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Platelet-rich fibrin (PRF) in implant dentistry in combination with new bone regenerative technique in elderly patients

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ContentslistsavailableatScienceDirect

International

Journal

of

Surgery

Case

Reports

jo u r n al ho me p a g e :w w w . c a s e r e p o r t s . c o m

Platelet-rich

fibrin

(PRF)

in

implant

dentistry

in

combination

with

new

bone

regenerative

technique

in

elderly

patients

Antonio

Cortese

(MD,

DDS)

(Professor

of

Maxillofacial

Surgery

at

University

of

Salerno)

a,∗

,

Giuseppe

Pantaleo

(DDS)

(Specialist

in

Oral

Surgery

at

University

of

Naples)

b

,

Antonio

Borri

(MD

University

of

Salerno,

Salerno)

c

,

Mario

Caggiano

(DDS)

d

,

Massimo

Amato

(MD)

(Professor

of

Dentistry

at

University

of

Salerno)

e

aDepartmentofMedicineandSurgery,UnitofMaxillofacialSurgery,UniversityofSalerno,Salerno,Italy

bDepartmentofNeurosciences,ReproductiveandOdontostomatologicalSciences,UniversityofNaplesFedericoII,Naples,Italy cUniversityofsalerno,Salerno,Italy

dDepartmentofMedicineandSurgery,UniversityofSalerno,Salerno,Italy eDepartmentofMedicineandSurgery,UniversityofSalerno,Salerno,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9July2016 Accepted10September2016 Availableonline22September2016 Keywords: PRF Splitcrest Elderlypatients Boneregeneration

a

b

s

t

r

a

c

t

INTRODUCTION:Somestudieshavedemonstratedthatplateletrichfibrin(PRF)isahealingbiomaterial withagreatpotentialforboneandsofttissueregeneration,withoutanyinflammatoryreactionsand maybeusedaloneorincombinationwithbonegrafts,promotinghemostasis,bonegrowth,and matu-ration.PRFappearsasanaturalandsatisfactoryaidinboneregenerativesurgeryinelderlypatientswith favorableresultsandlowrisks.

AIM:ThisstudywantstodemonstratehowPRFinassociationwithanewsplitcrestaugmentation techniquecanbeagreataidinimplantrehabilitation,especiallyintheelderlypatients,whenbone regenerationisrequired.

MATERIALSANDMETHODS:Tenpatientsweretreatedinthisstudy,fivefollowingtheflaplesssplitcrest newprocedureandotherfivepatientsfollowingtraditionalprocedurewithoutsplitcrestascontrol.Five patientswithanaverageagebetween50and60yearswereselectedtobeoperatedwithasplitcrest flaplessmodifiedtechniqueinordertooptimizetheregenerativeconditionswithaboneaugmentation andimplantinsertioninonesinglestageprocedure.ForallthepatientsautologousPRFhasbeenused tofillthesplitcrestgaporsimplyasregenerativematerial.Orthopantomography,intraoralradiography andCTDentaScan/CTConebeamwereperformedforeverypatientbeforethetreatmentandatfollow-up timeexeptionmadeforCT.

RESULTS:Allcasesweresuccessful,therewerenoproblemsatsurgerytime,atpost-operativeandat osteointegrationperiods.Allimplantsachievedosteointegration.Theseresultswereobtainedby accu-ratelymanagingimmediateandlatepostoperativeperiodinalloftheoperatedcases.Meandifference forheightbonelossbetweenthetwogroupsofpatientswas2.4mmatT1and2.2mmatT3.

DISCUSSION:Therationaleofthissplitcrestflaplessmodifiedtechniqueistoobtainaproperbuccal cortexexpansionpreservingitsvascularsupplyavoidingperiostealelevationforbettercorticalbone nourishing.Moreover,advantagesarereportedrelatedtotheuseofPRF.TheeffectivenessofPRFisshown inpromotingthehealingofsurgicalwounds,ithas,infact,plateletgrowthfactorsthatcanimprovethe vascularisationofthesurgicalsite,promotingneoangiogenesis.Furthermore,bysimplychangingthe settingsofthecentrifuge,itispossibletoobtainanormalgellingifithastobeusedasregenerativeand stimulatingmaterial,ormoreconsistentsubstancetobeusedasafillerinthesplitcrestgap.

CONCLUSIONS:Themainadvantagesinusingtheplatelet-richfibrinarehealingandboneregenerative propertiesincombinationwithitscompleteresorptionaftersurgery,thusavoidingasecondsurgery time,importantfactorintheelderlypatients.Currently,itisaminimallyinvasivetechniquewithlow risksandsatisfactoryclinicalresultssuchpreventingcomplicationsorimplantfailureparticularlyin elderlypatientsforagerelatedconditions.

©2016TheAuthors.PublishedbyElsevierLtdonbehalfofIJSPublishingGroupLtd.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗Correspondingauthor.

E-mailaddresses:ancortese@unisa.it(A.Cortese),giuseppepantaleo88@gmail.com(G.Pantaleo),antonio-borri@alice.it(A.Borri),dr.mariocaggiano@gmail.com(M. Caggiano),mamato@unisa.it(M.Amato).

http://dx.doi.org/10.1016/j.ijscr.2016.09.022

2210-2612/©2016TheAuthors.PublishedbyElsevierLtdonbehalfofIJSPublishingGroupLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

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1. Introduction

Platelets’regenerative potentialwasreportedinthe70’s[1], whenitwasobservedthattheycontaingrowthfactorsthatare responsiblefor increasecollagenproduction, cellmitosis,blood vesselsgrowth,recruitmentofothercellsthatmigratetothesite ofinjury,andcelldifferentiationinduction,amongothers[2].

Nowadaysinoralsurgerytherearetwokindofplatelet con-centratesforinvivotissueengineeringapplications:platelet-rich plasma(PRP)andplatelet-richfibrin(PRF).Plateletconcentrates areaconcentratedsuspensionofgrowthfactorsfoundinplatelets, whichactasbioactivesurgicaladditivesthatareappliedlocallyto inducewoundhealing[3].

PRFwasfirstusedspecificallyinoralsurgerybyDohanetal.[4]

andiscurrentlyconsideredasanewgenerationofplatelet concen-trate.Itconsistsofamatrixofautologousfibrin[5]andhasseveral advantagesoverPRP,includingeasierpreparationandnot requir-ingchemicalmanipulationoftheblood,whichmakesitstrictlyan autologouspreparation[6].Fortheseconsiderations,inourstudy wepreferredtousePRFprocedureinsteadofPRP.

PRF consists of an autologous leukocyte-platelet-rich fib-rin matrix [7] composed of a tetra molecular structure, with cytokines, platelets, and stem cells within it [5,8], which acts as a biodegradable scaffold [9] that favors thedevelopment of microvascularizationandisabletoguideepithelialcellmigration toitssurface[5,10].

Somestudies[11,12]havedemonstratedthatPRFisahealing biomaterialwithagreatpotentialforboneandsofttissue regener-ation,withoutinflammatoryreactionsandmaybeusedaloneorin combinationwithbonegrafts,promotinghemostasis,bonegrowth, andmaturation.

Thisautologousmatrixdemonstratedintheinvitrostudiesa greatpotentialtoincreasecellattachment[13]andastimulation toproliferateanddifferentiateosteoblasts[14].

Insurgicalprocedures,PRFcouldserveasaresorbable mem-branefor guided bone regeneration (GBR) [15], preventingthe migrationofnon-desirablecellsintobonedefectandprovidinga spacethatallowstheimmigrationofosteogenicandangiogenic cells permitting the underlying blood clot to mineralize [16]; moreover,anormalPRFmembranehasarapiddegradability(1–2 weeks)[17].

Fig.1.OrtopantomographyandCTDentascanofoneofpatientsselectedforsplit crestflapless,amoderateresorbededentulousridgeisshown.

Fig.2. Ortopantomographyofotherpatientselectedforsplitcrestflapless,there wasthefractureoftoothinregion1.1.

Fig.3. Ortopantomographyafterimplantinsertion.Attimeofinsertion,aresorbed edentulousridgewasshowninregion1.1.

PRFmembranehelpsinwoundhealing,protectingthesurgical site[18,19]promotingsofttissuerepair;whenmixedwithbone graft,itmayactasa“biologicalconnector”,whichattractsstem cell,favorsthemigrationofosteoprogenitorcellstothecenterof thegraft,andprovidesaneo-angiogenesis[19].

Inaddition,PRFmayactasabiologicadhesivetoholdthe parti-clestogether,facilitatingthemanipulationofthebonegrafts[20]. Inthisstudy,allofthefivepatientsofthefirstgroup(test)has beensubmittedtoanewsplitcrestflaplessmodifiedtechnique withtheuseofPRF.Plateletrichfibrincanbeusedbothasa regen-erativeandstimulatormaterial,bothasafillerinbonedefects;it dependsontype ofprotocolpreparationused.Inthistechnique boneregenerationissimilartoosteodistractioncreatinga regen-erativechamber withbone wallscovered bynativeperiosteum notcutorelevatedduringosteotomiespreservingfullabilityof feedingoftheunderlyingboneandcomparabletoreconstructive proceduresfortreatingperiodontalintraosseousdefects.

Inthisstudy,wewanttodemonstratehowPRFcanbeagreataid inimplantrehabilitation,especiallyintheelderlypatients,when boneregenerationisrequired.

2. Materialsandmethods

Tenpatientswithanagebetween50and60yearswerereferred totheMedicalDepartmentoftheUniversityofSalernofor eval-uation of a moderately reabsorbed edentulous ridgesecondary topreviousextraction. Fivepatientsweretreatedwiththesplit crestflaplessnewtechniqueforimplantinsertion,whilefiveother patients were treated with traditional technique with smaller implantstoovercomealveolarcrestthinness.Thetreatmentplan included rehabilitation with an implant-supported restoration (Figs.1–3).Thepatient’spastmedicalandsocialhistorywere non-contributory,andtheyhadgoodoralhygiene.Allthepatientshad nocontroindicationstoimplantplacement.

Thesurgerieswerenotperformedinpatientswithsystemicor psychologicaldisordersthatcontraindicateoralsurgery.

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Table1

Selectedelderlypatients(splitcrestflaplesstechnique)withimplantsdistribution,typeofPRFused,boneheightlossatT0(pre-operativetime),T1(immediatepost-operative

time),T2(3monthsaftersurgery),T3(6monthsaftersurgery).

Age Implantsplaced Implantlength,mm Torque,N.cm PRFtype Boneheightloss(T0,T1,T2,T3),mm

Patient1 53 1 11 35 Moreconsistent T0:0;T1:0;T2:0;T3:1

Patient2 59 4 ≥8; ≤13 40 Normalgel T0:0;T1:0;T2:0;T3:1

Patient3 60 2 11 35 Moreconsistent T0:0;T1:0;T2:1;T3:2

Patient4 57 1 8.5 35 Moreconsistent T0:0;T1:0;T2:0;T3:1

Patient5 55 2 ≥8; ≤11 40 Normalgel T0:0;T1:0;T2:0;T3:1

Table2

Controlpatientswithtraditionaltechniquewithimplantsdistribution,boneheightlossatT0(pre-operativetime),T1(immediatepost-operativetime),T2(3monthsafter

surgery),T3(6monthsaftersurgery).

Age Implantsplaced Implantlength,mm Torque,N.cm Boneheightloss(T0,T1,T2,T3),mm

Patient1 57 2 11 40 T0:0;T1:2;T2:3;T3:3 Patient2 60 1 8 35 T0:0;T1:3;T2:4;T3:4 Patient3 52 4 ≥11; ≤13 35 T0:0;T1:2;T2:2;T3:3 Patient4 55 2 11 40 T0:0;T1:3;T2:3;T3:4 Patient5 57 2 ≥8; ≤11 35 T0:0;T1:2;T2:3;T3:3 2.1. Surgicalprocedure

Afteradministrationofalocalanesthesia,apalatally(maxilla)or lingually(mandibular)shiftedincisionwasmadetogainaccessto underlyingboneridge:periosteumelevationwasperformedonly onthealveolarridgeuptothevestibularcorticalwall.Splitcrest wasperformedmakingathinmilling,andthencombiningsharping osteotomyandthansmoothosteotomesinsertionforexpansion.It continueswithrotatingdrillsoftheimplantkitsinthebasalbone preservinganadequatebonethicknessforbuccalandmedialbone wallsbysplittedwallsdivarication duringdrillingupto2.8mm burs.Topreservecorticalwallsintegrityfurtherimplantsite prepa-rationupto3.5mm.diameterwascarriedoutbyroundosteotomes (Sommers)orballburs.Withthismethodthereisapreservation ofthe vascular supply of thevestibular corticalwallliving the periostiumattachedonthevestibularside,withoutany vestibu-lar,mesialanddistalosteotomiesalsoachievingprimaryimplant stability.

Particularlytoobtainanoptimalimplantinsertion,atwolevels ofimplantsitepreparationwasperformed:1)splitcrestflapless techniqueforthealveolarandsmallamountofbasalboneand2) additionalandtraditionaldrillingpreparationbyimplantkitatthe basalbonelevel(usuallyshowingsufficientthickness).

In this way simultaneously were obtained optimal implant stability,adequatealveolarexpansion,boneheight-stabilityover timeforadequatecorticalwallsthicknessandoptimal nourish-mentofthesplittedvestibularwallsbynativeperiosteumwith attachedgingivacovering.Expansionwithsmoothchisel contin-ueduntilappropriatesitepreparationfortheselectedimplantwas obtained,beforeperformingthesteptwopreparationwith tradi-tionalimplantkitdrilling.Finalexpansionwasmaintainedbythe implantsitself.Theimplantswerepositionedatthesametimeof surgery,bonelevelimplantswerepreferredfortheprocedure.

TheVicrylpolyglactin(91,3/0)absorbablesuturewasusedto closetheflap.

Toobtainaprimaryclosureoftheflap,essentialforaproper healingoftheosteotomysite,partialreleasingincisionstechnique byscalpelatthemid-crestfibromucosawasperformedforflap elongation.Thisaspectis particularlyimportantbecause ofthe impossibilitytoapplya traditionalmembraneonthesplitcrest osteotomybecauseoftheflaplesstechnique.Inthis wayallthe bonelackattheosteotomysitewillbecoveredbyfibromucosa afterPRFapplyatosteotomyrimforbesthealing.

Forallofthefivepatientsofthefirstgroupautologousplatelet

rich fibrin (PRF) has been used to fill the osteotomy gap or

Fig.4.Splitcrestflaplesstechnique.Afterimplantinsertion,membranesofPRF

wereusedtoassureabettersofttissuehealing,makingthewoundhealingfaster.

ThePRFwasusedinaregenerativechamberofnativebonefedbyperiosteumto

testtheosteoinductiveandosteoconductivepower.

simplyasregenerative material.It dependsontype ofprotocol preparation used. PRF protocolrequires only centrifuged blood withoutanyadditionofanticoagulantandbovinethrombin.Then, abloodsampleistakenwithoutanticoagulantin10-mLtubesin aglassorglass-coatedplastictube,thenimmediatelycentrifuged at2700rpmfor12min.Thisprotocolwasusedin2of5cases,in theother3cases,theprotocolwaschanged(3000rpmfor13min) toobtainamoreconsistentsubstancetouseasafillerinthesplit crestgap(Fig.4).

Thefiveotherpatientstreatedwiththetraditionaltechniqueas controlgroupwereselectedwithathinalveolarcrestinorderto allowsmallerimplantinsertionwithoutboneaugmentationata deeperboneposition.

Orthopantomography, intraoral radiographs and CT DentaS-can/CTConebeamwereperformedbeforesurgeryforeachpatient inorder tohavea preliminaryradiologicalinvestigationandto giveageneraloverviewofthejawboneand relevantanatomic landmarksin abidimensionaland tridimensional reconstructed planes.Intraoralandfacephotographsweretakenforaestheticand functionalevaluationofthepatientsstatus.Forallthepatientsa

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beta-lactamantibiotic(Amoxicillin)wasgivenorally2gr.onehour beforethesurgery.Thepost-operativetherapyrequiredgoodoral hygiene,rinsingwithmouthwashcontaining0.2%chlorhexidine solutiontwiceadayandaneveningapplicationofthesame prod-uctingelform,aswellastheadministrationofanon-steroidal anti-inflammatoryaid(Ketoprofen80mg)for threeconsecutive days.

Followupwascarriedoutpostoperativelyat30days(clinical), 3monthsand6months(ortopantomographyandintraoralX-ray). Thisstudywasperformedfollowingtheprinciplesofthe Dec-larationofHelsinkiregardingresearchonhumans;signatureofa writteninformedconsentformfromallpatientswasrequested. Writteninformedconsentwasobtainedfromthepatientsfor pub-licationofthiscasereportandaccompanyingimages.Acopyofthe writtenconsentisavailableforreviewbytheEditor-in-Chiefofthis journalonrequest

3. Results

Allcasesendedwell,therewerenoproblemsatsurgerytime, post-operativeandintheperiodofosteointegration.Allimplants achievedosteointegration.Inallimplantsagooddegreeofprimary stabilitywasachievedatthesurgerytime.

All patients underwent an uneventful implant surgery. All implantswereplacedaccordingtothemanufacturer’sinstructions andachievingprimarystability(≥35Ncm).Nointraoperative sur-gicalcomplicationswererecorded.

Althoughpostoperativecomplicationsweregenerallymodest. Theclinicalhealingwasoptimalintheshortterm,nodehiscence wasreported.

Theseresultswereobtainedbyaccuratelymanagingimmediate andlatepostoperativeperiodinalloftheoperatedcases.

Particularattentionwaspaidtooralhygiene,andto inappro-priateearlyclinicalloadingatimmediateandlatepostoperative time.

Themaincharacteristicsandresultsachievedofthe10patients belongingtothestudyarepresentedinTables1and2.

ComparingresultsshowninTables1and2forheightdecrease atT0,T1,T2andT3betweenthenewandtraditionaltechnique itisevidentlessboneheightlossforthenewsplitcrestflapless technique.Boneheightwasmeasuredfromlowerborderofthe mandibleandfromthenoseorsinusfloorofthemaxillaeupto thealveolarbridgeatthepre-operativetimeanduptothemost coronallevelofbonetoimplantcontactatthepost-operativetime. AtT1meanheightlosswasof0mmandof1.2mmatT3forgroup 1(newflaplesstechnique)whileforgroup2patients(traditional implantinsertiontechnique)meanheightbonelosswas2.4mmat T1andof3.4atT3.

Meandifferenceforheightbonelossbetweenthetwogroupsof patientswas2.4mmatT1and2.2mmatT3.Inthiswayboneheight losswascalculatedalsoreferring topre-operativetime:heavier boneheightloss incontrolgroup canbeexplainedconsidering deeperimplantinsertionnecessityinthinalveolarcrestwithout expansionbysmallerimplantselectionwithrelatedworseningof thefinalaestheticresult.

4. Discussion

UseofPRFinoralandmaxillofacialsurgeryhasbeenimplicated indifferentproceduressuchassocketpreservation,sinusliftand boneaugmentation,rootcoverageprocedures,andhealingindonor sitewithgoodresults[6,7].

Someadvantagesarereportedintheliteraturerelatedtothe useofPRF,suchasthefollowing:

its preparation is a simplified and efficient technique, with centrifugationinasinglestep,freeand openlyaccessibleforall

clinicians[21,22];itisobtainedbyautologousbloodsample[10]; minimizedbloodmanipulation[23];itdoesnotrequirethe addi-tionofexternalthrombinbecausepolymerizationisacompletely naturalprocess,withoutanyriskofsufferingfroman immunolog-icalreaction[4,23].

Ithasanaturalfibrinframeworkwithgrowthfactorswithinthat maykeeptheiractivityforarelativelylongerperiodandstimulate tissueregenerationeffectively[13].

Itcanbeusedsolelyorincombinationwithbonegrafts, depend-ingonthepurpose[8,21];increasesthehealingrateofthegrafted bone[8,23];itisaneconomicalandquickoptioncomparedwith recombinantgrowthfactorswhenusedinconjunctionwithbone grafts[24]

Whenusedasamembrane,itavoidsadonorsitesurgical pro-cedureandresultsinareductioninpatientdiscomfortduringthe earlywound-healingperiod[25].

PRFmaypresentsomedisadvantagesasfollows:

thefinalamountavailableislowbecauseitisautologousblood

[8];thesuccessofthePRFprotocoldependsdirectlyonthe han-dling,mainly,relatedtobloodcollectiontimeanditstransference forthecentrifuge[4];needofusingaglass-coatedtubetoachieve clotpolymerization[22];possiblerefusaloftreatmentbythe punc-turerequiredforbloodcollectionatsurgerytime[21].

Thisprocedureonlyneedsaminimalexperienceofclinicianfor PRFmanipulation[7,21].

Withthisflaplesstechniquewithlingualorpalatalmucosa inci-sion, buccalalveolar cortex are preserved shiftingincision and periostealelevationonpalatalorlingualsideswherecorticalwall arethickerandmoreresistanttoreabsorption.Furthermore,this typeoftechniquereducesthepossibilityofvestibularbone fen-estration,avoidingperiimplantitisandmedicolegalconsequences, especiallyinelderlypatientswheresurgicalproblemscould hap-penmoreoftenfortheconsiderableanatomicaldifficulties[26]. Comparingthetwogroupsofpatientsoperatedforthisstudy,we canstatethatwiththisnewtechniqueitispossibletopreserve alve-olarboneheightatimplantinsertionwhileachievingtransversal alveolarexpansionbyproperboneregenerativeconditions.

In literature, in addition tothe split crest, other innovative andminimallyinvasivetechniquesfortheexpansionofthejaws aredescribed,thatareabletoimprovetheaestheticsoftheface

[27–30].

Recent literaturesuggests it isessential to leaveunchanged thevascularityofthesite,asevidencedbySchwartz-Arad[31].To allowproperosseointegration withgood healingofthesurgical sites and toassure primary closure of thesurgicalsite, releas-ing incisions byscalpel at themid crestcollapsedfibromucosa wereperformedobtainingsufficient flapelongationforprimary closureandosteotomysitecoveragewithadherent(firm) fibro-mucosa.

TopromotebonehealingattheosteotomysitesPRF(Platelet RichFibrin)canbeused.PRFseemedtoassureabettersofttissue healing,makingthewoundhealingfaster[8].EvenincaseofPRF membraneexposure,thereisnoriskofmembraneinfectionorbone loss,asPRFcanassurealsoasecondintentionhealingofthesoft tissues.Moreover,severalclinicalworkshave demonstratedthe effectivenessofPRFinpromotingthehealingofsurgicalwounds; thePRFhas,infact,plateletgrowthfactorsthatcanimprovethe vascularisation of the surgical site, promoting neoangiogenesis

[4].

The PRF if used in a regenerative chamber of native bone fedbyperiosteum,itmayobtainregeneration,whilenothaving osteoinductiveproperties.The osteoconductivepower,over the accelerationhealingprocessalreadydemonstratedintheliterature, hasrecentlybeenprovedinthearticleofSchwarz-Arad[31].

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5. Conclusions

Thebiggestadvantageinusingtheplatelet-richfibrinisforits completeresorption,thusavoidingasecondsurgicaltime, abso-lutelyacrucialfactorintheelderlypatients.

Moreover,bysimplychangingthesettingsofthecentrifuge,itis possibletoobtainanormalgellingifitistobeusedasregenerative andstimulatingmaterial,oramoreconsistentsubstancetouseas afillerinthesplitcrestbonegap.

Currently,itseemstobeaminimallyinvasivetechniquewith lowrisksandsatisfactoryclinicalresultssuchpreventing compli-cationsorimplantfailureparticularlyinelderlypatientsforage relatedconditions.

Writteninformedconsentwasobtainedfromthepatientsfor publicationofthiscasereportandaccompanyingimages.Acopyof thewrittenconsentsisavailableforreviewbytheEditor-in-Chief ofthisjournalonrequest.

Authorcontributions

Allauthorscontributedsignificantlytothepresentresearchand reviewedtheentiremanuscript.

AC:Partecipatedsubstantiallyinconceptionandexecutionof thestudyandintheanalysisandinterpretationofdata;also parte-cipatedsubstantiallyinthedraftingandeditingofthemanuscript. GP:Partecipatedsubstantiallyinexecutionofthestudyandin theanalysisandinterpretationofdata;alsopartecipated substan-tiallyinthedraftingandeditingofthemanuscript.

AB:Partecipatedsubstantiallyintheanalysisandinterpretation ofdata;alsopartecipatedsubstantiallyinthedraftingandediting ofthemanuscript.

MG:Partecipatedsubstantiallyintheanalysisand interpreta-tionof data;alsopartecipated substantially inthedrafting and editingofthemanuscript.

MA:Partecipatedsubstantiallyintheanalysisand interpreta-tionof data;alsopartecipated substantially inthedrafting and editingofthemanuscript.

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References

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