RESEARCHARTICLE Experimental Diabetes Alters the Morphology and Nano-Structure of the Achilles Tendon RodrigoRibeirodeOliveira1,2,3*,RoˆmuloMedinadeMattos4,LucianaMagalhãesRebelo5, FernandaGuimarãesMeirelesFerreira6,7,FernandaTovar-Moll6,7,LuizEuricoNasciutti1,4, GerlyAnnedeCastroBrito1 1 Inter-institutionalDoctoratePrograminMorphologicalScience,FederalUniversityofCeara´/Federal UniversityofRiodeJaneiro,RiodeJaneiro,Brazil,2 DepartmentofPhysicalTherapy,FederalUniversityof Ceara,Fortaleza,Ceara´,Brazil,3 TendonResearchGroup,Fortaleza,Ceara´,Brazil,4 Instituteof BiomedicalSciences,FederalUniversityofRiodeJaneiro,RiodeJaneiro,Brazil,5 DepartmentofPhysics, a1111111111 FacultyofPhysics,FederalUniversityofCeara,Fortaleza,Ceara´,Brazil,6 D’OrInstituteforResearchand a1111111111 Education(IDOR),RiodeJaneiro,Brazil,7 NationalCenterforStructuralBiologyandBioimaging,Federal a1111111111 UniversityofRiodeJaneiro,RiodeJaneiro,Brazil a1111111111 a1111111111 *[email protected] Abstract OPENACCESS Althoughofseveralstudiesthatassociatechronichyperglycemiawithtendinopathy,the connectionbetweenmorphometricchangesaswitnessedbymagneticresonance(MR) Citation:OliveiraRRd,MedinadeMattosR, MagalhãesRebeloL,GuimarãesMeirelesFerreira images,nanostructuralchanges,andinflammatorymarkershavenotyetbeenfullyestab- F,Tovar-MollF,EuricoNasciuttiL,etal.(2017) lished.Therefore,thepresentstudyhasasahypothesisthattheAchillestendonsofrats ExperimentalDiabetesAlterstheMorphologyand withdiabetesmellitus(DM)exhibitstructuralchanges.Theanimalswererandomlydivided Nano-StructureoftheAchillesTendon.PLoSONE 12(1):e0169513.doi:10.1371/journal. intotwoexperimentalgroups:ControlGroup(n=06)injectedwithavehicle(sodiumcitrate pone.0169513 buffersolution)andDiabeticGroup(n=06)consistingofratssubmittedtointraperitoneal Editor:HaraldStaiger,MedicalClinic,University administrationofstreptozotocin.MRwasperformed24daysaftertheinductionofdiabetes HospitalTuebingen,GERMANY andimageswereusedformorphometryusingImageJsoftware.Morphologyofthecollagen Received:March9,2016 fiberswithintendonswasexaminedusingAtomicForcemicroscopy(AFM).Anincreasein thedimensionofthecoronalplaneareawasobservedinthediabeticgroup(8.583±0.646 Accepted:December19,2016 mm2/100g)whencomparedtothecontrolgroup(4.823±0.267mm2/100g)resultingina Published:January17,2017 significantdifference(p=0.003)uponevaluatingtheAchillestendons.Similarly,ouranaly- Copyright:©2017Oliveiraetal.Thisisanopen sisfoundanincreaseinthesizeofthetransversesectionareainthediabeticgroup(1.328± accessarticledistributedunderthetermsofthe 0.103mm2/100g)incomparisontothecontrolgroup(0.940±0.01mm2/100g)p=0.021. CreativeCommonsAttributionLicense,which permitsunrestricteduse,distribution,and Thetendonsofthediabeticgroupshowedgreatirregularityinfiberbundles,includingmodi- reproductioninanymedium,providedtheoriginal fiedgraindirectionandjaggedjunctionsanddeformitiesintheformofcollagenfibrilsbulges. authorandsourcearecredited. DespitethemorphologicalchangesobservedintheAchillestendonofdiabeticanimals,IL1 DataAvailabilityStatement:Allrelevantdataare andTNF-αdidnotchange.OurresultssuggestthatDMpromoteschangestotheAchilles withinthepaperanditsSupportingInformation tendonwithimportantstructuralmodificationsasseenbyMRandAFM,excludingmajor files. inflammatorychanges. Funding:ThisworkwassupportedbyConselho NacionaldeDesenvolvimentoCient´ıficoe Tecnolo´gico(CNPq—Universalnu477692/2011- 7)andbyafellowshipfromtheCAPESforInter- institutionalDoctoratePrograminMorphological Sciences—FederalUniversityofCeara—UFC/ FederalUniversityofRiodeJaneiro—UFRJ— PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 1/13 DiabetesAltersStructureoftheTendon Brazil(Nu23038044935/2009-12).Thefundershad Introduction noroleinstudydesign,datacollectionand Tendinopathyisamajorhealthprobleminpeopleolderthan25years.Themainsymptomis analysis,decisiontopublish,orpreparationofthe manuscript. paininthetendonthatunderminesperformance.Thistypicallyresultsfromexcessiveuse,the severitydependingonthemagnitude,frequencyanddurationofthestimulusoverloadingthe CompetingInterests:Theauthorshavedeclared tendon[1].Thetendon,inthepresenceofpathologicalprocesses,showsalteredmorphology. thatnocompetinginterestsexist. Inmostcases,itischaracterizedbyintratendinousdegenerationanddisorganizationofcolla- Abbreviations:AFM,AtomicForceMicroscope; genfibers.Macroscopically,itpresentsitselfasmucoiddegeneration,withfriable,disorga- AGE,AdvancedGlycatedEnd-Products;CG, nizedtissueofabrownishcolor.Microscopically,itispossibletoconfirmthatthestructure ControlGroup;DG,DiabeticGroup;DM,Diabetes mellitus;ECM,Extracellularmatrix;GAG, showsdisorganizationandmicro-rupturesofcollagenfibers[2–4].Thetendontissuelosesthe Glycosaminoglycans;IL1,Interleukin1;MMP, parallelorganizationofitsfibersandpresentscellularincrement.Anincreaseinproductionof Metalloproteinases;MR,Magneticresonance;PBS, collagenfibersoccurs;however,duetotheirdisorganizedpattern,thetendonfibersarefriable phosphatebufferedsaline;STZ,Streptozotocin; andpronetoprematurerupture[5,6].Nevertheless,itisnotonlythecharacteristicofstress TNF-α,Tumornecrosisfactor-alpha. thatmayresultintendoninjury[7,8]. Twosystematicreviewsofliteraturehaveindicatedthatthereissubstantialevidenceofa linkbetweenDiabetesMellitus(DM)andtendinopathy[9,10].Recently,ithasbeendemon- stratedthatDMleadstomodificationsintheAchillestendonwhicharecompatiblewith chronictendinopathy.Thestateofchronichyperglycemiahasbeenassociatedwithsignifi- cantincreaseinmastcellsnumbers,avascularhyperplasiainthecross-sectionaltransverse areaofvesselsintheAchillestendonsaswellasanincreaseinvascularendothelialgrowth factor,type1collagen,andNF-κBexpressionwhencomparedtothetendonsofcontrolani- mals[7]. Furthermore,tothemanifestationsinthestructureoftendonsindiabeticpatients,ithas beenstatedthatthebiomechanicalpropertiesoftendonsindiabeticratshavealterationswhen comparedtohealthyanimals;changesinthevisco-elasticcapacityoftendonsmaydecrease thelimitofenergytransmissiontotheperipheryandinducethetendontoprematurerupture duetomechanicalstress[11,12]. However,regardlessofseveralstudiesthatassociatechronichyperglycemiawithtendinopa- thy,morphologicalchangesobservedinMRimages,nanostructure,andpro-inflammatory markershavenotyetbeenestablished.Therefore,thepresentstudyhasasahypothesisthatthe AchillestendonsoftheDMgroupexhibitcharacteristicsofmorphologicalandstructuralalter- ationsasinclassicaltendinopathy.ThisstudyrevealsnewinformationregardinginvivoMR imagesandnanostructureoftheAchillestendonindiabeticrats. MaterialsandMethods Animals MaleWistarrats(Rattusnorvegicus)wereused,withaninitialweightbetween300and350g, fromFederalUniversityofCeara.Theanimalswerekeptincollective,plasticcages(maximum of5animals/cage)inanenvironmentwithatemperatureof23±1˚C,12-hlight/darkcycle andwithfreeaccesstoamaintenancediet(Labina1—PurinaPetCareCompany)andwater adlibitum.Theanimalsweremonitoredandassesseddailytoensurethatanychangesinan animal’sconditionweredetectedearly. Theproceduresforhandlingandcareoftheanimalswereinaccordancewithinternational standardsestablishedbytheNationalInstituteofHealthGuideforCareandUseofLaboratory AnimalsandwereapprovedbytheCommissionofEthicsinAnimalExperimentation—Fed- eralUniversityofCeara/UFC,underprotocol51/2011. PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 2/13 DiabetesAltersStructureoftheTendon Experimentalgroupsandinductiontodiabetes Theanimalswererandomlydividedintwoexperimentalgroups:ControlGroup—CG (n=06)consistingofhealthyrats;DiabeticGroup—DG(n=06)consistingofratsinducedto DiabetesMellitus. Theexperimentaldiabetes,equivalenttoTypeI,wasinducedbyintraperitonealadminis- trationofstreptozotocin(SigmaChemicalCo.,USA)afterfastingfor14h.Thestreptozotocin (STZ)wasdilutedin10mMsodiumcitratebufferatpH4.5,inasingledoseof60mg/kgof animalweight,measuredcarefullyinaprecisiondigitalscale.Controlanimalssimilarly receivedequivalentdose(60mg/kg)ofsodiumcitratebuffersolution,and30minaftertreat- ment,theanimalswerefednormally[13]. Bloodglucose Verificationofbloodglucoseoccurredatthefollowingstagesoftheexperiment:1—afterthe 14hfastthatprecededtheinductionofdiabetes;2—sevendaysafterinduction,aimingto checktheinclusioncriteriafordiabetes,sinceonlyanimalsthathadbloodglucoselevelsabove 200mg/dL(Accu-ChekActivGlucometerkit)wereincluded;3—onday24afterdiabetes induction,inordertoevaluateglycemicexpressiononthedayoftendoncollection.Reagent stripswereused(Accu-ChekActiv)fordeterminationofbloodglucosefromadropofblood fromthetipoftheanimal’stail. InvivoMRimagesandMorphometry Magneticresonance(MR)imageswereacquired24daysafterinductionofDM.Theanimals wereanesthetizedwithisoflurane(1–2%formaintenance;upto3%forinduction)(E-ZAnes- thesia1Systems).Theimageswereacquiredina7-TMRscanner(7T/210HorizontalBore MagnetASRMRISystem,AgilentTechnologies).Theimagesoftendonswererecordedapply- ingaT1weigthedspin-echosequence(TR/TE:350/15ms;GAP:0),intheaxialplane(FOV:50 x80mm;matrix:192x192,slicethickness:1.0mm;10averages),coronal(FOV:70x85mm; matrix:128x128,slicethickness:0.5mm;10averages)andsagittal(FOV:80x50mm;matrix: 128x128,slicethickness:0.5mm;10averages)beforeandaftertheinjectionofgadolinium. Foreachdataset,theimageswerevisuallyinspectedforartifacts.Forimageprocessing, MRIcroNsoftwarewasused,latertheAchillestendonareawasmeasuredusingImageJsoft- ware.Tocomparetendondimensionsbetweentheanimalsofvariousbodysize,tendonCSA datawerenormalizedtobodyweight[7,11].Theassessmentofthemorphologicalcharacteris- ticsandmeasurementoftheareawereperformedbytwoexperiencedresearchersandcom- paredbetweenthegroups. ForthequalitativeanalysisofinvivoMRimages,anadditionalmethodofevaluationwas performedconsideringtheimagesbeforeandaftertheinjectionofgadolinium,inwhichthe tendonwasconsidereddamaged(positive)ornormal(negative).Tobeconsideredpositive, thetendonmustshowevidentdisorganizationoftissueand/orgadoliniumenhancementin thetendinouscore. CollectionofsamplesofAchillestendon FollowingMRimagesacquisition,onthetwenty-fourthdayaftertheinductionofDM,the animalsofbothgroupswereanesthetizedwithxylazinesolution(Rompum1—Bayer) (10mg/kg)andketaminehydrochloride(Ketalar1)(25mg/kg),0.10mlforeach100gof weightandanincisionwasperformedintheposteriorregionofthehindlegstoallowcollec- tionoftheAchillestendonfromitsoriginsandinsertions. PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 3/13 DiabetesAltersStructureoftheTendon DeterminationoflevelsofInterleukin-1andTNF-α QuantificationofIL1andTNF-αwereconductedbytheELISAmethodwiththeDuoSet kits(R&DSystems).Platesof96wellswerefilledwith50μLofprimaryantibodydilutedin phosphatebufferedsaline(PBS)andincubatedfor18hat4˚C.Theplatewaswashedthree timeswith0.05%TweenPBS.Next,200μL/wellof1%PBS/BSAwereaddedtoblocksites, for1hatroomtemperature.Afterincubationtheplateswerewashedagain.Thesamples andrecombinantcytokinesindilutionofknownconcentrationwerelabeledin100μL/well, incubatedfortwohoursat37˚C.Afterwashingtheplates,50μLofbiotinylateddetection antibodyforeachcytokinewereaddedforonehourat37˚C.Afterwashing,theplateswere incubatedwithperoxidase-conjugatedstreptoavidindilutedinPBS1:200(50μL)for30min atroomtemperature.Theplateswerewashedandincubatedwithasolutionoftetramethyl- benzidinefor20min.Thereactionoccurredwiththeadditionof25mL/wellof2Nsulfuric acid.TheopticaldensityofthesampleswasdeterminedbyanELISAreaderwitha450nm filter.Thecytokineconcentrations(pg/mL)foundinthetendonswerenormalizedbytotal proteinconcentrations. AtomicForceMicroscopy-AFM ToevaluatethemorphologyofthecollagenfibersandmeasurethefrequencyofDinterbands ofthefibrilstendonswereprocessedand1μm-thicktransversalhistologicalsectionswere madeandmountedonslides.Tocapturetheimages,asteeldiskwasaddedandsampleswere placedintheAtomicForceMultimodalmicroscope(DigitalInstruments,SantaBarbara,CA, USA),equippedwithNanoscopeIIIacontroller.Samplesweremeasuredinbothcontactand intermittent(tapping)modesofaround0.01nNattractiveforce.Thedatawereacquiredand theimagesprocessedusingascanningsystemwithresonantfrequencyperprobeandusinga siliconcantilever(Veeco-Probes)withanintegrated,triangular-shapedtiparadiusof15nN. Alltendonimageswerescannedin512x512size. Theintermittentcontactmode(tappingmode)oftheAFMwasutilizedfortheassessment oftheviscoelasticpropertiesofthefibrilssurface.Inthismode,thestemoscillatedclosetoits resonancefrequencybyasmallpiezoelectricelementfixedatthetipoftheAFM.Thesignal acquiredfromthedetectorsmeasuredtheoscillationmotionatthetipofthecantilever,sothat generatingaphasesignal(phaseimage),andaphasedifference(variationintheheightofcan- tileverinthezaxis)thatwasformedfromthedifferentinteractionsbetweenthetipandthe sample,indicatingtheviscoelasticpropertiesofthesurfaceofthetissue.Thephaseangle(θ) initiallyconsideredwas90˚,orθ=+90˚,sothat,inphaseimages,thedarkershadesimplya softerregion,andthelightershadesimplyastifferregion. Spectralanalysis(powerspectraldensity)wasperformedtocheckthestandardsofperiodic- ityofDcollagenbandsintheY-coordinate.ThedatawereexportedtoMatLabinorderto eliminatebackgrounds.LorentzianFitwasdoneontheimagestoobtaintheaveragesofthe spacingbetweenthelinksofthecollagen. Statisticalanalysis Todescribethecharacteristicsofthesampledescriptivemeasureswereused,suchas:mea- sureofcentraltendency(mean)anddispersion(standarddeviation).Forcomparisonofthe averagesofthenumericalvariablesbetweenthevarioustreatmentsemployed,theStudent’s t-testwasusedforindependentsamplesandcomparisonbetweenthecontrolgroupanddia- beticgroup.ThedatawereanalyzedintheSPSSsoftware.Asignificancelevelof5%was admitted. PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 4/13 DiabetesAltersStructureoftheTendon Results Thecontrolanimalsmaintainedstablebloodglucoselevels(below100ml/dL)duringallthe analyses;however,animalsinthegroupinducedtoDMshowedaconsistentincreaseofglu- coselevelsinthemeasurementsseven(352.1±46.3ml/dL)andtwenty-four(423.7±52.9 ml/dL)daysaftertheinductionofDM.ThegroupinducedtoDMrepresentedasignificant decrease(p=0.01)inweightaftertheinductionofDMintheseventhandthetwenty-fourth day(221.7±2.5g),whencomparedwithcontrolgroup(361.2±3.3g). MorphometryofinvivoMRimages Thediabeticgroup(8.583±0.646mm2/100g),whencomparedtothecontrol(4.823±0.267 mm2/100g),representedanincreaseinthesizeofthecoronalplane(p=0.003)uponevaluat- ingtheareaofAchillestendons,normalizedbyweight.Similarly,thediabeticgroup (1.328±0.103mm2/100g)incomparisontothecontrolgroup(0.940±0.01mm2/100g) increasedinthetransversesectionareaoftheAchillestendon(p=0.021).However,nodiffer- encewasobservedbetweenthegroupsinthenormalizedareaofthetendonsinthesagittal plane(Fig1). QualitativeanalysisofinvivoMRimagesbeforeandaftergadolinium injection Inthequalitativeevaluationofthemacroscopicorganizationofthetendonsofthecontrol groupnochangeswerenotedinthecontrolgroup(0/6).However,whenassessingtheDG,2/6 tendonshadalterationsinthemorphologicalorganization(p=0.222)asindicatedbyT1 weightedMRimage.Noanimals,ofeithergroup,presentedsignalenhancementafterGd administration(Fig2). MorphologicalandtopographicalcharacterizationwithAFM Thetypicalstructureofthecollagenfiberscanbeobservedthree-dimensionallybyAFMand thetopographicalevaluationsdemonstratedwell-organizedarrangementandagooduniaxial orientationofthehealthyAchillestendons,while,intheAchillestendonsofdiabeticanimals, thereisanotablelackofpatternanddisorganizationshowingchangesinfibrillarcollagen nano-structure.Inthequalitativeassessment,itwasobservedthatthecollagenfibershad alteredtheircylindricalshapeandexhibitedimportantdeformationanddiscontinuityofthe tendonfibersinthediabeticgroup(Fig3B,3Dand3F).Fig3Bdemonstratesgreatirregularity ofthefiberbundleswithbreaksinaspectofanabysswithdiscontinuities.Fig3Ddemonstrates Fig1.MeasurementofAchillestendonarea.A—Areainthecoronalplane;B—Areaintransversesection;C—Areainthesagittal plane.CG—controlgroupandDG—DiabeticGroup.*—p<0.05. doi:10.1371/journal.pone.0169513.g001 PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 5/13 DiabetesAltersStructureoftheTendon Fig2.DetailofSagittalSpinEchoimage—T1weightedof7TMRimages.A—TendonoftheControlGroup;B—TendonoftheDiabeticGroup showingincreasedareawhencomparedtothecontrol,inviewoftheweight.ThearrowindicatesthelocationoftheAchillestendondisorganization. doi:10.1371/journal.pone.0169513.g002 themorphologicaldisorganizationofthebundles,withmodificationofthegraindirectionof thebundlesandjaggedjunctions.Fig3Fshowsdeformitiesintheformofbuglescollagen fibrils. TheFig4showsthechangeindirectionofthecollagenfibersintheAchillestendonofthe diabeticgroup.Thefibersweremultidirectionalformorbifurcate,resultinginlossofmorpho- logicalandcollagennanostructurefeature. PeriodicityofBandD ThefrequencyofDinterbandsofthefibrilsofthediabetictendon(65.5±0.7)showednodif- ferencefromhealthytendons(65.3±2.8),andthefrequencyoftheDbandstayedwithinthe benchmark.However,itisimportanttostressthatthefibrilswithstructuralchangeswerenot measured,astheyfrequentlydidnotpresentanano-structureorganizedinauniaxialplaneor sometimeshadruptureofthefibrilsandabsenceoftherings,whichdidnotoccurinthe healthygroup(S1Fig). Phaseimageofsurfacetopographyoffibrils Fromthephaseimages,itwaspossibletoverifythatthediabeticgrouphadchangesinthe mechanicalproperties,i.e.elasticitymeasuredbyAFM,astherewasmorevariationinphase anglesinvariousregionsstudied.Intheinterfibrilspace,wheretheproteoglycans,glycosami- noglycans(GAG)chainsandthecovalentbonds(crosslinks)arelocated,thephaseangle increased.Thisfindingisconsistentwithmoreelasticstructures.Qualitatively,wecouldsay thattheinterfibrilstructureshaveincreasedelasticmodulus(Young’smodulus)inthetendons ofdiabeticanimals.Meanwhile,fibrillarregionsshowedwidevariationofphaseanglewith someregionsmorerigidandothersmoreelastic,whencomparedtothehealthygroup(Fig5). PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 6/13 DiabetesAltersStructureoftheTendon Fig3.CharacterizationofsurfaceroughnessusingAtomicForceMicroscopy.3Drepresentationofthetopographyofthe surfaceofthefibersandfibrilsoftheControlGroup(A,CandE)andtheDiabeticGroup(B,DandF)withanareaof30μm,5μm and2.5μm.TheControlGroup(A,CandE)showsnanofibersasuniaxiallyalignedandwellorganizedfibers.TheDiabetic Group(B,DandE),however,showslackofauniaxialpatternincollagenfibrillarnano-structure.Fig3Bdemonstratesgreat irregularityofthefiberbundleswithbreaksinaspectofanabysswithdiscontinuities.Fig3Ddemonstratesthemorphological disorganizationofthebundles,withmodificationofthegraindirectionofthebundlesandjaggedjunctions.Fig3Fshows deformitiesintheformofbuglescollagenfibrils. doi:10.1371/journal.pone.0169513.g003 PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 7/13 DiabetesAltersStructureoftheTendon Fig4.3DrepresentationofthesurfacetopographyofthefibrilsoftheAchillestendonofaDiabeticGroupanimal,showingthechangein directionofthecollagenfibers.Thefibersweredisplacedinoppositedirectionsorexhibitedbifurcations,resultinginlossofmorphologicandcollagen nano-structurefeatures,withchangesinthedirectionofthecollagenfibrilsindicatedbyarrows. doi:10.1371/journal.pone.0169513.g004 Inflammatoryaspects TheAchillestendonsofthediabeticgroupsdidnotpresentincreasedlevelsofInterleukin1— IL1(Fig6A)andtumornecrosisfactor-alpha—TNF-α(Fig6B). Discussion Thisstudyintendstoindicatehowthestateofchronichyperglycemia,derivingfromexperi- mentalTypeIDiabetesMellitus,mayinfluencethehomeostaticdisproportionofthetendon and,consequently,headstofeaturesofchronictendinopathy.Therefore,theAchillestendon waschosen,duetoitsimportantbiomechanicalfunction,especiallyinwalking,anditssuperfi- cialposition[14].Inaddition,somestudieshaveestablishedevidenceforbiomechanicaland PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 8/13 DiabetesAltersStructureoftheTendon Fig5.Phaseimageofsurfacetopographyofthefibrils.A—ControlGroupandB—DiabeticGroup,presentingchangesintheinterfibrilspace,where theproteoglycans,GAGchainsandthecovalentbonds(crosslinks)arelocated,thephaseangle(dark)wasindicatedbyredarrows.Thisfindingis consistentwithmoreelasticstructure.Meanwhile,fibrillarregionsshowedwidevariationofphaseangle,whencomparedtothehealthygroup,suggesting majorchangesintheviscoelasticpropertiesofthediabetictendon. doi:10.1371/journal.pone.0169513.g005 histologicalalterationsintheAchillestendonofdiabeticanimals[11,15,16]andanotherstudy describedthemorphologicalalterationsinthestructureoftheAchillestendonwhichcanpre- disposethepatienttodevelopadiabeticfoot[17]. TheinductionmethodofDMwassimilartothestudiesbydeOliveira[7,11,16],asingle intraperitonealadministrationofSTZsolution,consideredaverifiedandwell-definedproce- durebytheliteratureforthestudyofcomplicationscausedbychronichyperglycemiasimilar toType1DM[18]. TheincrementoftheAchillestendonareaisaclassicfeatureofchronictendinopathyand, typically,isrelatedtooveruseofthestructureinsportsand/orworkactivities[19,20].Itis oftenpossibletoverifythechangeintendonthicknessinclinicalinspection;however,isoften revealedbyimagingtests,suchasultrasound,computedtomographyandMR. Fig6.InflammatoryAspects.QuantificationoftheconcentrationofIL-1AandTumorNecrosisFactor-alfaimageBthroughtheenzyme-linked immunosorbentassay—ELISA.Nostatisticaldifferenceswerefoundbetweenthegroups.Valuesexpressedasmeanandstandarddeviation—P>0.05. doi:10.1371/journal.pone.0169513.g006 PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 9/13 DiabetesAltersStructureoftheTendon Inthisstudy,weobservedthatinthepresenceofchronichyperglycemia,anincrement occurredintheAchillestendonarea,detectedbyinvivoMRexamination,whichwascon- firmedthroughanalysisofthicknessofthetendonbythehistomorphometricmethod. Similarchangesofthicknessmeasuredbydifferentinvivoimagingmodalitieswereprevi- ouslyreportedintendonsofvariousbodyregionsandrelatedtothestateofchronic hyperglycemiainhumanType2diabetics[11,21–24], aswellasinanimalsinducedto Type1DMusingmacrometricstudy[12,17].However,theMRimagesandhistologicalfea- turearemoreaccuratethanotherassessmenttechniquesformeasurementsofthetendon area[24].Therefore,thepresentstudyoriginallyusedtheseevaluationsfortheAchilles tendontoaddressthethemeinexperimentalType1diabetesassuggestedbysystematic review[9]. AmongthefactorsthatcanfavorthethickeningoftheAchillestendonpresentinthedia- beticgroupisthedisorganizedarrangementofcollagenfibersandfibrils.Thisoutcome— observedinthisinvestigationwithinvivoMRandpreviouslyviahistopathologicalexamina- tions[7,24]—wasconfirmedbynanostructuralanalysisindiabeticanimals.Inadditionto these,thedisorganizationofcollagenwasconfirmedbyanotherstudywithatomicforce microscopy,wherethecollagenofrattailswasexposedinvivoandinvitrotohighconcentra- tionsofglucose[25],andalsobystudiesevaluatingMRimagesofhumanswithType2DM [24],reaffirmingourconsiderations. However,despiteobservationsofthechangesinstructureandarrangement,thefibrilsdid notshowchangesintheiraxialstructure,especiallyintheperiodicityoftheDbands.Similarly, otherstudieswithtendinopathy[26]andwithanalysisofcollagenofrattailtendonexposedto chronichyperglycemia[25,27]havenotedsimilarfindings. Mechanicalmodificationspresentedbythediabeticgroupinthisstudywerecharacter- izedbyreducedqualityoftendonpropertiesinresponsetotheoscillatingstimulusofthetip andcantileverofAFM,notablyintheproteoglycansandGAGchainsareawherethecova- lentbonds(cross-links)arelocated.Thisstatecouldleadtofragilecovalentbondsand reductionofstiffnessinitsmatrix,makingthetendonfriable.ThestudybyOdetti,Aragno etal.(2000)[25]assertedthatthesechangescanbeexplainedbythepresenceofnon-enzy- maticglycation(AdvancedGlycatedEnd-Products:AGE)inthecollagenfibrilsindiabetics. Inturn,theincreasingcomplacencyoflinkingstructuresbetweenthecollagenfibrils, observedinourstudy,canexplainthemechanicalchangesandearlyfailurefoundindiabetic animalsbyinvestigationswithmechanicaltractiontestsoftheAchillestendon[11,16]and patellartendon[12]. Toestablishaninflammatoryresponse,tenocytesproducepro-inflammatorycytokines, suchasIL1andTNF-α,whichinturnstimulatethesynthesizecollagen[28].Pro-inflamma- torycytokinesstimulatetheexpressionofmetalloproteinases(MMP1,MMP3,MMP13) whichdegradetheextracellularmatrix(ECM)oftendons.DespitethechangesintheECMof theAchillestendonofdiabeticanimalsobservedinthisstudy,therewasnoincreaseinIL1 andTNF-αlevelsinanimalssevenand24daysafterinductionofDM.Infact,theinflamma- torystatehasnotbeenfoundinconditionsofchronictendinopathy[29,30].Ontheother hand,itisimportanttorememberthattheabsenceofinflammatorymediatorsinthephases investigateddoesnotmeanthattheywerenotpresentinearlystages.However,chronictendi- nopathycanoccurwithlittleornoacuteinflammatoryexpression[31]. ThisstudyevaluatestherelationshipofDMwithchangestotheAchillestendonoveralland doesnotestablishcausalityofthephenomenapresented.Invitroandinvivoanalysesatdiffer- entpointsintimeafterinductioncanhelpclarifythecascadesthatmodifythetendonofthe subjectwithDM.Thereisaneed,however,toconductstudiesthatcouldbetterclarifythe causalpathwaysofthechangestotheAchillestendonpresentedbydiabetics. PLOSONE|DOI:10.1371/journal.pone.0169513 January17,2017 10/13