Impact of orthodontic retainers on periodontal health status assessed by biomarkers in gingival crevicular fluid

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1 Originl Article Impct of orthodontic retiners on periodontl helth sttus ssessed by biomrkers in gingivl creviculr fluid Wellington J. Rody Jr ; Hengmeh Akhlghi b ; Sercn Akylcin c ; Willim A. Wiltshire d ; Mnjul Wijegunsinghe e ; Getulio Nogueir Filho f ABSTRACT Objective: To evlute whether biomrkers of inflmmtion nd periodontl remodeling re differentilly expressed in the gingivl creviculr fluid (GCF) of ptients wering different types of orthodontic retiners. Mterils nd Methods: Thirty-one dult subjects (17 men nd 14 women with n ge rnge of 20 to 35 yers) were llocted to three different groups. Group 1 consisted of 10 ptients wering fixed retiners, group 2 included 11 ptients using lower removble retiners, nd group 3 comprised 10 ptients without retiners (control). Periodontl helth ssessment nd GCF collection were crried out t two sites per subject: the lingul side of centrl lower incisor nd the lingul side of lower second premolr. Aliquots from diluted GCF were screened for the presence of biomrkers using microrry technique. Results: Group 1 ptients exhibited higher percentge of sites with visible plque in the incisor region thn the other groups (P 5.03); no differences were noted in gingivl bleeding nd probing depths. The medin concentrtions (pg/ml) of interferon-gmm nd interleukin-10 were significntly higher in the premolr sites of ptients in group 2 (P 5.01 nd P 5.04, respectively), wheres the concentrtion of mtrix metlloproteinse-9 ws significntly higher t the incisors of ptients wering fixed retiners (P 5.02). A significnt difference between the two sites ws seen only in group 2. Conclusions: The presence of different orthodontic retiners my promote specific ltertions in the GCF composition. With retention periods potentilly becoming longer, this finding my be of clinicl significnce. (Angle Orthod. 2011;81: ) KEY WORDS: Gingivl creviculr fluid; Orthodontic retiners; Periodontl disese; Biomrkers; Protein microrrys Assistnt Professor, Division of Orthodontics, School of b Privte Prctice, Vncouver, BC, Cnd. c Clinicl Assistnt Professor, Dentl Brnch, The University of Texs Helth Sciences Center, Houston, TX. d Professor nd Hed, Division of Orthodontics, School of e Reserch Technicin, Division of Orthodontics, School of f Associte Professor, Division of Periodontology, School of Corresponding uthor: Dr Wellington J. Rody Jr, Division of Orthodontics, Deprtment of Preventive Dentl Science, D Bnntyne Avenue, University of Mnitob, Winnipeg, MB, Cnd R3E 0W2 (e-mil: rody@cc.umnitob.c) Accepted: April Submitted: Jnury Published Online: June 9, 2011 G 2011 by The EH Angle Eduction nd Reserch Foundtion, Inc. INTRODUCTION Alignment instbility is one of the mjor pitflls of orthodontic tretment. As result, orthodontists tend to recommend long-term use of retiners for enhnced stbility, which my require yers or even decdes of retiner wer. 1 A recent systemtic review 2 stted tht there re insufficient reserch dt on which to bse clinicl decisions regrding retention. Accordingly, the potentil increse in durtion of retention mkes it importnt to evlute the outcome of long-term retention on surrounding tissues, with prticulr emphsis on periodontl disese. Periodontl destruction is induced by the deleterious effects of inflmmtory meditors tht pper s result of bcteril plque buildup round the tooth. 3 Interestingly, the mjority of published studies 3 6 focused on bcteril plque ccumultion nd clinicl DOI: /

2 1084 RODY, AKHLAGHI, AKYALCIN, WILTSHIRE, WIJEGUNASINGHE, FILHO Figure 1. Collection sites for GCF in group 1 (fixed retiner), group 2 (removble retiner), nd group 3 (no retiner). Site A indictes gingivl sulcus t the lingul side of the mndibulr centrl incisor; site B, gingivl sulcus t the lingul side of the mndibulr second premolr. periodontl helth ssessment in the presence of retiners. Clinicl methods to dignose periodontl disese re very cost-effective, but they do not distinguish between disese-ctive nd disese-inctive periodontl sites. On the other hnd, biochemicl nlysis of the gingivl creviculr fluid (GCF) offers noninvsive mens to ssess the host response in periodontl disese. 7 GCF is complex biologicl fluid tht contins mixture of serum-derived nd inflmmtory proteins. 8 The constituents of GCF rise from vriety of sources, including microbil plque, host cells, nd serum-derived fctors; thus, number of biomrkers re vilble to exmine in the GCF. 7,8 Mtrix metlloproteinse-9 (MMP-9) is derived predominntly from monocytes nd mcrophges, nd its levels in GCF re higher in ptients with periodontitis; therefore, MMP-9 my serve s biomrker of periodontl disese nd id in erly detection of periodontitis. 9 An intermedite mechnism tht lies between bcteril stimultion nd tissue remodeling is the production of cytokines. Interferongmm (IFN-c) is proinflmmtory cytokine tht increses in experimentl gingivitis nd periodontitis. 8 On the other hnd, interleukin-10 (IL-10) is n ntiinflmmtory cytokine tht inhibits the relese of proinflmmtory nlytes. 10 All these cytokines ply key roles in immune nd inflmmtory responses, nd infection outcome my be ttributble to the blnce in the reltive rtios mong ll of them. The specific im of this project ws to evlute whether biomrkers of inflmmtion nd periodontl remodeling, such s MMP-9, IFN-c, nd IL-10, re differentilly expressed in the GCF of ptients who hve worn retiners for n extended period of time. The uthors hope to provide clinicins with better understnding of the effect of long-term retiner use on periodontl tissues, which will enble them to mke better decisions for their ptients. MATERIALS AND METHODS Thirty-one individuls (17 men nd 14 women with n ge rnge of 20 to 35 yers) were included in the study. To be included, subjects needed to hve completed full fixed orthodontic tretment t lest 4 yers before the initition of the study, to be free of systemtic disese, nd to hve received no periodontl tretment in the lst 6 months. Smokers nd drug users were excluded from the study. Written informed consent ws obtined from the prticipnts, nd ethicl pprovl ws grnted from the Helth Reserch Ethics Bord t the University of Mnitob (reference number H2009:15). Subjects were llocted into three groups. Group 1 consisted of 10 ptients who wore retiners bonded to the lingul surfces of both lower cnines. The fixed mndibulr retiners in this group hd been plced upon the completion of tretment nd hd been constructed of nonbrided inch round stinless steel wire. Group 2 consisted of 11 ptients who used removble lower Hwley-type retiners regulrly on dily bsis. Ten postorthodontic ptients wering no retiners were plced in the control group (group 3). The men period of retention ws 5.6 yers (rnge, 4 to 10 yers). Clinicl periodontl helth ssessment nd GCF collection were crried out t two sites per subject: the lingul side of lower centrl incisor nd the lingul side of lower second premolr (Figure 1). It ws deemed necessry to choose those teeth becuse different biologicl processes might be involved t sites directly influenced by the fixed retiner, such s the gingivl sulcus on the lingul side of the lower incisors. Probing depth (PD), mesured with stright periodontl probe, ws recorded s the distnce in millimeters from the gingivl mrgin to the most picl prt of the sulcus. 11 Presence or bsence of bleeding

3 IMPACT OF RETAINERS ON PERIODONTAL HEALTH STATUS 1085 Tble 1. Demogrphic Dt, Periodontl Prmeters, nd Gingivl Creviculr Fluid (GCF) Volume (men 6 SD) of the Studied Subjects Group 1 (n 5 10) Group 2 (n 5 11) Group 3 (n 5 10) Gender (M:F) 3:7 9:2 5:5 Age (y) PD, incisors (mm) PD, premolrs (mm) PA, incisors (%) * PA, premolrs (%) BOP, incisors (%) BOP, premolrs (%) GCF volume, incisors (ml) GCF volume, premolrs (ml) PD indictes probing depth; PA, plque ccumultion; nd BOP, bleeding on probing. * Sttisticlly significnt difference (P #.05). on probing (BOP) nd plque ccumultion (PA) were recorded in dichotomous mnner (0 or 1): If bleeding occurred within 15 seconds fter retrievl of the probe, the site ws recorded s BOP-positive, nd the presence of visible plque fter running the probe long the gingivl mrgin ws recorded s positive. 11,12 Before GCF collection, suprgingivl plque ws removed with plstic scler; then, ech site ws gently dried for 10 seconds with compressed ir nd isolted from sliv with cotton roll. GCF collection t ll sites ws performed using pper strips (Periopper, Orflow Inc, Plinview, NY). Two strips were inserted for 30 seconds into the gingivl sulcus of ech selected tooth, with 30 seconds between smplings. The volume of GCF ws determined by positioning the strips between the upper nd the lower counterprts of the preclibrted Periotron 8000 (Periotron 8000, Orflow Inc). The pper strips were then plced in single lbeled Eppendorf tube contining 80 ml phosphte-buffered sline (Invitrogen, Cmrillo, Clif), seled, nd immeditely sent to the lbortory. Superntnt smples from ll ptients were stored t 280uC for subsequent biomrker nlysis. Biomrker Anlysis To ensure tht the smples included enough proteins to be quntified, 5 ml of diluted GCF from ll sites were submitted to generl protein quntifiction ssy, s previously described by Desjrdins et l. 13 Subsequently, smple liquots from diluted GCF from ll groups sites were screened for the presence of specific proteins, including IL-10, IFN-c, nd MMP-9, with customized Quntibody Arry (RyBiotech, Norcross, G). One stndrd glss slide ws spotted with 16 wells of identicl biomrker ntibody rrys. Ech ntibody, together with the positive nd negtive control, ws rryed in qudruplicte. 14,15 Then, fter 30 minutes incubtion with smple diluent, the glss chips were wshed, nd ech well ws overlid with 50 ml of diluted GCF. After overnight incubtion t 4uC nd extensive wshing, the detector ntibody ws dded for 1 hour nd then wshed wy; Alex fluor 555-conjugted streptvidin ws then dded for 2 hours t room temperture. The signls (Cy3 wvelengths: 555 nm excittion, 655 nm emission) were scnned nd extrcted with Genepix 4000B lser scnner (Axon Instruments, Foster City, Clif). Both the totl mounts nd concentrtions of biomrkers were evluted in this study. The concentrtion levels, expressed in picogrms per milliliter, were clculted with RyBiotech Q Anlyzer softwre ginst stndrd curve set for ech biomrker. The totl mount of ech biomrker ws determined in picogrms by timing the concentrtion with smple volume. Sttisticl Anlysis Dt ws nlyzed with GrphPd prism softwre, version 5 (GrphPd Softwre Inc, L Joll, Clif). Nonprmetric tests were used to compre the differences between groups nd sites (P,.05). The Kruskl-Wllis nd Person s chi-squre tests were used to compre differences between the three groups in ech region for quntittive nd qulittive comprisons, respectively. When there ws significnt difference, multiple-comprison post hoc test (Dunn s test) ws used to determine the pirwise differences. The Wilcoxon signed rnk test ws used to compre differences between sites (incisor versus premolr) within ech group for quntittive vribles. Fisher s exct nd McNemr s tests were used for the sme purpose for the comprison of qulittive vribles between the sites. RESULTS Tble 1 presents the men demogrphic dt, s well s the men clinicl prmeters nd GCF volume, for the three groups. Age nd gender distribution did not differ significntly between clinicl groups. The

4 1086 RODY, AKHLAGHI, AKYALCIN, WILTSHIRE, WIJEGUNASINGHE, FILHO Tble 2. Concentrtion nd Totl Amount of Biomrkers by Group nd Site Concentrtion (pg/ml) Totl Amount (pg) MMP-9 IFN-c IL-10 MMP-9 IFN-c IL-10 IC* PM IC PM* IC PM* IC* PM IC PM* IC PM Group 1 Men 16,644 21, SD 13,327 12, Medin 20,300 23, Group 2 Men , SD Medin , Group 3 Men 14,512 18, SD , Medin 14,620 15, IC indictes incisor region; PM, premolr region; MMP-9, mtrix metlloproteinse-9; IFN-c, interferon-gmm; nd IL-10, interleukin-10. * Sttisticlly significnt difference between groups (P #.05). fixed retiner group exhibited more PA in the incisor region (P 5.03). In ddition, the GCF volume ws higher in the incisor region of group 1 thn in the other groups, but this difference ws not sttisticlly significnt. No significnt differences between groups were observed for the remining clinicl prmeters. Tble 2 provides the concentrtion nd totl mount of ech biomrker by group nd site. The concentrtion of certin biomrkers differed significntly in the GCF between the three groups; significnt differences between groups were detected for IL-10, IFN-c, nd MMP-9. The medin concentrtions of IL-10 nd IFN-c were significntly higher in the premolr sites of group 2 ptients (lower removble retiners) (P 5.04 nd P 5.01, respectively), wheres the medin concentrtion of MMP-9 ws significntly higher (P 5.02) in the GCF smples collected from the lower incisors of group 1 ptients (fixed retiners) (Figure 2). The totl mount of MMP-9 t incisor sites differed significntly between the three groups (P 5.017), s did the totl mount of IFN-c t premolr sites (P 5.012). No sttisticlly significnt results were observed for the totl mount of IL-10 t both premolr nd incisor sites (Tble 2). Although our dt showed firly consistent trend towrd incresed concentrtions of the trgeted biomrkers in GCF smples obtined from premolr sites, significnt difference between the two sites ws found only in group 2 (Tble 3). Interestingly, in ptients with removble retiners, the medin concentrtions of IL-10 (35.1 pg/ml), IFN-c (200 pg/ml), nd MMP-9 (12,700 pg/ml) in premolr sites were significntly higher when compred to the incisor sites (Figure 3). DISCUSSION It is resonble to stte tht the clinicl periodontl helth of our subjects ws not ffected by bonded lingul retiners, despite the fct tht significntly incresed PA ws found in the lower incisor region in group 1 (Tble 1). Störmnn nd Ehmer 16 lso found tht ptients with fixed retiners hd incresed PA over 24-month follow-up period, lthough regulr orl hygiene instruction ws given to the ptients. In ddition, our study confirms nd extends the results of Heier et l., 5 who found no differences in gingivl inflmmtion in spite of slightly incresed PA in Figure 2. Box plots displying biomrker concentrtions in GCF smples obtined from premolr nd incisor sites of ech group. *Significnt differences between groups.

5 IMPACT OF RETAINERS ON PERIODONTAL HEALTH STATUS 1087 Tble 3. Comprison of Biomrker Concentrtions (Medin Vlues, Expressed in pg/ml) Between Sites Within Ech Group Site A (IC) Site B (PM) P Group 1 IL NS (.45) IFN-c NS (.57) MMP-9 20,300 23,210 NS (.31) Group 2 IL * IFN-c * MMP , * Group 3 IL NS (.17) IFN-c NS (.14) MMP-9 14,620 15,450 NS (.7) Site A (IC) indictes incisor region; Site B (PM), premolr region; MMP-9, mtrix metlloproteinse-9; IFN-c, interferon-gmm; nd IL-10, interleukin-10. * Sttisticlly significnt t P #.05. NS indictes non sttisticlly significnt differences. ptients with fixed retiners during the first 6 months of retention. Few studies 5,17 hve investigted the impct of removble retiners on periodontl helth. Heier et l. 5 evluted the differences between fixed nd removble retiners, but only the segment from cnine to cnine ws nlyzed. The present study fetured more comprehensive evlution since premolr site ws lso included in the study design. One importnt finding is the fct tht group 2 ws the only group to exhibit sttisticlly significnt intrgroup differences, with higher concentrtion of biomrkers in the premolr sites versus the incisor sites (Figure 3). There re two possible explntions for this finding. Perhps the posterior crylic resin portion of the removble retiner irrittes the periodontl tissues, or perhps the GCF levels of biomrkers re elevted in the premolr region s result of tooth movement induced by the removble retiner. The results from previous studies, which hve shown elevted expression of MMP-9, IL-10, nd IFN-c in the periodontium during orthodontic movement, together with the results from the current study indicting tht periodontl clinicl prmeters (PA, PD, nd BOP) in premolr sites re not different between groups, support the ltter. Nevertheless, the two possibilities re by no mens mutully exclusive. Not only periodontl disese but lso tooth movement cn induce differentil expression of cytokines in the periodontium. Grlet et l. 19 demonstrted tht IL- 10 is up-regulted in the tension zone, wheres Alhshimi et l. 18 found higher IFN-c expression in the compression zone during orthodontic tooth movement. As result, it is possible tht the different biomrker profile found t premolr sites under the influence of removble retiners my be consequence of relpse or nturl dpttion of the occlusion in the posterior segment of the rch. The fixed retiner extends from cnine to cnine only, wheres the removble retiner evluted in our study is the Hwley-type, which extends distlly up to the molr region. Since group 2 ptients were wering retiners only t night, it is possible tht the pplince ws constntly moving those teeth bck to their originl position fter dy of posterior teeth drifting cused by the lck of retiner wer. As result, we my hypothesize tht the posterior teeth re constntly under light pressure during nighttime removble retiner wer, nd this my explin the elevted concentrtions of biomrkers in the GCF collected from premolr sites in group 2. It ws intriguing to observe tht the sme behvior ws not observed in the nterior region of the removble retiner group, despite the fct tht lower incisor movement is the most common mnifesttion of orthodontic relpse. 1 This my help provide better understnding of the mechnism of ction of lower removble retiners. From our biologicl observtions, we cn speculte tht the posterior segment of the lower rch is criticl re, where the removble retiner countercts the forces tht ct on the teeth throughout the dy, thus Figure 3. Box plots displying biomrker concentrtions in GCF smples obtined from premolr nd incisor sites of group 2. *Significnt differences between sites.

6 1088 RODY, AKHLAGHI, AKYALCIN, WILTSHIRE, WIJEGUNASINGHE, FILHO voiding nterior crowding by preventing mesil drifting of the posterior teeth. Our reserch confirms the findings of previous studies 3 6 tht found no reltionship between fixed retiners nd periodontl disese. However, it ws interesting to observe the significntly elevted concentrtion of MMP-9 ssocited with higher percentge of sites with visible plque in the incisor region of the fixed retiner group. It is lso importnt to note tht there ws trend towrd n incresed volume of GCF in the incisor region of group 1, lthough this ws not sttisticlly significnt (Tble 1). MMP-9 ctivities from polymorphonucler neutrophil sources hve been shown to be mrkedly incresed in humn gingivl tissue, GCF, nd sliv. 9,20,21 This mens tht dentl plque my trigger the relese of MMPs in GCF, regrdless of clinicl inflmmtion. It hs to be stted tht the mere elevtion of MMP-9 in periodontlly helthy sites of ptients using fixed orthodontic retiners my or my not be risk fctor for future periodontl bone loss in the incisor region. A recent study 22 supports the biologicl plusibility of n ssocition between GCF levels of MMP-9 nd progression of periodontitis in immunocompromised ptients; however, further studies re needed to vlidte MMP-9 s prognostic fctor for periodontitis in helthy individuls. Despite the difficulties nd uncertinties in this field of reserch, it should be kept in mind tht high lods of MMP-9 my be complicting fctor with potentilly longer orthodontic retention periods. Although promising, the study of GCF composition is still chllenging reserch field. The sensitivity of the ssy methods hs improved since reserch on the constituents of GCF first begn; nevertheless, it is still fr from being idel. The min limittion of the present study resides in the smll number of subjects in ech group, which, long with the symmetry of the dt sets, my hve resulted in lck of sttisticl power. In fct, the inclusion criteri were n importnt limiting fctor in terms of smple size for group 2, becuse most ptients were not wering removble retiners on dily bsis fter some yers in retention. In ddition, mesurement of biomrkers in GCF is not n esy tsk becuse the volumes of GCF re usully low. CONCLUSIONS N The dt indicted specific ltertions in the GCF composition s result of the presence of different orthodontic retiners. N Incresed GCF levels of MMP-9 from lower incisor sites in the fixed retiner group suggested subclinicl inflmmtion. N The elevted concentrtions of IL-10 nd IFN-c t premolr sites in the removble retiner group my be relted to the restrining effect exerted by the pplince on the posterior segment of the rch. ACKNOWLEDGMENT This work ws funded by the University of Mnitob Reserch Grnts Progrm. 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