Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier for Cellular Bioprocesses Re

20 Pages·2016·4.14 MB·English

RESEARCHARTICLE
Hyaluronan Hybrid Cooperative Complexes
as a Novel Frontier for Cellular Bioprocesses
Re-Activation
AntoniettaStellavato1,LuisanaCorsuto1,AntonellaD’Agostino1,AnnalisaLaGatta1,
PaolaDiana1,PatriziaBernini2,MarioDeRosa1,ChiaraSchiraldi1*
1 DepartmentofExperimentalMedicine,SectionofBiotechnology,MedicalHistologyandMolecular
Biology,SecondUniversityofNaples,Bioteknet,Naples,Italy,2 IBSAFarmaceuticiItaliaSrl,Lodi,Italy
*[email protected]
a11111
Abstract
HyaluronicAcid(HA)-baseddermalformulationshaverapidlygainedalargeconsensusin
aestheticmedicineanddermatology.HA,highlyexpressedintheExtracellularMatrix
OPENACCESS (ECM),actsasanactivatorofbiologicalcascades,stimulatingcellmigrationandprolifera-
tion,andoperatingasaregulatoroftheskinimmunesurveillance,throughspecificinterac-
Citation:StellavatoA,CorsutoL,D’AgostinoA,La
GattaA,DianaP,BerniniP,etal.(2016) tionswithitsreceptors.HAmaybeusedintopicalformulations,asdermalinducer,for
HyaluronanHybridCooperativeComplexesasa woundhealing.Moreover,intradermalHAformulations(injectableHA)provideanattractive
NovelFrontierforCellularBioprocessesRe-
tooltocounteractskinaging(e.g.,facialwrinkles,dryness,andlossofelasticity)and
Activation.PLoSONE11(10):e0163510.
restorenormaldermalfunctions,throughsimpleandminimallyinvasiveprocedures.Bio-
doi:10.1371/journal.pone.0163510
logicalactivityofacommerciallyavailablehyaluronicacid,Profhilo®,basedonNAHYCO™
Editor:NikosKKaramanos,UniversityofPatras,
technology,wascomparedtoH-HAorL-HAalone.Theformationofhybridcooperative
GREECE
complexeswasconfirmedbythesuddendropinη valuesintherheologicalmeasure-
Received:May26,2016 0
ments.Besides,hybridcooperativecomplexesprovedstabletohyaluronidase(BTH)
Accepted:September9,2016
digestion.Usinginvitroassays,basedonkeratinocytes,fibroblastscellsandonthePhe-
Published:October10,2016 nion®FullThicknessSkinModel3D,hybridcooperativecomplexeswerecomparedtoH-
Copyright:©2016Stellavatoetal.Thisisanopen HA,widelyusedinbiorevitalizationprocedures,andtoL-HA,recentlyproposedasthe
accessarticledistributedunderthetermsofthe mostactivefractionmodulatingtheinflammatoryresponse.Quantitativereal-timePCR
CreativeCommonsAttributionLicense,which
analyseswereaccomplishedforthetranscriptquantificationofcollagensandelastin.
permitsunrestricteduse,distribution,and
Finallyimmunofluorescencestainingpermittedtoevaluatethecompletebiosynthesisofall
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. themoleculesinvestigated.AnincreaseintheexpressionlevelsoftypeIandtypeIIIcolla-
geninfibroblastsandtypeIVandVIIcollageninkeratinocyteswerefoundwiththehybrid
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation cooperativecomplexes,comparedtountreatedcells(CTR)andtotheH-HAandL-HA
files. treatments.Theincreaseinelastinexpressionfoundinbothcellularmodelandinthe
Funding:ThisresearchworkwasfundedbyMIUR Phenion®FullThicknessSkinModel3Dalsoatlongertime(upto7days),supportsthe
PON03PE_00060_3:"designanddevelopmentof clinicallyobservedimprovementofskinelasticity.Thebiomarkersanalyzedsuggestan
moleculeswithnutraceuticalandcosmeceutical
increaseoftissueremodelinginthepresenceofProfhilo®,probablyduetothelonglasting
function".InadditionitwassupportedbyIBSA
FarmaceuticiItaliaSrl,Lodi,Italy,Europe,that releaseandtheconcurrentactionofthetwoHAcomponents.
providedthepharmaceuticalgradeproductsand
APIusedintheexperimentsandprovidedsupport
fortheEnglishcopyeditingofthefinalmanuscript.
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HAHybridCooperativeComplexesInduceBioremodeling
CompetingInterests:CS,MDR,ADAandALGare Introduction
inventorsofapatentonhybridcooperative
Asaresultofitsphysico-chemicalandrheologicalpropertiessuchasviscoelasticityandcapac-
complexes,whileacompanyistheassignee.PBis
affiliatedwithIBSAasMedicalMarketing.She itytoretainwater,HyaluronicAcid(HA)playsapivotalroleinthecontroloftissuehydration
contributedintheinitialdesignoftheexperimental andpermeabilitytosmallorlargemolecules,andthesepropertiescontributetoitsexcellent
workandsupportwiththecompanytheEnglish biocompatibilityandimmunebarrierfunction.Inthehumandermis,thehighpercentageof
finalcopyediting.IBSAalsosuppliesreagentsand
HAallowshydration,maintainingatthesametimeapropertissuevolumewhichbuffersskin
materialforthestudybutdidnothaveanyother
cellsfrommechanicaldamage.Aloneorincombinationwithothermolecules,HAaccelerates
additionalrolein,datacollectionandanalysis,
decisiontopublish,orpreparationofthe invitroprocessesrelatedtowoundhealing[1]andinvivotissueregeneration(e.g.,burns,
manuscript.Thisdoesnotalterouradherencetoall ulcers)[2].Furthermore,HAcanprovidebothanti-inflammatoryandbio-stimulatingeffect,
thePLOSONEpoliciesonsharingdataand andalsoactivateothersignalingpathwaysthroughtheinteractionwithcellmembranerecep-
materials.
torssuchasCD44,TLR-4,andRHAMM[3].Besides,hyaluronansrecentlyprovedtheability
Abbreviations:HA,HyaluronicAcid;ECM, topreventROSdamage[4].Moreover,HAisamoleculecriticallyinvolvedinskinaging.All
ExtracellularMatrix;BTH,BovineTesticular theabovementionedpropertiesmakeHAanexcellentdermalagent,abletocorrectsofttissue
Hyaluronidase;H-HA,highmolecularweightHA;L-
defectsofthefaceandpromptcollagenbiosynthesis,leadingtorejuvenationeffects[5].The
HA,lowmolecularweightHA;H/L-HA,high/low
agingprocessisaffectedbyenvironmentalfactors(asfreeradicalsformation)andindividual
HAhybridcooperativecomplexes;qRT-PCR,
geneticbackground.Inparticular,skinagingprocesscouldbeviewedasanalterationoccurring
quantitativereal-timePCR;CD44,Clusterof
Differentiation44;TLR-4,Toll-likereceptor4; incollagen,elastinand,mainly,inHAcontent[6].Theskinmatrixisindeedresponsiblefor
RHAMM,Hyaluronan-mediatedmotilityreceptor; structuralintegrity,mechanicalelasticity,stabilityandmanyotherfunctions.HAdegradation,
ROS,Reactiveoxygenspecies;SEC-TDA,Size togetherwiththeboneresorption,playsamajorroleintheformationofwrinklesandother
ExclusionChromatography-TripleDetectorArray;
signsofaging,andstructuralproteinsareessentialtoskinhealthandyouthfulness[7,8].Skin
PBS,phosphatebufferedsolution;HaCat,human
agingcanalsoberelatedtovarioustissuechangesindifferentskinlayers,suchasdestruction
dermalkeratinocytes;HDF,humandermal
fibroblasts;DMEM,Dulbecco’sModifiedEagle oftheepidermal–dermalinterface,dermalatrophy, andlossofelastictissueinthesub-epider-
Medium;FBS,FetalBovineSerum;COLIA1,typeI malelastinfibersnetwork[9,10].Theratiobetweendifferentcollagentypesmayalsochange,
collagen;COLIIIA1,typeIIIcollagen;COLIVA1, affectingseveralskinfunctions.Withaging,typeIandtypeIIIcollagenproductionandepider-
typeIVcollagen;COLVIIA1,typeVIIcollagen;ELS,
malmatrixturnoverratearereduced[9].OtherscientistsfoundtheratiooftypeIIItotypeI
elastin;HPRT,hypoxanthineguanine
collagenincreased,principallyduetoadropofcollagenIbiosynthesisinelderlywomen[11,
phosphoribosyltransferase;NAHYCO,NA
12].TypeIVandVIIcollagenareimplicatedinskinhomeostasis[13]:typeIVcollagenis
(sodium)HybridComplex.
foundprimarilywithinthebasementmembranezoneandplaysakeyroleinmaintaining
mechanicalfirmnesswhiletypeVIIcollageniscriticalforanchoringfibrilsofthebasement
membranetotheunderlyingdermaltissue.Itwashypothesizedthatwrinklesformationisalso
drivenbythereductionintheagedskinoftheseabovementionedtypesofcollagen[14].Asfor
skinhomeostasis,thereductionofHAmayleadtophysiologicalalterationsofkeratinocytes
andfibroblasts[15,16].TheuseofHAmoderatelyimprovedepidermalhomeostasisbarrier
andtissuethicknessinmurinemodels[17].Sincetheearly1980s,allthesedatahasledtothe
extensiveuseofHAinjectionsforcorrectionofwrinklesandrejuvenationprocedures[12].
Currently,alotofdermalhyaluronicacid-basedmedicaldevicesareusedintheaestheticmedi-
cine,mostofwhicharebasedonchemicalcross-linking;thischemicalmodificationimproves
stability,rigidity,andelasticity(elasticmodulusG’andviscousG”),butsubstantiallymodifies
thenaturalmoleculestructure.Topical orinjectedH-HAproductshavehadvariableeffectsin
restoringaphysiologicalandhydratedmicroenvironmenttypicalofyouthfulskin[18,19].In
thisstudy,weevaluatedtheinvitrocellularandmolecularchangesbothindermalkeratino-
cytesandfibroblastsandinfollowingtheuseofanewhyaluronicacid-basedformulation
namedProfhilo1,incomparisontoHigh(H-HA)andLowMolecularWeightHA(L-HA)
gels.TypeI,III,IVandVIIcollagenandelastingeneexpressionwereevaluatedbyquantitative
real-timePCRinkeratinocytesandfibroblastsmonocultures.Besides,weusedimmunofluores-
cencestainingtoassesstheexpressionandlocalizationofECMproteins,suchastypeIandIII
collagenandelastin,inkeratinocytesandfibroblastco-culturestreatedwithhybridcooperative
complexes,comparedtoH-HA,L-HAandtountreatedcells.Inadditionwerunafullsetof
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HAHybridCooperativeComplexesInduceBioremodeling
experimentsusingthePhenion1FullThicknessSkinModel3Dforgeneexpressiononall
markersat24hand7daysandimmunofluorescencestaining(forElastin)alreadyperformed
onco-culturescellmonolayer.
MaterialsandMethods
Materials
High-(H-HA;MW1200±100kDa)andlow-molecularweighthyaluronicacid(L-HA:
Mw=100±10)wereproducedbyAltergons.r.l.,Italy, fromStreptococcusequissp.equiasa
fermentativeultrapurepharmaceuticalgradeHA(i.e.purity>95%,watercontent<10%,EU/
mg<0.05,extremelylowheavymetalscontent).Theproductunderinvestigationhasapat-
entedtechnology(WO/2012/032,151),namedNAHYCO™technology[20],basedonthermal
proceduresfortheformationofhybridcooperativecomplexesofhyaluronicacid,starting
fromaninitialmixtureofanequalamount(ratio1:1)ofH-HA(Mw=1200±100kDaMw/
Mn=1.4)andL-HA(M =100±10kDa,Mw/Mn=1.4).Thefinalconcentrationusedinthese
w
experimentswas32g/L:32mgH-HA+32mgL-HAin2mLvolume,providedinprefilled
syringes.
Rheologymeasurements
DynamicviscositymeasurementswerecarriedoutusingaPhysicaMCR301oscillatoryrheom-
eter(AntonPaar,Germany)equippedwithacoaxialcylindersgeometryandaPeltiertempera-
turecontrol.Theflowcurveswerebuiltwithfiftymeasurementsat25°C,inarangeofshear
rate0.001-300s-1.Fromeachflowcurve,thezero-shearviscosity(η )wastheviscosityvaluein
0
therangeofNewtonianplateau.Flowcurveswereregisteredfordifferentsampletypes:
1. H-HA(16g/L,preandpostthermaltreatment):highmolecularweighthyaluronicacid,sol-
ubilizedatroomtemperature,subjectedornottothermaltreatment.
2. H-HA+L-HAwithoutthermaltreatment(16g/L+16g/L):highandlowmolecularweight
hyaluronicacidsolubilizedatroomtemperature(32g/Lfinalconcentration).
3. H-HA/L-HAhybridcooperativecomplexes:highandlowmolecularweighthyaluronicacid
solubilizedatroomtemperatureandsubmittedtothermaltreatment(32g/Lfinalconcen-
tration,initialH/Lratio1:1).
4. H-HA+L-HAmixedafterseparatethermaltreatment:highandlowmolecularweighthyal-
uronicacidsolubilizedindifferentvials(eachcontaining32g/LeitherofH-HAorof
L-HA),subjectedtothermaltreatmentsseparatelyandthen,aftercoolingtoroomtempera-
ture,mixed1:1(v/v)(finalconcentration32g/L).
Stabilitytohyaluronidase
SamplesoftheH-HA/L-HAhybridcooperativecomplexesweredilutedto4mg/mLinphos-
phatebufferedsolution(PBS)andincubatedinthepresenceofBTH(1U/mL)at37°Cunder
stirringconditions(1000rpm).Atdifferentincubationtimes,rangingfrom0.5hto10days,the
sampleswerewithdrawn,boiledfor10mintoinactivatetheenzyme,andthenappropriately
dilutedforchromatographicanalysis.Inparticular,aSizeExclusionChromatography-Triple
DetectorArray(SEC-TDA)equipment(Viscotek,LabServiceAnalyticaS.R.L.,Italy)wasused.
AdetaileddescriptionoftheSEC-TDAsystemandanalysisconditionsarereportedelsewhere
[21,22].Samplemolecularweight(M ,M ,M /M ),molecularsize(hydrodynamicradius-
w n w n
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HAHybridCooperativeComplexesInduceBioremodeling
Rh)andintrinsicviscosity([η])distributionswerederived.Thedegradationwasmonitoredby
followingthedecreaseinsamplefraction(w/w%)havingM higherthan1MDa.Alinear
w
H-HA(M =1120±100kDa;M /M =1.5±0.1)wasusedascontrol.
w w n
Cellcultures
To evaluatethebiologicalpropertiesofHAhybridcomplexes,humandermalkeratinocytes
(HaCat)andfibroblasts(HDF)cultures,wereusedascellularmodelsofhumanskin.HaCaT, a
spontaneouslytransformednon-tumorigenichumankeratinocytescelllinewasprovidedby
IstitutoZooprofilattico,Brescia,ItalyandthecellswereculturedinDulbecco’s ModifiedEagle
Medium(DMEM),supplementedwith10%(v/v)heatinactivatedFetalBovineSerum(FBS),
penicillin100U/mlandstreptomycin100μg/ml.DMEM,FBS,Pen-StrepPBSandTrypsin
wereprovidedbyGibcoInvitrogen(Milan,Italy).Ahumandermalfibroblastscelllineimmor-
talizedwithhTERT (HDFcells,BJ-5ta,ATCCCRL-4001),wasculturedina4:1mixtureof
DMEMandMedium199supplementedwith0.01mg/mlhygromycinBand10%(v/v)FBS.All
materialsforHDFculturewerepurchasedfromATCC(USA).Thecellsweregrownontissue
cultureplates(BDFalcon,Italy),usinganincubatorwithahumidifiedatmosphere(95%air/
5%CO v/v)at37°C.Forthegeneexpressionanalyses,humankeratinocytesandfibroblasts
2
weregrownindifferentcellcultures.Moreindetail,3.75x104cells/cm2inastandard24-well
cultureplatewereseeded.Fortheimmunofluorescencestaining,HaCat/HDFco-cultureswere
used.Inthismodel,humankeratinocytesandfibroblastswereseeded,culturedtogetherand
thenanalyzedonaglassmicroscopeslide.μ-dish(35mm,high)culture-insert(Ibidi,Inte-
gratedBioDiagnostics,Munich,Germany)wereusedtoseparatethetwocellpopulationsand
toallowcellinteraction.Cellswereseededdirectlyonchamberaffixedtoaspeciallyglass
microscopeslide(BDFalcon™BDBiosciences)ataratioof1:1.25HaCat:HDF(1x103/cm2and
1.25x103/cm2respectively).Inbothmodels,cellsweretreatedwithH-HA1400kDa(0.16%w/
w),L-HA100kDa(0.16%w/w)andhybridcooperativecomplexes(H-HA/L-HAcomplexes
0.16%w/w).Treatments lasted4and24hforthegeneexpressionanalysesand24hforthefluo-
rescencestaining.
3DSkinModel
ThePhenion1FullThicknessSkinModel,producedbyHenkel(Düsseldorf,Germany,diame-
ter1.3cm)wasusedforthestudy.Inthismodel,epidermalkeratinocytesanddermalfibro-
blasts(derivedfrombiopsymaterialfromhealthydonors)formamultilayeredskinequivalent
undercultureconditionsthatresembleshumanskinmultilayeredstructureandtissuefunc-
tionality[23].Uponarrival,theFTmodelswereremovedimmediatelyfromthesemi-solid
transportmedium.ThreelowerhalvesofsmallsizePetridishesfilledwith2sterilemetalsup-
portswereplacedinalargePetridish(F=100mm).ThesmallPetridisheswerefilledwith5
mlofALI1mediumprovidedwiththeskinmodelswereimmediatelytransferredinanincuba-
tor(37°C,5%CO2).6smallinjectionsof50μlofHybridcomplexes(Profhilo1gel)andH-HA
orL-HAinsixdifferentpointsofthemodeljustbelowtheepidermalsurfaceinthedermal
compartmentwereperformed.Thesampleswerethenincubatedfor24hand7days.Control
samples,wereFT-SKINinjectedwithPBSsolution(6injectionsof50μl).TheeffectofProf-
hilo1formulation,H-HAorL-HAalone,wereanalyzedfortheireffectonproductionoftypeI
collagen(COLI),typeIIIcollagen(COLIII),typeIVcollagen(COLIV),typeVIIcollagen
(COLVII)andElastin(ELS).
At24hand7daystheFTmodelswereopportunelycutwithasterilebladeindifferentsec-
tionsandimmersedinappropriatelybufferforsuccessiveexperiments,inparticularrealtime
PCRandimmunofluorescencestaining.
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HAHybridCooperativeComplexesInduceBioremodeling
RNAextractionandquantitativereal-timePCRoncellmonolayer
TheexpressionofeachmRNAencodingECMproteinssuchastypeI(COLIA1),III
(COLIIIA1),IV(COLIVA1) andVII(COLVIIA1)collagensandelastin(ELS)wasassayedby
quantitativereal-timePCR(qRT-PCR).Total RNAwasextractedfromhumandermalkerati-
nocytesandfibroblastsusingTRIzol1(Invitrogen,Milan,Italy),accordingtothemanufactur-
er’s proceduresfullydescribedinD’Agostinoetal.(2015).Inbrief,oneμgofDNase-digested
totalRNA(DNA-freekit;Ambion-AppliedBiosystems)wasconvertedtocDNAusingReverse
TranscriptionSystemKit(Promega,Milan,Italy).PCRwasthenperformedusingiQ™SYBR1-
GreenSupermix(Bio-RadLaboratoriess.r.l.,Milan,Italy)toanalyze,withtheappropriate
primerpairs,theexpressionlevelsofcollagenandelastin.Theprimersequencesandamplifica-
tionprotocolarereportedinTable1.Allreactionswereperformedintriplicate,andtherelative
expressionofspecificmRNAwasdeterminedbynormalizingtohypoxanthineguaninephos-
phoribosyltransferase(HPRT)housekeepinggene.Thefold-changeofgenesexpressionwas
calculatedbyusingthecomparativethresholdmethod(ΔΔCt=differenceofΔCtbetweenHA-
treatedcellsandcontrol)andtheresultswereexpressedasnormalizedfoldexpressioncom-
paredtocontrols,calculatedbyusingtheBio-RadiQ™5software(Bio-RadLaboratoriesSrl),as
theratioofcrossingpointsofamplificationcurvesofseveralgenesandinternalstandard[24].
Westernblottinganalysisoncellmonolayer
WesternblotanalyseswereperformedaftersevendaysoftreatmentwithH-HA,L-HAandH/
L-HAhybridcomplexes.CellswerelysedinRIPAbuffer(CellSignalingTechnology).Protein
concentrationwasdeterminedusingtheBradfordmethod[25]and80μgintracellularproteins
wereloadedandresolvedusing10%SDS–PAGE.Theseparatedproteinswerethentransferred
tonitrocellulosemembrane(Amersham).Themembranewasblockedin5%milkdissolvedin
Tris-bufferedsalineand0.05%Tween-20.Elastin(60kDa)primaryantibodywasusedat1:250
dilutions.Immunoreactivebandsweredetectedbychemiluminescenceusingcorresponding
horseradishperoxidase-conjugatedsecondaryantibody(SantacruzBiotechnology;1:5000dilu-
tions)andreactedwithanECLsystem(Chemicon-Millipore).Proteinlevelswerenormalized
respecttothesignalofantiactinpolyclonalantibodyusingashousekeepingprotein(Santacruz
Biotechnology;1:1000dilutions).Thesemi-quantitativeanalysisofproteinlevelswascarried
outbytheGelDoc2000UVSystemandtheGelDocEZImager(Quantityonesoftware,Bio-
RadLaboratories).
Immunofluorescencestainingoncellmonolayer
TheexpressionofthreeECMproteinsintheskin,collagentypeI,collagentypeIIIandelastin,
wasevaluatedonfixedHaCat/HDFco-cultures.After24hand7daysofincubationonlyfor
elastinantibody,cellco-cultureswerestainedsimultaneouslytoensureuniformconditionsfor
fluorescenceanalyses,fixedin4%paraformaldehydefor15min,rinsedinphosphate-buffered
Table1. PrimersequencesforspecificbiomarkersemployedfortheqRT-PCR.
Gene ForwardPrimer ReversePrimer ATPCR
HPRT 5'-TGACCTTGATTTATTTTGCATACC-3' 5'-CGAGCAAGACGTTCAGTCCT-3' 55˚C
COLIA1 5'-CCAGAAGAACTGGTACATCA-3' 5'-CCGCCATACTCGAACTGGAA-3' 55˚C
COLIIIA1 5'-TGGTCCCCAAGGTGTCAAAG-3' 5'-GGGGGTCCTGGGTTACCATTA-3' 55˚C
COLIVA1 5’-GGATCGGCTACTCTTTTGTGATG-3' 5'-AAGCGTTTGCGTAGTAATTGCA-3' 55˚C
COLVIIA1 5'-CGGAACTGACCATCCAGAAT-3' 5'-AATAGGGTGCTCACGGTCAC-3' 55˚C
Elastin 5'-AGGTGTATACCCAGGTGGCGTGCT-3' 5'-CAACCCCTGTCCCTGTTGGGTAAC-3' 60˚C
doi:10.1371/journal.pone.0163510.t001
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HAHybridCooperativeComplexesInduceBioremodeling
saline(PBS)andblockedbyusingblockingsolution(1XPBS/5%normalserum/0.3%tritonX-
100)for60min.Cellco-cultureswerethenincubatedovernight(4°C)withprimaryantibodies:
typeIcollagen(monoclonalrabbitantibody,1:100;Abcam,Cambridge,MA,USA),elastin
(monoclonalmouseantibody,1:50;SantaCruzbiotechnology,USA)andtypeIIIcollagen
(polyclonalmouseantibody,1:100;NovusBiologicals,CambridgeUK).Afterthreewashings,
glassmicroscopeslideswereincubatedwithGoatanti-RabbitIgG(H+L)SecondaryAntibody,
AlexaFluor1488conjugate(1:100;LifeTechnologiesItaly),goatanti-MouseIgG(H+L)Sec-
ondaryAntibody,AlexaFluor1568conjugate(1:100;LifeTechnologiesItaly).To visualize
actinfilamentscellswerestainedwitha50mg/mlfluorescentphalloidinTRITCconjugate
(Sigma-Aldrich,Italy)solutioninPBSfor40minutesatroomtemperature.Nucleiwerecoun-
terstainedbyHoechstfor10min(0.5μg/mLSigma-Aldrich,Italy).Coverslipslideswere
obtainedbyusingProLong™antifademountant(LifeTechnologiesItaly).
QuantitativeRealTimePCR(qRT-PCR)onFT-skinmodel
Total RNAwasextractedfromtheFT-skinmodelsusingTRIzol1(Invitrogen,Milan,Italy)
andTissueRuptorhomogenizer(Qiagen,HildenGermany)accordingtothemanufacturer’s
proceduresandc-DNAretrotrascriptionwasperformedusingReverseTranscriptionSystem
Kit(Promega,Milan,Italy).PCRwasthenperformedasdescribedinthepreviousparagraph.
Inparticular,thesamplesanalysedwerethe3DfullSkinmodelsinjected:a)withPBSsolution
(CTR),b)withH/L-HAhybridcomplexes,c)withH-HAandd)L-HA.Thefold-changeof
genesexpressionwascalculatedbyusingthecomparativethresholdmethod
(ΔΔCt=differenceofΔCtbetweenHA-treatedcellsandnegativecontrolFTmodelinjected
withPBSsolution)andtheresultswereexpressedasnormalizedfoldexpressionwithrespect
tothehousekeepinggene,calculatedbyusingtheBio-RadiQ™5software(Bio-RadLaboratories
Srl).
ImmunofluorescencestainingofelastinonFT-skinmodel
Aftertheincubationperiod(24hand7days),theFT-Skinmodelswereimmersedinformalin
4%v/vinPBSovernight.
TheFTsectionswereappropriatelydehydratatedwithincreasingethanolconcentration
(until95%v/v),followedbyisopropylalcohol,thentreatedwithxylenesolventovernighttobe
finallysuitableforparaffininclusion;thetissuesectionswerethencutwithamicrotome(10–
20μmthick)inslicesfortheelastinantibodyincubation.
Forimmunefluorescencesteps,deparaffinationandrehydrationwereperformed,followed
by8minat99°Cincubationincitratebufferasantigenretrieval,thenblockingwith5%BSAin
PBSfor60minutesfollowedbyincubationwithprimaryprimaryantibody:elastin(monoclo-
nalmouseantibody,1:50;SantaCruzbiotechnology,USA).Afterwhich,thesectionswere
incubatedwithappropriatesecondaryantibody,AlexaFluor488-conjugatedsecondaryanti-
body(Invitrogen)for1h.To visualizeactinfilamentscellswerestainedwitha50mg/mlfluo-
rescentphalloidinTRITCconjugate(Sigma-Aldrich,Italy)solutioninPBSfor40minutesat
roomtemperature.NucleiwerestainedwithHoechstfor10min(0.5μg/mLSigma-Aldrich,
Italy).SampleswerethenexaminedundertheNikonfluorescencemicroscopeandanalyzedby
Nikon(MulticolorPackage,Leica).
StatisticalAnalysis
Allexperimentswereperformedintriplicate.Student’s t-testwasusedforstatisticalevaluation.
Thelevelofsignificancewassetatp<0.01.
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HAHybridCooperativeComplexesInduceBioremodeling
Fig1.Flowcurves(ηvsshearrate)forH-HApreandpostthermaltreatment(bluesymbolsvoidandsolid,
respectively),H+L-HApre-thermaltreatment(voidgreentriangle),H/L-HACOMPLEXEShybrid
cooperativecomplexes(greensolidtriangles),H+L-HAMIX:H-HAandL-HAseparatelythermallytreated
andthenmixedaftercooling(solidyellowsymbols).
doi:10.1371/journal.pone.0163510.g001
Results
Dynamicviscositymeasurements
RheologyflowcurvesaredisplayedinFig1.Inparticular,theflowcurvesforH-HA(point1in
the“rheologymeasurements”paragraph),H-HA+L-HAmixedpreorpostthermaltreatment
(points2and4,asabove)andforH-HA/L-HAhybridcooperativecomplexes(point3,as
above)areplotted.
Thezero-shearviscosity(η )valuesforthetestedsamplesarereportedinTable 2.
0
Allsamplesshowedashearthinningbehaviour,butwithaconstantη intheshearrateof
0
0,01to0,5s-1.Therewasamarkeddecreaseofdynamicviscosity,higherthan20fold,related
tothehybridcooperativecomplexesformation(Table 2).Indeed,whentheη ofthesample2
0
(mixoftheH-HAchainsandthelowHAchainsatroomtemperature),wascomparedtothe
η ofthesample4(thetwosamesolutionsundergoingthethermaltreatment,andmixed
0
after),onlya4.7foldviscositydecreasewasobservedwhilea3.2-folddecreaseinviscositywas
observedwiththesample1(H-HA).
Table2. Rheologicalvalues(η )ofH-HA(rawmaterialsofPROFHILOdevices);H+L-HAMIXandH/L-HA(COMPLEXES),pre-andpost-thermal
0
treatment(12minutesat120˚C)andthecorrespondingfolddecreasevalues.
η η0(Pa×sec)Prethermaltreatment η0(Pa×sec)Postthermaltreatment Folddecrease
HHA(16mg/L) 74.4±0.3 23.0±0.2 3.2
H+L-HAMIX(16+16mg/L) 120.0±0.5 28.3±0.4 4.7
H/L-HACOMPLEX(16+16g/L) 120.0±0.5 5.5±0.0 21.9
doi:10.1371/journal.pone.0163510.t002
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HAHybridCooperativeComplexesInduceBioremodeling
Thelowη valueforhybridcooperativecomplexesgive(confer)theproducteasierinject-
0
ability(30gaugeneedlesaresuggested),andexpectedbetterdiffusibilitywithinthetissue.
Enzymaticstability
ResultsofenzymaticdegradationstudiesarereportedinFig2.Theresidualfraction(w/w%)
withmolecularweighthigherthan1MDa,afterincubationwithBTH1U/mL,wasevaluated.
Indetails,HAhybridcooperativecomplexesandthelinearH-HAwerestudiedafter0.5hto
10daysofincubationwiththeenzyme(Fig2Aand2B).Values werenormalizedfortheinitial
compositionatt=0(beforetheincubation).HAhybridcooperativecomplexesshoweda
significantlyhigherresistancetoBTHactionthanH-HA.Afteronedayofincubation,BTH
actionreducedtheresidualfractionoftheH-HAfraction(theHAhybridcooperative
Fig2. ResultsofsensitivitytoenzymaticdegradationforthehybridcooperativecomplexandHHA:normalized
samplefraction(w/w%)showingMwhigherthan1MDaduringBTH1U/mLactionupto24h(a)and10days(b).
doi:10.1371/journal.pone.0163510.g002
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HAHybridCooperativeComplexesInduceBioremodeling
complexes)toabout35%oftheinitialconcentrationandlessthan10%oftheH-HA.Nofur-
thersignificantdegradationcouldbedetectedinbothsampleswhenincubationlastedupto
tendays.
AnalysisofcollagensbyqRT-PCRoncellmonolayer
Geneexpressionanalysesaboutcollagens(typeI,III,IV, VII)andelastinwereperformedby
quantitativereal-timePCR(qRT-PCR),andtheresultswerepresentedasnormalizedfold
expressionincreasecomparedtocontroluntreatedcellsandhypoxanthineguaninephosphori-
bosyltransferase(HPRT)housekeepinggene.GeneexpressionlevelsoftypeIandIIIcollagen
inbothkeratinocytesandfibroblastscellculturesareshowninFig3.Theexpressionlevelsof
collagenIweretwofoldshigherforthekeratinocytesafter4hoursofincubationinpresenceof
HAhybridcooperativecomplexescomparedtocontrolcellsandH-andL-HA-treatedcells,
whichvaluesweresubstantiallysimilartothecontrolcells(halfandone-and-halffoldsrespec-
tively)(Fig3A).Infibroblasts,H-HAtreatmentpromptedgeneexpressionforcollagenI
(>twofoldincrease)at4hours,and,after24hoursinpresenceofHAhybridcooperative
complexes,asevenfoldshighergeneexpressionforcollagen-IwasfoundcomparedtoH-HA
andL-HAtreatments(Fig3B).AsforcollagenIIIgeneexpression,inkeratinocytes,after24
hoursinpresenceofHAhybridcooperativecomplexes,anincreaseoftwofoldsrespecttoH-
andL-HA-treatedcellswasfound(Fig3C),whileinfibroblaststhisincreasewastwelvefolds
(Fig3D).
InFig4thenormalizedmRNAexpressionlevelsofCOLIV, COLVII,andELSinkeratino-
cytesandELSinfibroblastswerereported.After4hoursincubation,H/L-HAhybridcoopera-
tivecomplexessignificantlyincreasedCOLIV, COLVII,andELSexpressioninkeratinocytes
Fig3.qRT-PCRrelativetoCOLIandCOLIIIexpression.TranscriptionalanalysisrelativetoCOLIA1and
COLIIIA1genesin(A-B)keratinocytesand(C-D)fibroblasts,inpresenceofH-HA,L-HAandH-HA/L-HA
complexes0.16%(w/w)after4and24h.Valuesarecalculatedasmean±SDofthreedifferentexperimentsand
areexpressedasmRNAnormalizedfoldincreaserespecttoCTR(1).Student’st-test(*p<0.01).
doi:10.1371/journal.pone.0163510.g003
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HAHybridCooperativeComplexesInduceBioremodeling
Fig4.qRT-PCRrelativetoCOLIV,COLVIIandELSexpression.TranscriptionalanalysisrelativetoCOLIVA1
andCOLVIIA1andELSgenesin(A)keratinocytesat4hand(B-C)fibroblasts,inpresenceofH-HA,L-HAand
H-HA/L-HAcomplexes0.16%(w/w)at4and24h.Valuesarethemean±SDofthreedifferentexperimentsandare
expressedasmRNAnormalizedfoldincreaserespecttoCTR(1).Thesamples(H-HA/L-HAcomplexesrespectto
H-HA)aresignificantlydifferentinaccordingtoStudent’st-test(*p<0.01).
doi:10.1371/journal.pone.0163510.g004
(Fig4A),whileinfibroblastsCOLIVandCOLVIImRNAlevelswereslightlylessorsimilarto
controlcells.After24hoursinpresenceofHAhybridcooperativecomplexes,ELSwassignifi-
cantlyincreased(twelvefolds)(Fig4B).
ElastinWesternblottinganalysis
AsshowninFig5,westernblottinganalysesshowedasignificantelastinproteinincreaseparticu-
larlyinpresenceofH/L-HAhybridcomplexesrespecttoH-HAandL-HAandrespecttoCTR.
Immunofluorescencestainingofcollagensandelastinproteinsoncell
monolayer
Atproteinlevel,collagen(COLIandCOLIII)andelastin(ELS)productionwerehighlightedby
immunofluorescencestainingofkeratinocytesandfibroblastsco-cultures.Microscopefluores-
cenceobservationsshowedthattypeIcollagenproteinsynthesiswashigherinfibroblaststhanin
keratinocytes(Fig6).Also,theresultssuggestedadifferentexpressionandlocalizationofCOLI
whenthecellsweretreatedwithHAhybridcooperativecomplexescomparedtoH-HAand
L-HAtreatments.After24hofincubation,thefluorescenceintensityofCOLIIIwashigherin
presenceofHAhybridcomplexes,comparedtocontrolcellsandH-andL-HA-treatedcells(Fig
7).TheimagesinFig8displayedahigherelastinexpressionwhenkeratinocyte-fibroblastco-cul-
turesweretreatedwithhyaluronanhybridcooperativecomplexesthanhighandlowmolecular
weightHA,respectively.Besides,intheFig9theimmunofluorescencestainingofelastinafter7
daysofincubationwasreported.ThisdataconfirmedthatHAhybridcomplexesenhanceelastin
proteinexpressionrespecttoCTRandH-HAandL-HAsingletreatmentalsoatlongertime.
AnalysisofcollagensbyqRT-PCRonFT-skinmodel
Inthe3Dskinmodelallthebiomarkersalreadymeasuredinthe2Dinvitroexperimentson
HaCatandFibroblasts,wereevaluatedafter24hand7daysofculture.AsshowninFig10A
and10B,qRT-PCRprovedasignificantlyhigherexpressionofCOLI,COLVIIandelastinfor
thefullthicknessskinmodelsamplestreatedwithHybridcomplexes.Thehigherexpression
levelwasnotonlyrecordedrespecttothecontrol(injectedPBS)butalsorespecttothesamples
treatedwithL-HAandH-HA.HoweverforCOLIIIandCOLIVthisexpressionwasonly
slightlysuperior,evenifstillsignificant.Overallthesedataseemtobeinagreementwithour
findingsonthemonolayerbasedhumandermalcellmodel.Inthe3Dskinmodelatlonger
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