Efficacy of a membrane composed of polyvinyl alcohol as a vehiclefor releasing of wound healing proteins belonging to latex of Calotropis procera

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ContentslistsavailableatScienceDirect

Process

Biochemistry

j ourna l h o m e pa g e : w w w . e l s e v i e r . c o m / l o c a t e / p r o c b i o

Efficacy

of

a

membrane

composed

of

polyvinyl

alcohol

as

a

vehicle

for

releasing

of

wound

healing

proteins

belonging

to

latex

of

Calotropis

procera

Ingrid

Samantha

Tavares

de

Figueiredo

a

_,

_Márcio

_Viana

_Ramos

b

_,

Nágila

Maria

Pontes

Silva

Ricardo

c

_,

_Maria

_Leônia

_da

_Costa

_Gonzaga

c

_,

Rachel

Sindeaux

Paiva

Pinheiro

d

_,

_Nylane

_Maria

_Nunes

_de

_Alencar

d,∗

a_Centro_{Universitário}_Estácio_do_Ceará_Via_Corpus_Rua_Eliseu_Uchoa_Becco,_n◦600–BairroÁguaFria,CEP:60810-270,Fortaleza,Ceará,Brazil

b_Departmento_de_Bioquímica_e_Biologia_{Molecular/UFC,}_Campus_do_Pici,_Caixa_Postal_6033,_60451-970_Ceará,_Brazil c_Departmento_de_Química_Orgânica_e_{Inorgânica/UFC,}_Campus_do_Pici,_Caixa_Postal_6021,_60455-760_Ceará,_Brazil d_Departmento_de_Fisiologia_e_{Farmacologia/UFC,}_Coronel_Nunes_de_Melo,₁₁₂₇_Rodolfo_Teófilo,_60430-270_Ceará,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27September2013

Receivedinrevisedform2December2013 Accepted27December2013

Availableonline18January2014

Keywords:

Drugdelivery Fibroplasia Inflammation Infrared Thermogravimetry

a

b

s

t

r

a

c

t

ThesolubleproteinsextractedfromthelatexofCalotropisprocera(LP)havebeenshowntopowerfully amelioratedifferentinflammatorydisorderssuchasarthritis,cancerandsepsis.Inthisstudy,we devel-opedapolyvinylalcohol-basedmembraneasanLPdeliverysystemanddeterminedtheefficacyofLPin tissueremodelingandhealingupontopicaltreatmentinmice.Aqueouspolyvinylalcohol(PVA,1%,w/v) solutionwasmixedwithLP(0.2%or1%)whilestirringtoobtainabiomembrane,whichwasthenapplied towounds.PVAandLP-containingPVAmembraneswerestructurallycharacterizedandstandardizedby infraredabsorptionspectroscopyandthermogravimetry.Woundhealingparametersincludedtimeof healingprogressandtissueremodeling(fibroplasiaandcollagen).LP-containingPVAmembrane(0.2% and1%),butnotPVAmembrane,significantlyacceleratedwoundhealingthroughfasterneo-tissue for-mation.Thisprocesswasaccompaniedbyintensifiedfibroplasiaandcollagendepositionasrevealedby microscopicanalyses.ItisthereforeconcludedthattopicalapplicationofLPonwoundsstimulatesthe healingprocessandthattheuseofPVAmembranestocarryLPisareliableapproachfortopicaldelivery ofpharmacologicallyactiveproteins.

1. Introduction

CalotropisproceraisashrubintheApocynaceaefamilythatis nativetotropicalregionswhereitisexploitedbyindigenous peo-ple.Itisalsousedasamedicinalplantinseveralcountries,most notablyinIndia,Pakistan,EgyptandMalaysia,andtoalesserextent inNortheasternBrazil.C.proceraisamilkweedplant,andthepartof theplantmostusedforfolkmedicinalpurposesisitslatex.Thelatex istraditionallyusedtotreatskininfections,inflammatory touch-iness,andotherrelatedailments[1,2].Scientificstudieswiththe wholelatexorpartiallypurifiedlatexfractionshavestrengthened thepharmacologicalpotentialofC.proceraand,toacertainextent, havevalidateditsethnopharmacologicalusages[3,4].

Chemicalanalysisofthelatexrevealedthepresenceofcalactin, calotropin,uscharin,sitosterol, andcalotoxinas phytochemicals

∗Correspondingauthor.Tel.:+558533668339.

E-mailaddresses:nylane@ufc.br,ingridfarma@gmail.com(N.M.N.deAlencar).

[5,6].Proteins,suchasantioxidativeenzymesand pathogenesis-related proteins (i.e. proteins with fungicidal or insecticidal activities),havebeenreported[7,8].Recently,proteaseshavebeen discoveredinlaticiferfluids[9],andproteasesofC.proceralatex havebeeninvestigatedinsomedetail[10].

C.proceralatexexhibitsanti-andpro-inflammatoryactivities, andtheanti-inflammatoryfractionhasbeenshowntoinhibitthe effectofthepro-inflammatoryfraction[11].Theanti-inflammatory fraction,whichiscomposedofthemajorwater-solubleproteins of the latex, has successfully reduced inflammation in several standard models of inflammation, suchas peritonitis and paw edema[12].Later,thepharmacologicalpotentialofthis fraction wasreliablyconfirmedbyitsabilitytomodulateinvivo inflamma-toryconditionsinducedbynociception,cancer,arthritisandsepsis

[13–16].

The use of pharmacologically active proteins as drugs is of interestduetotheirputativelylowefficacyassociatedwith molec-ularinstability,susceptibilitytoproteolysisandlimiteddiffusion becauseof their molecularsize. In this study,both thewound

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healingpotentialoftheproteinfractionofC.proceraandthe effi-cacyofapolyvinylalcohol-basedmembraneasadeliveryvehicle forthelatexfractionweredetermined.

2. Materialsandmethods

2.1. Chemicals

PolyvinylalcoholwaspurchasedfromSIGMA(SãoPaulo,Brazil), ketaminechloridrateandxilazinechloridratewaspurchasedfrom VETBRANDS(Paulínia,SãoPaulo,Brazil),iodopovidonewas pur-chasedfromADV(SãoPaulo,Brazil),andhematoxylin–eosinwas purchasedfromMerckMillipore,SãoPaulo,Brazil.Allother chem-icalswereofanalyticalgradeunlessotherwisespecified.

2.2. Latexproteins

Latexproteinsusedinthisstudywereobtainedusingmethods describedbyAlencaretal.[11].Thesolubleproteinsrecoveredwere lyophilizedandstoredat25◦_C _until_use._Biochemical_and

phar-macologicalcharacterizationofthissamplehavebeenpreviously reportedindifferentmanuscripts[11–14].

2.3. Polyvinylalcohol-basedmembrane

BiomembranecomposedofLP/PVAwastentativelypreparedby fixingPVAat1%mixedwithdifferentpercentofLPstartingat0.1% upto2%.Twofinalcompositionswerechosenforusinginbiological assays.Therefore,basedoninfraredmicroscopydataand thermo-graphyteststhebetterfilm-formingwasobtainedatthemaximum concentrationof1%LPassociatedto1%PVA.0.2%LPin1%PVAwas usedforcomparativepurpose.Thesebiomembraneswereprepared asfollows.

Initially,aPVA(1%,w/v)solutionandLP(0.2%or1%)with10mL ofdistilledwaterwereprepared.Aftercompletehomogenization, PVAwasmixedwithLPtoobtainthefollowingfinalsolutions:1% PVA+1%LPor1%PVA+0.2%LP.Thesesolutionswerestirredat 50◦_C_for₂_h._The_resulting_mixtures_were_filtered_using_a_funnel

withasinteredglassplateundervacuumandthenpoured(20mL) intopetriplates(13.0cm diameter/height1.0cm).Themixtures weredried in anair circulation ovenat 40◦_C _until_the_solvent

wascompletelyevaporatedandadequatelyprotectedtoprevent

contamination.ThePVAmembranescontainingLP werenamed

BioMemPVA/LP0.2%or1%.TheBioMemsweresterilizedby ultra-violetlight(UV)irradiationpriortofurtherexperiments.

2.4. Chemicalmeasurements

2.4.1. Infraredspectroscopyanalysis

Fourier transform infrared spectroscopy (FTIR-ATR) of the BioMemsampleswasrecordedwithaspectrophotometer(Perkin Elmer2000)atambienttemperature(25◦_C)._The_resolution_was

4cm−1_in_the_range_of_4000–400_cm−1_._Samples_were_dried_and

powderedinthepresenceofliquidnitrogenbeforeanalyses.Allof theexperimentswereperformedasdescribedbyGomesetal.[17].

2.4.2. Thermography

FilmsofBioMemsampleswerecharacterizedintermsoftheir thermalstabilityusingmethodsreportedinCasariegoetal.[18]. ThermogravimetriccurveswereobtainedusingaShimadzu(model TGA-50H)thermogravimetricanalyzerwithatemperaturerange of25–800◦_C,_nitrogen_flow_rate_of₂₀_mL/min_and_a_heating_rate_of

10◦_C/min.

2.5. Biologicalassays

2.5.1. Animals

AdultmaleSwissmice,12weeksoldandweighing25±3.0g, wereobtained fromtheCentral Animal Houseof the Universi-dadeFederaldoCeara,Brazil.Theanimalswerekeptinindividual plasticcagesundercontrolledenvironmentalconditions(12/12h light/darkcycles,temperatureof25◦_C_and_humidity₅₅_±_10%)_with

waterandfoodadlibitum(commercialsterilediet–Purina, Paulí-nia,SP, Brazil).Themicewerehandledaccordingtothecurrent guideforthecareanduseoflaboratoryanimals,andassayswere approvedbytheEthicalCommitteeforAnimalUseofthe Univer-sidadeFederaldoCeara(24/2009).

2.5.2. Wound-inducedmodel

Micewereanesthetizedbysubcutaneousinjectionswith10% ketamine chloridrate (115mg/kg) and 2% xilazine chloridrate (10mg/kg)beforetheirsurgicalprocedure[19].Aftershavingthe dorsalsurfaceskin,theregionwaspreparedforasepticsurgery using1%iodopovidonefollowedby70%ethanol.Onewound,either incisionalorexcisional,wasintroduced.

Incisional wounds (0.5cm) were created by a single liner cut. Excisional wounds (1cm2₎ _were _performed _with _a _tissue punch.AftersubcutaneousimplantationofthedifferentBioMems (1.2cm2_), _incisional _wounds _were _sutured _with _mononylon 5-0 (PolySuture, Brazil) so that the healing occurred by primary intention.Excisionalwoundswerenotsuturedsothatthehealing occurredbysecondaryintention.

Animalswererandomlydividedintogroups(n=15pergroup),

as follows: (i) animals with 1% polyvinyl alcohol membrane

implantedintoincisionalor(ii)excisionalwounds,(iii)animals with BioMem PVA/LP 0.2% used in incisional or (iv)excisional wounds,(v)animalswithBioMemPVA/LP1%usedonlyinincisional wounds,and(vi)animalssubjectedonlytosurgicalproceduresthat didnotreceivedanyBioMem(SHAM).

2.5.3. Macroscopicanalyses

Macroscopicdifferencesinneoformedtissueinducedby

sub-cutaneously implanted BioMem were monitored 2, 7, and 14

daysaftersurgery[20].Underanesthesiaaspreviouslydescribed, the entire dorsal skin was removed, and neoformed tissues of incisional wounds were evaluated using the following scoring system: (0): absent; (1): mild; (2): moderate and (3):intense. Excisionalwoundswereevaluatedevery48haftersurgery consid-eringthefollowingaspects:edema,hyperemia,woundareaand re-epithelialization.Inflammatoryclinicalsignals,suchasedema andhyperemia,werescoredas(0):absent;(1):mild;(2):moderate and(3):intense.Woundareasofexcisionalwoundsweremeasured usingapachometerandwerecalculatedasfollows:A=×R×r, whereA,Randrarearea,largerayandsmallray,respectively[21]. Re-epithelializationwasnotedwheneverascabwasnotcovering thewounds[22].

Aftermacroscopicstudy,4mmpunchbiopsiesfromneoformed tissue and excisional wounds with adjacent normal skin were removed (n=5/group)for histological study.Then, theanimals weresacrificedwithananestheticoverdose.

2.5.4. Microscopicanalyses

Neoformed tissuesofincisional andexcisional woundswere fixedinformaldehyde(10%,v/v)andresuspendedin0.01MPBS,pH 7.2over24hinpreparationforhistopathologyprocedures.Sections (5␮m)werestainedwithhematoxylin–eosintoevaluatefibroblast numberandre-epithelialization.Mallory’trichromewasusedto examinecollagenfiberdeposition.

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DFC280camera.Imagesof10randomfieldsofconnectivetissue werecapturedat400×,and20 regionsofinterest (ROI)ofsize 800×1536pixels (=11␮m×21␮m) were extracted toestimate thenumberoffibroblasts.Collagenfiberdepositionwas quanti-fiedinimagesobtainedfrom6randomfieldsat200×in24regions of374×260pixels(=10␮m×7␮m).ImageJ® 1.46software(U.S. NationalInstitutesofHealth,USA)wasusedtocountfibroblasts andevaluatecollagenesisbyusingthe“cellcounter”and “thresh-oldcolor”plug-ins,respectively[20].Thethicknessofthenewly formedepidermisofexcisionalwoundswasmeasuredusing18 imagesof655␮m×492␮minwoundsectionsfromday14after surgery[23].

2.6. Statisticalanalyses

All results are expressed as mean values of groups

(n=5)±standard error of the mean (S.E.M.). Statistical signif-icancewasassessedbyANOVAfollowedbyBonferroni’stestfor multiplecomparisonsofmediaorKruskal–Wallisformedian.The levelofsignificancewasdeterminedasP<0.05usingGraphpad prism,version3.02.

3. Resultsanddiscussion

Poly(vinyl alcohol) (PVA) is a synthetic water-soluble poly-mer,non-toxic,biocompatible,biodegradablewithexcellentfilm formingproperties,which hasbeenstudied widelyfor medical andbiomedicalapplications[24,25].Furthermore,reportsclearly demonstratethedevelopmentof biocompatibleand biodegrad-ableprotein-coatedPVAmatrices,includingblendedwithproteins ofvarioussources,suchasPVA/wheat-gluten,PVA/collagenand PVA/gelatin[26–28].Unprecedented,thispaperdemonstratedthe abilityofPVAinfilm-formingwithpharmacologicallyactive lati-ciferproteins,whichefficientlyimprovewoundhealing.

InfraredspectraobtainedofLP,PVA1%andPVA/LP1areshown inFig.1.Spectra(B)and(C)showfrequenciesthatarecharacteristic ofPVAandLP,respectively,whichdemonstratesthequalityofthese substances.

Thesignalobservedatafrequencyof1735cm−1_is_from_a car-bonylgroup(C O).In thePVA spectrum,thevibrations witha frequencyof854cm−1_are_due_to_C _H_bonds,_while_the_vibrations

as1544cm−1_in_the_LP_spectra_come_from_N _H_and_C _N_bonds

andarecharacteristicoftheproteins.Otherfrequenciesrecorded

Fig.1.FTIRspectra:(A)BioMemPVA/LP1%,(B)PVA1%,and(C)LP.

inthespectra(B)and(C)arecommontobothPVAandLPwithonly minorvariationsofvalues,whichiscommonforthetypeofapplied analysis.

Asimilarspectroscopicprofilewasobservedinthespectrum oftheBioMemPVA/LP1%(spectrumA),inwhichbothsubstances’ spectroscopicsignalsareobserved.Notably,thefrequencies asso-ciatedwithC O,C H,C NandN Hareidentifiable.Inaddition, somefrequenciesappearinthespectrumoftheBioMemPVA/LP1% butinneitherspectrum(B)or(C),mostlikelyindicatingthe occur-renceofVanderWaalsinteractionsandhydrogenbonding(butnot covalentbonding)betweenthetwosubstances.Therefore,thereis indicationthatPVA/LPhasreachedastabledispersion,preventing thecoagulationofproteinandkeepingitcrystalline.

OnlysmalldifferencesinthebandsoftheBioMemPVA/LP1% spectrumareobservedwhencomparedtotheisolatedsubstances’ spectra.Thisobservationconfirmsthatnosignificantchangesoccur uponformationofBioMem(Table1).

ThermogramsofBioMemsamplesunderanitrogenatmosphere arepresentedinFig.2.Thefirstmasslossobservedat50◦_C_is

asso-ciatedwithadsorbedwater(asobservedintheinfraredspectra), whilesampledecompositionoccursat200,300and330◦_C_in_PVA

1%,LP1%andBioMemPVA/LP1%,respectively.Therefore,mixing ofLPinPVAcausesBioMemPVA/PL1%tobecomemorethermally stable.Moreover,theadditionofLPinPVAledtoincreasedfilm flexibility,lesstransparencyandasallowercolor,asviewed macro-scopically.

Previousreportsdemonstratedthatdailytopicalapplicationof watersolublefractionsoflatexfromC.procerareducestheareaof excisionalwounds[29].However,thiseffectwasobtainedusingan aqueousextractofnaturallatexinsteadofapurifiedmoleculeor specificfraction,andnoreportshaveattributedthehealingeffectof thelatexfromC.proceratobioactiveproteinsexistinginthesoluble fraction.

In the present work, a BioMem was made by combining

polyvinylalcohol and soluble proteinsobtainedof the latex of

C.procera(BioMemPVA/LP)toinvestigate theefficacyofthese BioMemsasdrugdeliverysystems.Theeffectsoflatexproteinson thetissueneoformationtogetherwithwoundhealingwere evalu-ated.Initially,concentrationsof0.2%and1%ofLPwerebasedonthe effectiveconcentrationoflatexfractionsreportedpreviously[29]

andtheconcentrationsneededtoformstabledispersionsbetween thecompounds(LP)withinthePVABioMem.

SubcutaneouslyimplantingLP-containingBioMeminincisional woundsinducedneoformedtissue(Fig.3andTable2).No signif-icantdifferencewasfoundbetweentheSHAMand PVAcontrol

groups. BioMem PVA/LP (0.2% and 1%), however, induced the

appearanceofagreateramountofneoformedtissuebythesecond dayaftersurgerywhencomparedtothePVAandSHAMcontrol groups(P<0.05).Afterlongerperiodsoftimefollowing surgery (especiallyafter14days),areductioninthequantityoftissuewas observed,butthedifferencebetweentheexperimentaland con-trolgroupswasmaintainedthroughout.Theseresultsaresimilarto

Table1

Assignmentsofthemeaningbandsoftheinfraredspectra.

Wavenumber(cm−1₎ _Assignments

3428 ␯O H(A,B)

2926 ␯C HofCH2andCH3(A,B)

1725 ␯C O(A)

1643 ␯asCOO−_(A,_B)

1540 ␯CN,␦NH(B)

1267 ␯CN,␦NH(B)and␯CO(A,B)

1095 ␯(C C)(A,B)

852 ␦C H(A)

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Fig.2.Thermogram(A)andderivative(B):PVA1%,LPandBioMemPVA/LP1%.

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Fig.4.Healingprogressonexcisionalwounds:(A)macroscopicaspects,(B)woundareaand(C)progressofre-epithelizationonwoundingarea.Imageswereselectedfrom animalsbelongingtothecorrespondingexperimentalgroups.Dataaremeanofthreeindependentexperimentsandareexpressedasmean±standarderrormean(S.E.M.)

ofaveragewoundarea.*P <0.05indicatesstatisticaldifferencecomparedwiththeSHAMgroupand#_P_<_0.05_with_PVA_group._{ANOVA–Bonferroni}_test;_{Kruskal–Wallis}_test followedbyDunn’stest.

thoseobservedusingnaturallatexbiomembranefromtherubber

treeHeveabrasiliensis,inwhichlatexprotein-dependentinduction

oftissueneoformationwasalsodemonstrated[20].

Astheamountofneoformedtissuewassimilarinboth0.2%and 1%BioMemPVA/LP,thelowestdoseofLPwaschosentoevaluate theeffectonexcisionalwoundhealing.Onalternatedays follow-ingthesurgery,woundsweremacroscopically evaluatedbythe presenceofflogisticsigns,woundareasizeandpresenceofcrust. Duringtheinflammatoryperiod (Day2),edema andhyperemia wereobservedinallexcisionalwounds.Animalsimplantedwith BioMemPVA/LP0.2%showedmoderateedemainrelationtothe

Table2

Semi-quantitativeevaluationofneoformedtissue.

Groups

Days SHAM PVA PVA/LP0.2% PVA/LP1%

2 0(0–1) 1(0–2) 3(2–3)* ₃_(2–3)*

7 0(0–1) 0(0–2) 3(1–3)*# ₃_(2–3)*#

14 0(0–1) 1(0–1) 2(2–3)* # ₂_(1–3)*

Datarepresentthemedianandrangeofscoresfromtwoseparateexperiments:(0) absent,(1)mild,(2)moderateand(3)intensetotissueneoformed.

* _P_<_0.05_indicates_statistical_difference_compared_with_the_SHAM_group₍_n₌₁₅ animals/group,Kruskal–WallistestfollowedbyDunn’stest).

#_P_<_0.05_indicates_statistical_difference_compared_with_the_PVA_group₍_n₌₁₅ animals/group,Kruskal–WallistestfollowedbyDunn’stest).

SHAMandPVA controlgroups (P<0.05),withnodifferencesin hyperemia(Fig.4AandTable3).

A significant difference in wound size reduction in animals

implantedwithBioMemPVA/LP0.2%whencomparedtocontrol

groups(P<0.05)(Fig.4B)wasobservedontheseventhdayafter surgeryandpeakedontheninthday.Moreover,ahigher percent-ageofre-epithelializedwoundareawasmacroscopicallyobserved 2weeksaftersurgeryintheexperimentalgroupascomparedto controlgroups(P<0.05)(Fig.4C).

Thepost-inflammatoryphaseofhealingisfollowedby fibropla-siaandisclinicallycharacterizedbygranulationtissueformation andfibroblastaccumulation[30].Fibroblastsarethemost com-monresident cells responsiblefor themajorityof collagenand elastinsynthesis,andtheyevenorganizetheextracellularmatrix

Table3

Semi-quantitative evaluation of macroscopic flogistic signs induced by biomembranes.

Flogisticsigns SHAM PVA BioMemPVA/LP0.2%

2nd day

Edema 1(0–1) 1(0–2) 2(2–3)*#

Hyperemia 1(0–1) 1(0–2) 2(1–2)

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Fig.5.Fibroplasiaonincisionalwoundhealing.On14thdayaftersurgery,animalsweresacrificedandsamplesofsubcutaneoustissuewereremovedtoperformhistological analyses.Hematoxylin–eosinstainedsectionswereemployedtoestimatefibroplasia.(A)Evaluationoffibroplasiaprogressamongthedifferentexperimentalgroups.Data aremeanofthreeindependentexperimentsandareexpressedasmean±standarderrormean(S.E.M.)ofthenumberoffibroblasts.*P<0.05indicatesstatisticaldifference comparedwiththeSHAMgroupand#_P_<_0.05_with_PVA_group_{(ANOVA–Bonferroni}_test)._Histological_views_of_fibroplasia_on_SHAM_(B),_PVA_(C),_BioMem_PVA/LP_0.2%_(D)_and 1%(E)experimentalgroups(400×magnification).

components [31]. Therefore, some fibroblasts differentiate into myofibroblasts,whichperformwoundcontraction[22,32].

Histological evaluation of incisional wounds showed that BioMemPVA/LPprovidedstimulusforfibroplasia,asevidencedby ahighernumberoffibroblastswhencomparedtocontrolgroups onDay14(Fig.5A–E).Accordingly,thesametrendwasobserved inexcisionalwounds(Fig.S1A–D).Moreover,slidesstainedwith Mallory’strichromewereusedtoestimatethepercentageof colla-gendeposition.AsshowninFig.6A–EandFig.S2A–D,therateof collagendeposition,estimatedaspercentageperarea,wasgreater inneoformedtissueandexcisionalwoundswhenstimulatedwith BioMemPVA/LP(P<0.05).

Previous reports have shown an effective communication

betweenmast cells and fibroblasts through gapjunction inter-cellularcommunication.Physicalorchemical factorsmayresult frommastcellsdegranulation,whichisassociatedwithfibroplasia andcollagendepositionduringcicatricialprocess[33–35].Thus, thestimulusgivenbyBioMemPVA/LPontheinflammatoryphase

mostlikelyinvolvesmacrophageactivationandmastcell degranu-lation,whichmaythereforeberelatedtotheobservedintensified fibroplasiaandcollagendeposition.Measurementofcellularand molecularmarkersinvolvedinthecourseofhealing,suchas neu-trophilinfiltration,mastcelldegranulation,macrophageactivation, and inflammatory mediator levels (e.g. cytokines and nitrites), wouldaidinunderstandingthemechanismunderlyingLPactivity inthecourseofwoundhealing.

This studyconcluded that theprotein fraction of C. procera

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Fig.6. Collagendepositiononincisionalwoundhealing.On14thdayaftersurgery,animalsweresacrificedandsamplesofsubcutaneoustissuewereremovedtoperform histologicalanalyses.Mallory’strichromestainedsectionswereemployedtoestimatecollagendeposition.(A)Evaluationofcollagendepositionamongthedifferent exper-imentalgroups.Dataaremeanofthreeindependentexperimentsandareexpressedasmean±standarderrormean(S.E.M.)ofthecollagencontentestimatedbythetotal numberofpixelsinafixedarea(10␮m×7␮m),expressedinpercent.*P<0.05indicatesstatisticaldifferencecomparedwiththeSHAMgroupand#_P_<_0.05_with_PVA_group (ANOVA–Bonferronitest).HistologicalviewsofcollagendepositiononSHAM(B),PVA(C),BioMemPVA/LP0.2%(D)and1%(E)experimentalgroups(200×magnification).

cycleoffemalescouldinterferewiththemechanismoftissuerepair

[36].Thecompletepharmacologicalbenefitsofthissystemonthe courseofhealingarecurrentlyunderevaluation.Thesuccessof thisfirstbiotechnologicalapproachwillpermitfurtherevaluation oftheefficacyofpolyvinylalcohol-basedmembranesforLP deliv-eryastreatmentforcutaneouslesionsofleprosypatientsinthe future.

Acknowledgements

The study has been supported by grants from the

follow-ing Brazilian Agencies: Concelho Nacional de Desenvolvimento CientíficoeTecnológico(CNPq)andFundac¸ãoCearensedeApoio

ao Desenvolvimento Científico e Tecnológico (FUNCAP). The

Incentive of International Foundation for Science (IFS) support provided toMVR at thebeginning of his careeris also greatly

appreciated. M.V.R. and N.M.N.A. are senior researcher fellows ofCNPq.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.procbio.2013.12.015.

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