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ORIGINAL ARTICLE
Physical, mechanical, andorthodontic wires: An in-
Shubhaker Rao Juvvadi,a Vignesh Kailasam,b Sridevi Pad
Hyderabad and Chennai, India
Introduction: Understanding the biologic requirements of orttion studies of new archwire alloys. The aims of this study wer
ainlem-mel, cus oas
ce ptingesuvaluictionsmoith iture,lloy w
tensilearacter-towardtuationson.e popu-ction ofith tita-, corro-et al1
ficantly. Theyduringtinol ormodu-ice that
of nitinol and less than half that of SS. Its stiffness
to develop required force magnitudes. Furthermore,
Although various archwire alloys are available for
Kamala Nagar, Dilsukh Nagar, Hyderabad, India.
Sri Ramachandra University, Porur, Chennai, India.
0889-5406/$36.00retraction of teeth, SS archwires have always been theCopyright 2010 by the American Association of Orthodontists.doi:10.1016/j.ajodo.2009.01.032
623Kapila and Sachdeva3 stated that the relatively lowforces generated by TMA wires would imply that thecounterproductive forces generated by TMA wires canbe counteracted by smaller forces that those requiredfor SS wires.
The authors report no commercial, proprietary, or financial interest in the
products or companies described in this article.
Reprint requests to: Vignesh Kailasam, Department of Orthodontics, Faculty of
Dental Sciences, Sri Ramachandra University, Porur, Chennai 600116, India;
e-mail, theorthodontist@gmail.com.
Submitted, May 2008; revised and accepted, January 2009.makes it ideal in applications where less force than steelis required but the lower modulus would be inadequate
bProfessor, Department of Orthodontics, Faculty of Dental Sciences, Sri
Ramachandra University, Porur, Chennai, India.cProfessor and head, Department of Orthodontics, Faculty of Dental Sciences,Tooth movements and the associated changes arethe result of an applied force system and the tis-sue response to it. Orthodontic wires, which gen-
erate the biomechanical forces, communicate throughbrackets for tooth movement and are central to the prac-tice of orthodontics. Changes in the field of mechano-therapy have largely been made possible by neworthodontic materials. Selecting the appropriate arch-wire requires a thorough knowledge of the biomechan-ical and clinical applications of various archwires.
Archwire alloys require proper characterization topredict their outcome when used clinically. Most ofthe time, the exact compositions and material propertiesare not specified and, in some cases, are even not
available from the manufacturers. Evaluatingproperties, bending characteristics, frictional chistics, and surface properties are the initial stepsunderstanding archwire behavior in clinical siand are integral to archwire alloy characterizati
Titanium-based alloys have gained immenslarity over the years, mainly with the introdunickel-titanium and beta-titanium (TMA), wniums superior properties of biocompatibilitysion resistance, and low stiffness. Garnerreported that stainless steel (SS) provided signiless frictional resistance than nitinol and TMAsuggested that, when high stiffness was requiredsliding mechanics, SS should be used over niTMA. Burstone and Goldberg2 reported that thelus of elasticity (E) of TMA is approximately tw
aAssistant professor, Department of Orthodontics & Dentofacial Orthopedics,
Panineeya Mahavidhyalaya Institute of Dental Sciences & Research Centre,materials and to compare their properties with those of ststraight lengths of 3 types of wires: stainless steel, titaniuproperties were evaluated: wire dimension, edge bevcharacteristics, ultimate tensile strength (UTS), moduldeflection characteristics. A toolmakers microscope wfluorescence was used for composition analysis. Surfawere used for surface evaluation. A universal tescharacteristics, tensile strength, and 3-point bending. Rhad the lowest friction and spring-back values and highmolybdenum alloy was the roughest wire; it had high frUTS values. The beta-titanium alloy was intermediate forspring-back. Conclusions: The beta-titanium alloy wsuggesting that it would have greater resistance to fractitanium-molybdenum alloy wires. The beta-titanium aOrthod Dentofacial Orthop 2010;138:623-30)flexural properties of 3vitro study
manabhan,b and Arun B. Chitharanjanc
hodontic patients requires proper characteriza-e to evaluate properties of wires made of 2 newss steel.Methods: The sample consisted of 30olybdenum alloy, and beta-titanium alloy. Eightomposition, surface characteristics, frictionalf elasticity (E), yield strength (YS), and loadused to measure the edge bevel, and x-rayrofilometry and scanning electron microscopymachine was used to evaluate frictional
lts: Stainless steel was the smoothest wire; ites for stiffness, E, YS, and UTS. The titanium-and intermediate spring-back, stiffness, andothness, friction, and UTS but had the highestncreased UTS and YS had a low E value,thereby overcoming a major disadvantage ofire would also deliver gentler forces. (Am J
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Calif); group II, TMA (SDS Ormco, Glendora, Calif);
madewith a surface profilometer (Taly surf 50 profilom-
624 Juvvadi et al American Journal of Orthodontics and Dentofacial Orthopedicsand group III, beta CNA (Ultimate Wireforms, Bristol,CT, USA; Libral Traders, Delhi, India).
The wire dimension used in this study for all typeswas 0.43 3 0.64 mm (0.017 3 0.025 in), and the wireswere tested under similar conditions.
The height and width of each wire were measured tothe nearest 0.001mmwith a digital micrometer accurateto 61 mm (Mitutoyo, Kyoto, Japan). Five wires weretaken from each group, a total of 15 readings with 3such readings along each segment were measured, andthe arithmetic mean was used in the subsequentcalculations.
The transverse section of wire from each specimenwas viewed with a tool makers microscope (Mitutoyo)with 150 times magnification, measured to the nearest5 m, and readings were taken. The values were fed intosoftware (version 2000, Auto CAD, Autodesk, CA,USA) from which cross-sectional dimensions and theradius of the edge bevel were obtained. All 4 cornerswere measured, and, from these measurements, themean was calculated.
The various wire specimens were examined by us-ing x-ray fluorescence spectroscopy (Alpha-2000, serialmainstay for this phase of treatment. Titanium-basedarchwires, also used for this purpose, are available invarious forms and brands. TMA, for example, has anexcellent balance of properties, including high spring-back, low stiffness, high formability, and the ability ofdirect welding. However, a major drawback is its highcoefficient of friction. Also, some batches of TMA arch-wires are susceptible to fracture during clinical manip-ulation.4 TMA wires demonstrate higher levels ofbracket wire friction than SS or cobalt-chromiumwires.1,5
In the past few years, a relatively new entrant,Connecticut new archwire (CNA),6 a beta-titanium alloy,has gained immense popularity. However, little infor-mation is available in the orthodontic literature regard-ing its mechanical properties. The aims of this studywere therefore to evaluate and compare the followingproperties: wire dimensions, edge bevel, composition,surface characteristics, frictional characteristics, ulti-mate tensile strength (UTS), E, yield strength (YS),and load deflection characteristics of 3 wires used inorthodontics: SS, TMA, and CNA.
MATERIAL AND METHODS
Thirty straight lengths of each wire brand weredivided into group I, SS (Ortho Organizers, Carlsbad,number 8325, Innov-X Systems, Woburn, Mass) toevaluate the composition of the wires.eter, FTSI-6960, and Ultra Analysis program, version5.5.4.20, Taylor Hobson, Leicester, United Kingdom)with speeds of 0.5 mm per second (0.02 in/s) alongthe length of the 0.64-mm side over a distance of10 mm. Proprietary software was used to calculate thearithmetic average roughness, Ra (the arithmetic meanof the absolute departures of the roughness profilefrom the mean line) and Rz (the maximum peak-to-valley height of the roughness profile).
Surface characteristics were studied with a scanningelectron microscope (SEM) (QUANTA 200, FEI, Eind-hoven, Netherlands).
Frictional measurements weremadewith a universaltesting machine (Autograph AG 2000G, Schimadzu,Kyoto, Japan) with a 400-kg load cell at a crossheadspeed of 10 mm per minute set in standard tensilemode and force levels required to pull the wire throughthe bracket (022 3 028-in slot, Gemini maxillary rightfirst premolar bracket, Roth prescription, 3M Unitek,Monrovia, Calif), which was fixed on a metal sheet.The archwires were ligated by using 0.012-in elasto-meric ligatures. The archwire and bracket were testedso that a new wire and a new bracket with fresh ligationwere used for each combination and then discarded toeliminate the influence of wear.
Sample wires from each group were pulled throughthe bracket by a distance of 20 mm, the force levelsneeded for the movement of the wire were recordedfrom the digital readout, and the averagewas calculated.All tests were conducted in dry conditions.
We tested the mechanical properties as follows.A standard tensile test with each archwire from the 3groups was performed in the universal testing machine.A full-scale load of 1000 N was set in the machine witha crosshead speed of 1 mm per minute. The span of thewire between the grips was standardized at 40 mm. Theload taken to break the wire divided by the cross-sectional area of the wire gave the value for UTS.Youngs modulus (E) was then calculated from theload deflection data obtained from the tensile testing.
For the load deflection test, the wire samples fromeach group were tested by using a specially made3-point wire-testing jig attached to the testing machine.The load deflection characteristics were evaluated bya 3-point bend test described by Miura et al7 andmodified by Krishnan and Kumar.8
Statistical analysisSurface roughness measurements of each wire were
November 2010A standard statistical software package (version 10,SPSS, Chicago, Ill) was used for data analysis. Absolute
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to calculate the P values. The Mann-Whitney U test
skal-
D
0031
0057
0060
osition
ium
4
75
Table III. Means, standard deviations, and P values bythe Kruskal-Wallis 1-way ANOVA between groups forsurface roughness (mm)
GroupSurface roughness-Ra
Mean 6 SDSurface roughness-Rz
Mean 6 SD
Group I 0.0286 6 0.0030 0.2788 6 0.016 0.0001
Group II 0.2321 6 0.0082 1.9541 6 0.033
Group III 0.1739 6 0.0015 1.7945 6 0.063
American Journal of Orthodontics and Dentofacial Orthopedics Juvvadi et al 625was used to identify the significant groups at the 5%priori level of significance (P #0.05).
RESULTS
The results of the measurements for wire dimen-sions and edge bevels are given in Table I. There wereand percentage mean errors, standard deviations, and95% confidence levels were calculated. The Kruskal-Wallis 1-way analysis of variance (ANOVA) was used
Table I. Means, standard deviations, and P values of Krusions (mm)
Group
Measured dimensions
Stated wire dimensions Height Width
Height Width Mean 6 SD Mean 6 S
SS 0.4318 0.635 0.4333 6 0.0025 0.6387 6 0.
TMA 0.4318 0.635 0.4252 6 0.0031 0.6263 6 0.
CNA 0.4318 0.635 0.4333 6 0.0025 0.6304 6 0.
Table II. Compositions of the wires
Group
Comp
Iron Chromium Nickel Titan
SS 72.39 18.17 9.02 0
TMA 0 0 0 78.
CNA 0 0 0 80.
Volume 138, Number 5highly significant differences in height of the wires be-tween groups I and II, and groups II and III (P\0.001)but no significant difference between groups I and III(P .0.05). There were highly significant differencesin the widths of the wires between groups I and II, andgroups I and III (P\0.001) but no significant differencebetween groups II and III (P .0.05). There were alsohighly significant differences in the edge bevel betweengroups I and II, and groups II and III (P\0.001) butno significant difference between groups I and III(P .0.05).
The CNAwires had more titanium but less molybde-num, zirconium, and tin (Table II).
Surface roughness measurements with surfaceprofilometry (Table III) showed a highly significantdifference between the 3 groups (P\0.001). The sur-face topography evaluation of the wires with SEM pho-tomicrographs showed that the SS wire (Figs 1, A, 2, A,3, A, and 4, A) was the smoothest, and the TMA wire(Figs 1, B, 2, B, 3, B, and 4, B) was the roughest,whereas the CNA wire (Figs 1, C, 2, C, 3, C, and 4, C)was intermediate.Wallis 1-way ANOVA between groups for wire dimen-
Difference
Height Width Edge bevel
Mean 6 SD Mean 6 SD Mean 6 SD P value
0.0015 6 0.0003 0.0038 6 0.0005 0.0616 6 0.0045 0.001
0.0066 6 0.0031 0.0088 6 0.0031 0.0786 6 0.0065
0.0015 6 0.0006 0.0046 6 0.0007 0.0582 6 0.0008
(%)
SumMolybdemun Zirconium Tin
0 0 0 99.58
11.33 6.28 4.05 100.06
9.78 5.75 3.78 100.06The results of frictional characteristics are given inTable IV. There were highly significant differences instatic friction between the 3 groups and in kinetic fric-tion between groups I and II (P\0.001), and significantdifferences between groups II and III, and groups I andIII (P\0.05).
The results of E, YS, UTS, and YS:E are given inTable V. There were highly significant differences inE, UTS, and YS between groups I and II, and groups Iand III (P\0.001) but no significant difference betweengroups II and III (P .0.05). There was a highly signif-icant difference in the stiffness strength between groupsI and II, and groups I and III (P\0.001), but no signif-icant difference between groups II and III (P\0.05).
The results of the load deflection characteristicsare given in Table VI. There were highly significant dif-ferences in the load deflection characteristics duringloading at 0.5 mm between groups I and II, and groupsI and III (P\0.001) but no difference between groups IIand III (P .0.05). There were highly significantdifferences in the load deflection characteristics during
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and C
626 Juvvadi et al American Journal of Orthodontics and Dentofacial OrthopedicsFig 1. Photomicrographs: A, SS; B, TMA;loading at 1 mm between all groups (P\0.001). Therewere also highly significant differences in the load de-flection characteristics during unloading at 0.5 mm be-tween groups I and II, and groups I and III (P\0.001),and a significant difference between groups II and III(P\0.05).
DISCUSSION
The performance of an archwire in torsion dependson wire material and cross-sectional geometry. Smallerarchwires are selected to ensure lower force at the initialstage of fixed appliancemechanotherapy, but this resultsin inadequate control of tooth movement, since therewould be much play between the wire and the bracket.The development of titanium-based archwires made itpossible to use larger wires, since they offer similarranges of forces but better bracket engagement. Thus,wire dimension is a critical component in force delivery.Among the various wires measured, SS was the closestto the stated dimensions, followed by CNA and TMA.There was no statistical difference between the SS andCNAwire dimensions.
Fig 2. Photomicrographs: A, SS; B, TMA; and C, CNA. Original magnification, 500 times.
November 2010Therefore, with SS and CNA, there would be lessvariation with the expected force delivery, therebyensuring greater predictability of tooth movement. Allwires measured in this study were within the range of60.005 in (0.127 mm) as stated by Meling et al.9
Variations of 60.001 in (0.0254 mm) would result inconcomitant loss of 2 to 4 of effective torque.10 Thiswould imply that, with the wires tested, the maximumloss of torquewould be between 1 and 2, with the leasttorque loss for SS and CNA. However, wires areproduced in various batches, resulting in interbatchvariations, but the results obtained in this study amongthe various wire specimens were consistent throughout,implying that the variations in individual specimenswere negligible.
The edge bevel is a critical factor for the actualtorque expressed by a specific square or rectangularwire and bracket combination, since the edges of thearchwire first engage the bracket for torque delivery.The increased bevel of TMA wires in this study is inaccordance with the findings of Meling and Odegaard11
and Sebanc et al,12 who stated that the inability of the
, CNA. Original magnification, 1000 times.
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nd C
American Journal of Orthodontics and Dentofacial Orthopedics Juvvadi et al 627Fig 3. Photomicrographs: A, SS; B, TMA; a
Volume 138, Number 5manufacturer to better approximate the square cornerfor the beta-titanium wire during rolling might be dueto the mechanical and wear properties of this alloy,thereby resulting in increased edge bevels. Sernetz13
stated that the edge bevel should be at least 0.04 mmfor patient comfort. Deviations from stated wire dimen-sions and edge bevel will influence torsional clearanceand stiffness.11,14 Rucker and Kusy15 reported that an8% decrease in cross-sectional area (ie, 2% from eachcorner) could result in nearly a 20% decrease in stiff-ness. However, each corner of the wire in the cross-section contributes minimally to torsional stiffness,since stress to the corner of the rectangular wire intorsion is absent.
The composition of SS was similar to the valuesreported by Brantley,4 whereas the composition ofTMA was similar to the values reported elsewhere inthe literature.4,16,17 The compositions of TMA andCNA wires were similar to the values reported byKusy et al.18 The CNA wires had more titanium com-pared with TMA wires. The zirconium in the TMAand CNA wires contributes to increased strength and
Fig 4. Photomicrographs: A, SS; B, TMA; C,, CNA. Original magnification, 2000 times.hardness and prevents the formation of an embrittlingomega phase during processing at elevated tempera-tures.4 Less zirconiummight contribute to the decreasedstiffness of the CNAwires.
Surface topography can critically affect estheticsand performance of orthodontic components.19 The sur-face profilometry showed that the SS is the smoothestwire. Of the 2 titanium-based wires, CNAwas smootherthan TMA. The Ra and Rz values showed highly signif-icant differences among the wires tested (P\0.001).
SEM examination of beta-titanium orthodonticwires showed rough surfaces. This surface roughness,along with localized sites of cold welding or adherenceby the wire to the bracket slots, could contribute tothe increased archwire-bracket sliding friction seenwith titanium-based archwires.4 SEM examinationconcurred with the surface profilometry test regardingthe roughness of the various wires tested. The resultsof the profilometry and SEM agreed with otherstudies.16,18-21
Many variables can affect the magnitude of the fric-tional force between the bracket and the wire.19,20,22-37
CNA. Original magnification, 5000 times.
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628 Juvvadi et al American Journal of Orthodontics and Dentofacial OrthopedicsStatic frictional forces were generally greater thankinetic frictional forces; these results agreed withthose of Kusy et al.18 Both static and kinetic frictionalforces were the lowst for SS followed by CNA andthen TMA, which showed the greatest amount of fric-tion. Similar results with regard to SS and TMA werefound by others.1,5,19,21,22,25,26,38-40 Ogata et al25 withSS and Angolkar et al23 with SS and TMAwires foundfrictional values that compared favorably with our re-sults. Static and kinetic frictional values showed statis-tically significant differences between all 3 wires tested.Kusy et al41 stated that in the dry state there was lowerfriction for SS. The friction in the dry state with beta-titanium wires was greater than that of SS. This couldbe because the titanium-rich layer breaks down, reacts,adheres, and breaks away, resulting in a stick-slip phe-nomenon. When saliva was introduced, friction fortitanium-based wires decreased to the levels of SS.41
The method of ligation used might also affect friction;steel ligatures will decrease friction when comparedwith elastomers.30,33-35
The E or Youngs modulus corresponds to the elasticstiffness or the rigidity of the material.4 Increased valuesindicate stiffer wires.42,43 The results of the E of SS in thisstudy are in accordance with other studies.3,8,44-46 Thereported modulus values for TMA also agreed withmany studies,47-52 whereas higher values were reported
Table IV. Means, standard deviations, and P value byKruskal-Wallis 1-way ANOVA between groups forstatic and kinetic friction (cN)
GroupStatic frictionMean 6 SD (g)
Kinetic frictionMean 6 SD (g) P value
Group I 0.783 6 0.003 0.729 6 0.007 0.0001
Group II 1.02 6 0.006 0.970 6 0.020
Group III 0.87 6 0.022 0.835 6 0.011in other studies.4,53 Goldberg and Burstone17 stated thatbeta-titanium alloys had great potential in orthodontic de-signs. TheYS andUTS ofCNAwere intermediate amongthe wires compared. SS had the highest YS and UTS.TMA had the lowest values.54 The results of this studyagree with the finding that YS and UTS values go handin hand, meaning that if the YS of 1 wire is more thanthat of the other, the UTS of the former is also morethan the latter. The ratioYS:E is considered a useful indexof wire performance.8,55 This ratio indicates the clinicalperformance of wires in terms of working range.
In our study, CNA (0.0158) was the least stiff wirefollowed by TMA (0.0138) and SS (0.0097), whichhas the greatest stiffness (Table V). Statistically, therewere significant differences between SS and CNA andbetween SS and TMA (P\0.001), and between CNAand TMA (P\0.05). When YS and E were comparedindividually, there was no statistically significant differ-ence between TMA and CNA, but the ratio YS:E wasstatistically significant because of increased YS anddecreased E. The significance of this observation wouldrequire clinical evaluation.
Upon tensile evaluation, SS was the strongest alloywith high values for UTS, E, and 0.02% offset YS. Thiswas followed by TMA and CNA, respectively. Theincrease in the UTS value for CNA implies greaterfracture strength than TMA.
Load deflection properties are critical in determin-ing the biologic nature of tooth movement.42 The resultsof this study with CNA, followed by TMA and SS, asevidenced from the low values needed to deflect thewire, agreed with the results of Krishnan and Kumar,8
who evaluated SS, TMA, and Timolium and clearlyindicated the favorable nature of titanium-based arch-wires. This would be a benefit in a clinical situationwhere engagement of the wire in the bracket of a mala-ligned tooth would deliver controlled forces to the toothand supporting tissues.
Evaluation of unloading characteristics showed theresilient and consistent nature of CNA compared withthe TMA and SS wires. SS was the most rigid, withhigh loading values and lower spring-back properties.TMA wires were intermediate. CNA had low stiffnessand therefore higher spring-back, and thus was superiorcompared with TMA and SS.
In the repeated-measures ANOVA, the 3 groups hadstatistically significant results for load deflectioncharacteristics. This indicated that the hysteresis (en-ergy loss on unloading) is associated with all 3 arch-wires and is higher with SS, followed by TMA andCNA. The unloading characteristics showed that theCNA wires deflected the greatest amount of the wires(1507 g) tested, indicating that thesewires had the great-est resilience or spring-back (Table VI).
This study indicates that the CNA was the bestamong the wires compared in terms of the 3 charac-teristics of deflection, stiffness, and formability andcan be considered a superior alternative to TMAwires. This, we believe, could be because of variousfactors such as greater accuracy in wire dimensions,edge bevel, variation in composition, surface smooth-ness, lower friction, and greater strength and resil-ience. However, since the stiffness was less thanTMA, torque expression might be affected. Hence,clinical studies are warranted to examine whether
November 2010this decreased stiffness would alter clinical torqueexpression.
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We thankMMurugan, Prof and Head, T Venkatesan,
skal
timateth (Uean
7.67
1180
1287
American Journal of Orthodontics and Dentofacial Orthopedics Juvvadi et al 629Table V. Means, standard deviations, and P values by Kruand maximum strain
Wire
Flexural modulus ofelasticity (E) (GPa)
Mean 6 SD
0.2% offset yieldstrength (YS) (Mpa)
Mean 6 SD
Ulstreng
M
Group I 194.33 6 11.15 1886.33 6 23.86 204
Group II 81 6 6.56 1116.67 6 93.78
Group III 73 6 4.00 1156.33 6 54.15
Table VI. Load deflection characteristics of wires at 0.5and 1 mm loadings, and 0.5 mm unloading
Sample Mean 6 SD (g) P value
0.5 mm loading
Group I 2350 6 91.65 0.0001
Group II 1627 6 55.08
Group III 1437 6 41.63
1 mm loading
Group I 3480 6 115.33 0.0001
Group II 2697 6 116.76
Group III 2143 6 50.32
0.5 mm unloading
Volume 138, Number 5CONCLUSIONS
1. The measured dimensions of SS, TMA, and CNAwires varied from the dimensions stated by themanufacturers. SS and CNA wires approximatedtheir stated dimensions more closely than did TMA.
2. Edge bevel was the greatest for TMA and the leastfor CNA.
3. The composition of SS closely approximated thevalues in the literature. The compositions of TMAand CNAwere different, with CNA having less zir-conium but more titanium; this could be a reasonfor the decreased stiffness when compared withTMA.
4. SS had the smoothest surface, followed by CNAand TMA.
5. The frictional force levels showed an increasedvalue for TMA compared with CNAwith the lowestvalue for SS wire.
6. The UTS and YS values were highest for SS, fol-lowed by CNA and TMA. The increase in UTSvalue for CNA shows that it would have greater re-sistance to fracture, thereby overcoming a majordisadvantage of TMA.
7. Ewas the highest for SS followed byTMAandCNA.
Group I 947 6 58.6 0.0001
Group II 1323 6 61.1
Group III 1507 6 55.07
Kruskal-Wallis 1-way ANOVA.Lecturer andRTamilselvan, Lab Technician,Departmentof Metrology and Metallurgy, Crescent EngineeringCollege, Vandalur, Chennai; Prof B Kothandaraman,P SSampath , and GunaSekharan,Department ofRubberand Plastics Technology, MIT, Chennai; Bala Murali,M R Ramesh, and Uma Ravichandran, Micro Labs,Vanagaram, Chennai; and A Rajaraman, Departmentof Bio-Physics, Central Leather Research Institute,Chennai, for use of the facilities, proper guidance,and help for completion of our study.
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Physical, mechanical, and flexural properties of 3 orthodontic wires: An in-vitro studyMaterial and methodsStatistical analysis
ResultsDiscussionConclusionsReferences
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