Release of metal ions from fixed orthodontic appliance An in vitro study in continuous flow system

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1 Originl Article Relese of metl ions from fixed orthodontic pplince An in vitro study in continuous flow system Mrcin Mikulewicz ; Ktrzyn Chojnck b ; Pulin Wołowiec c ABSTRACT Objective: To evlute the relese of metl ions from fixed orthodontic pplinces. Mterils nd Methods: A new system for in vitro testing of dentl mterils ws constructed nd consisted of thermosttic glss rector tht enbled immersion of the studied mteril. Experimentl conditions reflected the humn orl cvity, with temperture of 37uC nd sliv flow rte of 0.5 ml/min. The simulted fixed orthodontic pplince mde of stinless steel ws evluted. Smpling ws performed t severl time points during the 28-dy study, nd the metl ion concentrtion ws determined by inductively coupled plsm opticl emission spectrometry. Results: The totl mss of relesed metl ions from the pplince during 4 weeks of the experiment ws s follows: nickel 18.7 mg, chromium 5.47 mg, copper 31.3 mg. Conclusions: The estimted doses of nickel, chromium, nd copper (determined by extrpoltion of experimentl dt) relesed during the tretment period were fr below the toxic dose to humns. This shows tht orthodontic tretment might not be significnt source of exposure to these metl ions. (Angle Orthod. 2014;84: ) KEY WORDS: Metl ions; Orthodontic pplince; Artificil sliv INTRODUCTION Recent decdes hve seen gret interest in the biocomptibility spects of biomterils, whether internlly or externlly pplied. Mterils pplied in dentistry hve to pss specific biocomptibility tests to meet regultory stndrds. 1 Some metls (eg, nickel [Ni], chromium [Cr], coblt (Co)) tht re components of orthodontic lloys re well known to be llergenic, cytotoxic, nd mutgenic. 2,3 Applinces nd dditionl devices used during orthodontic tretment re exposed to different fctors, such s temperture, ph, mechnicl stress (corrosion), nd microflor (biocorrosion). 4 All Assistnt Professor, Deprtment of Dentofcil Orthopedics nd Orthodontics, Wroclw Medicl University, Polnd. b Professor, Institute of Inorgnic Technology nd Minerl Fertilizers, Wroclw University of Technology, Polnd. c Doctorl Student, Institute of Inorgnic Technology nd Minerl Fertilizers, Wroclw University of Technology, Polnd. Corresponding uthor: Dr Mrcin Mikulewicz, Deprtment of Dentofcil Orthopedics nd Orthodontics, Wroclw Medicl University, ul Krkowsk 26, , Wroclw, Polnd (e-mil: mikulewicz.mrcin@gmil.com) Accepted: Februry Submitted: November Published Online: Mrch 11, 2013 G 2014 by The EH Angle Eduction nd Reserch Foundtion, Inc. these fctors my led to relese of toxic metl ions from lloys. The biocomptibility of orthodontic fixed pplinces (brckets, bnds, wires) might be determined by evluting the mount of metl ions tht re relesed during the process of dissolution s the consequence of corrosion in in vitro nd in vivo studies. 5 In vivo studies provide informtion on the behvior of mteril under the conditions for which the mteril is designted. However, smple collection of invsive mtrices (blood) is difficult becuse of the need for ptient consent. Noninvsive mtrices such s urine or sliv re esier to collect, but it must be emphsized tht ll mtrices (blood, urine, sliv) reflect only momentry exposure. Thus, the methodology of smple collection nd the results obtined to simulte the verge orthodontic tretment time of 2 yers re not s meningful s tht which occurs in chronic exposure. 5 7 On the other hnd, in vitro studies re performed in controlled lbortory environment but do not reflect the conditions previling in the orl cvity. 8 Mny studies of the corrosion behvior of orthodontic devices in rtificil sliv did not tke into ccount the equilibrium reched in btch experiments; tht is, during the experiment, the sliv ws not replced or llowed to flow through the system, s in the humn orl cvity This my led to results tht 140 DOI: /

2 METAL IONS RELEASE FROM ORTHODONTIC APPLIANCE 141 Figure 1. Schemtic digrm of the system for the evlution of ion relese from dentl mterils. The instlltion consisted of: (1) glss rector with wter jcket, (2) wter bth with n externl circuit to enble opertion of the rector under temperture-controlled conditions, (3) multichnnel peristltic pump to feed the solution into the rector, (4) glss tnk plced on thermosttic mgnetic stirrer, nd (5) the collecting vessel. The receiving solution flowed down grvittionlly. The vrious strems of solutions were distributed through the system by flexible, chemiclly resistnt hoses mde of Norprene tubes (Msterflex, Vernon Hills, IL, USA) for use with the peristltic pumps. Adequte flow rte ws chieved by the dimeter of the tubing. The mteril ws plced in glss tnk, which ws immersed in the solution, which flowed into the system. do not ccurtely reflect the ctul degree of ion relese from the evluted mteril, nd thus it is lso difficult to ssess the dose to which ptient is exposed during orthodontic tretment. The im of the present study ws to evlute the relese of metl ions from fixed orthodontic pplinces in vitro in continuous-flow system. Another gol ws to elborte new, more relible, nd more meningful technique for evlution of the relese of potentilly toxic chemicls from dentl mterils. MATERIALS AND METHODS In Vitro System The experimentl conditions were selected to reflect s closely s possible (in n in vitro system) the environment of the humn orl cvity with n orthodontic pplince. The experiments were crried out in specilly designed thermosttic glss rector (Figure 1), in which the orthodontic pplince ws submerged in rtificil sliv, which ws flowing through the system t rte reflecting the flow of sliv in humns (0.5 ml/min). 12 The durtion of the experiment ws 4 weeks (28 dys). The smples were collected over time, which enbled us to investigte the kinetics of metl ion dissolution from prts of the fixed orthodontic pplince. The experiment durtion enbled n ttempt to extrpolte the dt nd estimte (fter subtrction of the results from the control group) the overll dose to which ptient is exposed during orthodontic tretment. The system consisted of mgnetic stirrer with heting nd contct thermometer with glss-coted sensor (Heidolph MR Hei-Tec nd Heidolph EKT Hei- Con G, (Heidolph: Schwbch, Germny), multiplechnnel peristltic pump (Heidolph Peristltic Pumps PD 5201 MC, (Heidolph: Schwbch, Germny), wter bth with n externl circuit (LAUDA ECO E 10 S, Lud, Lud-Koenigschofen, Germny), nd thermosttic glss rector with cover (Figure 1). Tested Mteril The evluted mterils were brnd new prts of the orthodontic pplince. Wires, brckets, nd bnds were ll mde of stinless steel, wheres elstic ligtures were fbricted of polymer. The following elements of the orthodontic pplince were tested: inch rchwires (Americn Orthodontics, ref no , ), size 37+ bnds, Sheboygn, Wis, USA). (3M Unitek, Victory Series brckets (3M Unitek, Monrovi, Clif, USA), nd elstic ligtures (Americn

3 142 MIKULEWICZ, CHOJNACKA, WOŁOWIEC Tble 1. Totl Mss (mg) of Metl Ions Relesed During the Experiment Metl Ion Relesed Mss, mg Ni Cr Cd Cu Fe Mn Mo Si Ni indictes nickel; Cr, chromium; Cd, cdmium; Fe, iron; Mn, mngnese; Mo, molybdenum; nd Si, silicon. Orthodontics, Sheboygn, Wis, USA). The orthodontic fixed pplince consisted of two wires, four bnds, 20 brckets, nd 20 elstic ligtures to correspond to the upper nd lower dentl rches during prticulr phses of orthodontic tretment. Experimentl Setup The pplince ws plced in the thermosttic glss rector with cover nd incubted (immersed) in the modified rtificil sliv solution 13 t 37uC for 28 dys. The solution of rtificil sliv ws spiked with Ni 2+ to ensure physiologicl concentrtion similr to tht of humn sliv. The concentrtion ws clculted s the verge from the vlues found in the vilble literture The feed solution of rtificil sliv ws pumped into the glsswre in continuous mnner. The verge flow rte ws ml/min ( g/min, d g/ml). The recycle rtio ws 1:1. The smples (50 ml) were collected directly from the outlet vessel. At the beginning of the experiment, smpling ws done more frequently to investigte the initilly (ccording to literture reports) more intense metl ion dissolution Figure 2. () Kinetics of relese of Ni ions from the orthodontic pplince into the rtificil sliv. (b) Kinetics of relese of Cr ions from the orthodontic pplince into the rtificil sliv. (c) Kinetics of relese of Cd ions from the orthodontic pplince into the rtificil sliv. (d) Kinetics of relese of Cu ions from the orthodontic pplince into the rtificil sliv.

4 METAL IONS RELEASE FROM ORTHODONTIC APPLIANCE 143 Figure 2. Continued. (e) Kinetics of relese of Fe ions from the orthodontic pplince into the rtificil sliv. (f) Kinetics of relese of Mn ions from the orthodontic pplince into the rtificil sliv. (g) Kinetics of relese of Mo ions from the orthodontic pplince into the rtificil sliv. (h) Kinetics of relese of Si ions from the orthodontic pplince into the rtificil sliv. during the preliminry phse (first 12 hours: every 50 minutes; first week: every 12 hours; second week: every 24 hours; third week: every 3.5 dys; fourth week: t the end of the experiment). As control, rtificil sliv ws mde to flow through the system under the sme conditions s the experimentl set, lthough in the bsence of the orthodontic pplince. Tble 2. Results nd Sttisticl Anlysis of the Experimentl nd Control Groups (n 5 38) Experimentl, mg/l Control, mg/l Metl Ion Men Medin SD Min Mx Men Medin SD Min Mx Ni * Cr * Cd Cu * Fe Mn Mo Si Ni indictes nickel; Cr, chromium; Cd, cdmium; Fe, iron; Mn, mngnese; Mo, molybdenum; Si, silicon; min, minimum; mx, mximum; nd SD, stndrd devition. P

5 144 MIKULEWICZ, CHOJNACKA, WOŁOWIEC Tble 3. Interelement Correltion Mtrix for Experimentl (Exp) vs Control (Contr) Groups Ni-Exp Ni-Contr Cr-Exp Cr-Contr Cd-Exp Cd-Contr Cu-Exp Ni-Exp P Ni-Contr P.285 Cr-Exp P Cr-Contr P Cd-Exp P Cd-Contr P Cu-Exp P Cu-Contr P Fe-Exp P Fe-Contr P Mn-Exp P Mn-Contr P Mo-Exp P Mo-Contr P Si-Exp P Si-Contr P Ni indictes nickel; Cr, chromium; Cd, cdmium; Fe, iron; Mn, mngnese; Mo, molybdenum; nd Si, silicon. Anlyticl Methods A smple (50 g) ws purified from orgnic mtter with concentrted nitric cid (69% m/m; 5 ml), which ws spectrlly pure (Suprpur, Drmstdt, Germny), in Teflon bombs in microwve oven (Milestone Strt D, Sorisole, Itly). The selected prmeters of the process ensured complete digestion of the smples. After minerliztion, the smples were diluted with redeminerlized wter (Millipore Simplicity, Molsheim, Frnce) to 50 g. Minerlized smples underwent multielementl nlysis. The concentrtions of Ni, Cr, cdmium (Cd), copper (Cu), iron (Fe), mngnese (Mn), molybdenum (Mo), nd silicon (Si) were deter- Tble 4. Relese of Metl Ions from n Orthodontic Applince in the Present Study nd Reported in the Literture in n Artificil Sliv Environment (37uC, ph 7) References Number of Applince Elements Brckets Bnds Wires Prt of Whole Applince Evluted Mteril-SS Volume of Artificil Sliv, L Present study Yes b Brrett et l. (1993) (4 premolr) 1 1 Yes 0.10 Hwng et l. (2001) ,J Yes 0.05 Gursoy et l. (2004) J Yes, but wire Ni-Ti 0.05 Kuht et l. (2009) J Yes 0.05 Mikulewicz et l. (2012) Yes The presented vlues were obtined by multipliction of the concentrtion of metl ions (mg/l) 3 prt of the pplince 3 volume of incubtion solution (L); this enbled evlution of the mss (mg) of metl ions relesed from whole orthodontic pplince. b The experiment ws crried out on the whole pplince in continuous mode. The volume of sliv tht flowed through the system ws L. The mss of relesed ions in ll intervls is summrized. c The vlues in prentheses re the rel msses of relesed metl ions received by subtrction of control results (in the present work: Cu mg, Cr mg, Fe mg, Ni mg) from experimentl results.

6 METAL IONS RELEASE FROM ORTHODONTIC APPLIANCE 145 Tble 3. Extended Cu-Contr Fe-Exp Fe-Contr Mn-Exp Mn-Contr Mo-Exp Mo-Contr Si-Exp Si-Contr Ni indictes nickel; Cr, chromium; Cd, cdmium; Fe, iron; Mn, mngnese; Mo, molybdenum; nd Si, silicon. mined with n inductively coupled plsm opticl emission spectrometry device (ICP-OES Vrin-Vist MPX, Brisbne, Austrli) equipped with n ultrsonic nebulizer (CETAC U5000AT+, Omh, Neb, USA). Also, the presence of ny dditionl elements ws scertined. The nlyses were crried out in qulity mngement system ccording to PN-EN ISO/IEC 17025:2005. The smples were nlyzed three times, nd the rithmetic men nd reltive stndrd devition (SD) (,5%) were clculted. Tble 4. Extended Concentrtion of Metl Ions, mg/l Mss of Ions Relesed from the Applince, mg/pplince Cu Cr Fe Ni Cu Cr Fe Ni (31.3) c (5.47) c (214.56) c (18.7) c / / / / / / (2.900) c (2.314) c (32.05) c (13.96) c

7 146 MIKULEWICZ, CHOJNACKA, WOŁOWIEC Sttisticl Methods Sttisticl nlysis of the results ws performed by the softwre Sttistic (version 9.0, Tuls, Okl, USA). Descriptive sttistics (men, medin, SD, minimum [min], nd mximum [mx]) were reported. Sttisticl differences between the experimentl nd control groups were ssessed by the nonprmetric Wilcoxon test for pired smples. The sttisticl significnce of differences between the groups ws ccepted t the,.05 level. Anlysis of interelement correltions ws crried out by determintion of the Person coefficient. A correltion ws considered sttisticlly significnt t,.05. RESULTS In the present work, metl ion relese in the environment of rtificil sliv ws investigted in continuous mode with recycling (the rtio ws 1:1). The rtificil sliv ws collected from the outlet of the system, nd the concentrtions of Cr, Ni, Cd, Cu, Mn, Mo, Fe, nd Si ions were determined. In ll, 19.4 L of rtificil sliv flowed through the system during the 28 dys of the experiment. Tble 1 reports the totl quntity of metl ions (expressed in milligrms) tht ws relesed from the orthodontic pplince during the whole experiment. The reported dt re the result of subtrction of the control smples from the experimentl smples. The ions were relesed in the following order: Si.Cu. Ni.Cr.Mo.Mn.Cd. Fe ions were not relesed from the pplince. There ws no correltion between the content of given metl in n lloy nd the relesed quntity. The concentrtion of metl ions in the experimentl nd control smples is presented in Figure 2 h. In the cse of Ni, Cr, nd Cu ions, more intense relese ws observed during the first 2 hours of the process, in comprison to the concentrtion in the control smples. Tble 2 presents sttisticl elbortion of results from the experimentl nd control groups (n 5 38 smples drwn during the study in ech group) for ech evluted relesed metl ion. The concentrtion of ions ws in the following order: Si.Fe.Cu.Ni.Cr. Mo.Mn.Cd. The level of Cd ws close to the lower limit of detection. The concentrtions of the remining ions were detectble in the experimentl smples. The differences between the experimentl nd control groups were ssessed by the nonprmetric Wilcoxon test for pired smples. Sttisticlly significnt differences were found for Ni, Cr, nd Cu. The men concentrtions were 12%, 26%, nd 93% higher, respectively, in the experimentl group vs the control group. Interelement interctions were investigted in the experimentl nd control groups seprtely. Strong nd sttisticlly significnt correltions were found between the following elements in the experimentl group: Ni-Cr, Ni-Cu, Ni-Fe, Ni-Mn, Cr-Cu, Cr-Fe, Cr-Mn, Cu-Mn, Fe-Mn (Tble 3). It cn be concluded tht Ni, Cr, Cu, Fe, nd Mn were interrelted. The concentrtions of Cd, Mo, nd Si ions were not correlted with those of the other ions of metls in the lloys of the orthodontic pplince. DISCUSSION Mny in vitro studies tht evluted ion relese from fixed orthodontic pplinces used different methodologicl/mteril pproch from tht employed in the present study. Vrious types of immersion liquids, different nlyticl methods, nd mterils obtined from severl mnufcturers mke comprison of studies more difficult nd sometimes even impossible. It is hrd to drw meningful conclusions from comprison of the degree of relesed ions from vrious mterils. All vilble studies reported the results of in vitro experiments in rtificil sliv in btch mode. Tble 4 shows the results of those studies longside those of the present investigtion. A literture review retrieved five studies 9 11,22,23 tht reported on the relese of metl ions from fixed orthodontic pplinces (brckets, bnds, nd wires mnufctured from stinless steel) in controlled in vitro conditions, ie, rtificil sliv ( ml) t 37uC nd ph 7 with the experiment lsting dys. Brrett et l. 9 conducted study on simulted fixed pplince tht represented complete mxillry rch. The experiment lsted 28 dys; smples were collected t 1, 7, 14, 21, nd 28 dys; nd the nlyticl technique ws flme tomic bsorption specrometry. 9 Hwng et l. 10 performed study on simulted fixed pplince, which represented hlf of the mxillry rch; the experiment lsted 90 dys nd smples were collected t 1, 3, 7, 14, 21, 28, 56, nd 84 dys. The nlyticl technique ws inductively coupled plsm mss spectrometry. Two types of stinless steel wires were investigted. Brckets nd bnds were the sme in the experimentl groups. The elements of the pplince were soldered together. 10 Gursoy et l. 22 conducted study on hlf-rch of fixed pplince. The experiment ws crried out for 45 dys, nd the smples were collected on the lst dy. The nlyses were crried out by inductively coupled plsm opticl emission spectrometry. 22 Kuht et l. 11 lso used mxillry hlf-rch. The durtion of the experiment ws 28 dys, nd smpling times were 1, 7, 14, nd 28 dys. The nlyticl technique ws high-resolution inductively coupled

8 METAL IONS RELEASE FROM ORTHODONTIC APPLIANCE 147 plsm mss spectrometry. 11 Mikulewicz et l. 23 conducted study on complete pplince (representing upper nd lower rches) in 25 ml of rtificil sliv, in which the pplince ws incubted for 30 dys (smpling ws done on the lst dy). The nlyticl technique ws inductively coupled plsm mss spectrometry. 23 The different concentrtions nd msses of relesed metl ions from the pplinces re shown in Tble 4. Differences cn be explined by the slightly different experimentl methodologies nd mterils: proportions of the elements in the pplince, rtio of pplince to solution, mnufcturers of orthodontic prts nd sometimes mterils (Ni-Ti wire, solder), composition of rtificil sliv (which my hve contined trces of the studied metl ions), nd nlyticl techniques with different lower detection limits, interferences, nd mtrix effects. Also, the studies were crried out between yers 1993 nd The sensitivity of pplied nlyticl techniques hs improved significntly in recent yers. Finlly, in three of the studies, no control group ws used. The knowledge of the composition of the control solution enbles one to subtrct the quntity of the studied ions in the mtrix (solution of rtificil sliv, metl ions relesed from the system, glss, tubes). Consequently, if one is ttempting to clculte the mss of relesed ions, the ions present in the solution from other sources should be tken into considertion. Knowledge of the level of ions in the control group enbled us to report the ions relesed from the orthodontic pplince only. In in vitro experiments, the influence of single fctors cn be considered. In contrst, in in vivo studies, mny prmeters influence the obtined results nd mke it difficult to interpret the dt; however, they reflect ctul conditions Bsed on the mss of relesed metl ions in the continuous flow experiment, n ttempt to clculte the verge mss of metl ions relesed during potentil 2-yer tretment period ws undertken. The msses of metl ions were s follows: Cr 131 mg, Ni 444 mg, nd Cu 751 mg. The World Helth Orgniztion s recommended dily doses re mg for Cr nd mg for Ni. 27 The verge dily doses relesed from the pplince in this study were clculted s mg for Cr, mg for Ni,nd 1.12 mg for Cu; thus, ll vlues re fr below the recommended dily doses. It must be emphsized tht ion relese from metl orthodontic devices in vivo is ffected by vrious fctors, such s sliv composition, ph, diet, nd microflor. Also, there is wide rnge of pplied mterils with different compositions (which cn ffect ion relese). It is nerly impossible to tke into ccount ll those fctors in n in vitro study. On the other hnd, the results of n in vivo study which re typiclly bsed on noninvsive mtrices (sliv, urine, hir) re ffected by methodologicl spects: smpling regimen, other sources of exposure, nd multitude of other fctors relted to living hbits. CONCLUSIONS N The highest concentrtion of metl ions ws relesed t the beginning of the experiment. After this, pssivtion probbly occurred, which hindered further intense relese. It ws found tht the ions were relesed in doses tht re not toxic to humns. N Comprison with btch-testing dt reported in the literture showed tht, in fct, more ions were relesed from the mteril thn hd been previously reported. In the present pper, in contrst to other dt vilble in the literture, the conditions did not rech equilibrium. ACKNOWLEDGMENT This reserch ws finncilly supported by The Ntionl Centre for Reserch nd Development in Polnd (N R ). REFERENCES 1. Brntley WA, Elides T. Orthodontic Mterils: Scientific nd Clinicl Aspects. Stuttgrt, Germny: Georg Thieme Verlg; Kusy RP. Orthodontic biomterils: from the pst to the present. Angle Orthod. 2002;72: Elides T, Bouruel C. Introrl ging of orthodontic mterils: the picture we miss nd its clinicl relevnce. Am J Orthod Dentofcil Orthop. 2005;127: Elides T, Athnsiou AE. In vivo ging of orthodontic lloys: implictions for corrosion potentil, nickel relese, nd biocomptibility. Angle Orthod. 2002;72: Schmltz G, Arenholt-Bindslev D. Biocomptibility of Dentl Mterils. Heidelberg, Germny: Springer-Verlg Berlin; Amini F, Rkhshn V, Sdeghi P. Effect of fixed orthodontic therpy on urinry nickel levels: long-term retrospective cohort study. Biol Trce Elem Res Dec;150(1 3): DOI: /s Chojnck K, Mikulewicz M. Hir minerl nlysis in the ssessment of humn exposure to metls. In: Preedy V, ed. Hndbook of Hir in Helth nd Disese. Wgeningen, The Netherlnds: Wgeningen Acdemic Publishers; Mikulewicz M, Chojnck K. Trce metl relese from orthodontic pplinces by in vitro studies: systemtic literture review. Biol Trce Elem Res. 2010;139: Brrett RD, Bishr SE, Quinn JK. Biodegrdtion of orthodontic pplinces. Prt I. Biodegrdtion of nickel nd chromium in vitro. Am J Orthod Dentofcil Orthop. 1993;103: Hwng CJ, Shin JS, Ch JY. Metl relese from simulted fixed orthodontic pplinces. Am J Orthod Dentofcil Orthop. 2001;120:

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