Structural are crucial to the benefits of guar gum in experimental

ContentslistsavailableatScienceDirect

Carbohydrate

Polymers

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Structural

characteristics

are

crucial

to

the

benefits

of

guar

gum

in

experimental

osteoarthritis

Rondinelle

R.

Castro

_,

_Christine

_Maria

_M.

_Silva

_,

_Rodolfo

_M.

_Nunes

_,

Pablyana

L.R.

Cunha

_,

_Regina

_Celia

_M.

_de

_Paula

_,

_Judith

_P.A.

_Feitosa

_,

_Virgínia

_C.C.

_Girão

_,

Margarida

M.L.

Pompeu

_,

_José

_Alberto

_D.

_Leite

_,

_Francisco

_A.C.

_Rocha

a_{SuperiorInstituteofBiomedicalSciences,StateUniversityofCeará,Fortaleza60714-903,Brazil} b_{DepartmentofInternalMedicine,FederalUniversityofCeará,Fortaleza60430-170,Brazil}

c_{DepartmentofOrganicandInorganicChemistry,FederalUniversityofCeará,Fortaleza60451-970,Brazil} d_{DepartmentofMorphology,FacultyofMedicine,FederalUniversityofCeará,Fortaleza60430-170,Brazil} e_{DepartmentofPathology,FacultyofMedicine,FederalUniversityofCeará,Fortaleza60441-750,Brazil} f_{DepartmentofSurgery,FederalUniversityofCeará,Fortaleza60430-170,Brazil}

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t

i

c

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e

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Articlehistory:

Received2February2016

Receivedinrevisedform1May2016 Accepted11May2016

Availableonline13May2016

Chemicalcompoundsstudiedinthisarticle:

Galactomannan(PUBCHEMCID:439336) Chonsurid(PUBCHEMCID:24766)

Keywords:

Guargum Osteoarthritis Oxidation Pain Sulfation

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b

s

t

r

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c

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Protein-freeguargum(DGG)wasoxidized(DGGOX)orsulfated(DGGSU)byinsertionofnewgroupsin C-6(manose)andC-6(galactose),forDGGOXandDGGSU,respectively.Ratsweresubjectedtoanterior cruciateligamenttransection(ACLT)oftheknee,jointpainrecordedusingthearticularincapacitation test,andtheanalgesiceffectofintraarticular100␮gDGG,DGGOXorDGGSUsolutionsatdays4–7was evaluated.OthergroupsreceivedDGGorsalineweekly,fromdays7to70andjointdamageassessed usinghistologyandbiochemistryasthechondroitinsulfate(CS)contentofcartilage.Themolarmassof CSsampleswasobtainedbycomparingtheirrelativeelectrophoreticmobilitytostandardCS.DGGbut notDGGOXorDGGSUsignificantlyinhibitedjointpain.DGGsignificantlyreversedtheincreaseinCS, itsreducedelectrophoreticmobility,andhistologicalchangesfollowingACLT,ascomparedtovehicle. StructuralintegrityaccountsforDGGbenefitsinexperimentalosteoarthritis.

©2016ElsevierLtd.Allrightsreserved.

1. Introduction

Osteoarthritis(OA)isaleadingcauseofdisabilityandchronic painwithincreasingprevalenceandcoststohealthcaresystems. Therecognitionthatinflammatorycellsplay amajorroleinOA pathogenesishasledtotheconceptofanimportant immunome-diatedinflammatorycomponentinOApathogenesis(Berenbaum, 2013).OAtreatmentstillreliesonattemptstoprovidesymptom improvement whereas chondroprotective compounds, meaning treatmentstoalterOAprogressionand/oroutcome,areanunmet need(McAlindonetal.,2014).

Viscosupplementationhas beenproposedas a termto indi-catethe recoveryof theviscoelastic properties of thesynovial

∗Correspondingauthor.

E-mailaddress:arocha@ufc.br(F.A.C.Rocha).

fluidaftertheadministrationofhighmolarmasshyaluronicacid solutions or its analogues (Hunter, 2015). Despite a persistent debateontheclinicalefficacyofviscosupplementation,currentOA treatmentguidelinesrecommendthisstrategy,particularlytotreat patientswithkneeOA(Bruyèreetal.,2014;Hochbergetal.,2012; McAlindonetal.,2014).Systematicreviewsandmeta-analysisof dataonviscosupplementationefficacyarecontroversialprobably becauseofdifferentselectionofarticlesandalackof standardiza-tionofpatientselectionandevaluation(Hunter,2015).

Themechanismsresponsiblefortheviscosupplementation effi-cacy are yet to be demonstrated. Though some claim that the higher the molar mass of the agent, coupled to the gel state, thebettertheresults,there arenodefinitivedatatoprovethis assumption(Bannuruetal.,2015).Indeed,highmolarmasshylans (around 106_{g/mol) had similar efficacy,as compared to lower}

masscompounds (5–7.5×105_{g/mol)in providing pain reliefin}

OA(PasqualiRonchettietal.,2001).Thesustainedclinicalrelief

http://dx.doi.org/10.1016/j.carbpol.2016.05.031

Fig1. FTIRspectrumofdeproteinizedguargum(DGG),oxidized(DGGOX),and sulfated(DGGSU)derivatives.

thatmaybeachievedafterasetof3–5injectionsorevena sin-gleinjectionofthesecompoundsmaylastforupto8–12months (Kirwan, 2001) argues against a local rheological effect to explainthetherapeuticmechanism.Consideringthatendogenous hyaluronicacidclearancefromthejointfluidisestimatedat20h andin OAjointthisclearance timeis evenless (Hunter, 2015), thepossibility thatexogenously administeredhyaluronates last lessthan1dayinthejointisverylikely.Furthermore,theissueof whetherviscosupplementationagentsdisplayachondroprotective effectinOAisalsoyettobedefined.

Guargumisahighlyviscouspolysaccharidederivedfromthe seedendospermoftheplantCyamopsistetragonolobus.Itis pre-dominantlycomposedofagalactomannanwithalongcentralchain ofmanoseresidues,joinedbytype␤-(1→4)glycosidiclinksand residuesofgalactosejoinedbytype␣-(1→6)linksattachedtothe sidesofthemannancentralcore.Thisgumwaspurifiedbyfour dif-ferentmethodsinordertoreducetheimpurities,especiallyprotein residues(Cunha,Maciel,Sierakowski,dePaula,&Feitosa,2007).

UsinganexperimentalOAmodelinrats,wedemonstratedthat theintraarticular(i.a.)injectionofthepurifiedDGG, meaninga protein-free guargum derivative,provided analgesia similarto thatofHylanG-F20,acommerciallyavailable viscosupplementa-tionagent(Castroetal.,2006).Additionally,thegalactomannan providedanalgesiawhenitwasgivenasaviscousorsaline solu-tion.Thatviscouspreparationpresentsaviscositysimilartothat ofHylanG-F20,110and120Pas,respectively,buttheviscosityof thegalactomannansolutionwasonly3.6Pas,atthesameshearrate andtemperature(Cunha,Castro,Rocha,dePaula,&Feitosa,2005). Thus, weproposedthat viscosupplementationdoesnotdepend onlyontheviscoelasticpropertiesofthecompound.Rather,itcould derivefromalocalpharmacologicaleffectyettobedescribed.

Inthepresentstudy,throughderivatizationoftheDGG (oxida-tionorsulfation)weprovidefurtherevidencethatthebiochemical structure,ratherthantheviscosity,accountstoexplaintheclinical effectsofthispolysaccharideinexperimentalOA.Additionally,the datashowthati.a.administrationofaDGGsolutionpreventsjoint damageinanOAmodel.

Male Wistarrats(180–200g) fromourown animalfacilities were used throughout the experiments. Animals were housed in cages (6/cage) in temperature-controlled rooms with a 12h light/darkcyclewithfreeaccesstowaterandfood.Atthestartof anyexperiments,ratswere2.5monthsofage.Alleffortsweremade tominimizeanimalsufferingandthenumberofanimalsused.The experimentalprotocolwasapprovedbyourlocalethics commit-tee(protocolnumber113/07)thatfollowedtheguidelinesofthe BrazilianCollegeofAnimalExperimentation(COBEA).

2.2. Methods

2.2.1. Sulfationofguargum

Theprotein-freeDGGwassulfatedusingmethodologyearlier reported by Moura Neto, Maciel, Cunha, de Paula, and Feitosa (2011)andrecentlyusedforsulfationofa galactomannanfrom Dimorphandragardneriana(MouraNetoetal.,2014).Some mod-ificationswereperformed,asfollows:amassof3gofDGGwas dissolvedin225mLofformamideovernight,followedbythe addi-tionof60mLpyridine.Thesolutionwascooledto4◦_Cand18mLof chlorosulfonicacid(CSA)wasdroppedoveraperiodof3h.The pro-portionSR/SU(molarratioofsulfationreagenttosugarunit)was maintainedin14.6.After12hat13◦_{C,thesolutionwas} neutral-izedwithNaHCO3,dialyzedagainstwater,thesolidprecipitated,

washedmanytimeswithethanol,andfiltered.Thedryproductwas denotedasDGGSU.

2.2.2. Oxidationofguargum

The protein-free DGG was oxidized following the method reportedbySierakowski,Milas,Desbrièes,andRinaudo(2000)and Cunhaetal.(2007),asfollows:amassofDGG(2g)wasdissolved in1Ldistilledwater understirringovernight.Thesolutionwas cooledinanicebath,sodiumhypochlorite(9mL)wasadded,and pHadjustedto9.2.TEMPOreagent(18.4mg)andNaBr(160mg) wereadded.TheoxidationproceededatconstantpH,adjustedwith NaOH.Borohydride(50mL)wasincludedtostopthereaction.The pHwasdecreasedto7,thegumprecipitatedwithEtOH,andafter 12hinrefrigerator,filtered,andwashedwithEtOH.DGGOXisthe designationoftheproduct.

2.2.3. Characterizationofguargumderivatives

Fig.2.13_{CNMRspectrumofdeproteinizedguargum(DGG),oxidized(DGGOX),andsulfated(DGGSU)derivatives.}

ShimadzumodelIRPrestige21spectrophotometerbetween400 and4000cm−1_{.Thepositionoftheintroducedgroupswasdefined}

usingNuclear MagneticResonanceanalysis,withuni(RMN 1_H,

RMN13_{CandDEPT135)orbidimensionaltechniques}

(Heteronu-clearMultipleQuantumCoherence,1_H–13_{CHMQC).Thegumswere}

dissolvedindeuteratedwater for12handanalyzedinaBruker modelAvance-DMX-500spectrometer.

Thestaticlight scattering measurementswereperformed in a multi angle photometer fromMalls Dawn Wyatt Technology Corporation,workingwithaHe-Nelaser(632.8nm).Thegums con-centrationsvariedfrom3.0×10−4_to1.15

×10−3_g/mLinNaNO 3

0.1mol/L,usedafterfiltrationthrough0.2and0.45␮mMillipore membranes.

Theapparentviscositywasdeterminedin 1%(w/v)aqueous solutionsatshearrate200s−1_{,andtemperature25}◦_C,bytheuse ofaRheometerBrookfieldcone-platemodelLV-DVIII.The intrin-sicviscosity([␩])wascalculatedfromflowtimeofsolutionsina capillaryUbbelohdeviscometerfromCannonInstrumentsmodel 1I-71.Thesolventusedwas0.1mol/LNaCl,andthetemperature wascontrolledat25◦_{C.Hugginsplotwasappliedforthe[␩]} deter-mination.

Themolarmassdistributionwasestimatedbygelpermeation chromatographyin aShimadzuequipmentand refractiveindex detector(RID-6A).Anultrahydrogellinearcolumn(7.8×300mm, Exclusionlimit7.0×106_gmol−1_,WatersTM_{Corporation),0.1mol/L}

NaNO3 assolvent,flowat0.5mL/min,andambienttemperature

weretheexperimentalconditionsforGPCanalysis.

2.2.4. Analysisofglycosaminoglycansfromthearticularcartilage Thecartilageofthedistalfemoralextremitieswasexcisedwith asurgicalbladeimmediatelyaftersacrificeofanimalsandweighed afterovernightdrying(80◦_{C).Thematerialwassubjectedto} pro-teolysisusing PROLAV 750TM _{(Prozyn, SP, Brazil) suspended in}

Tris–HCl/NaCl50mmol/L/150mmol/Lbuffer(pH8.0),andfurther precipitatedinabsoluteethanol,followedbydilutionindistilled water. Thismaterial wasseparated ona 0.6% w/vagarose gel-electrophoresisindiaminopropaneacetatebuffer(50mmol/L,pH 9.0).Gelswerestainedwith0.1%w/vtoluidine-blue(Bezerraetal., 2004).Forcomparison,standardC4S,C6S,andheparansulfatewere subjectedtothesameprotocol.Quantificationwasmadeby densit-ometry(525nm).Datawereexpressedas␮gCS/mgdriedcartilage.

2.2.5. Anteriorcruciateligamenttransection(ACLT)model

Rats were anesthetized with i.m. ketamine (50mg/kg) and xylazine(10mg/kg).After preparingfor localaseptic surgery, a parapatellarincisionwasmade,followedbylateraldisplacementof thepatella,therebyprovidingaccesstothejointspace.Theanterior cruciateligamentistheneasilyvisibleandwassurgicallyexcised tryingtoavoiddamagetotheunderlyingcartilageandmeniscus. Theincreaseinanteriordisplacementofthetibiainrelationtothe femurwasusedtoassurethattheligamentwastransected.The jointcapsuleandskinweresuturedwithVycril(6-0)and monony-lon(4-0)threads,respectively.Ashamgroupwassubjectedtoskin incision,patelladisplacement,andexposureofthejointwithout damagetotheligaments,followedbywoundclosure.Anaivegroup receivednomanipulation.

2.2.6. Measurementofjointpain

Thearticularincapacitationmethod,asdescribedearlier,was usedwithmodifications(Castroet al.,2006).Theanimalswere puttowalkonasteelrotarydrum (30cmwide×50cm diame-ter),whichrotatesat3rpm.Speciallydesignedmetalgaiterswere wrappedaroundbothhindpaws.Afterplacementofthegaiters,the animalswereallowedtowalkfreelyforhabituation.Therightpaw wasthenconnectedviaasimplecircuittoamicrocomputerdata input/outputport.Thepawelevationtime(PET),asrecordedinthe presentstudy,isthetimeinsecondsthatduringa10-minperiod thehindpawisnotincontactwiththecylinder.Thearticular inca-pacitationmeasuredisinhibitedbyclassicalanalgesiccompounds leadingtotheassumptionthatitreflectsjointpain.Forcomparison betweentreatedgroups,resultsarereportedasthemeanofthePET obtaineddaily,startingatday4,untilday7.

2.2.7. Histopathology

Fig.3. Chemicalstructuresofdeproteinizedguargum(DGG),oxidized(DGGOX),andsulfated(DGGSU)derivatives.

forboth condylesandexpressedasone resultforeach sample. Semi-quantitativehistopathologicalgradingwasperformedbytwo independentpathologists(VCCG,MMLP)blindedtogroup alloca-tionaccordingtotheOsteoarthritisResearchSocietyInternational (OARSI)histopathologygradingandstagingsystem(Pritzkeretal., 2006).Themaximalpossiblefinalscore(meanofmeasuresmadeby thetwopathologists)was24.Resultsareexpressedasthemedian (interquartilerange-IQR)valuesforeachtreatmentgroup.

2.2.8. Pharmacologicalmanipulation

Inordertoinvestigatewhetherthebiochemicalstructureis rele-vanttotheanalgesiceffectprovidedbythegalactomannan,groups ofratssubjectedtoACLTreceivedintra-articular(i.a.)injectionsof 100␮gin50␮LsalinesolutionsofeitherDGG,DGGSUorDGGOX derivatives,atday4followingACLT.Thearticularincapacitation, asreflectingjointpain,wasmeasureddaily,untilday7.Inanother setofexperiments,in anattempttoevaluatea potential chon-droprotectiveeffect ofthe galactomannan,meaninga potential jointstructuralbenefit,groupsofratsreceivedeitherDGGsolution (100␮g/50␮Li.a.)orsalineonceaweek,startingatday7,during 10weeks.

2.2.9. Statistics

Resultsarepresentedasthemeans+95%CIforpain measure-mentsand medians(IQR)for histologymadeonsix animalsin

eachgroup.Assessmentofnormalityofthepainbehaviourdata wasdoneusingtheD’Agostino-PearsonOmnibustest.Differences betweenmeanswerecomparedusingone-wayanalysisofvariance (ANOVA)followedbyTukey’stest;P<0.05wasconsideredas sig-nificant.Forcomparisonofmedians,theKruskal–Wallistestwas used,followedbyDunn’stest;P<0.05wasconsideredsignificant.

3. Resultsanddiscussion

3.1. Characterizationofproteinfreeguargumandderivatives

TheFT-IRspectra(Fig.1)ofderivativesareverydifferentfrom thatoftheproteinfreeDGG.IntheDGGSUanewandintenseband appearsat1262cm−1_{,assignedtoasymmetricalstretchingofS O}

vibration.SpectraldifferencecanbeseeninDGGOXincomparison withunmodifiedDGG.AbandattributedtoC Ostretching vibra-tionisnowpresentinDGGOXspectrumduetotheinsertionof COOHgroup.

Fig.4.Gelpermeationchromatographycurvesofdeproteinizedguargum(DGG), oxidized(DGGOX),andsulfated(DGGSU)derivativessolution.

groupsper6carbons.FromdataontheSandCcontent,andusing Eq.(1)(Melo,Feitosa,Freitas,&dePaula,2002),theDSvalueof 0.60wascalculated.ThedegreeofsubstitutionfortheDGGOXwas 0.36,closetothevalueobtainedfortheoxidationof galactoman-nanfromLeucaemaleucocephalainsimilarcondition(Sierakowski etal.,2000).

DS=

( S%

atomicmassofS)

( C%

atomicmassofCx6)

=2.25(S%

C%) (1)

ThederivativeswereanalyzedbyNMRinordertoverifythe prefer-entialpositionofsulfationandoxidation.Comparisonbetweenthe

13_{CNMRspectrumofDGGOXandDGG(Fig.2)revealedtwomain}

differences:(a)theabsenceofthesignalat63.7ppmpresentinthe unmodifiedDGG,and(b)presenceoftwonewsignalsatthe177.5 and177.8ppm.TheabsenceofthesignalfromC-6offreemannose indicatesthatthismonosaccharidewastotallysubstitutedinthe oxidationreaction.ThenewsignalsareduetothepresenceofC O groupfromCOOH.

The1_H–13_{CHSQCspectra(SupplementaryinformationFig.S1)}

exhibittheshifttoalower␦valueoftheprotonH-1frommannose residueinDGGOX,whichconfirmsthesubstitutionatC-6from mannose.Newsignalsat70.3,75.6,and100.5ppmwereobserved in13_{CNMRspectrumofsulfatedgum(Fig.2).Thefirstoneisdueto}

theinsertionofsulfategroupintheprimarycarbon(C-6).Theproof thatthisnewsignalisattributedtosubstitutedCH2wasobtained

fromDEPT135(SupplementaryinformationFig.S2).Thesignals fromCH2appearinoppositeamplitudetothoseofCHandCH3.

Fig.5.Sulfationoroxidationofdeproteinizedguargum(DGG)abrogatesanalgesia inanosteoarthritismodel.RatsweresubjectedtoACLT.Jointpainwasassessed dailyastheincreaseinthepawelevationtime(PET),usingthearticular incapac-itationtest.AshamgroupwassubjectedtothesurgicalprocedurewithoutACLT andreceivedsalinebygavage.Thenon-treated(NT)groupreceivedsaline.Groups received100␮g/50␮Li.aofeitherDGG,sulfatedDGG(DGGSU)oroxidizedDGG (DGGOX),starting4daysafterACLT.ResultsarereportedasthemeanofthePET (s/10min)obtaineddaily,startingatday4,untilday7;n=6animals/group;*P<0.05 comparedtoNTusingone-wayANOVA,followedbyTukey’stest.

Thesignalsat75.6and 100.5ppmareattributedtoC-5andC-1 fromsulfatedgalactose,shiftedfromoriginal␦.

Thesulfationoccurredpreferentiallyinthecarbon6ofthe galac-toseresidueoftheoriginalgalactomannanwhereastheoxidation occurredpreferentiallyinthecarbon6ofthemannoseresidue,as showninFig.3.Theguargumderivativeswerealsocharacterized byviscosityand molarmassdetermination.Thegelpermeation curves (Fig. 4)indicate chain degradation in oxidized and sul-fated samples.The guar gumshows a monomodal distribution withelutionvolume7.6mL.Theoxidizedderivativealsopresents amonomodaldistribution,butwiththeelutionvolumeshiftedto highervolume(7.8mL).AlowermolarmassisobtainedforDGGOX. ThecurveprofileforDGGSUisdifferent.Amainpeakoccursat 7.8mL,buttwoshoulderscanbevisualized,at7.4and9.4mL.The distributionofmolarmassismoreheterogeneous,withlow frac-tionofunsulfatedmaterialandotherfractionwithintensechain degradation(9.4mL).Duetotheionicchargeofthederivativesand theunavailabilityofproperstandardsthemolarmasscannotbe quantifiedbythiskindofGPC/detector.Theweightaveragemolar mass(Mw)wascalculatedbylightscatteringexperiments,basedon

Zimmplots.Allderivativesshowlowermolarmassthanthe orig-inalguargum.TheDGGpresentsMw3.9×106_{g/mol,andDGGOX}

andDGGSUshow2.6×106_and2.8×106_{g/mol,respectively.The}

slightlyhigherMw valueofDGGSU,incomparisonwithDGGOX

maybeduetothepresenceofthefractionoflowelutionvolume, andtheinfluenceofhighdimensionchainovertheMw.

Table1

Maincharacteristicsofunmodifiedandmodifiedguargum.

Characteristics Guargum

Non-modified sulfated oxidized

Insertedgroup – OSO3Na COOH

Substitutiondegree 0.00 0.60 0.36

Insertposition – C-6-galactose C-6-mannose

Intrinsicviscosity(dL/g)a _{6.2 4.9 2.2}

Weightaveragemolarmass(g/mol) 3.9×106 _2.8

×106 _2.6

×106

Apparentviscosity(mPas)b _{58 39 9}

a_{Solvent0.1mol/LNaClat25}◦_C.

themeans±SDofCS(␮g/mgofdriedcartilage)ingroupsof6animals.*P<0.05comparedtosham;#_{P<0.05,comparedtoNT(one-wayANOVA,followedbyTukey’stest).}

(B)Cartilagesampleswereranona6%polyacrylamidegelelectrophoresis.Therelativemobilitywasobtainedbytheratiobetweenthepeakpositionofthesampleandthe peakpositionofstandardchondroitin-4-sulfate(C4S).TheMwoftheCSfromthevariousgroupsampleswasassessedbydeterminingitsrelativemobilitytostandardC4S

raninparallelona6%polyacrylamide-gelelectrophoresis.ValuesrepresenttheratioofthepeakpositionofthedigitalizedgelimagecurvesandthatofC4S,arbitrarilyset at1.0;n=6animals/group.#_{P<0.05comparedtoNT(one-wayANOVA,followedbyTukey’stest).}

Theintrinsicviscosityandtheapparentviscositywere deter-minedand thevalues showedinTable1.TheDGGOXpresents thelowestintrinsic(2.2dL/g)andapparentviscosity(9.0mPas). The unmodified DGG exhibits the highest values ([␩]=6.2dL/g and␩app=58mPas).IntermediatevaluesareobtainedforDGGSU

([␩]=4.9dL/gand␩app=39mPas).

3.2. Structuremodificationsoftheguargumabrogatejoint analgesia

As we have reported previously, a single administration of a DGG solution(100␮g/50␮L)at day4 following ACLT signifi-cantlyreducedjointpain,ascomparedtoanimalsthatreceived saline(Fig.5).AdministrationofeithertheDGGOXortheDGGSU preparationssignificantlypreventedjointanalgesiapromotedby the original DGG solution (Fig.5), meaning that the structural integrityofthepolysaccharideiscrucialtoitsanalgesiceffect.It shouldberemarkedthatthemolarmassofthecompounds,aswell astheirintrinsicviscosity,werenotsignificantlymodifiedafter oxi-dationorsulfationofthegalactomannan.Coupledtoourprevious reportshowingthatthegalactomannaninhibitionofjointpainin thisOAmodeloccursregardlessofusingagelorsalineformulation (Castroetal.,2006),thisfindingaddssupporttoourproposalthat pharmacologicratherthanrheologicalmechanismsaccountforthe analgesia.

We are not aware of similarmodifications in thestructural ofcommerciallyavailableviscosupplementationagents. Rheolog-ical properties of hyaluronic acid preparations are claimed to be responsible for the beneficial effect of those compounds in patientswithOA.Apparently,high-molarmassHApreparations improveviscoelasticitythusamelioratingjointlubrication. How-ever,invitrostudiesareusedtosupportthisassumption,whichis questionedforinstancebytheobservationthatasustainedreliefis experiencedbypatientsexposedtoviscosupplementation.In keep-ingwiththisskepticism,arecentinvivostudyinrabbitsshowed that theviscoelasticityofthe synovialfluidwassimilar in ani-mals that received saline, as well as low or high molar mass HA preparations, but only the high molecular weight HA was chondroprotective(Elmorsyet al.,2014).Thefactthat ourguar gumpreparationsaresaline-solubleand theirrheologic proper-tiesdifferfromthoseHApreparations,whichareviscous(Elmorsy etal.,2014),arguesthatmolecularstructure ratherthan intrin-sicviscosityaccountforthebiologicalactivityofintraarticularly administered polysaccharidesin OA. Thus, ifwe have toadmit

Fig.7.DGGsolutionpreventsjointdamageseenathistologyintheACLTmodel.Rats weresubjectedtoACLTandkilledafter70days.GroupsreceivedGG(100␮g/50␮L i.a)orsaline(50␮Li.a)(NT),weekly,startingatday7untilsacrificeatday70 afterACLT.Femoralextremitieswereexcisedandprocessedforhematoxylin–eosin andtoluidine-bluestaining;evaluationusedtheOARSIgradingandstaging sys-tem.Resultsaremedians(range)ofn=6animals/group.*P<0.05comparedtoNT; #P<0.05,comparedtoSham(Kruskal–Wallis,followedbyDunn’stest).

Fig.8.DGGsolutionpreventshistologicalalterationsintheACLTmodel.RatsweresubjectedtoShamoperation(AandD)orACLT(BandE).Thedistalfemoralextremities wereexcised70daysafterACLTandprocessedforhematoxylin–eosin(leftpanel)ortoluidine-blue(rightpanel)staining.Groupsreceivedsaline(BandE)or100␮g/50␮L DGG(CandF)i.art.,weekly,startingatday7untilsacrificeatday70afterACLT.Thisrepresentativeillustrationshowspreservationofthearticularcartilageliningand stainingintensityinshamgroup(AandD),ascomparedtothesalinegroup(BandE)thatshowsfissure,erosion(arrow)andareaofdenudation(arrow-head)thatarenot noticedinthegroupthatreceivedDGG(EandF)(originalmagnification×40).

as1.0.Themolarmass(MM)canthenbeestimatedasinversely relatedtotherelative mobility ofthesample. Asa comparison C6S(MM=5.88×104_{g/mol),underthesameconditions,presents}

arelativemobilityof0.708.

3.3. Galactomannanpreventsstructuraljointdamagein experimentalOA

Aswe showedpreviously (Silva et al., 2009), cartilage sam-plesfromratssubjectedtoACLTdisplayanincreaseintheGAG contentaswellasareducedelectrophoreticmobilityoftheGAG, as compared to sham-operated animals. In the present study, administrationoftheoriginallypurifiedDGGsolutionsignificantly reversedboththeincreaseinGAGcontentandthealtered elec-trophoreticmobilityoftheextractedGAG,ascomparedtoanimals thatreceivedsaline(Fig.6a–b),thusindicatingachondroprotective effectofthegum.

TheincreaseintheGAGcontentofthecartilagesamplesfrom theOAgroupwerereportedpreviouslyinourhands(Silvaetal., 2009)aswellasinotherstudies(Hosseininia,Lindberg,&Dahlberg, 2013).Ithasbeenproposedthatthisincreaserepresentsanearly

anabolicresponsetotheinjurythatsubsides,leadingplacetoa catabolicstageandprogressivedestructionofthejointcartilage. Byinterferingwiththeongoingprocess,theadministrationofDGG apparentlypreventedjointdamagetoanimalssubjectedtoACLT. Thoseeffects wereassociatedtoapronouncedinhibitionofthe structuraldamageathistopathology.Hence,inadditiontoprovide analgesia,theDGGsolutionhadajointprotectiveeffect.

3.4. Galactomannanadministrationpreventsjointdamageat histopathologyinexperimentalOA

Fig.7showsthehistologicalscoresatboththefemoraland tib-ialextremitiesofratssubjectedtoACLTwhich,asexpected,were clearlymoresevereandextensive,ascomparedtosham-operated animals. Administration of the purified DGG, using the same concentration(100␮g)thatwasanalgesic,significantlyprevented the severity and the extension of the joint damage seen at histopathology,ascomparedtoanimalsthatreceivedthevehicle.

ingredient in all commercially available viscosupplementation agents, wespeculate that structural integrity is crucialto their clinicalefficacy.

4. Conclusions

Tothebestofourknowledge,webelievethisisthefirststudy toshowthatstructuralmodificationsapartfromthemolarmass ofthecompoundarecrucialtodefineanalgesiaassociatedto vis-cosupplementation.SincewehavepreviouslyshownthatDGGis effectiveregardlessofbeingaviscousorsaline-solublepreparation, ourpresentdatafurtherindicatethatpolysaccharidesprovide anal-gesiaandchondroprotectioninOAthroughpharmacologicalrather thanrheologicalmechanisms.

Acknowledgements

This work was supported by grants 302218/2014-9 and 459334/2014-0 from CNPq (Conselho Nacional de Desenvolvi-mentoeTecnológico-Brasil).TheauthorsaregratefultoDr.Maria RitaSierakowski(UFPr)forhersupportwiththemodificationof thepolysaccharide.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.carbpol.2016.05. 031.

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