Preclinical Bioassay of a Polypropylene Mesh for Hernia Repair Pretreated with Antibacterial

17 Pages·2015·4.91 MB·English

RESEARCHARTICLE
Preclinical Bioassay of a Polypropylene Mesh
for Hernia Repair Pretreated with
Antibacterial Solutions of Chlorhexidine and
Allicin: An In Vivo Study
BárbaraPérez-Köhler1,4,FranciscaGarcía-Moreno1,4,ThierryBrune2,GemmaPascual3,4,
JuanManuelBellón1,4*
1 DepartmentofSurgery,MedicalandSocialSciences,FacultyofMedicineandHealthSciences,University
ofAlcalá.Madrid,Spain,2 Covidien–SofradimProduction,Trévoux,France,3 DepartmentofMedicineand
MedicalSpecialties,FacultyofMedicineandHealthSciences,UniversityofAlcalá.Madrid,Spain,
4 NetworkingResearchCenteronBioengineering,BiomaterialsandNanomedicine(CIBER-BBN),Madrid,
Spain
*[email protected]
Abstract
OPENACCESS
Citation:Pérez-KöhlerB,García-MorenoF,BruneT,
PascualG,BellónJM(2015)PreclinicalBioassayof
aPolypropyleneMeshforHerniaRepairPretreated Introduction
withAntibacterialSolutionsofChlorhexidineand
Prostheticmeshinfectionconstitutesoneofthemajorcomplicationsfollowingherniarepair.
Allicin:AnInVivoStudy.PLoSONE10(11):
e0142768.doi:10.1371/journal.pone.0142768 Antimicrobial,non-antibioticbiomaterialshavethepotentialtoreducebacterialadhesionto
themeshsurfaceandadjacenttissueswhileavoidingthedevelopmentofnovelantibiotic
Editor:DimitriosZeugolis,NationalUniversityof
Ireland,Galway(NUIGalway),IRELAND resistance.Thisstudyassessestheefficacyofpresoakingreticularpolypropylenemeshes
inchlorhexidineorachlorhexidineandallicincombination(anaturalantibacterialagent)for
Received:June15,2015
preventingbacterialinfectioninashort-timehernia-repairrabbitmodel.
Accepted:October27,2015
Published:November10,2015 Methods
Copyright:©2015Pérez-Köhleretal.Thisisan Partialherniadefects(5x2cm)werecreatedonthelateralrightsideoftheabdominalwall
openaccessarticledistributedunderthetermsofthe
ofNewZealandWhiterabbits(n=21).Thedefectswereinoculatedwith0.5mLofa106
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany CFU/mLStaphylococcusaureusATCC25923strainandrepairedwithaDualMeshPlus
medium,providedtheoriginalauthorandsourceare antimicrobialmeshoraSurgipromeshpresoakedineitherchlorhexidine(0.05%)orallicin-
credited. chlorhexidine(900μg/mL-0.05%).Fourteendayspost-implant,meshcontractionwasmea-
DataAvailabilityStatement:Allrelevantdataare suredandtissuespecimenswereharvestedtoevaluatebacterialadhesiontotheimplant
withinthepaper. surface(viasonication,S.aureusimmunolabeling),host-tissueincorporation(viastaining,
Funding:Covidien–SofradimProductionprovided scanningelectronmicroscopy)andmacrophageresponse(viaRAM-11immunolabeling).
supportintheformofafinancialresearchgrantand
salariesforTB,butthefundersdidnothaveany
Results
additionalroleinthestudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe ThepolypropylenemeshshowedimprovedtissueintegrationrelativetotheDualMeshPlus.
manuscript.Therearenopatents,productsin
BoththeDualMeshPlusandthechlorhexidine-soakedpolypropylenemeshesexhibited
developmentormarketedproductstodeclare.This
highbacterialclearance,withthelattermaterialshowinglowerbacterialyields.Theimplants
doesnotaltertheauthors'adherencetoallthePLOS
ONEpoliciesonsharingdataandmaterials. fromtheallicin-chlorhexidinegroupdisplayedaneoformedtissuecontainingdifferently
PLOSONE|DOI:10.1371/journal.pone.0142768 November10,2015 1/17
UseofAntisepticstoPreventImplantInfectionsafterHerniaRepair
CompetingInterests:Thecommercialaffiliationof sizedabscessesandlivingbacteria,aswellasadiminishedmacrophageresponse.The
TBtoCovidien–SofradimProductiondoesnotalter allicin-chlorhexidinecoatedimplantsexhibitedthehighestcontraction.
theauthors'adherencetoPLOSONEpolicieson
sharingdataandmaterials.Theauthorshave
Conclusions
declaredthatnocompetinginterestsexist.
Thepresoakingofreticularpolypropylenematerialswithalowconcentrationofchlorhexi-
dineprovidesthemeshwithantibacterialactivitywithoutdisruptingtissueintegration.Due
tothesimilaritiesfoundwiththeantimicrobialDualMeshPlusmaterial,thechlorhexidine
concentrationtestedcouldbeutilizedasaprophylactictreatmenttoresistinfectionbypros-
theticmeshduringherniarepair.
Introduction
Thesurgicalproceduresdevelopedtorepairabdominalwallpathologiessuchasherniashave
evolvedoverthelastdecades[1].Traditionalmethods,suchasautoplastyandsuturerepair,
havebeengraduallyreplacedbyprostheticimplantsthatachieveasignificantreductioninthe
incidenceofherniarecurrence[2,3].Nevertheless,theuseofbiomaterialstorepairhernia
defectsareassociatedwithsomepost-surgicalcomplications,withprostheticmeshinfection
presentingoneofthemostdevastatingeffectsonpatienthealthandalsoonthehealthcaresys-
tem[4].
Prostheticmeshinfectionratefollowingherniarepairvarieswiththesurgicalprocedure.
Incisionalherniashaveinfectionratesofapproximately3.6%whenrepairedwithlaparoscopic
surgeries[5]andapproximately10%followingopensurgeries[6].Regardlessoftheanatomical
locationofthedefectandthetypeofrepairapplied,thebacterialcontaminationofanimplanted
prostheticdevicedisruptsnaturaltissuerepairandremodelingprocesses[7],whichinturn
jeopardizestheintegrityoftheimplantandincreasestheriskofherniarecurrence[8].
Bacteriacanenterthewoundfromthepatient’sand/ortheclinicians’skin,fromsurgical
instruments,orfromtheenvironment;thisbacterialentrycanthentriggerasurgical-siteinfec-
tion(SSI)[9].Onceinsidethebody,bacteriainteractwiththesurfaceoftheimplantedmaterial
throughreversiblephysicochemicalprocessesandconsequentlycolonizetheimplantinan
irreversiblemannerduetoastrongadhesiontotheimplantedmaterialandtissuemediatedby
thebacterialadhesins[10,11].Theinitialmechanismsofbacterialadherencetothesurfaceof
theimplantedbiomaterialarethusakeystepinthepathogenesisoftheinfection.Staphylococ-
cusaureus(Sa)andS.epidermidis(Se)representthetwomajorsourcesofSSIandprosthetic
meshinfections[12].
Oneofthemainconsequencesofbacterialadherencetothemeshsurfaceisthedevelop-
mentofbiofilms.Theinteractionsamongbacteriaandtheirproductionofanexopolysacchar-
idecapsulepromotesthedevelopmentofcomplexbacterialcommunitiescharacterizedbya
potentadhesiontothesubstrateandanincreasedresistancetotheactionofdrugsandhost
immunecells[13–15]Moreover,thepresenceofforeignbodiessuchasimplantedbiomaterials
andsurgicalsuturesdecreasesthebacterialloadrequiredtopromoteaSSIbyapproximately
twothirds[16].
Inanattempttoreducetheriskofinfectionafterherniarepair,preoperativeprophylaxis
throughthesystemicadministrationofantibiotics[17]ortheimpregnationoftheprosthesis
inantibioticsolutionspriortotheimplantation[18]arecommonlypracticedinclinicalset-
tingsandarehighlyrecommendedinhigh-riskpatientsundergoingsurgery[18].Nonetheless,
insomecircumstancestheantibioticprophylaxisiscontroversial,particularlyinsurgical
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UseofAntisepticstoPreventImplantInfectionsafterHerniaRepair
proceduresforinguinalherniarepair[19,20],sincethisstrategycannotguaranteeabsolute
effectiveness[21,22].Furthermore,theextendedorinadequateuseofantibioticprophylaxis
canleadtoseveraladverseeffects[23]andpromotetheoccurrenceofnovelantibioticresis-
tances[24],sothelocaladministrationofantisepticsolutionsisbecomingmorefrequent.The
utilizationofantibacterialagentsorantisepticscouldavoidthedevelopmentofnovelresis-
tances[25]andtheirlocal/topicalapplicationcouldreducethesystemictoxicitycausedbyanti-
biotictreatment[26].
Oneofthemostwidelyutilizedantisepticsischlorhexidine(CHX).Thisisapotentantibac-
terialagentwithprovenactivityagainstgram-positive,gram-negativebacteriaandyeasts[27]
whichisfrequentlyusedforskindisinfection[28]andsoakingofsurgicaldevicessuchasvas-
cularcatheters[29].AtphysiologicalpH,thepositivechargesofCHXdestabilizethebacterial
wall,alteringtheosmoticequilibriumandinducingthebacterialdeath[27].
Moleculesofnaturalorigincanalsoexhibitantibacterialactivity.Historically,garlicandits
derivativeshavecausedstronginterestduetotheirantibacterial,antifungalandantioxidant
properties[30].Garlicclovescontainalliin,asulfoxidecompoundthatreactswiththeenzyme
alliinase(alliinlyase)whenclovesarecrushed,producingallicin[31].Allicinisanoxygenated
sulphurcompoundwhichexertsantibacterialactivitymediatedbychemicalinteractionswith
thesulfhydrylcompoundsofbacterialenzymes,provokingtheirinactivation[32].Thisanti-
bacterialagenthasactivityagainstawidebroadofgram-positive,gram-negativebacteria[30]
andresistantstrainssuchasmethicillinresistantS.aureus(MRSA)[31].
Inarecentinvitrostudy[33],ourgroupdemonstratedthatthepresoakingofreticularpoly-
propylene(PP)mesheswithalowconcentrationofCHXandacombinationofCHXwithalli-
cinefficientlyinhibitedtheadhesionofSatothematerialsurface.Giventhepositiveresults
obtained,theaimofthepresentworkistoevaluateinvivoinarabbitmodelofherniarepair
theeffectivenessofpresoakingPPreticularmesheswiththesetwoantisepticstocombatSa-
contamination.Theperformancesofthesemesheswerecomparedwiththebehaviorofacom-
merciallyavailablelaminarexpandedpolytetrafluoroethylene(ePTFE)meshthatexertsanti-
bacterialactivity.
MaterialandMethods
Preparationofthebacterialinocula
ThebacterialstrainSaATCC25923(subspeciesaureusRosenbach1884;CECT,Valencia,
Spain)wasutilized.AcryovialcontainingSawasthawed,platedonLysogenyBroth(LB)agar
platesandincubatedfor24hat37°C.Asinglecolonywasinoculatedinto25mLofLBmedium
andkeptovernightat37°C.Theabsorbance(OD600)wasreadbyspectrophotometryand
adjustedwithsterile0.9%salinetoanOD600equivalenttoapproximately1x108CFU/mL.
Twoten-foldserialdilutionswereperformedtogeneratea106CFU/mLinoculum.Thenum-
berofviablebacteriaintheinoculumwasdeterminedwiththespotplaquemethodintripli-
cate.Usingthe106CFU/mLinoculum,fiveten-foldserialdilutionswerepreparedand100μL
ofeachdilutionplatedonLBagarplates.After24hofincubationat37°C,theplateswere
countedandtheviableCFU/mLweredeterminedusingthefollowingcalculation:CFU/
mL=numberofCFUxdilutionfactor(10x)/volumeplated(0.1mL).
Antibacterialsolutions
Thefollowingsterileantimicrobialsolutionswereutilized:
- CHX:Solutionofchlorhexidinedigluconate(SantaCruzBiotechnology,Texas,USA)
dilutedto0.05%inultrapurewater.
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- Allicin-CHX:Mixedsolutioncontaining900μg/mLallicin(AllimedLiquid;AllicinInter-
nationalLtd.,Stratford,UK)and0.05%CHX.
Agarwelldiffusiontest
Theefficacyoftheantibacterialsolutionswastestedinvitrobymeansofanagarwelldiffusion
test.Usingsterileswabs,1mLoftheSa106CFU/mLinoculumwasutilizedtospreadbacterial
lawnsonto15LBagarplates.Circularwells(8mmindiameter,4mmindepth)werethen
punchedinthemiddleofeachcontaminatedplateandfilledwith100μLofthecorresponding
CHXorallicin-CHXsolution(n=5platespergroup),andplatesfilledwithsterile0.9%saline
wereusedascontrol(n=5).After24hofincubationat37°C,thezonesofinhibitionwere
recordedbymeasuringtwoperpendiculardiametersperplate.Theresultswereexpressedas
themeaninhibitionzone(mm)pertreatment.
Prostheticmaterialsandstudygroups
Thefollowingherniarepairprostheticmaterialswereutilized:
- GoreDualMeshPlusmesh(DM+)(W.L.Gore&Associates,Delaware,USA).Two-sur-
facelaminarantimicrobialePTFEmeshcoatedwithsilvercarbonateandchlorhexidine
diacetatewiththicknessesof1.5mm.
- Surgipromesh(PP)(Covidien,Dublin,Ireland).Heavyweight,monofilamental,non-
absorbablePPmesh(85g/m2)withaporesurfaceareaof0.26±0.03mm2.
Immediatelypriortotheimplantation,thePPmaterialsweresoakedinthedifferentanti-
bacterialsolutionsfor1min,andthefollowingstudygroupswereestablished:
- DM+(n=7)
- PP+CHX(n=7)
- PP+allicin-CHX(n=7)
Experimentalanimals
ThestudyprotocoladheredtotheAnimalsinResearch:ReportingInVivoExperiments
(ARRIVE)guidelinesforthepublicationofanimalstudies[34].Theexperimentalanimals
usedwere21maleNewZealandWhiterabbitsweighingapproximately3,000g,whichwere
randomizedintothedifferentstudygroups.Thestudywascarriedoutinstrictaccordance
withtheGuidefortheCareandUseofLaboratoryAnimalsoftheNationalandEuropean
InstitutesofHealth(Spanishlaw32/2007,SpanishRoyalDecree1201/2005,EuropeanDirec-
tive2010/63/UEandEuropeanConventionoftheCouncilofEuropeETS123).Thestudypro-
tocolwasapprovedbytheCommitteeontheEthicsofAnimalExperimentsoftheUniversity
ofAlcalá(registeredcode:CEI2013004).AllprocedureswereperformedattheAnimal
ResearchCenterofAlcaláUniversity.
Surgicaltechniquesandexperimentaldesign
Tominimizepain,theanimalswereadministered0.05mg/kgbuprenorphine(Buprecare,
DivasaFarmavic,Spain)1hbeforeand3daysafterthesurgicalprocedure.Theanimalswere
anesthetizedusingamixtureofketaminehydrochloride(Ketolar;Parke-Davis,Spain)(70mg/
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UseofAntisepticstoPreventImplantInfectionsafterHerniaRepair
kg),diazepam(Valium;Roche,Spain)(1.5mg/kg)andchlorpromazine(Largactil;Rhône-Pou-
lenc,Spain)(1.5mg/kg),whichwasadministeredintramuscularly15minbeforethesurgery.
Insomecases,anadditionaldoseofanestheticwasinjecteddirectlyintheabdominalcavity
duringthecourseofsurgery.
Usingasteriletechnique,anapproximately5-cmlongitudinallaparotomywasintroduced
ontherightsideofthemidline.Afterdissectingthesubcutaneoustissue,a5x2cmmusculo-
fascialdefectwascreatedata1-cmdistancefromthemidlinecomprisingthefasciaandoblique
muscleswhilesparingthetransversemuscleandperitoneum.Thedefectwastheninoculated
with0.5mLoftheSa106CFU/mLsuspensionpreviouslyprepared,andthecorrespondingbio-
materialwasfixedtothemarginsofthedefectbyrunningaPP4/0sutureinterruptedonlyat
theimplantcorners(Fig1).TheskinwasclosedovertheimplantbyrunningaPP3/0suture.
Atotalof7implantsperstudygroupweredeveloped.
Theanimalswereweigheddailytodetermineanyweightlossorincreaseandwerevisually
monitoredforsignsofsurgicalincisiondehiscence,fistulaorseromaformation,SSIand/or
othercomplications.After14post-operativedays,theanimalsweresedatedwithupto20mg/
kgofxylazine(Rompun;Bayer,Leverkusen,Germany)andtheneuthanizedinaCO chamber
2
withincreasingconcentrationsofCO .
2
Meshcontraction
Contractionofthemeshesat14dayspost-implantationwasdeterminedbyimageanalysisof
photographstakenatthemomentoftheeuthanasia.Thephotographswerescaledandthesur-
faceareaofeachmeshwasmeasuredusingimageanalysissoftware(ImageJ,NIH;http://
imagej.nih.gov/ij/).Thearearecordedforeachimplantwascomparedtotheinitialareaofthe
meshes(5x2cm).Theresultswereexpressedasthemeanpercentageofmeshcontractionsuf-
feredbyeachstudygroup.
Macroscopicobservations
Followingeuthanasia,theimplantswereexaminedtoscorethemostrelevantmacroscopic
observationsforeachspecimenusingthescoringsystemshowninTable1,specifically
designedforthispurpose.Ifseromaformationwasdetected,theliquidwasaspiratedwitha
sterilesyringe,storedat4°Cuntiltheendoftheintervention,and100μLofeachsamplewas
Fig1.Surgicaltechnique.Macroscopicimageofthebiomaterialsafterimplantintotheexperimentalanimal.(A)DM+implant,(B)PPimplantpresoakedin
eitherCHXorallicin-CHXsolutions.
doi:10.1371/journal.pone.0142768.g001
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Table1. Scoringsystemutilizedtoevaluatetheimplantsatthemomentoftheeuthanasia.
Infectionassessment—Mainfeatures
Skinnecrosis Absence Presence
Fistula Absence Presence
Swelling/Edema Absence SoftModeratearea SoftLargearea HardModeratearea HardLargearea
Purulentmaterial Absence Afewsitesalongthesutureline Allaroundtheimplant Covering<50%ofsurface Covering>50%ofsurface
Infectionassessment—Secondaryfeatures
Vascularization Normal Moderate Severe
Thrombosis Absence Moderate Severe
Tissueintegrationassessment
Encapsulation Absence Moderate Severe
Tissueintegration Complete Partialintegration Delamination
doi:10.1371/journal.pone.0142768.t001
platedonLBagarplatesandincubatedat37°Cfor24htodeterminethepresenceoffree-float-
ingbacteriaintheseroma.Aftermonitoringtheimplants,meshesplussurroundinghosttissue
wereharvestedfromtheinterfacesoftheprosthesis/visceralperitoneumandtheprosthesis/
subcutaneoustissueformicrobiological,morphologicalandimmunohistochemicalstudies,
accordingtothediagramshowninFig2.
Efficacyoftheantimicrobialcoatings
ToevaluatetheantibacterialeffectivenessoftheDM+andthePPmaterialsoakedinCHXor
allicin-CHX,meshfragmentswithdimensionsofapproximately2x1cmwereharvestedand
individuallytransferredtoglasstubescontaining20mLofsterileNeutralizingPharmacopoeia
Diluent(NPD)(8.5gNaCl,2.5mLTween-80,0.35glecithin,997.5mLdistilledwater).Under
Fig2.Experimentaldesign.Diagramshowingtheharvestingandprocessingofthetissuesamplestocarryoutthemacroscopic,microbiologicaland
histologicalevaluationofthedifferentimplants.
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sterileconditions,themeshwasseparatedfromthesurroundingtissueandbothsidesofthe
materialwerescrappedwithascalpelblade.TheglasstubescontainingtheNPD,explanted
meshandremainingtissueweresubjectedtoa10-minsonicationpulseat40kHzusingaBran-
sonic3800-CPXHultrasoniccleaningbath(BransonUltrasonics,Connecticut,USA).The
tubeswerethoroughlyvortexedfor1minandthesupernatantwasusedtoperformfive1/10
serialdilutions.Avolumeof100μLofeachdilutionwasseededontoLBagarplatesandincu-
batedfor24hat37°C.TheplateswerecountedandtheviableCFUpermeshfragmentwere
determinedusingthefollowingformula:ViableCFUpermeshfragment=viableCFU/mLx
20mL(volumeofNPDinthetube).Usingthesedata,thebacterialclearancewascalculatedas
thepercentageofanimalswithsterileculturesineachofthedifferentstudygroups.
Morphologicalanalyses
Forlightmicroscopy,thetissuespecimenswerefixedinF13solution(60%ethanol,20%meth-
anol,7%polyethyleneglycol,and13%distilledwater)andembeddedinparaffin.Tissuesec-
tions(5μmthickness)weresliced,stainedwithhematoxylin-eosinandMasson’strichrome
(Goldner-Gabe)andexaminedunderaZeissAxiophotlightmicroscope(CarlZeiss,Oberko-
chen,Germany).Forscanningelectronmicroscopy(SEM),thetissuespecimenswerefixedin
3%glutaraldehyde,placedinMillonigbuffer(pH7.3)anddehydratedinagradedethanol
series(30%,50%,70%,90%,and100%,eachincubatedfor15min).Thecriticalpointwas
reachedinanE-3000Polaroninstrument(PolaronLtd.,Newhaven,UK).Thepiecesweremet-
alizedwithgoldpalladiumandvisualizedwithaZeissDSM950scanningelectronmicroscope
(CarlZeiss).
Immunohistochemistry
Paraffin-embeddedtissuesectionswereutilizedtoevaluatethebacterialcolonizationofthe
mesh,thesurroundingtissue,andthemacrophageactivity.Thetissuesectionswerede-paraffi-
nizedinxyleneanddehydratedinagradedethanolseries(100%,96%,and70%,eachincu-
batedfor5min),hydratedandequilibratedinTris-bufferedsaline(TBS;pH7.4).Non-specific
proteininteractionswereblockedthroughincubationwith3%bovineserumalbumin(BSA)
for30minatroomtemperature.Thesectionswereincubatedwiththemonoclonalantibodies
againstSa(ab37644;Abcam,Cambridge,UK)andrabbitmacrophagesRAM-11(M-633;
Dako,Glostrup,Denmark)inthealkalinephosphatase-labeledavidin-biotinprocedure.The
methodincludedthefollowingsteps:incubationwithprimaryantibody(1:500foranti-Saand
1:50foranti-RAM-11,inTBS)for60min;incubationwithimmunoglobulinG(IgG)andbio-
tin(1:1,000inTBS)for45min;andlabelingwithstreptavidinalkalinephosphatase(1:200in
TBS)for60min.Thesestepswereconductedatroomtemperature.Negativecontrolswere
subjectedto3%BSAinsteadoftheprimaryantibodies.Theimageswererevealedusingachro-
mogenicsubstratecontainingnaphtholphosphateandfastred.Cellnucleiwerecounterstained
for1minwithacidhematoxylin.Thepresenceofbacteriawasqualitativelyevaluated.Labeled
macrophageswerequantifiedbyperformingcountsin10lightmicroscopyfields(magnifica-
tionx20)pertissuesample.Atotalof70fieldsperstudygroupwerecounted,andtheresults
wereexpressedasthepercentageofpositivelystainedcellsoutofthetotalnumberofcellnuclei
perfield.
Statisticalanalyses
Thedatacollectedfromthedifferentexperimentswererepresentedasthemean±standard
errorofthemean.Statisticalanalysiswasperformedusingone-wayanalysisofvariance
(ANOVA)andBonferroniasposthoctest.Allthestatisticalanalyseswereperformedusingthe
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GraphPadPrism5computerpackage(LaJolla,California,USA)forWindows.Thesignifi-
cancelevelwassetatp<0.05.
Results
Invitrostudy
TheSasuspensionusedtoinoculatetheexperimentalanimalsandtodeterminetheactivityof
theCHXandallicin-CHXsolutionsinvitrocontainedameanviablebacterialevelof1.45x106
CFU/mL.Theagarwelldiffusiontestconfirmedtheeffectivenessofthetwoantibacterialtreat-
mentstestedbecausebothprovokedthedevelopmentofinhibitionhalosinalltheSa-pre-lawn
agarplatesafter24hofthecontamination,whilealltheplatescontainingsalinewerefullycon-
taminated(Fig3).Themeasureofthehalodiametersrevealedsignificantlywiderinhibition
zonescreatedbytheallicin-CHXsolution(36.90±0.19mm)incomparisonwithCHX
(25.70±0.26mm)(p<0.001).
Postoperativefollow-up
Nomortalitywasobservedinanyofthestudygroups.Followingthesurgery,alloftheanimals
exhibitednobehavioralsignsthatmightimplyadecreaseinwelfare,regardlessofthe
implantedmesh.Therewasnowounddehiscence,inflammation,fistulaformation,skinery-
themaornecrosis.After14daysoftheimplantation,theanimalsfromthedifferentstudy
Fig3.Invitroagarwelldiffusiontest.Representativeimagesoftheinhibitionzonesafter24hofincubationat37°Cbythe(A)CHXand(B)allicin-CHX
solutionsutilizedtosoakthePPmeshes.(C)Sterilesalineusedascontroldidnotprovokebacterialgrowthinhibition.(D)Meandiameteroftheinhibition
zones(mm).Theresultsareexpressedasthemean±standarderrorofthemeanfor5samples.Theallicin-CHXsolutionprovokedsignificantlywider
inhibitionhalosthantheCHXtreatment.#:salinevsCHXandallicin-CHX(p<0.001);ф:CHXvssalineandallicin-CHX(p<0.001).
doi:10.1371/journal.pone.0142768.g003
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groupsshowednormalpercentagesofweightincrease,withvaluesof1.06±0.02%forDM+,
1.03±0.02%forPP+CHXand1.06±0.01%forPP+allicin-CHX.
Meshcontraction
Themeshcontractionmeasuredforthedifferentstudygroupsshowedmeanpercentagesof
6.38±1.18%forDM+,12.14±2.33%forPP+CHXand14.15±1.88%forPP+allicin-CHX
implants.WhilenosignificantdifferenceswereobservedbetweenthetwocoatedPPstudy
groups,thecontractionofthePP+allicin-CHXimplantswasstatisticallyhigherthanthe
recordedforDM+(p<0.05).
Morphologicalstudies
Atnecropsy,andpriortothesampleharvesting,thewoundareaoftheanimalswassubjected
toacomprehensivemacroscopicexaminationofthemeshandthesurroundingtissuestoeval-
uatetheresponseoftheimplanttothebacterialinfection(Table1).TheDM+implants(Fig
4Aand4B)partiallyintegratedinthehosttissueweresurroundedbyafibrouscapsulecontain-
ingtransparenttosemi-turbidexudate;fourofthemeshesshowedsmallamountsofpurulent
materialassociatedwiththePPsuture,andseromawascollectedfrom2specimensandwas
refrigeratedforfurtheranalysis.ThePP+CHXimplants(Fig4Cand4D)wereencapsulated,
totallyintegratedintothehosttissueandshowedmoderatevascularizationoverthemesh;as
observedpreviously,thepresenceofsmallpurulentmaterialattachedtothePPsuturefilament
wasdetected,andturbidseromawerecollectedfrom3specimens.TheobservationsofthePP
+allicin-CHXimplants(Fig4Eand4F)werecharacterizedbythepresenceofseveralamounts
ofpurulentmaterialcoveringthesuturefilamentandscatteredoverareasofthemesh;thebio-
materialsweretotallyintegratedinthehosttissueandnoneofthespecimensexhibitedseroma
formation.
TheseromascollectedfromboththeDM+andthePP+CHXgroupswereplatedonLB
agarplatesandincubatedat37°C.After24hoftheincubation,noneofthesamplesshowed
anycolonygrowth,andtheseromaswerethusconsideredfreeofbacteria.
Bacterialcolonizationontheimplantsurface
TheresultsfromthesonicationdemonstratedthegreatantibacterialactivityoftheDM
+implantswith86%ofbacterialclearancebecause6ofthesamplesweresterileandthelast
sampleshowed4.90x107CFUpermeshfragment.Thesamebacterialclearancewasrecorded
forthePP+CHXgroup;however,theonlycontaminatedspecimeninthisgroupshowed
lowerbacterialyieldsthanthecontaminatedDM+sample,with1.40x103CFUpermeshfrag-
ment.ThepoorestbehaviorwasrecordedforthePP+allicin-CHXgroup,with57%bacterial
clearanceand3contaminatedspecimensoutof7,withmeancountsof1.62x105CFUper
meshfragment.Therewerenostatisticaldifferencesamongthedifferentstudygroups.Table2
summarizesthebacterialcountsofthesamplesyieldedafterthesonication.
Histology
Consistentwiththemacroscopicoutcomes,thehistologicalevaluationofthetissuespecimens
evidencedthattheDM+implants(Fig5Aand5B)werenotfullyintegratedinthehosttissue.
Theseimplantsweresurroundedbyalayeroflooseconnectivetissue,whichgrewdenserinthe
areasofmeshanchorage.Theneoformedtissuewasslightlyvascularizedbelowtheimplant
andpresentedabarrierofinflammatorycellsalongthematerial,keepingthemeshwallfreeof
cellinfiltration.ThePP+CHXimplants(Fig5Cand5D)werecompletelyintegratedintothe
PLOSONE|DOI:10.1371/journal.pone.0142768 November10,2015 9/17
UseofAntisepticstoPreventImplantInfectionsafterHerniaRepair
Fig4.Macroscopicfindings.Appearanceofthedifferentmeshesafter14daysofimplantationandSacontamination.(A,B)DM+implantsshowingthick
fibrousencapsulation(*),seromaformation(▶)andthepresenceofdispersedpurulentmaterial(!)associatedwiththemeshanchorage,whilethemain
bodyoftheimplantremainsclean.(C,D)PP+CHXimplantsshowingsimilarbehaviortothepreviousones,withtheexceptionsofamoreintense
vascularizationandtotalmeshintegrationintothehosttissue.(E,F)PP+allicin-CHXimplantsshowlargeamountsofpurulentmaterial(!)covering
differentareasoftheimplantsurface.
doi:10.1371/journal.pone.0142768.g004
PLOSONE|DOI:10.1371/journal.pone.0142768 November10,2015 10/17