Braz. j. . vol.80 número4

Brazilian

Journal

of

OTORHINOLARYNGOLOGY

www.bjorl.org

ORIGINAL

ARTICLE

Spontaneous

healing

of

the

tympanic

membrane

after

traumatic

perforation

in

rats

夽

,

夽夽

Marcos

Miranda

de

Araújo

,

Adriana

Andrade

Batista

Murashima

,

Vani

Maria

Alves

,

Maria

Celia

Jamur

_,

_Miguel

_Angelo

_Hyppolito

a_{DepartmentofOphthalmology,OtorhinolaryngologyandHeadandNeckSurgery,FaculdadedeMedicinadeRibeirãoPreto,}

UniversidadedeSãoPaulo(USP),RibeirãoPreto,SP,Brazil

b_{DepartmentofCellandMolecularBiologyandPathogenicBioagents,FaculdadedeMedicinadeRibeirãoPreto,Universidadede}

SãoPaulo(USP),RibeirãoPreto,SP,Brazil

Received9August2013;accepted22March2014 Availableonline11June2014

KEYWORDS

Tympanicmembrane

perforation;

Woundhealing;

Histology

Abstract Themostcommonetiologiesoftympanicmembraneperforationareinfectionsand trauma.

Objective:Theobjectiveofthepresentstudywastoassessthehealingoftraumatictympanic membraneperforationinrats.

Methods:ThetympanicmembranefrommaleWistarratswasperforatedintheanteriorand posterior portions tothe handleofthe malleus. Five tympanic membraneswere evaluated 3 days after tympanic perforation; 5after 5days; 5 after 7 days; 3after 10 days; and4 after14days.Thetympanicmembranesweresubmittedtohistopathologicalevaluationafter hematoxylin---eosinstaining.

Results:Tympanicmembraneclosureoccurredatabout7---10daysafterinjuryandthehealing processwas completebyday14.Theproliferativeactivityoftheouterepitheliallayerwas presentclosetothehandleofthemalleusandtothetympanicannulus.

Conclusion:Thespontaneoushealingprocessofthetympanicmembranestartsfromtheouter epitheliallayer,withlaterhealingofthelaminapropriaandthemucosallayer.

© 2014Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.

夽 _{Pleasecitethisarticleas:AraújoMM.MurashimaAA,AlvesVM,JamurMC,HyppolitoMA.Spontaneoushealingofthetympanicmembrane}

aftertraumaticperforationinrats.BrazJOtorhinolaryngol2014;80:330---8.

夽夽

Institution:DepartmentofOphthalmology,OtorhinolaryngologyandHeadandNeckSurgery,FaculdadedeMedicinadeRibeirãoPreto, UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil.

∗_{Correspondingauthor.}

E-mails:drmarcosorl@usp.br,drmarcosorl@hotmail.com(M.M.Araújo).

http://dx.doi.org/10.1016/j.bjorl.2014.05.014

PALAVRAS-CHAVE

Perfurac¸ãoda

membranatimpânica;

Cicatrizac¸ão;

Histologia

Cicatrizac¸ãoespontâneadamembranatimpânicaapóssuaperfurac¸ãotraumática: estudoexperimentalemratos

Resumo As causas mais comuns de perfurac¸ões de membrana timpânica são infecc¸ões e trauma.

Objetivos: Avaliaroreparocicatricialdeperfurac¸õestraumáticasdamembranatimpânicaem ratos.

Método: AmembranatimpânicaderatosWistarmachos foramperfuradasnasporc¸ões ante-rioreposterioraocabodomartelo.Cincomembranastimpânicasforamavaliadas3diasapós perfurac¸ãotimpânica;5após5dias;5após7dias;3após10dias;e4após14dias.Asmembranas timpânicasforamsubmetidas àavaliac¸ãohistopatológicaapóscolorac¸ão com hematoxilina-eosina.

Resultados: O fechamentoda membrana timpânica ocorreu em torno de 7 a 10 dias após perfurac¸ãotraumática,eoprocessodecicatrizac¸ãoestavacompletono14◦ _{dia.Aatividade} proliferativa dacamadaepitelial externafoi identificadapróxima ao cabodomartelo eao ânulustimpânico.

Conclusão:O processode cicatrizac¸ãoespontânea damembrana timpânica se iniciacom a camadaepitelialexterna,composteriorcicatrizac¸ãodalâminaprópriaedacamadamucosa. ©2014Associac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicado por ElsevierEditoraLtda.Todososdireitosreservados.

Introduction

The tympanicmembrane (TM) is an anatomical structure

thatseparatestheouterearfromthemiddleear.TheTMis

responsibleforsoundamplificationandtransmissionthrough

theossicularchaintotheovalwindowandvestibularramp,

inadditiontotheprotectionoftheroundwindowandthe

tympanicramp.1

The ultrastructuralanatomyof TMconsistsof 3layers: theouterlayer,ofepithelial(ectodermal)origin;the mid-dlelayerorlaminapropria,ofmesodermalorigin;andthe innerlayer,ofendodermalorigin,comprisingthemiddleear mucosa.2

The outer layer consists of keratinized stratified squa-mousepithelium.3---5

The middle layer or lamina propria consists of loose subepithelial connective tissue, organized dense connec-tive tissueandsubmucosal loose connectivetissue. Loose subepidermalandsubmucosalconnectivetissueconsistsof looselyarrangedcollagenfibers,fibroblasts,nervefibersand capillaries. The dense connective tissue consists of more externallyorganizedradialcollagenfibersandmore inter-nally organized circular collagen fibers.2---5_{The inner layer}

consistsofsimplecolumnarepithelial tissue,whichis con-tinuouswiththemiddleearmucosa.2---6

ThemostcommonetiologiesofTMperforationsareotitis mediaandtrauma.7_{TraumaticTMperforationshavea78.7%}

rateofhealing.8_{FactorsthatpreventadequaterepairofTM}

intheabsenceofinfectionareyettobedefined.7,8

The objective of this study is to develop the two-dimensional studyof scar repair in traumaticTM perfora-tionsinrats.

Method

Theexperimentalstudywascarriedoutwith19maleWistar albinorats(Rattusnorvegicus),weighingonaverage280g

(range270---290g). The study followed the Ethical Princi-plesinAnimalResearchadoptedbytheBrazilianCollegeof AnimalExperimentation(COBEA)andwasapprovedbythe Ethics Committee on Animal Experimentation (CETEA) on 08/27/2007---protocolnumberforuseofanimalsinresearch No.082/2007.

Before the procedure, all animals were anesthetized with intramuscular ketamine hydrochloride (40mg/kg) (Ketamine 50mg/mL, Cristália Laboratory, São Paulo, Brazil)andintramuscularxylazine hydrochloride(5mg/kg) (Dopaser20mg/mL,HertapeCalier, Minas Gerais,Brazil). TheearsofallanimalswereassessedusingaDFVMU-M19 otomicroscope(DFV,RiodeJaneiro,Brazil)beforethe pro-cedure to rule out infection. A total of 19 animals were includedinthestudy,equivalentto23bullaewithnormal TMs.Bullae withinfectionwereexcludedfromthe proce-dure.

Traumatic perforation of the tympanic membrane was performedwitha30mm×0.8mmBDneedle(Becton Dickin-son,NewJersey,USA)anteriorandposteriortothemalleus handle,intheparstensaregionoftheTM(Fig.1).For histo-logicalevaluation,3animalswereeuthanized3daysafter theperforation(5bullae),4animalsafter5days(5bullae), 5animalsafter7days(5bullae),3animalsafter10days(3 bullae)and3animalsafter14days(4bullae).Oneanimal (1bulla)withintactTM wasassessedascontrol.The ani-malswereeuthanizedwithan intraperitonealinjectionof anoverdoseofthiopental(Thionembutal,Abbot,SãoPaulo, Brazil).

0.5 mm

Figure1 Schematicrepresentationoftympanic perforation inthe parstensaregionoftheratTM region,anteriorly and posteriorlytothemalleushandle.

handleasareference.Toallowhistologicalevaluation,the blockswerewornuntiltheyreachedapproximatelythesame perpendicularlevelrelativetothemalleushandle.Samples werestainedwithhematoxylinandeosin.

Histological evaluation was performed with an Olym-pusBX50microscope(OlympusAmerica,Inc.,Pennsylvania, USA),anddigitalhigh-resolutionimageswereacquiredwith aSpotRT3 camera(DiagnosticInstruments,Inc.Michigan, USA).The thicknessesoftheouterepithelial layer,lamina propriaand mucosa were measured using the Image-Pro-Plus® _{program, release 7.0 (Media Cybernetics Inc., MD,}

USA).InTMperforations,thicknessmeasurementswere per-formedatadistanceof50---100␮mfromtheborderofthe perforation in orderto evaluate thehealing activity near thelesion.9_{IntheintactTMs,thisassessmentwas}

standard-izedatadistanceof450---550␮mfromthemalleushandle, aregionthatcorrespondstothesiteofthepreviously per-formedTMperforation.

Results

Intacttympanicmembrane---control

ThethicknessoftheTMwasapproximately9.6␮m,havingas referencethedistanceof500␮mfromthemalleushandle. At adistance of 250␮m fromthemalleus handle, theTM thicknesswas 15.8␮m, andat 250␮mfrom thetympanic annulus,itsthicknesswas26.6␮m(Table1).

Thestratifiedsquamousepithelialtissueshowedfrom1 to2rowsofflattenedepithelialcellsthroughoutthelength oftheTM(Fig.2).Thethicknessoftheepitheliallayerwas 1.8␮m at a distance of 500␮m from the malleus handle (Table1).

This connective tissue present in its middle layer was organized,withoccasional flattenednucleus cells, proba-blyfibroblasts,andorganizedcollagenfibers,withlittleor noresponseofinflammatorycells(Fig.2).

Thecollagenfiberswereconnectedtothemalleus han-dle,andinthetympanicannulusregion,suchfibersformed the fibrocartilaginous ring of the tympanic annulus. Few capillaries without edema or vascular proliferation were locatednearthemalleushandleandwerenotfoundinthe intermediateportionsbetweentheannulusandmalleus han-dle.Thelaminapropriathicknesswas6.3␮m(Table1).

Themucosallayerhadonerowofflattenedcellsarranged assimplecolumnartissueandexhibitedcontinuitywiththe

EAC

EAC

EAC

EAC

Annulus m

m m

A

C

D

B

Table1 Meanthicknessoftheepithelial,laminapropriaandmucosallayers;andtotalthicknessofthetympanicmembrane (TM)throughoutthestudyondays0(intactTM),3,5,7,10and14.

Timeofeuthanization(days) Epitheliallayer, ␮m(%)

Laminapropria layer,␮m(%)

Mucosallayer,␮m (%)

Total,␮m(100%)

IntactTMa _{1.8(18.7%) 6.3(65.6%) 1.5(15.6%) 9.6}

IntactTMb _{2.6(16.4%) 11.9(75.3%) 1.3(8.2%) 15.8}

IntactTMc _{2.3(8.6%) 22.3(83.8%) 2(7.5%) 26.6}

3daysa _{18.9(48.4%) 14.3(36.7%) 5.8(14.9%) 39.0}

5daysa _{27.1(37.9%) 36.2(50.7%) 8.1(11.3%) 71.4}

7daysa _{37.5(42.1%) 46.1(51.7%) 5.5(6.1%) 89.1}

10daysa _{11.2(29.9%) 23.3(62.1%) 3(8.0%) 37.5}

14daysa _{5.7(27.9%) 12.5(61.3%) 2.2(10.8%) 20.4}

a _{IntheintactTMs,thethicknesswasmeasuredatadistanceof450---550}

␮mfromthemalleushandle.InperforatedTMs,measurements ofthicknesswereperformedatadistanceofbetween50and100␮mfromtheborderoftheperforation.

b _{Thethicknessmeasurementswereperformedatadistanceof250}

␮mfromthemalleushandleinanimalswithintactTMs.

c _{Thethicknessmeasurementswereperformedatadistanceof250}

␮mfromthetympanicannulusinanimalswithintactTMs.

middleearmucosa.Themucosallayerthicknesswas1.5␮m (Fig.2)(Table1).

Tympanicmembrane3daysaftertraumatic perforation

The mean thickness of TM was approximately 39␮m (Table1).

Three days after the traumatic perforation, therewas amoreproliferativeandhyperplasticepitheliallayer,with approximatelythreetofour rowsof epithelial cells, both

nearthemalleushandleandthetympanicannulus(Fig.3). Themeanepitheliallayerthicknesswas18.9␮m(Table1).

ThemiddlelayeroftheTMshowedthepresenceofcells withbasophilicnucleus,compatiblewithfibroblasts.These occasional disorganized fibroblasts did not overcome the limits of the ruptured collagen fibers in the TM perfora-tion.Therewasedemaintheloosesubepithelialconnective andsubmucosaltissuenearthemalleushandleandinthe tympanicannulus.

Therewasapredominanceofinflammationwith recruit-mentofpolymorphonuclearcellslocatedintheperivascular portion,andloosesubepithelialandsubmucosalconnective

EAC

A

C

B

D

EAC

EAC

EAC m

m

ep

ep ep

Annulus

Mucosa Lp

Lp

Figure3 HistologicalsectionimagesofratTM3daysaftertraumaticperforation,stainedwithHE.EAC,externalauditorycanal; m,malleushandle;ep,epitheliallayer;Lp,laminapropria;mucosa,mucosallayer.Image(A)showsamagnificationof20×;(Band

EAC

A

C

B

EAC

100µm

100µm

100µm

EAC

m

m Lp

Lp

ep

ep

Mucosa

Figure4 HistologicalsectionimagesofratTM5daysaftertraumaticperforation,stainedwithHE.EAC,externalauditorycanal; m,malleushandle;ep,epitheliallayer;Lp,laminapropria;mucosa,mucosallayer.Image(A)showsamagnificationof40×;(B)

100×and(C)400×.

tissue. Blood vessels with plethora or turgescence were present, close to the malleus handle and the tympanic annulusregion(Fig.3).Themeanlaminapropriathickness was14.3␮m(Table1).

Hyperplasiawasobservedinthemucosaltissuenearthe region of the perforation borders. A row of mucosal tis-suewithhyperplasticcellsandameanthicknessof5.8␮m (Table1)wasidentified.

Tympanicmembrane5daysaftertraumatic perforation

Fivedaysaftertheperforation,themeanthicknessofthe TMwasabout71.4␮m(Table1).

The epithelial layer showed hyperplasia in the region next to the malleus handle and the annulus region. The TMshowed areaswiththreetofiverowsof epithelial tis-sue (Fig.4). There wasformation of an epithelial bridge thatadvancedtowardtheperforationclosureinsomecases (Fig.4). The perforation closure started at the epithelial layer.Themeanthicknessoftheepitheliallayerwas27.1␮m (Table1).

Inthemiddlelayer,weobservedanincreaseinthe thick-nessoftheTMintheregionadjacenttotheperforationdue

tobasophilicnucleuscells,probablyfibroblasts.Clustersof redbloodcells,indicatingbloodcapillarieswerepresentin theconnectivetissueoftheTM(Fig.4).Themiddle layer thicknesswas36.2␮m(Table1).

Therewashyperplasiaofmucosaltissuewithtwotothree rowsofmucosalcells(Fig.4).Thethicknessofthemucosal layerwas8.1␮m(Table1).

Tympanicmembrane7daysaftertraumatic perforation

Seven days after the perforation, the repaired TM had a meanthicknessof89.1␮m(Table1).

TheepitheliallayeroftheTMshowedhyperplasia,with threetofiverowsthroughout theTMlength. Therewasa moremarkedepithelialreactionintheTMportionmidway betweentheannulusandmalleushandle,attheplacewhere theperforationwasperformed(Fig.5).Themeanthickness oftheepitheliallayerwas37.5␮m(Table1).

EAC EAC

A

_B

C

D

EAC

ep

ep ep

ep

Lp m

m

m

Lp

Mucosa

Figure5 HistologicalsectionimagesofratTM7daysaftertraumaticperforation,stainedwithHE.EAC,externalauditorycanal; m,malleushandle;ep,epitheliallayer;Lp,laminapropria;mucosa,mucosallayer.Image(A)showsamagnificationof100×;(B

andC)200×and(D)400×.

asthethickestregionoftheTM,occurredmidwaybetween theannulusandthemalleushandle(Fig.5).Themean thick-nessofthelaminapropriawas46.1␮m(Table1).

The mucosal layer showed a row of simple hyperpla-sic columnar cells in most of the tympanic membranes (Fig.5).Themeanthicknessofthemucosallayerwas5.5␮m (Table1).

Tympanicmembrane10daysaftertraumatic perforation

Tendaysaftertheperforation,therepairedtympanic mem-braneshowedthicknessof37.5␮m(Table1).

Epithelial tissue wasidentified withupto tworowsof cellsinmostoftheTM(Fig.6).Thethicknessofthe epithe-liallayerwas11.2␮m(Table1).

Thelaminapropriashowedanapparentreductioninthe numberoffibroblastsinallregionsofTM.Suchoccasional fibroblastsshowedamoreorganizedandaggregated distri-bution. There wasa trendof decreased blood capillaries (Fig. 6). The mean thickness of the lamina propria was 23.3␮m(Table1).

Themucosallayershowedonerowofflattenedmucosal cellsinmostoftheTM(Fig.6).Thethicknessofthemucosal layerwas3␮m(Table1).

Tympanicmembrane14daysaftertraumatic perforation

Fourteendaysaftertheperforation,therepairedTMhada thicknessof20.4␮m(Table1).

Theepithelialtissueshoweduptotworowsofflattened epithelialcellsthroughoutthelengthoftheTM(Fig.7).The meanthicknessoftheepitheliallayerwas5.7␮m(Table1). In the middle layer, basophilic nucleus cells, probably fibroblasts were present, but possiblyat smaller amounts whencomparedtotheshorterperiods (Fig.7). Themean thicknessofthelaminapropriawas12.5␮m(Table1).

The mucosal layer showed one row of flattened cells throughoutthelengthoftheTM(Fig.7).Themeanthickness ofthemucosallayerwas2.2␮m(Table1).

Closureofthetympanicperforation

The closure of the tympanic membrane occurred around 7---10daysaftertraumaticperforation,andthehealing pro-cesswascompleteonthe14thday.

Discussion

After the perforation, the healing process of the TM is typically described as occurring in three distinct phases, buttemporallyoverlapping:inflammatory,proliferativeand remodeling.3---7

In experimental skin studies, the inflammatory phase begins immediately after tissue injury and lasts for 4---6 days.10,11 _{This phase consists of a disarray of blood}

EAC

EAC EAC

EAC

m m

m

ep

ep

Lp _Mucosa

Annulus

100µm 100µm

100µm

100µm

A

C

B

D

Figure6 HistologicalsectionimagesofratTM10daysaftertraumaticperforation,stainedwithHE.EAC,externalauditorycanal; m,malleushandle;ep,epitheliallayer;Lp,laminapropria;annulus,tympanicannulus;mucosa,mucosallayer.Image(A)showsa magnificationof40×;(B)100×and(CandD)200×.

recruitedtothewound,whilemonocytesarerecruitedafter 48---96h.11

The proliferative phase is classically characterized by epithelialproliferation;proliferationoffibroblastswith col-lagen deposition; and by angiogenesis, with granulation tissueformation.11_{Theproliferativephaseisusuallypresent}

fromday4today14inanexperimentalstudyofskin.11

Onthe3rddayafterthetraumaticperforation,amore proliferative, hyperplastic epithelial layer, with approxi-mately 3---4 rows of epithelial cells, was observed in the TM, both near the malleus handleand close to the tym-panicannulus(Fig.3).After5---7days,theepithelialmitotic activityintensified,withportionsof5rowsofepithelialcells (Figs.4and5).Therewasatendencyforepithelialbridge formationthat advanced inthe direction ofthe tympanic perforationclosure.

Thisproliferativeactivityoftheouterepitheliallayerof TM hasbeen described asoccurring within thefirst hours aftertissueinjury.3,12,13_{Intheliterature,therearereports}

oftheexistenceofepithelialproliferativecentersnearthe malleushandleandthetympanicannulusregion.9,14,15

How-ever,onestudythatdescribedonlyonecenterofepithelial proliferationnearthemalleushandlewasfound.3

In experimental models of skin wound healing, the remodeling phase was described as starting after 8 days

oftissueinjuryandpersistingforafewmonths.Themain molecular events occur in the extracellular matrix of the laminapropria.11

InTM,epithelialremodelingwasobservedfromday10, which remained until the 14th day. The outer epithelial layerbecamethinner,withapproximately2rowsof epithe-lialcells.Inthemiddlelayer,fibroblasts wereobservedin increasingly smaller amounts, showing a flattened shape. Thetendencywasthereductioninthenumberof capillar-iesalongthetympanicmembrane.Themucosallayeralso becamethinner,withonerowofflattenedcellsthroughout thelengthoftheTM.

DuetothemorphologicalcharacteristicsoftheTM,the squamousepitheliumoftheTMouterlayerwouldbeinitially responsiblefortheperforationclosure,withtheformation ofanepithelialbridgeoverthelesionandonlysubsequently therewouldbetherestorationofthefibroustissueofthe middlelayerandmucosallayertissue(Fig.4).7

TheepitheliallayerwasthefirsttoclosetheTM,which doesnotmaketheeventsintheotherlayerslessimportant. At the moment when most cell proliferation and migra-tionoccurred,theepitheliallayershowedhighermetabolic activity,requiringgreatersupplyofoxygenandnutrients.16

EAC

EAC

A

B

C

EAC

m

m

m

ep

Mucosa Lp

100µm

100µm 100µm

Figure7 HistologicalsectionimagesofratTM14daysaftertraumaticperforation,stainedwithHE.EAC,externalauditorycanal; m,malleushandle;ep,epitheliallayer;LP,laminapropria;mucosa,mucosallayer.Image(A)showsamagnificationof40×;(B)

100×and(C)200×.

providingmolecularsupplyrequiredforthehealingevents tosatisfactorilydevelopintheTM.17---19

The lamina propria is responsible for the fibroelastic characteristics of the TM, such as vibration capac-ity for sound transmission and middle ear protection. In some cases, the newly formed TM can heal with only two layers, one outer epithelial and one inner mucosal layer, as it has been described in experimental studies.7

However,itispossible thatsuchneomembranesdonot have the ideal characteristics of sound transmission20 _or

thefibroelasticstructuralcharacteristicswhichenable with-standing air pressure variations, such as barotrauma or tubal dysfunctiondue to itsweaker thanusual structure, caused by deficient formation of the fibrous layer.8

Kris-tensenpointedtothepossibilityofatelectasisorretraction pouchesinatrophicneomembranesduetofibrouslayer defi-ciency.

Topreventtheoccurrenceofatrophicmembranesdueto lamina propria deficiency, treatments that increase fibro-blast activity and collagen production may be interesting to provide better organization of the lamina propria. In tympanoplasty surgery, whether with autografts such as temporalismusclefascia ortragal perichondrium,or even withtheuseofallografts,suchasAlloDerm®_,21_thepurpose

oftheseproceduresistorestoretheTMandthegraftsare

usedtorepairthefibrouslayer,participatinginthe forma-tionofthenewextracellularmatrix.

Aprevious studyreportedthatthe innermucosallayer contributedlittle to the healing process of the TM.17 _On

theotherhand, forWeinberger,Hawke andGotlieb,22 _the

medialmucosallayerofTMwouldbethefirstofthe struc-turestoundergo perforation closure.It wasnot possible, throughthemethodologyemployedinthisstudy,todefine theroleofthemucosallayerinthehealingprocessofthe TM.Moreover,eventhoughitisalayerwithalesscomplex histologicalarchitecturethantheothers,itisimportantto rememberthathealingeventsinthethreeTM layersmust beharmoniouslyorganized,sothatproperTMintegritycan berestored.

Conclusion

EvaluatingthenormalhealingprocessoftheTMinrats,we concludedthatthespontaneoushealingprocessisinitiated bytheTMouterepitheliallayer,withsubsequentclosureof thelaminapropriaandmucosallayer.

Funding

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

References

1.Wullstein H.Theoryand practiceoftympanoplasty. Laryngo-scope.1956;66:1076---95.

2.LimDJ.Tympanicmembrane.Electronmicroscopicobservation. I:parstensa.ActaOtolaryngolStock.1968;66:181---98.

3.SantaMariaPL,RedmondSL,AtlasMD,GhassemifarR.Histology ofthehealingtympanicmembranefollowingperforationinrats. Laryngoscope.2010;120:2061---70.

4.StenfeldtK,JohanssonC,HellstromS.Thecollagenstructure ofthetympanicmembrane.ArchOtolaryngolHeadNeckSurg. 2006;132:293---8.

5.WenigBM,MichaelsL.Theearandtemporalbone.In:MillsSE, editor.Histologyforpathologists.3rded.Philadelphia: Lippin-cottWilliams&Wilkins;2007.p.372---400.

6.LimDJ.Structureandfunctionofthetympanicmembrane:a review.ActaOthorhinolaryngolBelg.1995;49:101---15.

7.Gladstone HB, Jackler RK, Varav K. Tympanic membrane wound healing --- an overview. Otolaryngol Clin North Am. 1995;28:913---32.

8.KristensenS.Spontaneoushealingoftraumatictympanic mem-braneperforations.JLaryngolOtol.1992;106:1037---50.

9.GüneriEA,TekinS,YilmazO,ÖzkaraE,ErdagTK,IkizAO,etal. Theeffects ofhyaluronicacid,epidermalgrowth factor,and mitomycininanexperimentalmodelofacutetraumatic tym-panicmembraneperforation.OtolNeurotol.2003;24:371---6.

10.KoopmannCF.Cutaneouswoundhealing.OtolaryngolClinNorth Am.1995;28:835---45.

11.WitteMB,BarbulA.Generalprinciplesofwoundhealing.Surg ClinNorthAm.1997;77:509---28.

12.KobaR.Epidermalcellmigrationandhealingofthetympanic membrane:animmunohistochemicalstudyofcellproliferation

usingbromodeoxyuridinelabelling. AnnOtolRhinolLaryngol. 1995;104:218---25.

13.SpratleyJ,Hellstrom S,Eriksson PO,Pais-Clemente M.Early structural tympanic membrane reactions to myringotomy: a studyinanacuteotitismediamodel.ActaOtolaryngolStock. 2002;122:479---87.

14.KobaR, Yagi M,TabeH, KawabataI. Kineticanalysisofcell proliferationusingbromodeoxyuridine labelingin situ detec-tionofdyingcellsinthetympanicmembraneandmiddleear cholesteatoma.ArchHistolCytol.1996;59:339---46.

15.Somers TH, Houben V, Goovaerts G, Govaerts PJ, Offeciers EE. Histologyof the perforatedtympanic membrane and its muco-epithelialjunction.ClinOtolaryngolAlliedSci.1997;22: 162---6.

16.GilesB.Woundhealinginspontaneousperforationor myringo-tomy and middle ear reconstruction. Ear Nose Throat J. 2007;86:30---2.

17.JohnsonAP, SmallmanLA, Kent SE.The mechanismof heal-ing of tympanic membrane perforations: a two-dimensional histological study in guinea pigs. Acta Otolaryngol Stockh. 1990;109:406---15.

18.Makino K, Amatsu M. Epithelial migration on the tym-panic membrane and external canal. Arch Otorhinolaryngol. 1986;243:39---42.

19.Masutani H, NakaiY, Sugita M,Ohashi K, Moriguchi M, Mat-sunagaK. Microvasculatureofthetympanicmembrane. Acta OtolaryngolSupplStockh.1991;486:99---104.

20.O’Connor KN, Tam M, Blevins NH, Puria S. Tympanic mem-branecollagenfibers:akeytohighfrequencysoundconduction. Laryngoscope.2008;118:483---90.

21.LaiP,PropstEJ,PapsinBC.Lateralgrafttype1tympanoplasty usingAllodermfortympanicmembranereconstructionin chil-dren.IntJPedOtorhinolaryngol.2006;70:1423---9.