w w w . r b o . o r g . b r
Original
article
Effect
of
methylprednisolone
use
on
the
rotator
cuff
in
rats:
biomechanical
and
histological
study
夽
Gustavo
Vinícius
Ghellioni,
Lucas
Souto
da
Silva,
Anna
Paula
Piovezan,
Rafael
Olívio
Martins
∗UniversidadedoSuldeSantaCatarina(UNISUL),Tubarão,SC,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received14April2014 Accepted13June2014 Availableonline28May2015
Keywords:
Corticoids Rotatorcuff Orthopedics
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t
Objective:Toevaluatetheinfluenceoftreatmentwithdifferentdosesofmethylprednisolone onthemechanicalresistanceandpossiblehistologicalalterationsoftherotatorcufftendon inrats.
Methods:MaleWistarratsweredividedrandomlyintofourtreatmentgroups:sham,vehicle or0.6mg/kgor6.0mg/kgofmethylprednisolone.Changestomechanicalresistance(inN) andhistologicalparameters(fibrillarappearance,presenceofcollagen,edemaandvascular proliferation)oftherotatorcufftendonwereevaluated.Theanalyseswereconductedafter administrationofonetreatment(24hafterwards),twotreatments(7daysafterward)orthree treatments(14daysafterwards),intothesubacromialspace.
Results:Sevenandfourteendaysafterthetreatmentswerestarted,itwasfoundthatin adose-dependentmanner,methylprednisolonereducedthemechanicalresistanceofthe rotatorcufftendon(p<0.05inrelationtothevehiclegroup).Modificationstothe histologi-calparameterswereobservedonthe7thand14thdaysafterthefirstinfiltration,especially regardingthepresenceofcollagenandvascularproliferation,forthedoseof0.6mg/kg ofmethylprednisolone,andalsoregardingthepresenceofcollagen,edemaandvascular proliferationforthedoseof6.0mg/kgofcorticoid.
Conclusion:Theresultsobtaineddemonstratedarelationshipbetweenmethylprednisolone usethroughinfiltrationintothesubacromialspaceandreductionofthemechanical resis-tanceofandhistologicalmodificationstotherotatorcufftendoninrats.
©2014SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditora Ltda.Allrightsreserved.
夽
WorkdevelopedattheUniversityoftheSouthofSantaCatarina(UNISUL),Tubarão,SC,Brazil.
∗ Correspondingauthor.
E-mail:[email protected](R.O.Martins).
http://dx.doi.org/10.1016/j.rboe.2015.05.002
Efeito
do
uso
da
metilprednisolona
no
manguito
rotador
em
ratos.
Estudo
biomecânico
e
histológico
Palavras-chave:
Corticoides Manguitorotador Ortopedia
r
e
s
u
m
o
Objetivo: Avaliarainfluênciadotratamentocomdiferentesdosesdemetilprednisolona sobrearesistênciamecânica,bemcomopossíveisalterac¸õeshistológicasdotendãodo manguitorotador(MR)emratos.
Métodos: RatosWistarmachosforamdivididosaleatoriamenteemquatrogruposde trata-mento como sham, veículo, 0,6mg/kg ou 6mg/kg de metilprednisolona. Alterac¸ões na resistênciamecânica(emN)eemparâmetroshistológicos(aparênciafibrilar,presenc¸ade colágeno,edemaeproliferac¸ãovascular)dotendãodomanguitorotador(MR)foram avali-adas.Asanálisesforamfeitasapósotratamentocomuma(24horasapós),duas(setedias após)outrês(14diasapós)administrac¸õesnoespac¸osubacromial.
Resultados: Apóssetee14diasdoiníciodotratamentoametilprednisolonareduziu,de maneiradependentededose,aresistênciamecânicadotendãodoMR(p<0,05emrelac¸ão aogrupoveículo).Tambémforamobservadasmodificac¸ãoemparâmetroshistológicosnos diassetee14apósaprimeirainfiltrac¸ão,principalmentequantoàpresenc¸adecolágenoe proliferac¸ãovascularparaadosede0,6MG/kgdemetilprednisolonaepresenc¸adecolágeno, edemaeproliferac¸ãovascularparaadosede6mg/kgdocorticoide.
Conclusão:Osresultadosobtidosdemonstramumarelac¸ãoentreousodemetilprednisolona porinfiltrac¸ãonoespac¸osubacromialeareduc¸ãodaresistênciamecânicaemodificac¸ões histológicasnotendãodoMRderatos.
©2014SociedadeBrasileiradeOrtopediaeTraumatologia.PublicadoporElsevier EditoraLtda.Todososdireitosreservados.
Introduction
Oneofthemostfrequentcausesofshoulderpainis degener-ativetraumaticinjuriestotherotatorcuff,particularlyinthe avascularareaofthetendon,1whichmayaffectindividualsin
anyagegroupandmaybecomeworsewithagingandthrough workorrecreationalactivities.2,3
Approximately 54% of adults over the age of 60 years presentpartialtearingorcompleteruptureoftherotatorcuff, incomparisonwithonly4%ofthose agedbetween40and 60years.4Furthermore,aroundhalfofthesepatientsdonot
presenthistoriesoftrauma:thissuggeststhatinthesecases, the degeneration of the rotator cuff occurs gradually and resultsinincompletetearingandpossiblycompleterupture. Thiseventmayleadtolossofshoulderfunctiontovarying degrees.5,6Itcanalsooccuratdifferentlevels:shoulderpain,
weaknessinmakingarmabductionmovementsandlossof mobility.
Undertheseconditions,thetreatmentoptionsinclude con-servativemeasures(restandavoidanceofthecausalfactor), pharmacologicalmeasures(non-steroidalanti-inflammatory drugs)andrehabilitation(physiotherapy),alongwith infiltra-tionusingcorticoidsinthesubacromialspace(ICSS).
ICSSisusedintheinitialtreatmentofpathological condi-tionsoftherotatorcuffandmayproducegoodresultswith regard to pain relief and gains in range ofmotion, proba-bly becauseofthe anti-inflammatoryand analgesiceffects ofthesemedications,7,8resultingfromtheanti-inflammatory
effectofthecorticoids.Amongthecorticoidsused,theone seenmostoftenismethylprednisolone,whichisindicatedas short-termadjuvanttherapyforthis condition,particularly
forrelievingacutecrisesoravoidingexacerbation,becauseof itssolubilityandshort-termaction,whichcauseslesstissue damage.
However,thecurrent clinicalrecommendations forlocal useofthesemedicationsfortreatingpathologicalconditions oftherotatorcuffarethatthreeinfiltrationsoverthecourse ofayearshouldbethemaximumnumber,withaspacingofat leastthreemonthsbetweenthem.9Thisprecautionistaken
becauseofthesideeffectsreportedintheliterature,suchas tendonatrophy,alterationstothehealingprocess,structural alterations to the collagenfibers and metabolic alterations tocollagensynthesis.Thesecouldleadtodiminutionofthe biomechanicalpropertiesofthetendonandevencause com-pletetendonrupture.10,11
Fewstudieshaveexaminedthespecificclinicaleffectsof corticoidsontherotatorcuff.Theliteratureisbasically com-posedofcasereportsandexperimentalstudiesonanimals using the tendonofthe suraltriceps andthe patellar ten-don.Thelackofconsensusandthelackofspecificstudies evaluatingtheeffectofICSSonthetendonoftherotatorcuff strengthentheideathatthereisaneedtocomprehendand/or explainthemechanismsthatareimplicatedintheeffectsof corticoids.Currently,corticoidsare onlyusedempiricallyto treatthiscondition.12Therotatorcuffofratesisconsidered
tobeaveryusefulinvivomodelforstudyingthediseasesof therotatorcuff,13 althoughthesestudieshavenotyet
eval-uated the degreeof tendonresistance afterclinical use of ICSS.
cellularity,vascularizationandchangestocollagenfiber pat-ternsinthetendonoftherotatorcuffinrats.
Materials
and
methods
Seventy-five adult male Wistar rats (Rattus norvegicus) of averageweight300gwereused.Theywerekeptunder stan-dardizedlightingconditions(dark–light cyclesof12heach) and temperature conditions (22±2◦C), with free access to
waterandfeed.Theexperimentswereconductedinthe lab-oratoryofthesurgicalcenterofouruniversity,betweenthe hoursof08:00and17:00,aftertheanimalshadbecome accli-matized. The protocols were approved by our institution’s EthicsCommitteeforAnimalUse(no.12.016.4.01.111).
Thetotalsamplesizewassubdividedasfollows.A mini-mumgroupsizeofn=6wasrespected,whichwasconsidered to be the smallest number sufficient for the statistical analyses proposed,with regard to the species selected, as describedinthe literatureon thisfield.Thecalculationfor the number per group was made in accordance with the formula:[(Z˛:2+Zˇ)2p*(1−p*)2]:2ı; wherep*=(p0+p1)/2 and
ı (delta)=(p0+p1). Four treatment groups were evaluated, whichbeganwith18 animalseach.From thesegroups, six animalswereremovedatthedifferentobservationtimes,i.e. respectivelyatthetimesofthefirst,secondandthird admin-istrationsofthistreatment.Inadditiontotheseanimals,three rats were usedinitially, inapilotstudy to standardizethe techniqueforsubstanceadministrationandtorecognizethe animal’sanatomy.
Theanimalswererandomlydividedbetweenthegroups, which received the following treatments: group 1 (sham), whichdidnotundergoanyinterventionorreceiveany treat-mentoverthecourseoftheexperimentalperiodandservedas acontrolgroupfortheresistanceanalyses;group2(vehicle), whichreceivedtreatmentusingthe diluentofthecorticoid tested,inaccordancewiththemanufacturer’sspecification; andgroups3and4(experimental),whichreceived subacro-mialinjectionsoftwodifferentdosesofthecorticoidtested, respectively.
Thecorticoidinvestigatedwasmethylprednisoloneatthe dosesof0.6mg/kg(group3)or6mg/kg(group4).Thesedoses wereselectedbasedonareferencestudy14thatused0.6mg/kg
toobservedeleteriouseffectsfromsimilartreatmentonthe peritenon ofthesural tricepsofrats.Inaddition, pharma-cokineticissuesweretakenintoconsiderationandthedose of6mg/kgwaschosenwiththeaimofinvestigating possi-ble dose-effectcorrelations forthe parametersobserved in the present study,aswell aspossible adverse effectsfrom its administration. Thedrug wasadministered in the sub-acromial spaceofthe animals’right shoulder, ata volume of0.1ml/100goftheanimal’sweight.Threeadministrations wereperformed.Thetimeofapplicationofthefirstdosewas consideredtobetimezero,followedbyinjectionsatthetimes of24hand7daysafterthefirstadministration.
For theprocedure ofsubacromial injection, theanimals receivedanesthesiainducedbymeansofanintraperitoneal injectionofketaminehydrochlorideplusxylazine hydrochlo-ride(40–75mg/kgofketamine+5–10mg/kgofxylazine).The intra-articularinfiltrationsweremadeviaananteriorroute,
withtheanimalindorsaldecubitusonatablethatwastiltedat 45◦andtheupperrightlimbrotatedexternally.Followingthis, topographically, theapexoftheacromionandthehumeral headwerelocated.Theneedlewasintroducedbetweenthese two,alonganobliquepathgoingdownfromthehorizontal, untilitreachedcontactwiththehumeralhead.Itwasthen withdrawnslightlytoenableadministrationofthevolume.
Twenty-fourhoursafterthefirstapplication(day1),7days later(secondapplication)or14dayslater(thirdapplication), counting fromthe startofthe treatmentwith methylpred-nisolone, different groups ofanimals(n=6) were sacrificed and dissected.Thetendonofthe rotatorcuffwascarefully removedinordertoseparatethetendonfromthehumerus. Thiswasdone byraisingit fromits humeralinsertionand amputatingitatitsmuscle–tendonjunction,soastoforma unitthatwasthenevaluatedinaccordancewiththe method-ologydescribedbelow.
Mechanicalresistancetest
This test was conducted in accordance with what was recorded by other authors,15 using a bench with a
man-ual clamp. Its upper extremity was connected to a linear dynamometer and its lower extremity was connected toa receptacle.Theentireapparatuswassuspendedat approx-imatelyonemeterfromtheground.Forthisbiomechanical test,theforce(P=m×g)exertedonthetendonwasmeasured innewtons(N).Thetendonunitwasdissectedbymeansofan incision abovetheprotuberanceofthescapularspine,with accesssuperiorlytothesupraspinatusmuscle,togetherwith itstendon.Thiswasusedfortheresistancetestsand histologi-calevaluation.Thedistalportionofthetendonwasdeinserted fromthegreatertubercleofthehumerusandtenotomywas performedclosetothejunctionwiththemuscle.One extrem-ityofeachunitwasgrippedbythebenchclampandtheother end wasconnectedtothe dynamometer.Thereceptacleat thelowerextremitywasfilledwithdistilledwaterata con-stantflowrate.Inthismodel,asthewaterflowedin,theforce exertedonthetendonincreased,suchthatthecorresponding forceonthedynamometerproducedbymassofthereceptacle filledwithwatertogetherwithgravitycouldbereaddirectly fromthescale,innewtons.Themagnitudeoftheforceatthe momentwheneachanimal’stendonsnappedwasrecorded individually.
Histologicalanalysis
Forthehistologicalanalysis,theseparatedtendonunitwas embeddedinparaffinandsectionsofthicknessfivemicrons (5)werecutlongitudinallyandtransversally.Theslideswere
viewedatmagnificationsof100×,200×and400×afterthey had beenstainedusingapreparationofhematoxylin-eosin (HE),inordertoobservethealterationstothecollagenfiber bundlesandtothenucleiandcytoplasmofthefibrocytesand fibroblasts.Theparametersofcellularity,collagenthickness, occurrencesofedemaand vascularproliferationwere eval-uatedand categorizedasscoresof0,1,2or3,asdescribed inapreviousstudy.15Theseparametersorhistological
Table1–Influenceoftreatingtheanimalswithmethylprednisoloneontheresistanceoftheirrotatorcuffs,asassessed
usingamechanicalresistancetest.
Treatment ForceinNatthedifferentobservationtimes(numberofdaysafterthestart
ofthetreatment)
1 7 14
Vehicle 9.0±1.0 9.3±0.9 9.8±0.7
Sham 12.4±0.9 12.8±1.9 16.2±2.2
Methylprednisolone0.6mg/kg 7.9±1.0 8.5±1.6 9.6±1.4a
Methylprednisolone6mg/kg 10.6±3.7 6.4±0.7a 11.6±2.0a
Two-wayANOVAfollowedbyBonferroniforcomparisonbetweenallthegroups.p<0.05.
There were no statistical differences at the different observation times between the groups treated with the different doses of methylprednisolone.
a Inrelationtothevehiclegroup.
theanalyseswereperformedbyasinglepathologist,whowas unawareofthe respectivetreatmentgroupsoftheanimals fromwhichthesampleswereobtained.
Thedataarepresentedasthemean±SEM(standarderror ofthemean),forvaluesobtainedfromordinalvariables (ten-sioninN, toevaluatethe resistanceofthe tendon)or the medianforcategoricalvariables(degreeofinflammation).The analysis was performed using GraphPad Instat 4.0®, using
one or two-way ANOVA followed by the Kruskal–Wallis or Bonferronitest,respectively.p-Values<0.05weretakentobe significant.
Results
Theanalysisontheresistancetestsonthetendonsobtained from theanimals inthe differentgroups revealed thatthe treatmentsontheanimalsusingmethylprednisoloneatthe twodosesevaluatedinthisstudydidnotalterthetension(in N)thatwasneededtobreakthemwhentheywereevaluated within24hafterthefirstadministration(Table1).
However,ascanbeseeninTable1,atthetimesofseven and14daysafterthefirstsubacromialinfiltrationofcorticoid, significantdifferences(p<0.05)wereobservedinrelationto thecontrolgroup.Ontheseventhday,themeanvaluesforthe forceappliedtothetendonsinthegrouptreatedwith6mg/kg ofthesubstancewerelowerthanthoseobtainedfromanimals inthecontrolgroup(difference:−6.4N;95%CI:−13.3to0.4). Asimilareffectwasobservedonday14forthetwodosesof methylprednisoloneevaluated(0.6mg/kg:difference:−6.5N; 95%CI:−14.2to0.4;and6mg/kg:difference:−6.0N;95%CI:
−12.3to0.3).
Regarding the histological analysis comparing different parameters(asscores)inthetreatedgroupsinrelationtothe controlgroup,the evaluationunderthe opticalmicroscope revealed significant alterations (p>0.05) for the parame-ters evaluated, atthe different observationtimes afterthe treatmentwithmethylprednisolonestarted,aspresentedin
Table2.
Discussion
Thisstudydemonstratedthatthereisarelationshipbetween subacromialuse ofcorticoids and reductioninmechanical
resistanceandinthehistologicalparametersofthetendon oftherotatorcuff.Thisstrengthensthepreliminaryfindings fromsimilarstudies.15
Inrelationtothemechanicalresistanceoftherotatorcuff, alterationswereobservedstartingfromthethird administra-tion (afteraperiodof14days),forthe doseof0.6mg/kgof methylprednisolone.Forthehighercorticoiddose(6mg/kg), significantalterationswerealreadyobservedfromthesecond administration,afteraperiodof7days.
Conservative treatment using subacromial injections of corticoids for managingrotator cuff injuries is a common practicewithinthefieldofmedicine,butitssideeffectsmean thatgreatercareisrequiredwhenusingthis.Althoughseveral theoriesabouttheanti-inflammatoryeffectofthisdrugexist, thewayinwhichthedegenerationofthetendontissuetakes placehasnotbeenclearlyexpressed.
Inthepresentstudy,itcouldbeseenthat24hafter sub-acromial application of corticoid, there was no significant alteration(p>0.05)totheresistancebetweenthegroups stud-ied, independent of the dose used. Contrary results were observedinanotherstudy15thatalsoevaluatedtheinfluence
ofmethylprednisoloneonthetendonofthesuraltricepsof rats.INthelatterstudy,itwasnotedthattheforceneeded tobreakthetendondecreasedwithinthefirst24hafterfirst administrationofthedrug.Thiseffectwasmaintainedforat leastthenexttwoweeks.Thisdivergencebetweenthetwo studieswasprobablybecauseinthepresentstudy,unlikethe previousone,healthytendonswithoutanyinjurypriortothe treatmentwereevaluated,giventhatfragmentationofthe tis-suemightfacilitateitsrupture.
On theother hand,in thepresent study,from the time of the second administration, i.e. 7 daysafter the start of thetreatment,areductionintheresistanceofthetendonof therotatorcuffintheanimalstreatedwiththehigherdose ofmethylprednisolone(6mg/kg)wasnotedinrelationtothe controlgroup.Asimilareffectwasrecordedbyotherauthors14
T able 2 – Influence of tr ea ting the animals with meth ylpr ednisolone, with regar d to the resistance of the rota tor cuff, e v alua ted using differ ent histological par ameters. P ar ameter ev aluated Sham V ehicle Meth ylpr ednisolone 0.6 mg/kg Meth ylpr ednisolone 6 mg/kg 1 7 14 1 7 14 1 7 14 1 7 14 FA 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.2 0.0 ± 0.0 0.6 ± 0.2 a 0.3 ± 0.3 PC 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.6 ± 0.2 a,b 0.0 ± 0.0 0.8 ± 0.4 a,c 0.5 ± 0.2 ED 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.2 VP 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.3 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.7 ± 0.2 a 0.2 ± 0.2 0.6 ± 0.2 0.8 ± 0.4 a FA , fibrillar appear ance; PC, pr esence of colla g en; ED , edema; VP , v ascular pr olifer ation. Tw o-w a y ANO V A follo w ed by Bonferr oni for comparison betw een all the gr oups. p < 0.05. a In relation to the sham gr oup . b In relation to the v ehicle gr oup . c In relation to the meth ylpr ednisolone gr oup with 0.6 mg/kg.
According to the present findings, the adverse effects ofcorticoidsseemtofollowadose–dependentrelationship, given that although treating the animals with a dose of 0.6mg/kgpromotedsignificantalterationswithregardtothe parameters ofcollagen presenceand vascular proliferation afterthethirdadministration(day14)inrelationtothesham group,histologicalalterationswiththedoseof6mg/kghad alreadystarted tobeobservedfromthetimeofthe second administration(day7),regardingthefibrillarappearanceand presenceofcollagen.
In relation to the possible mechanisms through which these corticoid effectsmight be occurring, the histological alterationsseeninthetendonsoftheanimalstreatedwith methylprednisolone demonstrated possibledegenerationof thetendontissuecausedbythissubstance.Ithasbeen sug-gested thataftertissuedamage,vascularproliferation may subsequentlyoccur,withtheaimofsupplyingoxygenand cel-lularnutrition,inanattempttorepairthedamagedtissue.16
Thisnotionisinlinewithourhistologicalfindings,inwhich weobservedincreasedvascularproliferationonthe14thday afterthestartofthetreatment,withthetwocorticoiddoses evaluated.
Ontheotherhand,productionofcollagenbythe extracel-lularmatrixofthetendonisasourceofresistancetotendon traction.Simplydecreasingthequantityofthesefibersmay implydiminishedtendonstrength.17 Likewise,
methylpred-nisolonewasshowntoproducealowerquantityofcollagen and lower resistanceof the tendon ofthe rot, subsequent to partial injury induced by means of an incision.18 The
presentstudyreinforcesthesefindings,giventhatthe pres-enceofcollagenwasaffectedbybothdoses,ondays7and14, respectivelythroughtreatmentwiththedosesof6mg/kgand 0.6mg/kg.
Finally,inrelationtocellularity,itisknownthatthetendon isastructurecomposedmainlyofcollagenfibers,with rela-tively lowpresenceofcells,whichare mainlyfibroblasts.17
Although these cells were not investigated in the present study,thefindingsrelatingtothepresenceofcollagen indi-rectlysuggesttousthattheirpresenceatthesitesofcorticoid administrationincreased,startingfromthetimeofthe sec-ondadministrationofthehigherdosetested,giventhatthese cellsareresponsibleforproductionofthesefibers.Thisidea iscorroboratedbyastudyonthetendonsofthe semitendi-nosusmuscleofhumans,fromwhichit wasobservedthat increasedcellularitymightsuggestthatthetendonwasmore susceptibletoinjury.19
Conclusion
Theanalysisontheresultsobtainedfromthepresentstudy madeitpossibletodemonstratearelationshipbetween sub-acromial use of methylprednisolone and both diminished mechanicalresistanceandhistologicalalterationsinthe ten-donoftherotatorcuff,atthedosesandtimesevaluatedinthe presentstudy.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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