Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5

22 Pages·2017·6.75 MB·English

cells
Article
Injured Achilles Tendons Treated with
Adipose-Derived Stem Cells Transplantation
and GDF-5
AndreaAparecidadeAro1,2,*,GianeDanielaCarneiro1,LuisFelipeR.Teodoro1,
FernandaCristinadaVeiga3,DaniloLopesFerrucci1,GustavoFerreiraSimões1,
PriscylaWaleskaSimões4 ID,LúciaElviraAlvares3,AlexandreLeiteR.deOliveira1,
CristinaPontesVicente1,CaioPerezGomes5 ID,JoãoBoscoPesquero5 ID,
MarceloAugustoM.Esquisatto2,BenedictodeCamposVidal1andEdsonRosaPimentel1
1 DepartmentofStructuralandFunctionalBiology,InstituteofBiology,StateUniversityof
Campinas–UNICAMP,CharlesDarwin,s/n,CP6109,13083-970Campinas,SP,Brazil;
[email protected](G.D.C.);[email protected](L.F.R.T.);[email protected](D.L.F.);
[email protected](G.F.S.);[email protected](A.L.R.d.O.);[email protected](C.P.V.);
[email protected](B.d.C.V.);[email protected](E.R.P.)
2 BiomedicalSciencesGraduateProgram,HerminioOmettoUniversityCenter–UNIARARAS,
13607-339Araras,SP,Brazil;[email protected]
3 DepartmentofBiochemistryandTissueBiology,InstituteofBiology,StateUniversityof
Campinas–UNICAMP,CharlesDarwin,s/n,CP6109,13083-970Campinas,SP,Brazil;
[email protected](F.C.d.V.);[email protected](L.E.A.)
4 Engineering,ModelingandAppliedSocialSciencesCenter(CECS),BiomedicalEngineeringGraduate
Program(PPGEBM),UniversidadeFederaldoABC(UFABC),AlamedadaUniversidades/n,
09606-045SãoBernardodoCampo,SP,Brazil;[email protected]
5 DepartmentofBiophysics,FederalUniversityofSaoPaulo–Unifesp,PedrodeToledo,699,
04039-032SaoPaulo,SP,Brazil;[email protected](C.P.G.);[email protected](J.B.P.)
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:19July2018;Accepted:23August2018;Published:31August2018
Abstract: Tendon injuries represent a clinical challenge in regenerative medicine because their
naturalrepairprocessiscomplexandinefficient. Thehighincidenceoftendoninjuriesisfrequently
associatedwithsportspractice,aging,tendinopathies,hypertension,diabetesmellitus,andtheuse
ofcorticosteroids. Thegrowinginterestofscientistsinusingadipose-derivedmesenchymalstem
cells(ADMSC)inrepairprocessesseemstobemostlyduetotheirparacrineandimmunomodulatory
effectsinstimulatingspecificcellularevents. ADMSCactivitycanbeinfluencedbyGDF-5,whichhas
beensuccessfullyusedtodrivetenogenicdifferentiationofADMSCinvitro. Thus,wehypothesized
thattheapplicationofADMSCinisolationorinassociationwithGDF-5couldimproveAchilles
tendon repair through the regulation of important remodeling genes expression. Lewis rats had
tendonsdistributedinfourgroups: Transected(T),transectedandtreatedwithADMSC(ASC)or
GDF-5(GDF5),orwithboth(ASC+GDF5). Inthecharacterizationofcellsbeforeapplication,ADMSC
expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker,
CD45(7%). ADMSCwerealsodifferentiatedinchondrocytes,osteoblast,andadipocytes. Onthe
14thdayafterthetendoninjury,GFP-ADMSCwereobservedinthetransectedregionoftendons
intheASCandASC+GDF5groups, andexhibitedand/orstimulatedasimilargenesexpression
profile when compared to the invitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes
expressionincomparisontoTandASC+GDF5groups,whichcontributedtoalowerproteoglycans
arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC
group. TheapplicationofADMSCinassociationwithGDF-5down-regulatedDcn,Gdf5,Lox,Tgfb1,
Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration,
lower collagen fiber organization, and to an improvement of the rats’ gait 24 h after the injury.
Cells2018,7,127;doi:10.3390/cells7090127 www.mdpi.com/journal/cells
Cells2018,7,127 2of22
Inconclusion,althoughtheliteraturedescribesthebeneficeffectofGDF-5forthetendonhealing
process,ourresultsshowthatitsapplication,isolatedorassociatedwithADMSC,cannotimprove
therepairprocessofpartialtransectedtendons,indicatingthehighereffectivenessoftheapplication
ofADMSCininjuredAchillestendons. OurresultsshowthattheapplicationofADMSCininjured
AchillestendonswasmoreeffectiveinrelationtoitsassociationwithGDF-5.
Keywords: repair;extracellularmatrix;collagen;gait;biomechanics;geneexpression
1. Introduction
Tendoninjuriesareverycommonandtheirnaturalrepairprocessisextremelyslow,complex,
and inefficient due to their intrinsic hypocellularity and hypovascularity, representing a clinical
challenge to orthopedists mainly because these injuries often respond poorly to treatment [1].
As reviewed by Zabrzyn´ski et al. [2], the occurrence of tendon injuries is associated with sports,
aging,tendinophaties,hypothyroidism,hypertension,diabetesmellitus,arthropathies,corticosteroids,
vitaminCdeficiency,andothers. Collagenfibersarethemaincomponentoftheabundantextracellular
matrix(ECM)oftendons,andwiththeproteoglycans(PG),thecollagenfibersformahighlyorganized
supramolecularstructure,abletoattendtothebiomechanicaldemandsonthetissue[3,4]. Particularly,
smallleucine-richproteoglycans(SLRPs),suchasdecorin,fibromodulim,andbiglycan,arethemost
abundant PG in tendons, with decorin representing 80% of the total proteoglycan content of the
tissue[5]. SLRPsinteractwiththecollagenfibrils,actinginthefibrillogenesisofcollagen,andprobably
regulatingthegrowthindiameterofthesefibrils[6]. Besidestheparallelbundlesofpredominantly
type I collagen fibers and PG, non-collagenous glycoproteins, matrix metalloproteinases (MMP),
growthfactors,andcellsalsocompriseelementsofthetendons[5,7–9].
Thespecificmechanicaltendonpropertiesaredirectlyrelatedtothehighorganizationofcollagen
bundles[10]. Afterinjuries,thestructuralorganizationandcompositionoftendonsarenotcompletely
restored, withafibrousscarformationthatcancausesignificantdysfunctionandjointmovement
inability[11,12]. Aftertherepairprocess,tendonsbecomebiomechanicallyweakened,makingitmore
pronetore-rupture[9,13]. Clearly,newtreatmentsareneededwiththeobjectiveofimprovingtendon
repair,consideringthattheseinjuriesreachthepopulationthatiseconomicallyactive,andthattheuse
ofcelltherapyusingmesenchymalstemcells(MSC)couldbeagoodstrategy.
Adult tissues are attractive MSC sources, which are characterized as undifferentiated cells,
with mesodermal differentiation potential and self-renew and high proliferative capacities [14].
AdiposetissueisanalternativesourceofMSCthatcanbeobtainedbyalessinvasivemethodunder
localanesthesia,withlittleassociatedpatientdiscomfortandinlargerquantitiesascomparedwithbone
marrow[15]. Theeffectivenessofadipose-derivedmesenchymalstemcells(ADMSC)inregenerative
medicine seems to be due to their paracrine effects that stimulate specific cellular events, like the
growthfactorsandcytokinesdelivery[16],aswellastheirimmunomodulatoryeffectstopromote
tendonregeneration[17]. Besidesrecruitingprogenitorcellsfromsometissues,theADMSCcouldalso
differentiatespecificcellsduringtissuerepair[18–20]. However,theliteratureiscontroversialabout
theeffectsofADMSCtransplantationduringtendonrepair. Insomeanimalmodels,therewasgreater
structuralorganization,biomechanicalproperties,densityofcollagenfibers,collagentypesIandIII
genesexpression,andgrowthfactorssynthesisasfibroblastgrowthfactor(FGF),vascularendothelial
growthfactor(VEGF),andtransforminggrowthfactor(TGF-β)afterADMSCapplicationinrelationto
untreatedtendons[7,21–23]. Conversely,nodifferenceswereobservedafterthiscellulartherapyinthe
biomechanicalparametersorcollagenproduction[7,17,23].
It is widely accepted that the control of stem cell activity is influenced by several different
environmental factors [24], including growth factors [1]. To drive tenogenic ADMSC in invitro
differentiation, insulinlikegrowthfactor(IGF)-1orTGF-βinaco-culturewithprimarytenocytes
Cells2018,7,127 3of22
and growth differentiation factor-5 (GDF-5) have been used successfully [19,20]. GDF-5 belongs
totheTGF-βsuperfamilyanditisknownascartilage-derivedmorphogeneticprotein-1(CDMP-1)
and bone-derived morphogenetic protein-14 (BMP-14). Storm et al. [25] documented two other
membersofthissuperfamily,suchasGDF-6(BMP-13)andGDF-7(BMP-12). GDFfamilymembers
arealsocalledbone-derivedmorphogeneticproteins(BMP)becausetheyarefoundpredominantly
during the development of endochondral bones [26] and joint formation [27]. Wolfman et al. [28]
demonstratedthatthethreemembersofBMPinducetheformationofconnectivetissuerichincollagen
I,withanimportantroleintheprocessoftendonhealing[29,30]. Fromthisstudy,theBMPceased
tobeconsideredasonlychondrogenicandosteogenicfactors,assuggestedbythename,andwere
consideredtendinogenicfactors. Forslundandcolleagues[30],whoshowedincreasedAchillestendon
resistanceafterrupture,reportedtheinitialsuccessofBMP-12,-13,and-14intendonhealing.Inastudy
ofChhabraetal.[31],micedeficientintheGDF-5genehadapoorhealingprocess,withlesserstructural
organizationanddecreasedbiomechanicalpropertiesoftendons,evidencingtheimportanceofthis
growthfactorduringtendonrepairprocesses. Currently,celltherapyusingtheADMSCassociated
withtheexogenousapplicationofgrowthfactorsrepresentsagreatpotentialintheprocessoftendon
repair. Despitepromisingstudiesinanimals,notreatmentassociatedwiththeapplicationofADMSC
intendoninjurieshasbeenusedinclinicsduetothelackofknowledgeonmolecularaspectsinvolving
thosetherapies.
TheobjectiveofthepresentstudywastotestthehypothesisthattheapplicationofADMSCin
isolationorassociatedwithGDF-5couldimproveAchillestendonrepair. TheuseofGDF-5wasbased
ontheliteraturethatdemonstratesitsimportanceduringtendonhealingandtheroleofGDF-5in
modulatingADMSCtenogenicdifferentiationinvitro.Thus,thedown-orup-regulationofremodeling
genesexpressioninresponsetoADMSCandGDF-5applicationwereanalyzed,andtheinvolvement
ofthosegenesintherestorationofthestructural,biomechanical,andfunctionalpropertiesofAchilles
tendonsafterpartialtransection.
2. MaterialsandMethods
2.1. InVitroExperiments
2.1.1. IsolationofADMSCandCellCulture
TheprocedurewasdoneaccordingtoYangetal.[32]withsomemodifications. Adiposetissue
wasobtainedfromtheinguinalregionof10maleLewisratsbetween90–120days. Adiposetissuewas
cutandwashedinDulbecco’smodifiedphosphatebufferedsalinesolution(DMPBSFlushwithout
calciumandmagnesium)containing2%streptomycin/penicillintoremovecontaminatingbloodcells.
Then,0.2%collagenase(Sigma-Aldrich® Inc.,SaintLouis,MO,USA)wasaddedtodegradationofthe
ECMandthesolutionwasmaintainedat37◦Cundergentlestirringfor1htoseparatethestromal
cellsfromprimaryadipocytes. Dissociatedtissuewasfilteredusingcellstrainers(40µm)andthe
inactivationofcollagenasewasthendonebytheadditionofanequalvolumeofDulbecco’smodified
Eagle’smedium(DMEM)supplementedwith15%fetalbovineserum(FBS),followedbycentrifugation
at1800rpmfor10min. Thesuspendingportioncontaininglipiddropletswasdiscardedandthepellet
wasresuspendedinDMEM(containing50mg/Lpenicillinand50mg/Lstreptomycin)with15%FBS,
andtransferredto25cm2flasksfor48hours. Afterconfluence,cellsweretransferredto75cm2flasks
(1stpassage). Themediumwasreplacedafter48handthenevery3days. Culturesweremaintained
at37◦Cwith5%CO untilthe5thpassage(5P),alwaysatupto80%confluency.
2
2.1.2. FlowCytometry
ADMSC at 5P (n = 4) were trypsinized and centrifuged at 1800 rpm for 10 min, and counted
usingtheNeubauerchamber. 1×106ADMSCwereresuspendedin200uLofDMPBSFlushwith2%
BSA(bovineserumalbumin). Fortheimmunophenotypicpanel,thefollowingantibodieswereused:
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CD90-APC,CD105-PE,andCD45-APCdoubleconjugated(eBioscience®Inc.,SanDiego,CA,USA)
were diluted 1:200 and incubated for 40 min at room temperature. Subsequently, ADMSC were
washedtwicewith500µLofDMPBSFlushandcentrifugedat2000rpmfor7min. TheADMSCwere
resuspendedinDMPBSFlushwith2%BSA,followedbytheflowcytometryanalysis.
2.1.3. InVitroDifferentiationPotentialofADMSC
Usingdifferentmediaforeachtypeofdifferentiation,ADMSC(5P)werecultured(2×104cells)
according to Yang et al. [32] with some modifications. Osteogenic differentiation (n = 3): DMEM
supplementedwith10%FBS,0.1µMdexamethasone,200µMascorbicacid,and10mMβ-glycerol
phosphate. Adipogenic differentiation (n = 3): DMEM supplemented with 10% FBS, 1 µmol/L
dexamethasone,50µmol/Lindomethacin,0.5mM3-isobutyl-1-methyl-xanthine,and10µMinsulin.
Condrogenicdifferentiation(n=3): DMEMsupplementedwith10%FBS,acidfree15mMHEPES,
6.25µg/mLinsulin,10ng/mLTGF-β1,and50nMAsAP.Culturesweremaintainedat37◦Cwith5%
CO ,andthecompletemediumswerereplacedtwiceaweek. Attheendoffourweeks,cellswere
2
fixedwith4%paraformaldehydefor20minandstainedwith2%AlizarinRedS(pH4.1)during5min
forcalciumdetection,with0.025%ToluidineblueinMcIlvainebuffer(0.03Mcitricacid,0.04Msodium
phosphatedibasic,pH4.0)during10minforproteoglycansdetection,andwith1%SudanIVduring
5mintoshowlipiddroplets. SampleswereimagedontheAxiovertS100(ZEISS)invertedmicroscope.
2.1.4. CellViability
Flasksof75cm2 withADMSCin5P(n=3)weretrypsinizedandcentrifugedat1800rpmfor
10min.Thepelletobtainedfromeachflaskswasresuspendedin1mLofDMPBSFlush.Then,analiquot
of10µLofeachculturewasstainedwith0.4%TrypanBlue,andtheADMSCwereplacedforanalysis
intheCountessIIFL(LifeTechnologies®Inc.,Carlsbad,CA,USA)equipmentforcellconcentration
andviabilitymeasurements.
2.1.5. ContrastbyDifferentialInterference(DIC)
For the birefringence analyses of nuclei, ADMSC (5P) in culture (n = 3) were stained with
0.025%toluidinebluesolutionin0.1MMcIlvainephosphatebufferatpH4.3for30min,followedby
analysesintheOlympusBX-51(OlympusAmerica,CenterVallery,PA,USA)polarizingmicroscope
equippedwithaQ-color5camera(OlympusAmerica),andusingtheImagePro-plusv.6.3software
forWindows™(MediaCybernetics,SilverSpring,MD,USA)[4,33,34]. Withthemicroscopeandthe
software,itispossibletocarryoutanalysisofDICandtheanisotropicproperties,bothindividuallyand
incombination[34]. DICobservationswereperformedafteradditionofthetwocondenser’sWollaston
prisms in the light path. With DIC-PLM, the optical path differences in samples can be detected
throughtheslidingofanotherWollastonprismthatispositionedundertheanalyzer. Theresulting
colorsinthesamplearecomparedwiththecolorsinaMichel-Lévychart.
2.1.6. RealTime-PCRArrayforAnalysisofADMSCGeneExpression
For the total RNA extraction of the ADMSC (5P) using Trizol® reagent, Invitrogen,
Carlsbad, CA, USA, three different cultures were analyzed. A spectrophotometer (NanoDrop®
ND-1000, Thermo Fisher Scientific®, Waltham, MA, USA) was used to quantify the RNA in
each sample by determining the absorbance ratio at 260 and 280 nm. 0.5 µg from the total
extracted RNA of each sample was used for the synthesis of cDNA using the RT2 First Strand
Kit (QIAGEN®, Hilden, Germany) and thermocycler Mastercycler Pro (Eppendorf®, Hamburg,
Germany), also following the manufacturer’s instructions. The cDNA was frozen at −20 ◦C until
tested. The RT-PCR array reaction was performed using the RT2 Profiler PCR Arrays (A format)
kit in combination with the RT2 SYBR Green Mastermixes (QIAGEN®, Hilden, Germany) on the
thermocycler apparatus 7300 (ABI Applied Biosystems®, Foster City, CA, USA), following the
manufacturer’s instructions. For each culture sample, three types of reaction controls were used:
Cells2018,7,127 5of22
1. Positive PCR control; 2. Reverse transcriptase control; and 3. Control for contamination of rat
genomic DNA. The Glyceraldehyde-3-phosphate dehydrogenase (Gapdh, NM_017008) was used as an
endogenous control for each sample. The following genes were analyzed (QIAGEN®): Scleraxis
(Scx, NM_001130508); Tenomodulin (Tnmd, NM_022290); Tumor necrosis factor (TNF superfamily,
member2)(Tnf, NM_012675); Interleukin1beta(II1b, NM_031512); Transforminggrowthfactor, beta1
(Tgfb1,NM_021578);Matrixmetallopeptidase2(Mmp2,NM_031054);Matrixmetallopeptidase9(Mmp9,
NM_031055);Matrixmetallopeptidase8(Mmp8,NM_022221);TIMPmetallopeptidaseinhibitor2(Timp2,
NM_021989);Decorin(Dcn,NM_024129);Lysyloxidase(Lox,NM_017061);andGrowthdifferentiation
factor 5 (Gdf5, XM_001066344). Reactions were made in a single cDNA pipetting for each gene,
including the endogenous control. ∆CT values were obtained by the difference between the CT
valuesofthetargetgenesandtheGapdhgene. The2−∆CTmethod[35]wasusedtocalculatethegene
expressionforeachtargetgene.
2.2. InVivoExperiments
2.2.1. ExperimentalGroups
Atotalof110maleLewisrats(120-day-old),withfreeaccesstofoodandwater,weredividedinto
5experimentalgroups(22animalsforeachgroup):Normal(N):Ratswithtendonswithouttransection;
Transected(T):RatswithpartiallytransectedtendonsandtreatedwithtopicalapplicationofDMPBS
Flushinthetransectedregion;Mesenchymalstemcellsderivedfromadiposetissue(ASC):Ratswith
transectedtendonswithsubsequenttransplantofADMSC(3.7×105cells)inthetransectedregion;
GDF-5(GDF5): RatswithtransectedtendonsandtreatedwithtopicalapplicationofDMPBSFlushin
thetransectedregion+GDF5application(500ng)24hafterpartialtransection;andwithADMSCand
GDF-5(ASC+GDF5): RatswithtransectedtendonsandtreatedwithsubsequenttransplantofADMSC
(3.7 × 105 cells)inthetransectedregion+GDF5application(500ng)24hafterpartialtransection.
Animalswereeuthanizedonthe14thdayaftertransectionbyanoverdoseofanesthetic(Ketamine
andXylazine). AnimalsofgroupNwereeuthanizedat134daysandthetendonswithouttransection
werecollectedforanalysis.
2.2.2. PartialTransectionoftheCalcanealTendonandApplicationofADMSCandGDF-5
The animals were anesthetized with intraperitoneal injection of Ketamine (90 mg/Kg) and
Xylazine (12 mg/Kg), and the right lower paws submitted to antisepsis and trichotomy. For the
exposureofthecalcaneustendon,alongitudinalincisionwasmadeintheanimal’sskin,followed
byatransversepartialtransectionperformedintheproximaltendonregionlocatedatadistanceof
4mmfromitsinsertioninthecalcaneusbone[36,37]. Approximately3.7×105 ADMSC(5P)were
resuspendedin15µLofDMPBSFlushandtransplantedinthetransectedregionoftendonsinthe
ASCgroupusingapipette. 15µLofDMPBSFlushwasappliedintendonsoftheTgroup,andthe
GDF5grouptendonsreceivedanapplicationof15µLofDMPBSFlush+500ngofGDF-524hafter
the tendon transection. All applications were made in the region of the tendon where the partial
transectionwasperformed. Then,theskinwassuturedwithnylonthread(Shalon5-0)andaneedle
(1.5cm). AllsurgicalandexperimentalprotocolswereapprovedbytheInstitutionalCommitteefor
EthicsinAnimalResearchoftheStateUniversityofCampinas-UNICAMP-Brazil(Protocolnº2905-1).
2.2.3. PreparationofSectionsinFreezing
TendonswereplacedinTissue-Tek®,frozen,andcutincryostat(seriallongitudinalcutsof7µm
thickness). The sections were fixed using a 4% formaldehyde solution in Millonig buffer (0.13 M
sodiumphosphateand0.1Msodiumhydroxide,pH7.4)for20min,followedbybirefringenceand
lineardichroismanalysis.
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2.2.4. DAPIStaining
Immediately after fixation, the sections (n = 4) were incubated with DAPI
(4(cid:48),6-Diamidino-2-phenylindole dihydrochloride) (0.1 mg/mL in methanol) for 5 min at 37 ◦C.
Thesectionswereanalyzedbyfluorescencemicroscope(OlympusBX60)andtheimagescapturedby
theQCapture4.0program.
2.2.5. PolarizationMicroscopy: BirefringenceMeasurements
After fixation, image analyses of the tendons (n = 4) were evaluated to detect differences in
morphology based on the aggregation and organization of the collagen bundles, which reflect
the variation of birefringence intensity. Birefringence properties were studied using an Olympus
BX53 polarizing microscope and an image analyzer (Life Science Imaging Software, Version
510_UMA_cellSens16_Han_en_00). Because the birefringence appears visually as brilliance,
thisphenomenonwasmeasuredwithanimageanalyzerandexpressedasgrayaverage(GA)values
inpixels(8bits=1pixel). Thelargertendonaxiswaspositionedat45◦ tothecrossedanalyzerand
polarizer. Ascollagenbundlesexhibittwotypesofbirefringence,intrinsicbirefringence(Bi)andform
ortexturalbirefringence(Bf)[38],thetotalbirefringence(thesumofBiandBf)wasusedinthisstudy.
Measurementsofthetendonsineachexperimentalgroupweremadeafterimmersingthesections
in water, a condition in which total birefringence is highly detectable [4,33,34,38]. The number of
measurementsofGAwasrepresentedasthemedianandtheywerechosenatrandomin16sections
fromfourtendonsofeachgroup.
2.2.6. LinearDichroismMeasurements
Linear dichroism measurements (n = 4) were obtained from toluidine blue-stained sections.
LineardichroismmeasurementshaveshownthatGAGchainspresentincollagenfiberPGsarelinearly
distributedandpredominantlyparalleltothelongestfiberaxis[3,39,40]. Inthiscase,lineardichroism
is an extrinsic phenomenon, resulting from the arrangement of toluidine blue molecules that are
electrostaticallyboundtotheanioniclinksitesoftheorientedsubstrate. Thedichroicratio(DR=d/d⊥)
wasdeterminedbythetoluidineblueabsorbanceintheparallel(d)andperpendicular(d⊥)positions
ofthetendon’slongestaxis,withregardtothepolarizedlightplane(PLP)[39,40]. Lineardichroism
wasmeasuredusinganOlympusBX53polarizingmicroscope(Objective: OlympusUPlanFLN40×;
Camera:OlympusQ-color5;Polarizer:OlympusU-POT)andanimageanalyzer(LifeScienceImaging
Software,Version510_UMA_-cellSens16_Han_en_00). Thenumberofmeasurements(~100)ofGA
wasrepresentedasthemedianandtheywerechosenatrandomin16sectionsfromfourtendonsof
eachgroup.
2.2.7. RealTime-PCRArray
Thecollectedtendons(n=4)wereplacedinstabilizingsolution(RNA-later,QIAGEN®Hilden,
Germany) and maintained at −20 ◦C. For the total RNA extraction, the tendons were sprayed
using liquid nitrogen and then homogenized in a tube containing 5 stainless steel balls (2.3 mm
diameter,Biospec)bybeingshakeninaTissueLyserLTinstrument(QIAGEN® Hilden,Germany),
with2repetitions(60s)intercaletedwithicecooling(2min)betweeneachshakingstep(5). TotalRNA
was isolated from each sample using the RNeasy® Fibrous Tissue Mini Kit (QIAGEN® Hilden,
Germany),followingthemanufacturer’sinstructions. Aspectrophotometer(NanoDrop®ND-1000,
Thermo Fisher Scientific®, Waltham, Massachusetts, USA) was used to quantify the RNA in each
samplebydeterminingtheabsorbanceratioat260and280nm. 0.5µgfromthetotalextractedRNAof
eachsamplewasusedforthesynthesisofcDNAusingtheRT2 FirstStrandKit(QIAGEN® Hilden,
Germany)andthermocyclerMastercyclerPro(Eppendorf®Hamburg,Germany),alsofollowingthe
manufacturer’sinstructions. ThecDNAwasfrozenat−20◦Cuntiltested. TheRT-PCRarrayreaction
wasperformedusingtheRT2ProfilerPCRArrays(Aformat)kitincombinationwiththeRT2SYBR
Cells2018,7,127 7of22
GreenMastermixes(QIAGEN®Hilden,Germany)onthethermocyclerapparatus7300(ABIApplied
Biosystems®,FosterCity,CA,USA),followingthemanufacturer’sinstructions.Foreachanimalsample,
threetypesofreactioncontrolswereused: 1. PositivePCRcontrol;2. Reversetranscriptasecontrol;
and3. ControlforcontaminationofratgenomicDNA.TheGlyceraldehyde-3-phosphatedehydrogenase
(Gapdh, NM_017008) was used as an endogenous control for each sample. The following genes
were analyzed (QIAGEN® Hilden, Germany): Scleraxis (Scx, NM_001130508); Tenomodulin (Tnmd,
NM_022290); Tumor necrosis factor (TNF superfamily, member 2) (Tnf, NM_012675); Interleukin 1beta
(II1b, NM_031512); Transforming growth factor, beta 1 (Tgfb1, NM_021578); Matrix metallopeptidase 2
(Mmp2,NM_031054);Matrixmetallopeptidase9(Mmp9,NM_031055);Matrixmetallopeptidase8(Mmp8,
NM_022221); TIMP metallopeptidase inhibitor 2 (Timp2, NM_021989); Decorin (Dcn, NM_024129);
Lysyloxidase(Lox,NM_017061);andGrowthdifferentiationfactor5(Gdf5,XM_001066344). Reactions
weremadeinasinglecDNApipetteforeachgene,includingtheendogenouscontrol. ∆CTvalueswere
obtainedbythedifferencebetweentheCTvaluesofthetargetgenesandtheGapdhgene. Thesevalues
werenormalizedbysubtractingthe ∆CTvalueofthecalibratorsample(Tgroup)toobtain ∆∆CT
values. A2−∆∆CT methodwasusedtocalculatetherelativeexpressionlevel(foldchange)foreach
targetgene. Resultswererepresentedastherelativegeneexpressionincomparisontothecalibrator
samplethatisequalto1.
2.2.8. DosageofHydroxyproline
Hydroxyproline was used as an indicator of the amount of total collagen in the tendons of
thedifferentgroups(n=5)usedpreviouslyinthebiomechanicalassay. Thetendonswerecutand
immersedinacetonefor48h,followedbyasolutioncontainingchloroform:ethanol(2:1)alsofor48h.
Afterdehydration,thesampleswereplacedfordryingintheovenat37◦C.Sampleswereweighed
and hydrolyzed in 6N HCl (1 mL/10 mg of tissue) for 4 h at 130 ◦C according to Stegemann and
Stalder[41]withsomemodifications,andneutralizedwith6NNaOH.Theabsorbanceofthesamples
wasmeasuredat550nmusingamicroplatereader(ExpertPlus,Asys®,Holliston,MA,USA).
2.2.9. EvaluationoftheMaxContactIntensityoftheRatPawafterPartialTransection
The CatWalk system (Noldus Inc., Wageningen, The Netherlands) was used to analyze the
gait recovery of the animals (n = 5). In this protocol, the rats crossed a walkway (100 cm length,
5cmwidth,and0.6cmthickness)withaglassfloorilluminatedfromthelongedgeinadarkroom.
Dataacquisitionwasperformedwithahigh-speedcamera(PulnixTM-765ECCD),andthepawprints
wereautomaticallyclassifiedbythesoftware. Thepawprintswereobtainedduringthe3daysbefore
thepartialtransectionofthetendonstoassessthenormalstandardgaitoftheanimals,andtheywere
collectedagainafterthelesions. Post-operativedatawereassessedonthe1st,3rd,5th,7th,9th,11th,
and13thdaysfollowingsurgicallesion. Theparametersusedhereinwere“MaxContactIntensity”,
corresponding to the pressure exerted by the paw on the glass floor during gait. The intensity of
magnificationcanvaryfrom0to255pixels.
2.2.10. BiomechanicalParameters
Tendons from experimental groups (n = 5) were collected and stored at −20 ◦C until tested.
Beforethebiomechanicaltest,thetendonswerethawedandmeasuredwithpachymeter,considering
their length, width, and thickness. For the biomechanical assay, the tendons were maintained in
PBS to prevent their fibers from drying out. Then, the tendons were fixed to metal claws by the
myotendinousjunctionandbytheosteotendinousjunctionforcorrectalignmentintheequipment
(Texturometer, MTSmodelTESTSTARII).Ineachbiomechanicalassay, tendonsweresubjectedto
a gradual increase of load at a displacement velocity of 1 mm/s by using a load 0.05 N until the
tendonruptured. BiomechanicalparameterswereanalyzedaccordingtoBiancalanaetal.[42]and
Tomiossoetal.[9],suchasmaximumforce(N)andmaximumdisplacement(mm),whichwereused
tocalculatethemaximumstress(Mpa)andmaximumstrain(L)oftendonsfromtheexperimental
Cells2018,7,127 8of22
groups. The cross-sectional area of the calcaneus tendon was calculated by assuming an elliptical
approximation(A=πWd/4)usingmeasurementsofwidth(W)andthickness(d)valuesfromthe
sameSparrowetal.[43]. Themaximumstressvalue(MPa)wasestimatedbytheratiobetweenthe
maximumload(N)andthecross-sectionalarea(mm2). Themaximumdeformation(L)wascalculated
through (L = L − L/L), where (L ) is the value of the final length before rupture, and (L) is the
f i i f i
initialtendonlengthvalue. Stress-straincurveswereconstructedusingthemeanofeachmechanical
propertyobtainedforeachgroup.
2.2.11. StatisticalAnalysis
All results were presented as the mean and standard deviation for the values with a normal
distribution(orinterquartilerangeandmedianforthevaluesthatdidnotadheretotheGaussian
distribution). For the data with normal distribution, the analysis of variance (ANOVA) was used,
Cells 2018, 7, x 8 of 23
followedbytheTukeypost-hoctestforintra-groupanalysis(inthecaseofstatisticalsignificance),
or the Student’s t(LTi)e iss tthep irneiticael tdeneddon bleyngtthh vealuLe.e Svtreesns-estrTaien scut.rveFs owrered caontastruthcteadt udsinidg thne omteaand ofh eeacrhe to the Gaussian
mechanical property obtained for each group.
distribution,thenon-parametrictestoftheKruskal-Wallistestfollowedbythepost-hoctestofDunn
2.2.11. Statistical Analysis
forintra-groupanalysis(inthecaseofstatisticalsignificance)wasusedortheUTestofMann-Whitney.
All results were presented as the mean and standard deviation for the values with a normal
StatisticalanalysiswasperformedinthesoftwareStatisticalPackageforSocialSciences(SPSS)version
distribution (or interquartile range and median for the values that did not adhere to the Gaussian
22.0andforallthedisatrfibourtieomn). eFnort tihoe ndeatda wtieths tnsortmhael dsisitgribnuitfiionc,a thnec aenalleysvise olf αvar=ian0ce. 0(A5NaOnVdA) pwoasw useerd, ofthetestof95%
followed by the Tukey post-hoc test for intra-group analysis (in the case of statistical significance), or
wereconsidered. the Student’s t Test preceded by the Levene Test. For data that did not adhere to the Gaussian
distribution, the non-parametric test of the Kruskal-Wallis test followed by the post-hoc test of Dunn
3. Results for intra-group analysis (in the case of statistical significance) was used or the U Test of
Mann-Whitney. Statistical analysis was performed in the software Statistical Package for Social
Sciences (SPSS) version 22.0 and for all the aforementioned tests the significance level α = 0.05 and
3.1. InVitro power of the test of 95% were considered.
3. Results
3.1.1. InVitroDifferentiationPotentialofADMSCandFlowCytometry
3.1. In Vitro
Invitro ADMSC staining with Toluidine Blue, Alizarin Red S, and Sudan IV showed
3.1.1. In Vitro Differentiation Potential of ADMSC and Flow Cytometry
differentiation of ADMSC at the 5P in chondrocytes, osteoblasts, and adipocytes, respectively
In vitro ADMSC staining with Toluidine Blue, Alizarin Red S, and Sudan IV showed
(Figure 1A–C). Fldoifwferecntyiattioonm oef tArDicMSaCn aatl ythse is5P (iFn icghuonrderoc1yDtes,,E o)stesohbloaswts, eadnd tahdeipopcyrteess, erenspceectivoefly CD90 (90%) and
(Figure 1A–C). Flow cytometric analysis (Figure 1D,E) showed the presence of CD90 (90%) and
CD105(95%)positivesurfacemarkers,andthenegativemarker,CD45(75%).
CD105 (95%) positive surface markers, and the negative marker, CD45 (75%).

Figure 1. In vitro adipogenic (A), condrogenic (B), and osteogenic (C) differentiation of
Figure1.Invitroadipogenic(A),condrogenic(B),andosteogenic(C)differentiationofadipose-derived
adipose-derived mesenchymal stem cells (ADMSC) in 5P: Observe intracellular lipid droplets
mesenchymalstemstaicneedl lwsit(hA SuDdaMn IVS C(→)),i pnro5tePog:lyOcabnss setarivneed winitthr taolcueidlilnue lbalure (lip), iadndd exrtorapcellelutlasr scatlaciiunme dwithSudanIV
(→),proteoglycansstasinteadi nweithd alwizairtihn rteod lSu (id). iBnaers =b Alu aend( C(cid:73): 2)0,0a μnmd; Be: 1x0t0r μamc.e (lDlu) Hlaisrtogcraamlcsi udemmonssttaraitne tehde withalizarinred
x-axis fluorescence scale considered positive when the cell peak is above 101 (CD45) or 102 (CD105
S((cid:73)).Bars=AanadndC C:D2900)0. (µE)m C;onBtr:o1l 0fo0r µ–PmE .an(dD –)AHPCi s(twoitgh rvaemry slodwe mfluoorenscsetnrcae)t, ecotrhreespxon-daixngi stofl uorescencescale
consideredpositivneonw-mharekend tcehlles. (cFe) lFllopwe caytkomiestray bofo AvDeM1SC0 f1or( CCDD1045,5 C)Do90r, a1n0d2 CD(C45D su1rf0ac5e maanrkderCs. D90).(E)Controlfor
–PEand–APC (withverylowfluorescence),correspondingtonon-markedcells.(F)Flowcytometryof
ADMSCforCD105,CD90,andCD45surfacemarkers.
Cells 2018, 7, x 9 of 23
Cells2018,7,127 9of22
3.1.2. Fluorescence, Birefringence, and Contrast by Differential Interference (DIC)
3.1.2A. Fnlaulyosreiss ceunscien,gB irfelufroinregsecnecnec,ea nmdCicoronstcroasptyb ysDhoifwfeeredn tiAalDIMnteSrCfe-GreFnPc e(oDnI C5)P with fusiform
fibroblast-like morphology (Figure 2A). When DIC was used, nucleoli were observed (Figure 2B).
AnalysisusingfluorescencemicroscopyshowedADMSC-GFPon5Pwithfusiformfibroblast-like
On AT (pH 4.3) staining, most of the nuclei were stained in blue, with the presence of granules of
morphology(Figure2A).WhenDICwasused,nucleoliwereobserved(Figure2B).OnAT(pH4.3)
various sizes in some regions being highly stained in blue (Figure 2C). Under polarizing microscopy,
staining,mostofthenucleiwerestainedinblue,withthepresenceofgranulesofvarioussizesinsome
the granules exhibited abnormal interference colors due to differences in the high packing of DNA
regionsbeinghighlystainedinblue(Figure2C).Underpolarizingmicroscopy,thegranulesexhibited
(Figure 2D).
abnormalinterferencecolorsduetodifferencesinthehighpackingofDNA(Figure2D).

FFigiguurree 22.. MMoorrpphhoollooggyyo offA DAMDMSCSCon o5nP :5(AP:) A(AD) MASDCM-GSFCP-:GFFibPr:o bFliabsrto-lbiklaesmt-loikrpe hmoloorgpyh,woliothgya, fuwsiitfho rma
fushsaifpoerm.( Bs)hCapoen.t r(aBs)t CbyonDtrifafsetr ebnyt iDalifIfnetreernfetiraeln Icnete(DrfIeCre):nTceh e(DreIdC)t:o Tbhlue erbedan tdo sbhlouwe ibnagntdh eshinotwerinfegr etnhcee
ineftfeercfetroefntchee enfufecclte uofs tdhuee ntuochleiguhs edruceo tnoc ehnigtrhaetrio cnosnocfenmtraatteiroinals. oNf umclaetoelriiaplr. eNseunccleeoslei epnreinsetnhcee isneiteina lino f
ththee ibnliutiealb oafn tdh(e(cid:73) b)l.u(eC b)aAnDdM (SC). s(tCa)i nAeDdMwSitCh AstTai(npeHd 4w.3it)h: NAoTt e(pthHe 4p.r3e)s: eNnoceteo tfhneu pclreeissetnacien eodf innubclleuie
s(t→ain),eadn idn tbhleupe r(e→se)n, caenodf tghrea npurleessenofcev aorfi ogurasnsuizleess hoifg vhalyrisotuasin seidzeisn hbilguhelyin sstoaimneedr eigni obnluse( (cid:73)in) dsoumeteo
rtehgeiodniss p(on)i bdiluitey otof pthheo sdpihsaptoengibroiluitpys .o(fD p)hPooslpahriaztaet iognromupicsr. o(sDco) pPyo:lIanritzhaetidoent amileicdroimscaogpey,: nIont etthhee
abnormalinterferencecolorsduetoabnormaldispersionofbirefringencebecauseofthedifferences
detailed image, note the abnormal interference colors due to abnormal dispersion of birefringence
inthehighpackingofDNA.Bars=50µm(A),70µm(B,C),and35µm(D).(E)NumberofADMSC
because of the differences in the high packing of DNA. Bars = 50 μm (A), 70 μm (B,C), and 35 μm (D).
usedforapplicationintendons,presentingabout80%viabilityaftertripsinization.(F)RT-PCRarrayof
(E) Number of ADMSC used for application in tendons, presenting about 80% viability after
ADMSCon5PinvitroshowingtheexpressionprofileofthegenesLox,Dcn,Timp2,Mmp2,andTgfb1.
tripsinization. (F) RT-PCR array of ADMSC on 5P in vitro showing the expression profile of the
gNenoeesx Lporxe,s Dsiconn, Twiamspo2b, sMermvpe2d, faonrdS Tcxg,fbT1n. mNdo, eMxpmrpe9s,sGiodnf 5w,Tasn of,basnedrvIelbd1 fgoer nSecsx., Tnmd, Mmp9, Gdf5, Tnf,
and Ilb1 genes.
3.1.3. CellViability
3.1.3. FCieglul rVei1aEbislihtyo wedameanof3.7×105ADMSCusedfortendons’applicationoftheADMSCand
ASCF+iGguDrFe5 1gEr sohuopws,ewd iath maemane aonf 3o.f78 ×0 1%05o AfvDiaMbSleCA uDseMd SfCor( tFeingduorens2’E a)p. plication of the ADMSC and
ASC+GDF5 groups, with a mean of 80% of viable ADMSC (Figure 2E).
3.1.4. Real-TimePCRArray
3.1.4. IRneathl-eTiRmTe-P PCCRR aArrrarayya nalysis of ADMSC on 5P, the expression profile of the genes, Lox, Dcn,
Timp2,Mmp2,andTgfb1wasobserved. NoexpressionwasobservedfortheScx,Tnmd,Mmp9,Gdf5,
In the RT-PCR array analysis of ADMSC on 5P, the expression profile of the genes, Lox, Dcn,
Tnf,andIlb1genes. TheexpressionoftheMmp8genewasnotrepresentedbecauseonlyoneculture
Timp2, Mmp2, and Tgfb1 was observed. No expression was observed for the Scx, Tnmd, Mmp9, Gdf5,
expressedit(Figure2F).
Tnf, and Ilb1 genes. The expression of the Mmp8 gene was not represented because only one culture
expressed it (Figure 2F).
Cells2018,7,127 10of22
Cells 2018, 7, x 10 of 23
3.23..I2n. IVni vVoivo
3.23.1.2..I1m. Immumnuonflouflouroersecsecnecnece
InItnh ethpe rperseesnetnst tsutduyd,ys, isgingnifiifciacanntta altlteerraattiioonnss iinn tthhee EECCMM ooff tthhee AAcchhilillelse stetnednodno nwwereer oebosbersveervde d
aftaefrte1r4 1d4a dyasyssi nsicneceth tehep apratritailalt rtarannsseeccttiioonn,, bbootthh ffrroomm tthhee aapppplliiccaatitoionn oof fththe eAADDMMSCS Cisoislaotleadte idn itnhet he
injuinrjeudrerdeg rieognioann danfrdo mfrotmhe tahpe palpicpaltiicoantioonf tohfe tAheD AMDSCMaSsCs oacsisaotceidatwedit hwGithD FG-D5.FT-5h. eTcheell cmelilg mraitgiorantiaosns ay
demasosanys trdaetmedonthsteraptreeds etnhcee porfeAseDncMe SoCf -GADFPMiSnCt-eGnFdPo nins etcetniodnosn osfebctoitohnsg roofu bpos,thA SgCrouanpds, AASSCC+ GanDdF 5,
onAthSeC3+rGdDaFn5d, o1n4 tthhed 3aryds aanftde r14inthju drayy(sF aigftuerre in3j)u.ry (Figure 3).

Figure3.ADMSC-GFPMigrationtoTRonthe3rdand14thdaysafterinjury:Observethepresence
Figure 3. ADMSC-GFP Migration to TR on the 3rd and 14th days after injury: Observe the presence
ofADMSC-GFP(A,D,G,J)intheASCandASC+GDF5groups. Visiblehighernumbersofcellscan
of ADMSC-GFP (A,D,G,J) in the ASC and ASC+GDF5 groups. Visible higher numbers of cells can be
beobservedonthe14thdayinbothgroups. DAPI:Nucleimarking(B,E,H,K).Mergedimagesof
observed on the 14th day in both groups. DAPI: Nuclei marking (B,E,H,K). Merged images of
ADMSC-GFPwithnucleimarkedwithDAPI(C,F,I,L).Bar=50µm.
ADMSC-GFP with nuclei marked with DAPI (C,F,I,L). Bar = 50 μm.
3.2.2. Real-TimePCRArray
3.2.2. Real-Time PCR Array
ThTeh1e2 1g2e ngeenseesx pexrepsrseisosnioann aanlyaslyiss,iso, botbatinaiendedth trhoruoguhghth teheR TR-TP-CPRCRa rarraryay(F (iFgiugruere4 )4,)s, hsohwowededt htahtatt he
ADthMe SACDaMpSpCli caaptipolnicautpio-nre ugpu-lraetgeudlattheed etxhpe reexspsiroesnsioofnt hoef tLhoex L,oDxc, nD,cann, danTdg fTbg1fbg1e ngeensecso cmompapraerdedto tot he
Tgtrhoeu Tp ,garonudpo, fanthde oMf tmhep 2M,Tmipm2p, 2T,imanpd2, Ganddf5 Ggdefn5e gseinnerse ilna trieolnatitoont htoe tAheS CA+SGC+DGFD5Fg5r oguropu.p.