Pulp response to dentine adhesives: A study on mature human pulps

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1 Originl Article Pulp response to dentine dhesives: A study on mture humn pulps Pntelis Kouros 1, Eugeni Koliniotou Koumpi 1, Eliseth Koulouzidou 1, Mri Helvtjoglu Antonides 1, Dimitrios Tzifs 2 Correspondence: Dr. Pntelis Kouros Emil: kourosp@hotmil.com 1 Deprtment of Opertive Dentistry, Fculty of Dentistry, Aristotle University of Thessloniki, Thessloniki, Greece, 2 Deprtment of Endodontology, Fculty of Dentistry, Aristotle University of Thessloniki, Thessloniki, Greece ABSTRACT Ojective: To cliniclly investigte the nticteril effects of commercilly ville self etch 12 methcryloyloxydodecylpyridinium romide (MDPB) contining dhesive system in comprison with its respective non MDPB contining dhesive nd to evlute the pulp responses when in use on humn teeth. Mterils nd Methods: Sixty two vile humn teeth scheduled for extrction were used. Clss V cvities were prepred on the uccl surfces of the teeth nd filled with the tested mterils (Protect Bond/Clerfil AP X, SE Βond/Clerfil AP X nd Dycl/Ketc Fill Plus) s control group, ccording to mnufcturer s recommendtions. Rndomly divided to two groups teeth remined intr orlly for 4 nd 8 weeks. After extrction, teeth were declcified, sectioned nd stined using the Myer s hemtoxylin nd eosin, nd modified Brown Brenn s technique. Pulp responses were evluted microscopiclly under microscope nd remining dentine thickness mesured under stereomicroscope. Results: No sttisticlly significnt differences regrding pulp inflmmtion or cteril infiltrtion were found either for the mterils tested or for periods of post opertive evlution. Conclusions: The results suggested tht for short period of evlution there re no quntittive differences, regrdless to restoring mteril used. Key words: Adhesive, nticteril, dentine, 12-methcryloyloxydodecylpyridinium romide, pulp INTRODUCTION The rpid development of dhesive dentistry hs resulted to the introduction of new dhesive systems into the clinicl procedures. Although iologicl nd mechnicl properties of the mterils re constntly improving, prolems relted to polymeriztion construction of the composite resins my occur. Mrginl lekge nd post opertive cteril infiltrtion my ffect the longevity of the restortion nd the pulpl rection. In ddition, there is evidence tht the removl of the cries is most commonly incomplete. [1,2] The potentil of the pulp recovery fter plcing resin restortion seems to depend on severl fctors such s the pre opertive pulp sttus, the remining dentine thickness (RDT), the efficcy of tretment strtegy nd the pre opertive nd post opertive prevention of cteril infiltrtion. [3] It hs long een recognized tht the presence of cteri long the cvity wlls or within the dentinl tuules my e considered s the criticl determinnt in pulpl inflmmtory responses fter restortive procedures. [4] Restortive mterils in the new er tend to e io ctive, nd nticteril effects re highlighted s one of the most importnt properties. [5] The incorportion of nticteril gents into dhesive systems hs een proposed to eliminte residul cteri from dentine. Such n nticteril monomer, 12 methcryloyloxy dodecylpyridinium romide (MDPB), hs een developed nd incorported in self etch dhesive system, Clerfil SE Bond (Kurry Europe, GmH Dusseldorf Germny). The nticteril dhesive system employing the MDPB contining How to cite this rticle: Kouros P, Koliniotou-Koumpi E, Koulouzidou E, Helvtjoglu-Antonides M, Tzifs D. Pulp response to dentine dhesives: A study on mture humn pulps. Eur J Dent 2013;7: Copyright 2013 Dentl Investigtions Society. DOI: / S26

2 primer ws commercilized under the nme protect ond (Kurry Europe, GmH Dusseldorf Germny). Incorportion of the MDPB nticteril gent into dentine dhesive system resulted in strong nticteril ctivity ginst orl streptococci ex vivo. [6 8] The pulpl responses hve een further evluted histopthologiclly in vivo, in the dog s teeth. [9] The results indicted tht little or no inflmmtion ws present. Tzifs et l. hve further evluted the repir cpcity of the pulp dentine complex when protect ond system is plced on infected cvities in dog teeth. [10] However, niml test results cnnot e directly extrpolted to humns; therefore, mteril hs to e tested in clinicl conditions. Stndrdized in vivo studies re using young humn sujects which provide teeth with helthy pulps housed in lrge pulp chmers, for resons of comprison etween test nd control procedures. [3,11] Nevertheless, clinicl correltion hs to e undertken with cution. [3] Few studies hve ttempted to ssess the pulp s rection when it is in compromised stte. [3,11] The im of this study is to evlute pulp responses of mture humn pulps nd compre the short term nticteril potentil of two commercilly ville self etching dhesive systems; contining MDPB (Protect Bond) nd non MDPB contining (SE Bond) self etching dhesive system, when pplying those dhesives in deep cvities. The working hypothesis is tht there is no difference in pulpl responses of mture humn pulps in etween teeth treted with those dhesives nd the control group nd lso tht the short term nticteril potentil is equl for ech group of the study. MATERIALS AND METHODS A totl of 62 vile cries free single rooted humn teeth scheduled for extrction for periodontl resons from helthy ptients etween 40 nd 50 yers of ge, mles or femles were used for the purposes of the study. The vitlity of the teeth included in the study evluted using n electro pulp tester device. The experimentl protocol ws conducted ccording to ethicl guidelines for clinicl reserch in humns in the School of Dentistry, Aristotle University of Thessloniki. Ptients were informed with written sttement out the clinicl procedures nd of the possile inconvenience tht they were to experience. Those who greed to prticipte in the reserch signed n cceptnce form. The experimentl procedures should hve no effects on the therpeutic tretment of the ptients nd should not modify their tretment plns. Experimentl procedures On the dy of the opertive procedure ll teeth selected for the experiment were scled nd polished with ruer cup. Locl nesthesi ws provided prior to cvity preprtion (Scndonest 2%, Septodont). Clss V cvities (pproximtely 2.50 mm wide, 3.00 mm long) were prepred on the uccl surfce of teeth using tungsten cride per shped ur, ISO #330 L (SS. White, Lkewood NJ, USA), t ultr high speed with copious wter spry. A new ur ws employed on every fourth cvity to void excessive heting. Cvities were prepred ccording to the following protocols: 1. The preprtions were cut mm ove the cemento enmel junction 2. The floor of the cvity preprtions ws mintined curved nd prllel to the outer uccl surfce of the tooth 3. A evel ws mde t the enmel mrgin of the cvities using high speed dimond ur 4. Teeth were rinsed with sterile distilled wter nd isolted with ruer dm nd sliv ws controlled through high speed evcution. All procedures were performed y the sme opertor. Teeth rndomly divided into six experimentl groups, ccording to mterils treted nd the time period of evlution. The clcium hydroxide sed mteril Dycl (Dentsply) in comintion with glss ionomer cement Ketc Fill Plus (3M ESPE) ws used to form control group. Chemicl compositions of dhesive systems used re shown in Tle 1. Tle 1: Composition of the two step SE dhesives tested Adhesive (mnufcturer) Composition ph Btch numer Clerfill SE Bond (Kurry Europe GmH, Dusseldorf, Germny) Clerfill Protect Bond (Kurry Europe GmH, Dusseldorf, Germny) Primer: HEMA, MDP, hydrophilic dimethcrylte, Primer: 1, N, N diethndiol p toluidine, CQ, wter Adhesive: HEMA, MDP, hydrophilic dimethcrylte, N, N diethndiol p toluidine, CQ, silnized colloidl silic, BisGMA Adhesive: 2, 8 #1970 EU Primer: HEMA, MDP, hydrophilic dimethcrylte, MDPB, wter Primer: 1, Adhesive: HEMA, MDP, hydrophilic dimethcrylte, N, Adhesive: 2, 8 #1910 EU N diethndiol p toluidine, CQ, silnized colloidl silic BisGMA: Bisphenol A glycidyl dimethcrylte, THB: Toluene hydroxyutyrte, CQ: D,1 cmphorquinone, HEMA: 2 Hydroxyethyl methcrylte, MDP: 10 Methcryloxydecyl dihydrogen phosphte, MDPB: 12 Methcryloyloxydodecyl pyridinium romide, PEM F: Monofluoro phosphzene modified methcrylte, Piro EMA: Phosphoric cid modified methcrylcte, UDMA: Urethne dimethcrylte, SE: Self etching S27

3 Teeth were extrcted under locl nesthesi (Scndonest 2%, Septodont, Cedex, Frnce) nd prepred for histologicl nlysis. Immeditely fter extrction, roots were sectioned midwy etween the cemento enmel junction nd the pex. Teeth were fixed in 10% neutrl uffered formlin solution for 2 weeks. The specimens were then deminerlized in Morse s solution (50% formic cid + 20% sodium citrte) for 2 months. Finlly, the teeth were emedded in prffin nd serilly sectioned through the pulp t 5 μm thickness. All sections coming through the cvity floor were stined either with Myer s hemtoxylin nd eosin, stin to ssess soft tissue orgniztion nd tertiry dentine formtion or were sujected to modified Brown Brenn s technique to detect the presence of Grm positive nd Grm negtive microorgnisms. Histologicl ssessment The stereotypic connective tissue rections nd the cteril infiltrtion of the cvity were evluted ccording to the following criteri. Inflmmtory cell response Inflmmtory cell infiltrtion of the pulp tissue ws clssified s: 0 (none), sence of inflmmtory cells; 1 (slight/moderte), few scttered inflmmtory cells or moderte inflmmtory cell infiltrtion; 2 (severe), hevy inflmmtory cell infiltrtion of the coronl pulp round the exposure site (in exposed teeth) or scess formtion. Tissue disorgniztion Disorgniztion of pulp tissue ws clssified s: 0 (no), physiologicl ppernce of the pulp dentine interfce nd centrl pulp tissue; 1 (slight disorgniztion), reduction in cells in the odontolstic lyer eneth the cvities, ut norml centrl pulp (in non exposed teeth); 2 (severe disorgniztion) complete disorgniztion of the odontolstic lyer. dentine to the post opertive formed mtrix. [2] The minimum RDT ws estimted for every specimen. The 20 djcent sections were nlyzed twice, y two independent oservers who were lind to the tretment group. Inter oservtion vrition ws only noticed in scoring tissue disorgniztion of unexposed teeth. In these cses, the highest disorgniztion score ws finlly recorded. Rectionry dentine formtion The thickness of rectionry dentine ws mesured eneth the site where minimum RDT ws mesured nd lso t the opposite pulp wll [Figure 1, rrows]. Sttisticl methods The comprison of the nturl log trnsformed vlues of the minimum RDT etween the different groups ws performed using the two wy ANOVA. The ssumptions of normlity nd the homogeneity of vrinces were tested y the Shpiro Wilk test (smple size < 50) nd Levene s test, respectively. The Kruskl Wllis test ws used to comp re pulp degenertion, inflmmtory cell response nd tissue necrosis etween the three mterils within ech time period. Pir wise comprisons were performed with the Mnn Whitney test, wheres Bonferroni s method ws pplied for the djustment of Type I error. In ddition, the Mnn Whitney test ws used to compre 4 week nd 8 week period smples for ech mteril. All P vlues were lso computed y the Monte Crlo method sed on 10,000 smpled tles. The nlysis ws performed with SPSS 15.0 (Sttisticl Pckge for Socil Sciences, Chicgo, Illinois, USA) nd the level of sttisticl significnce ws set t P < Regrding rectionry dentine thickness, men nd stndrd devition were used for the description of Bcteril infiltrtion The presence of stined cteri in the pulp spce or long the cvity wlls/within the cut dentinl tuules clssified s: 1 (no), sence of cteri infiltrtion; 2 (on cvity wlls): Presence of cteri mong the cvity wlls; 3 (in the pulp): Presence of cteri inside the pulp tissue or the pulp chmer. Remining dentine thickness All stined sections were evluted nd the RDT ws mesured y mens of grticule under stereomicroscope (Olympus CO, Tokyo, Jpn) t 100 mgnifiction, etween the cvity floor nd the line of interfce from the pre opertive circumpulpl Figure 1: Mesurement sites of remining dentine thickness (ig rrow), rectionry dentine thickness djcent to cvity nd to the opposite dentine wlls (smll rrows) S28

4 the differences% (mesurement 1 mesurement 2)/ mesurement 2 100) etween the thickness elow the RDT site (mesurement 1) nd the thickness t the opposite pulp wll (mesurement 2). Dt were frctions hence the Arcsine of the squre root of ech vlue were computed. The ssumption of normlity ws tested y the Shpiro Wilk test (N < 50) nd the ssumption of homogeneity of vrinces ws tested y the Levene s test. Kruskl Wllis test nd Mnn Whitney test were used for the nlysis of the trnsformed dt. The nlysis ws performed on the Sttisticl Pckge for the Socil Sciences (SPSS 16.0, IBM) softwre nd the level of sttisticl significnce ws set t P < RESULTS The teeth used in this study were minly from periodontl ptients ged etween 40 nd 50 yers Tle 2: Men vlue of the minimum RDT nd SD vlues Groups of teeth N Men vlue of RDT (mm) Group 1: Clerfil Protect Bond/ Clerfil AP X/4 week evltion Group 2: Clerfil SE Bond/ Clerfil AP X/4 week evltion Group 3: Dycl/Ketc Fill Plus/4 week evltion Group 4: Clerfil Protect Bond/ Clerfil AP X/8 week evltion Group 5: Clerfil SE Bond/ Clerfil AP X/8 week evltion Group 6: Dycl/Ketc Fill Plus/8 week evltion SD (mm) RDT: Remining dentine thickness, SD: Stndrd devition, SE: Self etching old. As expected some reticulr degenertion ws oserved in the pulp tissue. This fct my ffect the pulp heling potentil or the inflmmtion intensity. The minimum RDT ws mesured nd compred mong the six groups. In Tle 2, the men nd stndrd devition of the minimum RDT in ech group of teeth re presented. Levene s method ws used to test the working hypothesis tht the error vrince of the dependent vrile (RDT) is equl cross the groups. This hypothesis ws not rejected (P = 0.070). In ddition, two wy ANOVA ws conducted to compre the men vlues of the RDT etween the groups of mterils (P = 0.447) nd the men vlues etween the levels of time (P = 0.482). Bcteril infiltrtion of the pulp ws not present in ny specimen of this experiment. In few numer of specimens cteri were found long the cvity wlls or within the cut dentinl tuules [Figure 2]. A smll numer of pulps demonstrted slight to moderte inflmmtory cell response. No pulp demonstrted severe inflmmtion. Slight tissue disorgniztion of coronl pulp in proximity to cvity wlls ws present in few specimens, ut of no sttisticl importnce. The mjority of specimens demonstrted no tissue disorgniztion or pulp inflmmtion [Figures 3 5]. Tests showed no sttisticl difference of importnce regrding to inflmmtory cell response nd tissue necrosis for ll groups of the specimens. Histopthologicl findings of this study re presented in detil in Tle 3. Tle 3: Frequency of scores for ech group of teeth fter tretment with the test mterils Test groups Protect Bond/Clerfil AP X SE Bond/Clerfil AP X Dycl/Ketc Fill Plus 4 weeks evl. 8 weeks evl. 4 weeks evl. 8 weeks evl. 4 weeks evl. 8 weeks evl. Inflmmtory cell response None Slight/moderte Severe Tissue disorgniztion No Slight Severe Bcteril infiltrtion No On cvity wlls In the pulp Pulp firous degenertion Slight Advnced S29

5 Figure 2: Presence () or sence of cteri () in cut dentinl wlls treted with protect ond, oserved 8 weeks post opertively (Brown Brenn s modified technique, originl mgnifiction 40) Figure 3: Cvities treted with SE Bond, evluted t 4 weeks () nd 8 weeks (). Degenertion of the pulp mesenchym is noticele ut dentinolstic lyer (D) is preserving its rchitecture (H nd E, 100) Figure 4: Cvities treted with protect ond, 4 weeks () nd 8 weeks () post opertively. Similr to those treted with SE Bond, dentinolstic lyer (D) ppers intct nd with no signs of inflmmtion. Pulp mesenchym is degenerted (H nd E, 100) DISCUSSION The results of this study showed tht there were no sttisticlly significnt differences etween groups of mteril tested regrding inflmmtory pulp response nd cteril infiltrtion of the cvity wlls. This is considering confirming study s working hypothesis. Inflmmtory pulp response ws not dependent of the time elpsed from the restortion to extrction nor the restortion mteril used. Most of the tooth pulp, demonstrted slight or no inflmmtion. Pulp inflmmtion tht occurs during restortion procedure is considered to e result of: () mechnicl irrittion of dentinolstic processes nd temperture rising during preprtion, () cteril infiltrtion in cvity wlls nd dentinl tuuli or pulp tissue nd (c) chemicl irrittion of non polymerized smll sized monomers, which cn penetrte towrd pulp chmer through dentinl tuuli. [11] Preventing of cteril infiltrtion in cvity wlls hs een chllenge for mnufcturers. The use of self etching dhesive system such s Clerfil SE Bond hs een shown to provide short term, mild nticteril effect, which hs een supposed to e due to the cidic nture of those mterils. [5,6,11,12] In ddition, n nticteril gent (MDPB) tht co polymerizes with other resin monomers hs een dded in Clerfil protect ond, in order to chieve long term residul cteri inhiition nd protection ginst cteril infiltrtion. In vitro studies hve shown tht non light ctivted mteril demonstrtes greter S30 Figure 5: No inflmmtion of dentinolstic lyer (D) ws oserved in cvities treted with C (OH) t 4 weeks () nd 8 weeks (). Pulp 2 degenertion is lso dvnced (H nd E, 100) nticteril ctivity. [13] This decrese of nticteril ctivity cused y polymeriztion, is supposed to occur due to entrpment of the nticteril molecule in the resin mtrix. [13] It seems tht the remining nticteril effect is cused y the unpolymerized dhesive ecuse there is never complete conversion of monomers to polymers minly due to the presence of n oxygen inhiited lyer. [9] On the other side, the cidity of the unpolymerized monomers contined in self etch dhesives could cuse inflmmtory rections when in direct contct or in close proximity with pulp tissue. Bcteril infiltrtion of the cvity wlls hs een estimted histologiclly with the use of modified Brown nd Brenn s technique. Ptients prticipting in this study suffered from severe periodontitis cused y poor hygienic hits. Restortions tht remin in such n environment re expected to e exposed to lrge mount of cteri, which potentilly could proliferte eneth the composite. On the contrry, histologicl exmintion showed tht only smll numer of cteri could e found in cvity wlls of few specimens. This proly demonstrtes the protection from cteril infiltrtion tht provides the dhesive with the nticteril molecule MDPB, ut it lso shows the short term nticteril ction of the non MDPB contining dhesive, which occurs due to its cidity. These nticteril properties of the dhesives used in this study hve een shown previously in vitro. [13] Bcteril infiltrtion ws not present either in the control group specimens. The nticteril properties of C (OH) nd glss ionomer 2 cements hve een well demonstrted in the pst. [14]

6 The presence of residul cteri within the dentinl tuules or long the cvity wlls is considered to e the most significnt fctor relted to the pulp rection under resin sed restortive mteril in non exposed cvities. Previous studies hve shown tht there precise method for clinicl dignosis of crious dentine is still not ville nd in most cses of cries tretment, there is infected dentine left on the cvity wlls. [15,16] Use of mterils with nticteril properties could inhiit the residul cteri or prevent the coloniztion of the tooth restortion interfce post opertively with eneficil results for the vitlity of the pulp nd the longevity of the restortion. The present experiment tests the short term performnce ginst cteril infiltrtion of such n dhesive nticteril fctor contining mteril in comprison with its predecessor dhesive from the sme mnufcturer. It is likely to ssume tht inflmmtion, in cses tht occurred, my e the result of clinicl procedures or result of diffusion of uncured residul monomers or oligomers with cytotoxic effects towrds the pulp tissue, rther thn result of relese of cids nd toxins y cteri, since the presence of cteri in cvity wlls hs een oserved only in few cvities. In the present study, RDT vlues rnged etween nd μm nd in etween the groups of teeth there were no sttisticlly significnt differences (P < 0.05) oserved. Chemicl irrittion of the pulp occurs due to the ility of monomers to penetrte into the pulp tissue. In vitro studies hve shown tht uncured residul monomers contining in primer or/nd the dhesive such s hydroxyethylmethcrylte (HEMA), 10 methcryloxydecylihydrogen phosphte (MDP), isphenol A diglycidylethermethcrylte (Bis GMA), my penetrte through the dentine towrd the pulp. This seems to e dependent of the RDT nd of the dentine permeility. [17,18] The potentil for this penetrtion increses s the RDT decreses. Should e noted tht dentine permeility depends lso on the width of dentinl tuuli, which is significntly smller in elder people. [9] Teeth used in this study were the origin from mture individuls ged etween 40 nd 50 yers old nd it is likely to ssume tht permeility of dentine is reduced. Pre opertive pulp tissue condition could e ffected y the fct tht the ptients were older nd suffered from periodontitis. In order to clirte the results, histologicl estimtion of the pre opertive pulp condition depending on the firous degenertion ws introduced in the study. The sttisticl nlysis of the results hs shown tht with the exception of the groups 1 nd 3 no sttisticlly significnt differences were found in pulp degenertion etween the groups. These results indicte tht the teeth used for this study hd similr pulp tissue condition prior to the restortion procedures. Our results re in greement with the results of previous studies, which tested the sme mterils in dog teeth pulps. [10,19 21] In conclusion, pulp inflmmtion or tissue necrosis is irrelevnt with ppliction of contemporry dhesives in deep non exposed cvities. The ppliction of n dhesive contining nticteril monomer (MDPB), demonstrtes no difference regrding pulp cell response when compred with n dhesive without nticteril monomer nd when plced in non exposed cvities. The thickness of rectionry dentine formtion ws lso mesured eneth the RDT site nd to the opposite pulp chmer wll on stright line [Figure 1], in order to evidence possile reound response of odontolsts. This response is ssumed to e cused y ctivtion of odontolstic cells due to inflmmtion. Dt in this study re in complince with the previous experiment dt were no evidence of such reound response ws present. [15] CONCLUSIONS The mjority of clinicl studies concerning the influence of restortive mterils to the pulp tissue re performed t helthy young pulps with lrge pulp chmers nd good heling potentil. However, the everydy clinicl relity stins from this sitution, since pulps of restored teeth hve een previously undergone cteril ttck, cusing ltertions in pulpl mesenchym nd pulp volume. The present study is n ttempt of estimting the sfety of restoring procedures with self etch dhesives in proximity to compromised pulp tissue. Within the limittions of reserch performed t teeth with pulps in compromised sitution, results showed tht plcing self etch dhesives in proximity to such pulps is sfe procedure. The pulp inflmmtory response seems to e non MDPB contining dependent nd so is the cteril infiltrtion of the cvity wlls since cidity of self etch dhesive system seems to provide short term nticteril ction. [13] REFERENCES 1. Anderson MH, Loesche WJ, Chreneu GT. Bcteriologic study of sic fuchsin cries disclosing dye. J Prosthet Dent 1985;54:51 5. S31

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