained was converted to a stress–straindiagram for each specimen, a representative of which is shown asFig. 1.

2.10. Toxicological evaluation

2.10.1. Sample preparationThe six groups of archwires were cut into small pieces and

ground to a powder using an eight flute tungsten carbide bur con-nected to a micromotor handpiece rotated at a speed of25,000 rpm. The powdered archwires were placed in air-tight ster-ilized glass test tubes and transferred to the laboratory to conductthe experiments.

generated from load deflection data.


2.10.2. Cell cultureSCC 172 cells, derived from poorly differentiated squamous cell

carcinoma of mandibular origin, were maintained in Dulbecco’smodified Eagle’s medium (DMEM) (Sigma, St. Louis, MO) containing10% fetal bovine serum (FBS) (Himedia, India) and 1% antibiotic/antimycotic cocktail (Gibco, Carlsbad, CA). MTT (3(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide) and Hoechst werepurchased from Sigma.

2.10.3. Cell viability assayThe MTT assay was carried out according to Edmondson et al.

[14]. Briefly, SCC 172 cells (7500 cells per well) were seeded in96-well plates and allowed to grow for 24 h. Cells were then incu-bated with the powdered b titanium archwires from all groups in adose-dependant manner. Different doses were arbitrarily fixed at1, 0.5 and 0.25 mg cm–2. The corresponding final concentrationsof the powdered materials were calculated to be 300, 150 and75 lg per well, respectively. The materials were incubated withthe cells for 72 h. Fifty lg of MTT in solution was then added toeach well and incubated for another 4 h. Mithochondrial dehydro-genase enzymes present in viable cells reduce MTT to blue forma-zan crystals. Thus, the amount of formazan formed is directlyproportional to cell viability. The wells were then aspirated and100 ll of isopropanol added in order to dissolve the formazan crys-tals. Absorbance was measured at 570 nm and the percentage via-bility of different groups of archwire powders calculated [14,15]and a graph constructed.

2.10.4. Nuclear condensation studyNuclear condensation is an early event in apoptosis. In order to

check whether apoptosis is induced in the presence of the pow-dered materials under test we performed nuclear condensationstudies using Hoechst dye nuclear staining. Briefly, SCC 172 cells(7500 cells per well) were seeded in 96-well culture plates andtreated with powdered b titanium archwires from all groups(300 lg per well) for up to 72 h. The cells were then incubated withHoechst 33342 (5 lg ml–1) for 15 min at 37 �C in the dark. Cellswere then observed using an inverted fluorescence digital micro-scope (Leica, Germany) for the presence of nuclear condensation.Different fields were randomly selected for each group and repre-sentative micrographs obtained.

2.10.5. Micronuclei (MN) assayMN induction is a measure of abnormal cell division and is con-

sidered an ideal tool to assess the toxicity of new agents. Briefly,SCC 172 cells (7500 cells per well) were seeded in 96-well cultureplates and treated with powdered b titanium archwires from allthe groups (300 lg per well) for up to 72 h. The cells were thenfixed with ice cold 70% ethanol for 10 min and stained using ethi-dium bromide and visualized using an inverted fluorescence digitalmicroscope for the presence of MN. MN identification was carriedout according to previously established criteria [16]. The numbersof MN were analysed microscopically from randomly selectedfields in 1000 cells. The experiment was done in triplicate. A pow-dered b titanium archwire was considered to be a positive inducerof MN if at least a twofold increase in the number of MN over thenegative control was observed.

2.11. Statistical analysis

Where appropriate, data were tabulated as means ± SD and sta-tistical significance was determined two-way ANOVA with Tukey’sHSD test for post hoc comparisons or Student’s t-test. Statisticalsignificance was established only if P < 0.05.

3. Results

3.1. Linear scratch test

Fig. 2 shows the Lc at which a sharp peak in the AE curve is ob-served for both TiAlN and WC/C coated b titanium archwires. Themean Lc values for TiAlN and WC/C coated b titanium archwiresare 7.23 ± 0.22 and 7.67 ± 0.17 N, respectively.

3.2. Electrochemical characterization

The OCP variation of three different samples in artificial salivawith respect to a SCE is shown in Fig. 3. All three groups of wires(groups 1, 3 and 5) exhibited a relatively nobler electrochemicalpotential in artificial saliva. At the initial time of immersion BTCT1exhibited a potential above 100 mV vs. SCE, with a further poten-tial shift anodically to 200 mV after 10 h. The electrochemical per-formance of the samples in a solution containing 2% NaF inartificial saliva is shown in Fig. 4. All three groups of samples re-vealed an extended potential shift due to the presence of fluoridein comparison with the artificial saliva solution. Although therewas corrosion in all three groups, the final potential of samplesof BTCT1 did not shift beyond –300 mV, even after 10 h immersion.

3.3. Surface characteristics

3.3.1. ProfilometryCompared with the uncoated form, the PVD-coated archwires

revealed a rough surface in the line profile measurements. Therewere further increases in Ra and Rp when these archwires weresubjected to fluoride attack. Statistical evaluation revealed signifi-cant differences between groups 1 and 2 and 5 and 6, while BTCT1(TiAlN coated) was found to be superior, in comparison with un-coated and WC/C-coated archwires, with only a slight increase inboth Ra and Rp after fluoride immersion treatment, which was sta-tistically insignificant (Table 1). Coating thickness evaluation byprofilometry revealed mean heights of 6.56 and 1.61 lm, respec-tively, for BTCT1 and BTCT2 specimens.

3.3.2. Surface morphology and elemental analysisThe surface changes induced by fluoride prophylactic agents on

the archwires of all three groups (groups 1, 3 and 5) are clearly de-picted in the SEM micrographs (Figs. 5 and 6). Both the coatedwires exhibited surface crystallization/homogenization after fluo-ride immersion. The coatings were found to be stable upon SEMevaluation even after making a right-angled bend (Fig. 7). EDSanalysis revealed the coating process to be continuous with no par-ent metal traceable externally. Coated wires showed peaks for tita-nium, aluminium and nitrogen (group 3) and tungsten and carbon(group 5) only (Fig. 8).

3.4. Microstructural characterization

Representative micrographs obtained by optical microscopy arepresented in Fig. 9. The difference in coating thickness between thegroup 3 and 5 wires is clearly visible in these micrographs. Further,the measurements for these coatings were found to correlate withthose obtained through profilometric evaluation. Corrosion wasclearly visible on the surface of the group 2 (uncoated and fluoridetreated) and group 6 (WC/C-coated and fluoride treated) wires.Clear pitting areas are visible on the group 2 wires, while on group6 wires the coating exhibited discontinuities along their length.Group 4 wires (TiAlN-coated and fluoride treated) showed no sur-face dissolution and the surface appeared to be smoother than be-fore fluoride immersion.

Fig. 2. Acoustic emission graph obtained through a linear scratch test.

Fig. 3. The variation in OCP of uncoated and coated wires as a function of time inFusayama–Mayer artificial saliva.

Fig. 4. The variation in OCP of uncoated and coated wires as a function of time in 2%NaF + Fusayama–Mayer artificial saliva solution.


Table 1Roughness parameters of the uncoated and coated archwires with and without fluoride immersion.

Ra(Mean ± SD) t value P value Statistical significance Rp (Mean ± SD) t value P value Statistical significance

BTUC 0.11 ± 0.0114.897 0.0006 Significant

0.25 ± 0.0265.65 0.0002 Significant

BTFL 0.16 ± 0.024 0.38 ± 0.050

BTCT1 0.24 ± 0.0922.198 0.0527 Not significant

0.58 ± 0.2091.688 0.1223 Not significant

BTCT1FL 0.33 ± 0.040 0.75 ± 0.131

BTCT2 0.23 ± 0.0674.787 0.0007 Significant

0.58 ± 0.1444.099 0.0021 Significant

BTCT2FL 0.44 ± 0.084 1.06 ± 0.248

P < 0.05, 95% confidence level.

Fig. 5. Surface analysis of uncoated and coated b titanium archwires before and after fluoride immersion by SEM at low magnification (300�). (a) BTUC; (b) BTFL; (c) BTCT1;(d) BTCT1FL; (e) BTCT2; (f) BTCT2FL.


3.5. Element release

ICP–AES evaluation revealed molybdenum release, although innegligible amounts, from wires immersed in fluoride mouthwash(groups 2 and 6), while no release was observed from group 4(TiAlN-coated) wires. The amount of molybdenum released washigher from group 6 (WC/C-coated and fluoride immersed) arch-

wires (0.002 mg/l) than from group 2 (uncoated and fluoride im-mersed) b titanium archwires (0.001 mg/l).

3.6. Load deflection characteristics

The loading and unloading moduli of elasticity of all groups ofarchwire samples are presented in Table 2. Statistical analysis

Fig. 6. Surface analysis of uncoated and coated b titanium archwires before and after fluoride immersion by SEM at higher magnification (1000�). (a) BTUC; (b) BTFL; (c)BTCT1; (d) BTCT1FL; (e) BTCT2; (f) BTCT2FL.


revealed a significant difference between the loading moduli ofelasticity of both the coated archwires (TiAlN and WC/C) beforeand after fluoride immersion, whereas no statistically significantdifference was obtained on comparison of uncoated b titaniumarchwires, before and after fluoride immersion. The unloadingmodulus values of all three groups (uncoated, TiAlN and WC/C) re-vealed no significant differences from their fluoride immersedcounterparts.

3.7. Toxicological evaluation

3.7.1. CytotoxicityThe MTT assay showed no radical differences in the viability of

SCC-172 cells exposed to any of the six groups of b titanium arch-wire powders at any concentration (75, 150 and 300 lg) comparedwith the control group (Fig. 10). The cells showed continuousgrowth and cell numbers had increased after 72 h. The percentage

cell viability for all groups except BTCT2FL reached almost 90% atthe higher doses. Statistical evaluation by one-way ANOVA(P > 0.05) showed no statistical difference between the six groupsin terms of cell viability at any concentration. Morphological exam-ination of the cells after incubation with the test materials indi-cated that the slight reduction in percentage viability (�10–20%)for all treatment groups was not due to cytotoxicity, but ratherto physical obstruction of the cells on the growth surface by thepowdered materials (Fig. 11). Together these results indicate thatthe powdered materials under test are not cytotoxic.

3.7.2. Apoptosis inductionOur results indicated no signs of nuclear condensation, an early

event of apoptosis, in any of the groups (Fig. 12) even after 72 htreatment. Moreover, there was evidence of cell growth over thepowder particles, to some extent, indicating no initiation of nucleardisintegration or breakdown.

Fig. 7. Surface analysis by SEM of uncoated and coated b titanium archwires after right angled bending to assess coating stability at low (250�) and higher (500�)magnification.


3.7.3. MN inductionOur results showed no increase in the number of MN in any of

the groups tested as evident from fluorescence microscopy(Fig. 13). These results again confirmed the biocompatibility ofthese powdered materials with the test cell line.

4. Discussion

The present research has attempted, for the first time in ortho-dontics, to produce two PVD coatings of TiAlN (through CA-PVD)and WC/C (through magnetron sputtering) on b titanium arch-wires. This was to prevent or minimize fluoride attack and the cor-rosion associated with it. The b titanium archwires in the uncoatedand two coated forms were evaluated for protection against fluo-ride-induced corrosion through their electrochemical behaviour,surface characteristics, mechanical properties, microstructure andtoxicology.

Thin film coatings with PVD are new to orthodontics and theadvantages that they offer are increased resistance to wear and

corrosion, increased lubricity, reduced galling and extended lifeof the substrate [8,9]. The mean values for coating thickness ob-tained over substrate b-titanium archwires were 6.56 and1.61 lm for TiAlN and WC/C, respectively, when evaluated by pro-filometry, which was further confirmed by the microstructures ob-tained. Thin film coatings are advantageous in orthodonticsbecause thicker coatings will result in binding of the archwire tothe bracket slot, increasing frictional resistance and impedingtooth movement. The coatings were found to be relatively stableon linear scratch test (more than 7 N to produce an initial distur-bance in acoustic emission) and even after bending through a rightangle (Fig. 7). Recent research suggests performing these two teststogether to assess coating stability [17]. The results clearly indicatethat these coatings have the capacity to withstand bending stressesas well as extended clinical use in the oral cavity.

Normally all types of titanium-based surfaces are expected toexhibit an anodic potential shift upon electrochemical potentialevaluation in test solutions such as artificial saliva due to theformation of a passive layer. Although uncoated b titanium alloy

Fig. 8. EDS analysis of all groups of archwires. (a) BTUC; (b) BTFL; (c) BTCT1; (d) BTCT1FL; (e) BTCT2; (f) BTCT2FL.


archwires revealed a substantial potenial shift in the anodic region,it did not increase above �150 mV vs. SCE, even after 10 h immer-sion. The more anodic equilibrium surface potential of TiAlN-coated archwires after immersion for 10 h revealed that thepassive layer formed on the substrate was not only nobler but alsomore corrosion resistant. The interaction between fluoride ionsand titanium results in changes in the protective passive layer ofthe metal. In fluoridated artificial saliva the potential of uncoatedb titanium archwires dropped sharply, to a value of approximately�700 mV, suggestive of complete dissolution of the protective pas-sive layer and a consequent reduction in the corrosion resistance.This susceptibility to localized corrosion of the passive film can beattributed to the higher number of imperfections or defectstructures in the oxide film, such as inclusions, mechanically formeddefects, grain boundary defects and structural crystallographic

defects. The results are in concordance with the previously pub-lished literature in this regard [18,19]. Coating with TiAlN was foundto result in superior corrosion resistance compared with WC/C interms of electrochemical behaviour, which can be attributed to astronger TiO2 layer formed on the coated surface. There is a furtherincrease in surface hardness of the TiAlN coating when the archwirealloy is subjected to fluoride attack, which is not apparent forWC/C-coated archwires, as is evident in the SEM micrographs.Instead, on WC/C-coated specimens there was evidence of surfacediscontinuity, making it more prone to corrosion.

For orthodontic purposes the unloading modulus of elasticity ismore important than the loading modulus because, once deflected,the wire has to release the energy absorbed and return to its origi-nal position, along with the required tooth movement [20]. Evalu-ation of the modulus of elasticity data indicated that fluoride has

Fig. 9. Microstructural characterization of all groups of archwires. (a) BTUC; (b) BTFL; (c) BTCT1; (d) BTCT1FL; (e) BTCT2; (f) BTCT2FL.

Table 2Loading and unloading moduli (E) of all wire groups as determined by three pointbending.

Wire group Elastic modulus (GPa)

Loading Unloading

Group 1 (BTUC) 58.23 ± 7.64 32.8 ± 2.15Group 2 (BTFL) 61.95 ± 9.08 30.02 ± 5.33Group 3 (BTCT1) 49.3 ± 6.54 38.24 ± 5.87Group 4 (BTCT1FL) 64.08 ± 5.37a 36.07 ± 3.84Group 5 (BTCT2) 47.81 ± 1.02 37.92 ± 2.19Group 6 (BTCT2FL) 53.53 ± 2.35b 35.82 ± 2.55

a Statistically significant difference in comparison with BTCT1, P 6 0.05.b Statistically significant difference in comparison with BTCT2, P 6 0.05.


only a negligible effect on this parameter. The unloading modulusshowed no statistically significant differences for all three archwiregroups (uncoated, TiAlN-coated and WC/C-coated), both beforeand after fluoride immersion. This indicates that neither the coat-ing process nor fluoride treatment is likely to affect the clinical per-formance of archwires.

It is well known that the corrosion properties of hard coatingslargely rely on their microstructure. Previously used monolayerPVD hard coatings for industrial purposes, particularly TiN coat-

ings, were usually grown as columnar structures. The columnarstructure leads to straight grain boundaries and open (through-coating) porosities, which provides efficient diffusion channelsfor the corrosive electrolyte to penetrate down to the substrate.With the incorporation of Al into the TiN the coating structure be-comes finer and finer, resulting in discontinuous crystallite bound-aries in the columnar structure. This decreases the opportunity forthrough-coating defects or open pores to form. Reactants diffusingthrough the finer grained coating must follow a convoluted route,such that the diffusion process is slow and difficult. This will resultin improved corrosion resistance of the finerstructured coating/substrate system [21]. Similarly, Ibrahim et al. [22] reported that‘‘alloying’’ a binary TiN coating with Al to form ternary TiAlN effec-tively improved the corrosion resistance of the as-derived coating/substrate system in both borate and NaCl aqueous solutions. How-ever, further increases in the Al content of the TiAlN coating in-creased the coating roughness, which is evident from theprofilometric evaluation in the present study. This can be attrib-uted to the increased incorporation of aluminium microdropletsemitted from the Al cathode due to its lower melting point.

Tungsten carbide/carbon (WC/C) thin films have classicallybeen used as protective hard coatings due to their good mechanicalproperties, high hardness and corrosion resistance and low wear

Fig. 10. Percentage cell viability for different treatment groups.

Fig. 11. Morphological examination of cells in the presence of test materials. MTT assay.


Fig. 12. Nuclear condensation analysis by Hoechst dye based nuclear staining.


properties, which are sustained up to 350 �C [23]. The WC phasewas grown using direct current magnetron sputtering, whereasthe carbon (C) phase was grown simultaneously from a hydrocar-bon (acetylene, C2H2) and argon plasma. All coatings had a rela-tively thin chromium layer next to the substrate to promoteadhesion. The surface roughness of the WC/C coating was compa-rably low compared with TiAlN, which was evident from SEM. Itwas possible to generate very thin (between 1 and 2 lm) WC/Ccoatings, but their effectiveness in protecting against fluoride-in-

duced corrosion was found to be low. This finding was reinforcedby the optical micrographs (Fig. 9), which clearly indicated surfacepitting of uncoated b titanium archwires and discontinuities alongwith surface pitting of the WC/C coating when subjected to fluo-ride treatment. In comparison, TiAlN-coated archwires exhibiteda smooth layer without any surface discontinuities or pitting afterfluoride immersion.

Fluoride treatment produced qualitative surface changes onall three groups of archwires. It is clearly evident from SEM

Fig. 13. MN induction analysis by ethidium bromide staining.


micrographs that there is an increase in roughness of all threegroups of archwires. Uncoated (Figs. 5b and 6b) and WC/C-coated(Figs. 5f and 6f) b titanium archwires showed cracks along the sur-face, which were deeper and accentuated after fluoride treatment.An improvement in the surface features of TiAlN-coated archwiresafter fluoride treatment (Figs. 5d and 6d) was evident, with morecrystallization changes and no evidence of cracking. A homogeni-

zation process was found to occur in TiAlN wires after fluoridetreatment, which was not apparent in the other two archwire spec-imens (uncoated and WC/C-coated archwires after fluoride treat-ment). Although only in trace amounts, molybdenum release, asdetermined by ICP–AES, from the WC/C coating (0.002 mg/l) wasmore than that from uncoated b titanium archwires (0.001 mg/l).There was no such element release from TiAlN-coated wires, which


makes them superior to uncoated as well as WC/C-coated b tita-nium archwires. The results further emphasize the fact that theTiAlN coating is less affected by corrosion compared with uncoatedand WC/C-coated archwires.

The majority of studies carried out to evaluate the toxicology ofdental materials have utilized fibroblasts, while there is an epithe-lial lining over connective tissue in the actual intra-oral environ-ment. In this study we utilized oral epithelial cells (SCC 172) fortoxicological evaluation, in order to produce more valid results.The MTT assay is a standard method to indirectly assess cell prolif-eration or growth rate through direct measurement of percentagecell viability. We could not observe any adverse effect on cellgrowth or viability with any of the b titanium archwire powders,even at the long exposure time of 72 h (Figs. 10 and 11). Eventhough statistically insignificant, there were slight differences incell growth when various groups are compared. The WC/C-coatedwires exhibited greater deviation compared with the others afterfluoride immersion. Further, examination of the cell morphologyin detail (Fig. 11) confirmed that this deviation in cell growth pat-tern is primarily due to physical obstruction, and not to cytotoxic-ity. This indicates that b titanium archwires are the mostbiocompatible as far as orthodontic mechanotherapy is concerned.

DNA condensation is an early event of apoptosis and Hoechstdye based nuclear staining is a widely used method to assess it[24]. Since this is an early event of apoptosis we analysed itsoccurrence in cells upon treatment with the powdered materials,to indicate the likelihood of apoptotic induction at later timepoints. Our results revealed no evidence of nuclear condensation,even after 3 days of cell culture with the test materials (Fig. 12),suggesting that the powdered materials are not apoptotic. MN aresmall round nuclei ranging from one-third to one-sixteenth thesize of the main nucleus. MN formation occurs due to incompleteseparation of the chromosomes during mitosis. This can be due tomalfunction of the spindle apparatus allowing some part of thechromosomes to lag behind, without attaching to the spindle,during chromosomal separation. They stain as intensely as themain nucleus and indicate whether cell division has proceededin the proper manner. Any agent that induces chromosome aber-rations can produce MN [16], and assessment of it is a widelyused tool to identify genotoxic agents. Our studies did not showany marked difference in MN formation between the controland different treatment groups of oral epithelial cells (Fig. 13),indicating that the materials used in the present study are com-patible with the oral epithelial environment.

In vitro cytotoxicity tests are designed to determine how amaterial affects a particular cell type. The results, however, yieldlittle information on how the sample affects a whole environment.On the other hand, it is known that the oral mucosa is generallymore resistant to toxic substances, compared with isolated cells,due to the presence of mucin and keratin layers [15]. However,the extrapolation of data obtained through this in vitro approachto in vivo conditions requires caution. Before clinical use of thematerial a secondary assay, such as a mucous membrane irritationtest or long-term cell culture experiments for genetic expressionstudies, may be warranted. Future study designs might include,apart from biocompatibility, frictional characteristics, as well asload deflection evaluation with various orthodontic loop designs,along with the actual clinical performance of both TiAlN- andWC/C-coated b titantium orthodontic archwires.

5. Conclusions

The present study, aimed at evaluating the fluoride-inducedcorrosion protection properties of two newly developed PVDcoated b titanium orthodontic archwires, revealed the following.

1. The two PVD coatings, TiAlN and WC/C, on b titanium ortho-dontic archwires were found to be stable, as observed withscratch tests and after bending through a right angle.

2. TiAlN coating by CA-PVD was found to be superior in terms ofcorrosion protection compared with WC/C coating by magne-tron sputtering, as shown by the electrochemical polarization,a lack ofsignificant element release and no visible surface disso-lution, in optical as well as SEM images.

3. Fluoride attack has a negligible effect on the load deflectioncharacteristics of uncoated, TiAlN-coated and WC/C-coatedarchwires, as shown by the statistically insignificant unloadingmodulus values.

4. Toxicological evaluation of the TiAlN- and WC/C-coated arch-wire specimens indicated that they are biocompatible.

Acknowledgements

We gratefully acknowledge Oerlikon Balzers Coatings India Lim-ited, especially M.R.K. Karanth and C.V. Gopinath, for helping us withthe PVD coating process. The input and guidance of Dr. Subramanian(VSSC, Trivandrum, Kerala, India), Dr. Muraleedharan, (SCIMST,Trivandrum, Kerala, India) and M.S.G.K. Pillai (NIIST, Trivandrum,Kerala, India) were very valuable throughout the study. The SCC172 cells were a kind gift from Dr. Susanne M. Gollin (University ofPittsburgh, PA).

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1, 2, 8, 9–13, aredifficult to interpret in black and white. The full colour images canbe found in the on-line version, at doi:10.1016/j.actbio.2010.11.026.

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Documents

Development and evaluation of two PVD-coated β-titanium orthodontic archwires for fluoride-induced corrosion protection