Novel rechargeable calcium phosphate nanoparticlecontaining

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1 (217) 9, OPEN ORIGINAL ARTICLE Novel rehrgele lium phosphte nnoprtileontining orthodonti ement Xin-Ju Xie 1,2, Dn Xing 2,3, Lin Wng 2,4, Hn Zhou 2, Mihel D Weir 2, Yu-Xing Bi 1 nd Hokin HK Xu 2,5,6 White spot lesions (WSLs), due to enmel deminerliztion, our frequently in orthodonti tretment. We reently developed novel rehrgele dentl omposite ontining nnoprtiles of morphous lium phosphte (NACP) with long-term lium (C) nd phosphte (P) ion relese nd ries-inhiiting pility. The ojetives of this study were to develop the first NACPrehrgele orthodonti ement nd investigte the effets of rehrge durtion nd frequeny on the effiy of ion re-relese. The rehrgele ement onsisted of pyromelliti glyerol dimethrylte (PMGDM) nd ethoxylted isphenol A dimethrylte (EBPADMA). NACP ws mixed into the resin t 4% y mss. Speimens were tested for orthodonti rket sher ond strength (SBS) to enmel, C nd P ion initil relese, rehrge nd re-relese. The new orthodonti ement exhiited n SBS similr to ommeril orthodonti ement without CP relese (P4.1). Speimens fter one rehrge tretment (e.g., 1 min immersion in rehrge solution repeting three times in one dy, referred to s ) exhiited sustntil nd ontinuous re-relese of C nd P ions for 14 dys without further rehrge. The ion re-relese did not derese with inresing the numer of rehrge/re-relese yles (P4.1). The ion re-relese onentrtions t 14 dys versus vrious rehrge tretments were s follows: 43min 2 times4443min 1 time4. In onlusion, lthough previous studies hve shown tht NACP nnoomposite reminerlized tooth lesions nd inhiited ries, the present study developed the first orthodonti ement with C nd P ion rehrge nd long-term relese pility. This NACP-rehrgele orthodonti ement is promising therpy to inhiit enmel deminerliztion nd WSLs round orthodonti rkets. (217) 9, 24 32; doi:1.138/ijos.216.4; pulished online 4 Novemer 216 Keywords: ond strength; lium phosphte nnoprtiles; lium phosphte ion rehrgeility; long-term ion relese; orthodonti ement; white spot lesions INTRODUCTION White spot lesions (WSLs) hve een reported s previling nd hllenging prolem in fixed orthodonti therpy. 1 4 The irregulr surfes of rkets, nds, wires nd other tthments provide res for iofilm/plque uild-up tht re diffiult to len. 1 3 These onditions promote the oloniztion of riogeni teri, whih produe ids to indue enmel deminerliztion nd n led to WSLs. 5 7 It ws reported tht 5% 7% of ptients with fixed orthodonti therpy hd WSLs, 8 9 nd tht it took only 1 month to develop WSLs. 1 Extensive efforts were mde to improve the properties of dentl mterils, inluding the use of nti-teril gents, reduing polymeriztion stresses, enhning dhesive systems, 2 22 nd improving linil operting methods Previous efforts lso inluded methods to omt WSLs in orthodonti tretment However, these methods rely on ptient ompline nd re thus unrelile. 27 Clium phosphte (CP) reminerliztion pprohes my e promising for WSL prevention nd enmel reminerliztion Dentl resins ontining CP prtiles were shown to relese C nd P ions They t s C nd P ion reservoir in plque nd on the tooth surfes nd n e relesed during n idi riogeni hllenge to prevent deminerliztion nd filitte reminerliztion. 31,33 34 However, trditionl CP omposites used CP prtile sizes of 1 55 μm nd exhiited poor mehnil properties. 31,34 Reently, novel nnoomposite ws developed using nnoprtiles of morphous lium phosphte (NACP) tht hd men prtile size of 116 nm. 35 The NACP nnoomposite relesed lium nd phosphte ions similrly to trditionl CP omposites, ut with twofold inrese in mehnil properties for lod-ering restortions The NACP omposite ould reminerlize the enmel lesions, rehing reminerliztion effiy tht ws fourfold tht of ommeril fluoride-relesing omposite. 37 Enmel minerl loss t the mrgins 1 Deprtment of Orthodontis, Shool of Stomtology, Cpitl Medil University, Beijing, Chin; 2 Biomterils nd Tissue Engineering Division, Deprtment of Endodontis, Periodontis nd Prosthodontis, University of Mrylnd Dentl Shool, Bltimore, USA; 3 Deprtment of Dentistry, Chin Rehilittion Reserh Center, Beijing, Chin; 4 VIP Integrted Deprtment, Stomtologil Hospitl of Jilin University, Chnghun, Chin; 5 Center for Stem Cell Biology nd Regenertive Mediine, University of Mrylnd Shool of Mediine, Bltimore, USA nd 6 Deprtment of Mehnil Engineering, University of Mrylnd, Bltimore County, USA Correspondene: Professor YX Bi, Deprtment of Orthodontis, Shool of Stomtology, Cpitl Medil University, Beijing 169, Chin E-mil: yuxing@263.net Professor HHK Xu, Biomterils nd Tissue Engineering Division, Deprtment of Endodontis, Periodontis nd Prosthodontis, University of Mrylnd Dentl Shool, 65 West Bltimore Street, Bltimore MD 2121, USA E-mil: hxu@umrylnd.edu Aepted 12 July 216

2 25 round the NACP nnoomposite ws one-third tht of ontrol omposite. 38 However, CP omposites exhiited C nd P ion relese tht lsted only few months efore eing diminished Reently, CP-rehrgele dentl resins were developed for the first time NACP ws mixed into resin onsisting of pyromelliti glyerol dimethrylte (PMGDM) nd ethoxylted isphenol A dimethrylte (EBPADMA). 39 The speimens were immersed in ph 4 solution to exhust C nd P ion relese. Then, the speimens were rehrged y immersing them in C nd P solutions, whih ould potentilly e inorported into n orl rinse solution. 39 The rehrged speimens were le to re-relese high levels of C nd P ions ontinuously, without further rehrge, for 7 dys. After 7 dys, the speimens were given seond rehrge, nd they were le to rerelese for 7 more dys. C nd P ions ould e repetedly rehrged for re-relese, with no derese in ion re-relese while inresing the numer of rehrge/re-relese yles The novel C nd P ionrehrgele resins promise to hieve long-term reminerliztion nd ries-inhiiting pilities. However, the pilot studies only tested one rehrge method of 3 min of immersion two times in one dy, 39 4 without testing other rehrge time durtions or frequenies. It would e desirle to shorten the immersion time from 3 to 1 min; however, it is not ler if this would ompromise the rehrge effiy. Furthermore, to dte, there hs een no report on the development of C nd P ion-rehrgele orthodonti ement to omt WSLs. Therefore, the ojetives of this study were to develop CPrehrgele orthodonti ement nd to investigte the effet of rehrging time durtion nd frequeny on the rehrge/re-relese effiy. The following hypotheses were tested: (1) new orthodonti ement ould e developed to e CP rehrgele, without deresing the enmel ond strength; (2) the re-relese of C nd P ions from the orthodonti ement would e mintined over time nd would not derese while inresing the numer of rehrge/re-relese yles; nd (3) the CP rehrge durtion nd frequeny would signifintly ffet the rehrge effiy of orthodonti ement. MATERIALS AND METHODS Development of CP-rehrgele orthodonti ement The resin onsisted of 44.5% (m/m) of PMGDM (Hmpford, Strtford, CT, USA), 39.5% of EBPADMA (Sigm-Aldrih, St Louis, MO, USA), 1% of 2-hydroxyethyl methrylte (HEMA) (Essteh, Essington, PA, USA), nd 5% of isphenol A glyidyl dimethrylte (Essteh, Essington, PA, USA), s desried in previous study. 39 Then, 1% of phenylis (2,4,6-trimethylenzoyl) phosphine oxide (Sigm-Aldrih, St Louis, MO, USA) ws dded for photopolymeriztion. 39 PMGDM nd EBPADMA were used euse they hd ytotoxiity vlues similr to those of other dentl dimethryltes. 41 PMGDM is n idi dhesive monomer nd n helte with lium ions from the rehrging solution to filitte ion rehrge. 39 HEMA ws dded to improve the flowility nd hydrophiliity, s reported in previous study. 44 BisGMA ws dded euse it ould improve the rosslink of monomers nd enhne the onding to tooth strutures. 45 This resin is referred to s PEHB. NACP [C 3 (PO 4 ) 2 ] were mde vi spry-drying tehnique s desried previously. 35,37 Briefly, lium ronte nd dilium phosphte nhydrous were dissolved in n eti id solution. The onentrtions of C nd P ions were 8 nd mmol L 1, respetively, yielding C/P molr rtio of 1.5. The solution ws spryed into heted hmer to evporte the wter nd voltile id. The dried NACP ws olleted using n eletrostti preipittor. The NACP men prtile size ws previously mesured to e 116 nm. 35 NACP ws mixed with the PEHB resin t n NACP mss frtion of 4% to form the CP orthodonti ement (referred to s PEHB+ NACP). The 4% NACP filler level yielded flowle pste, with high levels of C nd P ion relese nd good mehnil properties, s shown in previous studies Trnsond XT (3M, Monrovi, CA, USA) is used s n orthodonti ement nd served s ontrol (referred to s the TB ontrol). Aording to the mnufturer, the TB ontrol onsisted of silnetreted qurtz (7% 8% y weight), isphenol A diglyidyl ether dimethrylte (1% 2%), isphenol-a-is (2-hydroxyethyl) dimethrylte (5% 1%), silne-treted sili (o2%) nd diphenyliodonium hexfluorophosphte (o.2%). Vitremer (3M, St Pul, MN, USA) served s nother ontrol nd ws referred to s the VT ontrol. The VT ontrol is resin-modified glss ionomer ement (RMGI) tht onsists of fluoroluminosilite glss nd light-sensitive, queous polylkenoi id. Inditions inlude lss III, V nd root-ries restortions nd lss I nd II lesions in the primry teeth. VT ws seleted euse RMGIs hve een used s orthodonti ements. 46 A powder/liquid mss rtio of 2.5/L ws used for the VT ontrol ording to the mnufturer. Testing of orthodonti rket SBS to enmel Three ements (PEHB+NACP, TB ontrol, VT ontrol) were tested using n enmel sher ond strength (SBS) method. 47 Sixty extrted humn first premolrs were rndomly divided into three groups of 2 teeth eh. The riteri for tooth seletion inluded intt ul enmel tht hd not een pre-treted with hemil gents, no visile rks nd no enmel irregulrities. 48 All teeth were stored in.1% thymol solution t 4 C nd used within 1 month fter extrtion. Eh tooth ws emedded vertilly in self-uring ryli resin (Lng Dentl, Wheeling, IL, USA), onsidering the ul xis of the linil rown, suh tht their lil surfes were prllel to the fore during the sher ond test. 47 The oronl portion ws sumitted to prophylxis with oil-free pumie nd ruer ups t low speed for 1 s. For TB ontrol, the ul surfes of teeth were ethed for 3 s with 35% phosphori id (Sothond; 3M, St Pul, MN, USA), wshed nd dried until they quired frosty white pperne. Trnsond XT primer (3M Unitek, Monrovi, CA, USA) ws pplied to the ethed surfes in thin, uniform ot. TB ontrol ement pste ws pplied to the se of the premolr metl orthodonti rkets (Ormo Series 2; Syron Dentl, Ornge, CA, USA), whih ws pled on the entre of the tooth surfe. Exessive ement round the rket ws removed. The ement ws photoured with Optilux VCL 41 (Demetron Kerr, Dnury, CT, USA) for totl of 4 s on ll four sides (mesil, distl, olusl nd gingivl) of the rket, with 1 s on eh side. 47 For PEHB+NACP ement, the ething nd onding proedures were the sme s those used for the TB ontrol. For the VT ontrol, ording to the mnufturer nd the literture, RMGI n e used s n orthodonti ement without ething the enmel Therefore, the onding proedure onsisted of polishing the enmel surfe for 1 s with flour pumie, followed y rinsing with wter for 1 s. Then, the VT pste ws pplied to the rket se nd the rket ws positioned nd onded to the enmel. 5,52 The smple ws then light ured for totl of 4 s s desried ove. 5,52 For eh of the three groups (PEHB+NACP, TB ontrol, VT ontrol), speimens were divided into two sugroups of 1 smples eh. A sodium hloride (NCl) solution (133 mmol L 1 ) ws prepred nd uffered to ph 4 with 5 mmol L 1 lti id s riogeni, low ph ondition. 35 One sugroup of eh ement ws immersed in this solution for 1 dy, nd the other sugroup for 7 dys. Thus, 3 2 full ftoril design ws tested with three

3 26 ements (PEHB+NACP, TB ontrol, VT ontrol) nd two immersion times (1 dy, 7 dys). Eh sugroup of eh ement ws stored in 5 ml of NCl solution in seled polyethylene ontiner. 53 For the 7-dy sugroups, the solution ws hnged one every week. At the end of the immersion period, the SBS ws mesured following previous studies. 51,54 A hisel on Universl Testing Mhine (MTS, Eden Pririe, MN, USA) ws positioned on the upper prt of the rket se nd prllel to the onded interfe. A lod ws pplied t displement rte of.5 mm min 1 until the ond filed. The SBS = lod t filure/rket surfe re, following the previous studies. 47 Eh tested tooth surfe ws exmined using stereomirosope (Lei Zoom 2; Lei, Wetzler, Germny). The dhesive remnnt index (ARI) ws sored to ssess the remining ement on enmel using the following riteri: 48 = no ement remined on enmel; 1 = less thn hlf of the ement remined on enmel; 2 = more thn hlf of the ement remined on enmel; nd 3 = ll the ement remined on enmel. Mesurement of initil C nd P ion relese from PEHB+NACP orthodonti ement The PEHB+NACP ement ws tested for C nd P ion relese. The NCl solution t ph 4 ws used to mesure the ion relese in simulted riogeni onditions. 35 As in previous studies, 32,35 three speimens of pproximtely 2 mm 2 mm 12 mm were immersed in 5 ml of solution to yield speimen volume/solution of 2.9 mm 3 ml 1. This ws similr to speimen volume per solution of ~ 3. mm 3 ml 1 reported in previous study. 32 The onentrtions of C nd P ions relesed from the speimens were mesured t 1, 3, 5, 7, 14, 21, 28, 35, 42, 49, 56, 63 nd 7 dys. 35 At eh time period, n liquot of.5 ml ws removed nd repled with fresh NCl solution. The ph of the solution ws monitored nd djusted to ph 4 with 5 mmol L 1 lti id using omintion ph eletrode (Orion, Cmridge, MA, USA). 36 The liquots were nlysed for their C nd P ion onentrtions vi spetrophotometri method (DMS-8 UV-visile; Vrin, Plo Alto, CA, USA) using known stndrds nd lirtion urves. 35 This represented the virgin ion relese from the ement nd ws termed initil relese to differentite from the susequent rehrge nd re-relese. Rehrge of PEHB+NACP orthodonti ement The proedures of rehrge nd re-relese re illustrted in Figure 1. First, PEHB+NACP ement speimens were immersed in the ph 4 solution for 7 dys, s desried ove for the mesurement of initil ion relese. The speimens were then removed from the solution nd ultrsonited in distilled wter for 3 min. These speimens were denoted fter 7-dy relese, s indited y the lower left rrow in Figure 1. Then, these speimens were immersed in fresh ph 4 solution for C nd P ion mesurement for 7 dys, whih onfirmed tht indeed the ion relese ws exhusted nd there ws no further relese, s indited y the lower middle rrow in Figure 1. Then, these exhusted speimens were used for C/P ion rehrge. The lium ion rehrge solution onsisted of 1 mmol L 1 of CCl 2 nd 5 mmol L 1 of 4-(2-hydroxyethyl)-1-piperzineethnesulfoni id (HEPES) uffer. 34,55 The phosphte ion rehrge solution onsisted of 6 mmol L 1 of K 2 HPO 4 nd 5 mmol L 1 of HEPES uffer. Solutions were djusted to ph 7 using 1 mol L 1 of KOH. 34,55 Three speimens of B2 mm 2 mm 12 mm were immersed into 5 ml of rehrge solution nd gently shken using mixing mhine (Anlog Vortex Mixer; Fisher Sientifi, Wlthm, MA, USA) for 1, 2 or 3 min s listed elow. This immersion nd shking tretment C ion re-relese/(mmol. L -1 ) st yle of rehrge nd re-relese Dy 3 Dy 1 Dy 5 Dy 14 2nd yle After 7 d relese Relese ws exhusted 3rd yle Ion re-relese time/d Rehrged, then pled in fresh solution to test re-relese for 14 dys s 1 yle Figure 1 Illustrtion of C nd P ion rehrge nd re-relese. NACPontining ement speimens were first immersed in ph 4 solution for 7 dys to exhust the ion relese, s indited y the lower left rrow. Then, the speimens were immersed in fresh ph 4 solution to onfirm tht the ion relese ws indeed exhusted, s indited y the lower middle rrow. The exhusted speimens were rehrged in rehrge solution. The rehrged speimens were tested for C nd P ion re-relese for 14 dys, s indited y the lower right rrow. This onstituted the first rehrge/rerelese yle. This ws repeted for three yles. C, lium, NACP, nnoprtiles of morphous lium phosphte; P, phosphorus. represented the movement in the mouth-rinsing proess when CP mouth rinses ould e used. 39 Then, the speimens were rinsed with distilled wter to remove ny loosely tthed deposits on surfes suh tht only the ions rehrged into the interior of the ement were mesured in the susequent re-relese test. Severl different rehrge methods were tested. For exmple, for the rehrge using 1-min immersion nd repetition three times in one dy (referred to s ), the forementioned rehrge proess ws repeted three times t 9:, 13: nd 17: hours. Then, strting on the next dy, the speimens were tested for ion re-relese for 14 dys ontinuously (without ny rehrge during those 14 dys). Nine rehrge durtions nd frequenies were tested: 1. 1 min immersion, 1 time dy t 9: hours (referred to s ) min immersion, 2 times dy t 9: nd 17: hours (referred to s 1min 2 times ) min immersion, 3 times dy t 9:, 13: nd 17: hours ( ) min immersion, 1 time dy t 9: hours ( ) min immersion, 2 times dy t 9: nd 17: hours ( 3min2 times ) min immersion, 3 times dy t 9:, 13: nd 17: hours ( 3 min 3 times ) min immersion, 1 time dy t 9: hours ( ) min immersion, 2 times dy t 9: nd 17: pm ( 6min 2times ) min immersion, 3 times dy t 9:, 13: nd 17: ( 6min 3times ).

4 27 Dye-ssisted rehrge solution testing As n initil sreening test of the effiy of the nine rehrge methods, rhodmine B fluoresent dye (Aldrih, Milwukee, WI, USA) ws dissolved in the lium rehrge solution t onentrtion of 1%. 56 The exhusted speimens were immersed in this rehrge solution with rhodmine B fluoresent dye. After eh of the nine rehrge tretments, eh speimen ws ut in the middle to expose its 2 mm 2 mm ross-setion using dimond sw (Isomet, Buehler, Lke Bluff, IL, USA). Eh ross-setion ws exmined using n epifluoresene mirosope (Elipse TE2-S; Nikon, Melville, NY, USA) to exmine the dye penetrtion depth into the speimen. 56 Mesurement of C nd P ion re-relese from rehrged PEHB+ NACP ement A preliminry dye-ssisted rehrge solution test showed tht the penetrtion depths in ement speimens were sttistilly the sme for these four rehrge tretments:, 3 min 3 times, 6 min 2 times nd 6 min 3 times. The 3 min 3 times, 6 min 2 times nd 6 min 3 times tretments took longer to perform ut exhiited the sme rehrge effiy s. Therefore, ws seleted from these four tretments. The 3 min 3 times, 6 min 2 times nd 6 min 3 times tretments were not inluded in susequent tests owing to longer rehrge times with no signifint enefit. Therefore, six rehrge tretments were tested for C nd P ion re-relese: (1) ; (2) ; (3) ; (4) ; (5) ; nd (6). The rehrged speimens were immersed in 5 ml of ph 4 solution to mesure the C nd P ion re-relese using the method desried ove, s indited y the third rrow in the ottom of Figure 1. The re-relese ws tested for 14 dys ontinuously, nmed yle 1. After 14 dys of re-relese, the speimens reeived the seond rehrge nd were then tested for re-relese ontinuously for 14 dys, nmed yle 2. This ws repeted for three yles in the present study, s illustrted in Figure 1. Sttistil nlysis The Kolmogorov Smirnov test nd Levene s test were performed to onfirm the normlity nd equl vrine of the dt. The results of SBS, rehrge solution diffusion depth nd C/P ion relese/re-relese were nlysed with two-wy nlyses of vrine. Post ho multiple omprisons were performed using the Tukey s honestly signifint differene test. The results of ARI were evluted using the χ 2 test. Enmel sher ond strength/mp TB ontrol, 1 d, TB ontrol, 7 d VT ontrol, 1 d Figure 2 Enmel sher ond strengths of orthodonti ements fter immersion in ph 4 solution for 1 dy or 7 dys. Different letters indite signifint differenes (Po.5). Men 7 stndrd devition, n = 1. NACP, nnoprtiles of morphous lium phosphte; TB, Trnsond XT; VT, Vitremer. Sttistil signifines were preset t Po.5 using the SPSS 14. softwre pkge (SPSS, Chigo, IL, USA). RESULTS The orthodonti SBSs to enmel fter immersion for 1 or 7 dys is plotted in Figure 2 (men ± stndrd devition; n = 1). TB ontrol nd PEHB+NACP ement hd similr ond strengths (P4.1). VT ontrol hd reltively lower ond strengths (Po.5). For eh mteril, there ws no signifint differene etween 1 nd 7 dys (P4.1). The ARI sores re listed in Tle 1. Most of the speimens filed t the rket ement interfe. VT ontrol hd lower ARI sores (Po.5). There ws no signifint differene etween immersion for 1 or 7 dys for eh ement (P4.1). The PEHB+NACP ement ws tested for initil C nd P ion relese, nd the results re shown in Figure 3: () C ion relese nd () P ion relese (men ± stndrd devition; n = 6). The relesed ion onentrtions inresed with time from 1 to 35 dys; fter 35 dys, the ion onentrtion inrese ws slowed, whih indited reltively less ion relese from 35 to 7 dys. Rhodmine B fluoresent imges of dye penetrtion in PEHB +NACP during rehrge re shown in Figure 4 4i. The outer red, VT ontrol, 7 d, PEHB+NACP, 1 d, PEHB+NACP, 7 d 14 7 Initil C ion relese/(mmol. L -1 ) Initil P ion relese/(mmol. L -1 ) Immersion time/d Immersion time/d Figure 3 Initil C nd P ions relese from the PEHB+NACP orthodonti ement. () Cion;() P ion. Men ± stndrd devition; n = 6. C, lium; NACP, nnoprtiles of morphous lium phosphte; P, phosphorus.

5 28 Tle 1 ARI sores of orthodonti ements ARI sores (n = 1)* Group Solution ging SIG PEHB+NACP 1 dy A TB ontrol 1 dy A VT ontrol 1 dy B PEHB+NACP 7 dys A TB ontrol 7 dys A VT ontrol 7 dys B ARI, dhesive remnnt index; NACP, nnoprtiles of morphous lium phosphte; SIG, sttistil signifine; TB, Trnsond XT; VT, Vitremer. *ARI: = no dhesive left on the tooth surfe; 1 = less thn hlf of the dhesive ws left on the tooth surfe; 2 = hlf or more of the dhesive ws left on the tooth; 3 = the entire dhesive ws left on the tooth surfe. Different letters (A, B) inditing signifint differenes in the ARI sores (Po.5). 5 µm 3min 1 time 3 min 3 times 6 min 2 times 6 min 3 times 12 Dye penetrtion depth in PEHB+NACP/µm d 3 min 3 times s 6 min 2 times 6 min 3 times Figure 4 Typil imges of rhodmine B fluoresent dye penetrtion for different rehrge groups. () ; () ; () ; (d) ; (e) ; (f) 3 min 3 times; (g) ; (h) 6 min 2 times nd (i) 6 min 3 times. The imge shows the ross-setion of the r of ~ 2 mm 2 mm. The red re indites the depth of penetrtion. (j) Quntittive mesurement of the dye penetrtion depth (men ± stndrd devition, n = 6). Dissimilr letters indite vlues tht differ from eh other (Po.5). NACP, nnoprtiles of morphous lium phosphte.

6 29 edge shows the externl surfe of the speimen, inditing the ~ 2 mm 2 mm ross-setion of the r. The thikness of the red line indites the dye penetrtion depth, whih inresed from 1 to 6 min of immersion durtion. At eh durtion, the penetrtion depth inresed from 1 time to 3 times of rehrge in dy. The quntittive penetrtion depth is plotted in (J) (men ± stndrd devition, n = 6). The, 3 min 3 times, 6 min 2 times nd 6 min 3 times tretments yielded no signifint differene (P4.1). When repeting the rehrge for 3 times dy, inresing the immersion durtion from 1 to 6 min produed no signifint inrese in penetrtion depth (P4.1) The PEHB+NACP rehrge nd re-relese results re plotted in Figure 5: () C nd () P ion re-relese (men ± stndrd devition, n = 3). The rehrged speimens were immersed in fresh ph 4 solution nd the re-relese ws mesured for 14 dys, s one yle. Therefore, eh rehrge tretment (whih ws done in one dy) ws tested for 14 dys of ontinuous ion relese. Three suh rehrge/rerelese yles were plotted in Figure 5. For ll groups, fter eh rehrge tretment, the ion relese onentrtion inresed rpidly in the first week, nd the relese slowed or plteued in the seond week. The ion re-relese onentrtion t 14 dys showed the order of 1 min 3 times43min 2 times446min 1 time43min 1.5 Cyle 1 Cyle 2 Cyle 3.4 C ion re-relese/(mmol. L -1 ) Cyle 1 Cyle 2 Cyle 3.3 P ion re-relese/(mmol. L -1 ) Immersion time/d Figure 5 Re-relese from the exhusted nd rehrged PEHB+NACP orthodonti ement fter different rehrge tretments. () C ion; () P ion. Men ± stndrd devition, n = 3). Three rehrge/re-relese yles were tested, nd eh re-relese ws mesured for 14 dys. The ion re-relese t ws the gretest. There ws no derese in re-relese when inresing the numer of rehrge/re-relese yles from 1 to 3 (P4.1). C, lium; NACP, nnoprtiles of morphous lium phosphte; P, phosphorus.

7 3 C ion re-relese in yle 3/(mmol. L -1 ) P ion re-relese in yle 3/(mmol. L -1 ) Figure 6 The C nd P ion re-relese onentrtions of different rehrge groups t 14 dys of the third yle. () Cion;() P ion. Men ± stndrd devition, n = 3. In eh plot, dissimilr letters indite vlues tht re signifintly different from eh other (Po.5). time4. There ws no derese in ion re-relese from rehrge/re-relese yle 1 to yle 3. For the different rehrge tretments, the C nd P ion onentrtions t 14 dys of the third rehrge/re-relese yle re plotted in Figure 6: () C nd () P ion onentrtions (men ± stndrd devition, n = 3). The tretment hd the highest rerelese ion onentrtions, lthough it ws not signifintly different from those of (P4.1). However, the tretment totlled only 3 min of immersion in rehrge, wheres the tretment totlled 6 min of immersion in rehrge. The tretment exhiited signifintly higher re-relese ompred with the first four groups in Figure 6 (Po.5). DISCUSSION An orthodonti ement ontining NACP with C nd P ion rehrge nd re-relese pilities ws developed for the first time. The hypotheses were proven nd showed tht the new orthodonti ement with CP rehrge did not ompromise the enmel ond strength; the re-relese of C nd P ions from the ement ws mintined over time nd did not derese while inresing the numer of rehrge/rerelese yles; further, the rehrge immersion time nd frequeny signifintly ffeted the rehrge effiy of the orthodonti ement. This new orthodonti ement with CP rehrge pility ws promising to exhiit long-term C nd P ion relese to omt enmel deminerliztion nd inhiit WSLs during orthodonti tretments.,d d,e e The reommended ond strengths of metl rkets to enmel were ~ 8 MP or greter to provide dequte dhesion to enmel in orthodonti tretments. 57 In the present study, the ond strength of PEHB+NACP fter 24 h of immersion ws 13 MP, similr to tht of the TB ontrol nd higher ompred with tht of the VT ontrol. A onern ws whether the ond strength of PEHB+NACP would derese over time euse of the relese of C nd P ions. After 7 dys of immersion in ph 4 solution, whih exhusted the C nd P ion relese, there ws no signifint derese in ond strength ompred with tht t 1 dy. The smll prtile size of NACP likely ontriuted to the good enmel ond strength, s lrger CP prtiles in resins ould led to poor mehnil properties for resins with ion relese. 31,34 Further study is needed to investigte the enmel ond strength of the PEHB+NACP orthodonti ement t times longer thn 7 dys, suh s 1 yer nd 2 yers. PMGDM nd EBPADMA re the mjor monomers used in the PEHB resin. PMGDM is n idi dhesive monomer tht n hemilly helte with C ions euse of its roxylte groups. 42 EBPADMA is monomer tht is used in dentl omposites nd hs reltively flexile struture, lower vinyl group onentrtion nd lower visosity thn BisGMA systems. 44 Both HEMA nd BisGMA re trditionl monomers tht re widely used in dentl resins. 45 BisGMA ontins ester linkges tht onnet isphenol A segments to the polymerizle vinyl segments. 45,58 BisGMA hs isphenol A s the ore of its hemil struture, whih mkes it stiff moleule tht produes mehnilly strong polymer. In ddition, BisGMA hs two pendnt hydroxyl groups tht n form strong hydrogen onds with the hydroxyl groups on djent BisGMA moleules. Furthermore, the moleulr weight of BisGMA is high, whih promotes exellent mehnil properties. 45,58 However, BisGMA- nd HEMArih onding gents my rise the onerns of moderte hydrolysis nd the potentil degrdtion of the onded interfe Therefore, the quntities of BisGMA nd HEMA need to e ontrolled euse they my influene the onding durility. 61 The present study used only smll mounts of BisGMA nd HEMA (5% nd 1%, respetively) to minimize ny potentil negtive effet on the onding durility. This PEHB resin omposition yielded the est CP rehrgeility, s shown previously in omprison with severl other resin mtries. 39 In the present study, this resin yielded new orthodonti ement with sustined C nd P ion relese, whih is promising to inhiit WSL in orthodonti tretments. The rehrgeility of PEHB+NACP orthodonti ement likely depend on two mehnisms First, the roxylte groups of PMGDM n helte with C ions in the rehrge solution. There likely exists dynmi equilirium etween the heltion of lium ions to the PMGDM monomer nd the relese of lium ions from the monomer, whih is dependent on the lol ph of the immersion solution. The solution for rehrge (simulting mouth rinse) hd ph of 7. The PMGDM in the ement my helte with the lium ions diffusing from the rehrge solution into the resin. After the rehrging proess, during the rerelese, the ond etween PMGDM nd lium ions my e severed in the ph 4 solution in whih the re-relese ws mesured, simulting lol riogeni ph from iofilm ids. The seond ftor tht ontriuted to the rehrge my e its spe-oupying effet. After the initil C nd P ion relese, whih exhusted the ion relese, the sites tht were previously oupied y the C nd P ions eme ville for the inoming C nd P ions from the rehrge solution. Rhodmine B fluoresent dye hs een widely used in onded interfe nd mirolekge studies. 56,62 The deepest lyer tht the diffusion of the rehrge solution rehed ws ~ 1 μm sshownin

8 31 Figure 4j, no mtter how long of the totl rehrge durtion ws. At 14 dys of the third yle, the tretment, with n umultive totl rehrge durtion of 3 min, relesed C onentrtion of.43 mmol L 1 nd P ion onentrtion of.3 mmol L 1. They were higher ompred with those of the 6-min, 1-time tretment (with totl rehrge durtion of 6 min), whih yielded C onentrtion of.33 mmol L 1 nd P ion onentrtion of.22 mmol L 1. These results indite tht longer totl rehrge durtion does not lwys yield greter ion relese, nd the rehrge effiy is not diretly proportionl to the totl rehrge durtion time. Teengers my hve reltively poor ompline in hygiene nd diet ontrols, ut they re the mjor group of orthodonti ptients. Therefore, the CP rehrge proedure for the PEHB+NACP orthodonti ement should e user friendly nd require miniml time to perform. Two points should e mde. First, the dy tretment required totl of 3 min, whih is shorter thn 6 min 3 times, 6 min 2 times or 3 min 3 times. However, the 1 min, 3 times tretment rehed similr degree of rehrge nd re-relese. Therefore, the tretment ppered to e the preferred method. Although it required the 1-min immersion to e repeted three times, this yielded ontinuous relese of C nd P ions for t lest 7 dys. Therefore, the ptient ould potentilly use mouth rinse for 1 min eh fter rekfst, lunh nd dinner on Sundys to mintin sustined high level of C nd P ion relese for the whole week. Therefore, the ptient only needs to rehrge one week insted of one dily. Seond, the re-relese ws mesured with the speimen immersion in solution with ph 4 ontinuously s n elerted test. Clinilly, the Stephn urve shows tht the plque ph following gluose intke stys in the riogeni re of ph 4.5 for o3 min, nd then the ph inreses k to sfe ph of 6 or higher, fter the teri hve ompleted their metoliztion of gluose nd sliv hs uffered the id. 63 Therefore, insted of eing immersed in ph 4 solution for 24 h every dy, the orthodonti ement in vivo would likely exhiit ph lose to 4 for only ouple of hours per dy, thus preserving the ion reservoir in the ement from eing quikly exhusted. This mens tht fter one rehrge tretment, the PEHB +NACP orthodonti ement ould potentilly re-relese C nd P ions for muh longer thn 2 weeks, perhps even llowing the ptient to undergo rehrge tretment () only one per month. Further study is needed to investigte the re-relese of C nd P ions from the PEHB+NACP orthodonti ement under ph onditions onsistent with the Stephn urve s trend of orl plque ph, to determine the re-relese longevity fter eh rehrge tretment. Further study is lso needed to evlute the inhiition of WSL in enmel using the PEHB+NACP orthodonti ement under simulted onditions in vivo. CONCLUSIONS In this study, we developed the first orthodonti ement ontining NACP with C nd P ion rehrge nd re-relese pilities. The new orthodonti ement with C nd P ion rehrge/re-relese hd n enmel ond strength similr to ommeril ement without CP relese. The NACP orthodonti ement ould e repetedly rehrged to mintin the long-term relese of C nd P ions, nd the ion relese did not derese when inresing the numer of rehrge/re-relese yles. The rehrge method of 1 min of immersion three times dy ppered to e optiml, yielding the sustntil nd ontinuous relese of ions for t lest 1 week fter just one rehrge tretment. 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The influene of hemil struture on the properties in methrylte-sed dentin dhesives. Dent Mter 211; 27(11): Profet A, Mnnoi F, Foxton R et l. Experimentl eth-nd-rinse dhesives doped with iotive lium silite-sed miro-fillers to generte therpeuti resin dentin interfes. Dent Mter 213; 29(7): Thylstrup A, Fejerskov O. Textook of Clinil Criology. Copenhgen: Munksgrd, This work is liensed under Cretive Commons Attriution 4. Interntionl Liense. The imges or other third prty mteril in this rtile re inluded in the rtile s Cretive Commons liense, unless indited otherwise in the redit line; if the mteril is not inluded under the Cretive Commons liense, users will need to otin permission from the liense holder to reprodue the mteril. To view opy of this liense, visit r The Author(s) 217