International Journal of Pediatric Otorhinolaryngology (2007) 71, 1129—1137

www.elsevier.com/locate/ijporl

Healing time, long-term result and effects ofstem cell treatment in acute tympanicmembrane perforation

Anisur Rahman a,*, Magnus von Unge a, Petri Olivius a, Joris Dirckx b,Malou Hultcrantz a

aCenter for Hearing and Communication Research and Department of Otorhinolaryngology,Karolinska University Hospital and Institute, 17176 Stockholm, SwedenbBiomedical Physics Group, University of Antwerp, Belgium

Received 8 February 2007; received in revised form 4 April 2007; accepted 5 April 2007

KEYWORDSLaser;Moire interferometry;Morphology;Myringotomy;Rat;Stem cell

Summary

Objective: The incidence of otitis media in children between the age of 2 and 6 yearsis well documented. Repeated attacks may cause acute and chronic perforations. Thesurgical treatment for repairing chronic perforation is quite uncomfortable for thepatients of this age group because of the invasiveness of this treatment. The aim ofthis study was to determine the long-term influence of embryonic stem cells on acuteperforations and the effect of gelatin as a vehicle for applied stem cells. Thepossibility of teratogenic effects of the stem cells was also observed.Methods: Bilateral laser myringotomy was performed in 17 adult Sprague—Dawleyrats, divided into two groups. Gelatin, a substance suitable as vehicle for bioactivematerial was used bilaterally around the perforation in group A, to serve as a scaffoldfor repairing tissue. The stem cells were used in the right tympanic membraneperforation leaving the left tympanic membrane as a control. The animals in groupB received the same treatment except for the use of gelatin and in addition receivedan immuno-suppressive agent. After half a year of observation the mechanicalstiffness of the tympanic membrane was measured by moire interferometry for groupB and the morphological study was performed by light microscopy for both groups Aand B and electron microscopy for group A.Results: Stem cell treated ears did not show any enhanced healing of the perforationalthough a marked thickening of the lamina propria was observed compared withcontrol group. After half a year the strength and the stiffness of the tympanicmembrane was almost the same for both treated and untreated ears. No evidence

of teratoma was found after half a year.

* Corresponding author. Tel.: +46 8 5177 9344; fax: +46 8 5177 6267.E-mail address: anisur.rahman@ki.se (A. Rahman).

0165-5876/$ — see front matter # 2007 Published by Elsevier Ireland Ltd.doi:10.1016/j.ijporl.2007.04.005

1130 A. Rahman et al.

Conclusion: This study suggests that the stem cells stimulate the proliferation ofconnective tissue and fibers in the lamina propria, possibly mediated by secretedsubstances, although the stiffness properties do not seem to be altered. The use ofgelatin does not seem to enhance the healing process of the tympanic membraneperforation.# 2007 Published by Elsevier Ireland Ltd.

1. Introduction

Perforation of the tympanic membrane (TM) is animportant clinical problem worldwide, especially inchildren. It can cause a conductive hearing loss andrepeated infections. It is often the result ofrepeated otitis media or trauma or as sequel aftertreatment with ventilating tubes [1]. Traumaticacute perforations heal spontaneously in a relativelylarge number of cases whereas chronic perforationgenerally requires surgical repair with a tissue graft.Surgical treatment (myringoplasty) is successful in88—95% of the cases but can cause operative risks,causes discomfort for the patient and costs forsociety [2]. Millions of people from developingand under-developed countries have not got accessto such treatment and are left with having theproblem life-long. Repairing a perforation by officetechniques has been successful for a few cases forsmall traumatic perforations [3]. These attemptshave however been disappointing in case of non-traumatic or large perforations. The healing processof TM perforations has been subjected to severalinvestigations with the purpose of finding bettertreatment of chronic TM perforations [4].

Because of a broad clinical potential, it is impor-tant to test the healing enhancing capability ofvarious substances that can be applied to the per-foration, so that conventional surgerymight becomeunnecessary. Studies have been reported on enhan-cing the healing with the use of hyaluronic acid [5—7], blood [8] and growth factors [9]. So far, no finalsolution for a simple out-patient procedure of clos-ing chronic TM perforations has been established.

Since the first successful derivation of embryonicstem (ES) cells from the blastocyst-stage, these cellsare in the center of tremendous interest over thepotential application in the newly emerging field ofregenerative medicine. ES cells are pluripotent andthey can proliferate infinitely in an undifferentiatedstate in vitro. In a previous study the application ofmouse ES cells in a gerbil acute TM perforationmodel appeared to enhance the healing [10].

The purpose of the present study is to test andcompare the capability of applied mouse ES cells toenhance the closure of acute perforations with andwithout the use of gelatin.

2. Material and methods

Twenty adult female Sprague—Dawley rats, weigh-ing between 250 and 300 g, were used. Three ratsdied during anesthesia and the 17 remaining ratswere divided in two different settings, 10 rats ingroup A and seven rats in group B. The animals werebred at Biomedicinskt Centrum, Uppsala, and werekept in the animal department during the experi-ments. The animals were kept in a single-species,temperature-controlled, 12 h light/dark cycle facil-ity. Food and water were provided ad libitum. Theresearch protocol was approved by StockholmsNorra Djurforsoksetiska Namnd (N344/02).

The Tau-GFP-labeled mouse ES cells were col-lected from the laboratory of Prof. John O. Mason,department of biomedical sciences and center fordevelopmental biology, Edinburgh, United Kingdom.The cells were dispended in physiological salinegiving a solution of approximately 1 � 104 cells/mLin each application.

Myringotomy was performed bilaterally on all 17rats, under general anesthesia of 100 mg ketaminehydrochloride (Ketalar, Pfizer AB, Taby, Sweden) and10 mg xylazine hydrochloride (Rompun, BayerHealth Care AG, Germany) intraperitoneally. Themyringotomy was done under an operating micro-scope using a KTP laser beam, directed through a0.2 mm fiber in group A and a 0.4 mm in group B thatwere entered via the external auditory canal. 0.5 ssingle pulses of 1 W were given repeatedly. Themyringotomy was made in the postero-superiorquadrant of the pars tensa. It was verified undera scaled ocular microscope that the fiber diametercoincided with the created perforation size (Fig. 1).

In group A, a droplet of gelatin was appliedbilaterally on the TM around the perforation. Thepurpose of putting gelatin over the perforation wasto produce a platform for the ES cells to migrate orproliferate upon. Ten minutes after the application,the gelatin has formed a layer which spans theperforation. Thereafter the solution of ES cellswas applied to the perforation site on the rightTMs and the same solution without ES cells was usedin the left, control side.

The seven animals in group B were treated in thesame way as described above, except for the gelatin

Tympanic membrane perforation and stem cells 1131

Fig. 1 0.4 mm perforation observed by otomicroscopyimmediately after laser myringotomy. Margin of perfora-tion (arrow) and handle of malleus (M) are indicated.

application, and a larger diameter of laser fiber of0.4 mm was used. In order to minimize the immu-nological rejection, the transplanted host animalsreceived injections of cyclosporine (Novartis, Swe-den, 0.56 mg/100 gm body weight) at every alter-nate day from the day of transplantation up to 2weeks.

Postoperatively, all TMs in groups A and B weremonitored with otomicroscopic examination dailyfor three consecutive days followed by every secondday until the perforations were closed, and at thestudy end after 6 months. Observation was maderegarding the presence of a perforation, blood clot,infection, myringosclerosis and thickened TM.

The animals in group A were euthanized underCO2 anesthesia after 6 months. Two ES cell treatedTMs were dissected out along with the annulus,placed on a glass slide and photographed usingfluorescence light under a Zeiss Axioplan. Theremaining ES cell treated right ears, as well asthe non-treated, left ears were fixed in 2.5% glutar-aldehyde for 24 h. The tympanic bulla was opened,the external ear canal and the cochlea were

Table 1 Accumulated number of closed TMs at various da

Group A (gelatin)

Day Control Treated

6 0 08 1 0

10 4 112 6 614 8 8

removed and the temporal bone was trimmed.Post-fixation was done in osmium tetroxide for1 h. The specimens were then embedded accordingto the standard methods in agar-resin for light- andtransmission electron microscopy. Thickness mea-surements were made at 10 different places in TMsections based on electron micrograph pictures.

In group B the animals were sacrificed at 6monthsafter myringotomy and the fresh TMs were preparedaccording to the protocol for moire interferometry[11,12] to assess the mechanical strength and stiff-ness. The displacement of the TM was measuredduring sequences of static pressures applied to themiddle ear in the range between�350 to +350 daPa.At first a positive pressure cycle was run, startingfrom 0 to +350 daPa and then in reverse sequencefrom +350 back to 0 daPa. A negative pressure cyclefrom 0 to�350 daPa and back to 0 was performed inanalogy with the positive one. After measurementsthe TMs were prepared for light microscopy asdescribed above.

3. Results

3.1. Otomicroscopy

Two rats from group A died during the experimentfrom theeffects of anesthesia. Closure timeof theTMperforations was recorded in all other ears (seeTable 1). In both groups the control side closedearlierthan the ES cell treated side, indifferent of whetherthe immunosuppressive agent was administered ornot. These differences, however, were minute andinsignificant. It was obvious that the TMs of group B,larger perforation (0.4 mm) without gelatin, tendedto close earlier than those of group A.

The 16 TMs of group A and the 14 of group Bshowed neither signs of blood clot, nor infection orthickening of the TM. At 1 month after myringotomymost of the TMs of both groups showed a visible scar,as an opalescent ring in the postero-superior quad-rant of the pars tensa. Visible scars were no longerfound at the end of the study where all TMsappeared normal.

ys after myringotomy

Group B (no gelatin, immuno-suppressive agent)

Control Treated

1 07 67 67 67 7

1132 A. Rahman et al.

3.2. Fluorescence microscopy

Two treated TMs of group A were investigated forfluorescent labeled cells and a faint staining ofunspecific origin could be found. It was not possibleto correlate the staining to any specific cell orstructure in the TM.

3.3. Light and electron microscopy

For both groups A and B, light microscopy haveshowed a similar thickening of the TM at and aroundthe site where the myringotomy was done. Thisthickened area covered almost the entire postero-superior quadrant (Fig. 2a and b). The anteriorquadrants appeared completely unaffected. Elec-tron microscopy performed in group A revealed thatthe thickening mostly consists of changes in thecollagen layers of the TM lamina propria. The thick-ening was more pronounced in the ES cell-treatedTMs than in the controls. The mean TM thickness asmeasured at different places in a few treated and afew control ears were approximately 36 and 28 mm,respectively. The normal thickness is approximately5 mm [11]. The thickening was in both groups char-acterized by edema with dispersed and discon-nected fiber bundles running in diverse directions(Fig. 3a and b). There was also an abundance ofcells, mainly fibroblast and vessels in the fibrouslayer (Fig. 3c). In some TMs the keratin layer was

Fig. 2 Light microscopy photograph of healed (a) acontrol and (b) a stem cell treated tympanic membraneat 6 months after myringotomy. The myringotomy site(arrow), handle of malleus (h), middle ear cavity (ME),external auditory canal (EAC) are indicated. Note that thethickness increase is largest in the treated tympanicmembrane.

found unevenly thickened. Myringosclerosis was notfound in any of the TMs.

3.4. Moire interferometry

3.4.1. Displacement of control earsMoire interferometry allows to measure TM defor-mation over the entire surface, and was described inour previous paper [11]. Successful moire interfero-metry measurements were obtained from six out ofthe seven TMs in group B. One specimen leakedbefore starting the pressure cycle. One TM rupturedat 160 daPa while the others went through thecomplete positive and negative pressure sequencesof 350 daPa. The appearance of the moire fringeswas normal (Fig. 4a) [13].

In Fig. 5a the peak displacement values calcu-lated from the interferograms of a control ear areplotted versus the applied pressures, and the curveshows an ‘‘S’’ shape which is typical for visco-ealsticmaterial. The displacement is more extensive in thenegative pressure zone as compared to the corre-sponding positive pressure zone.

In a mean peak displacement curve plot for theentire group of control ears the ‘‘S’’ shape becomeseven smoother (Fig. 5b). The mean peak displace-ment for the control group at +350 daPa pressurewas 2.65 � 10�4 m with a S.D. of 0.68 � 10�4 m andat �350 daPa was 3.45 � 10�4 m with a S.D. of0.10 � 10�4 m.

3.4.2. Displacement of treated earsAll seven treated TMs were successfully measuredduring the pressure sequences between 0 and�350 daPa. Moire fringes showed similar patternsas compared with the control ears (Fig. 4b). Thedisplacement versus pressure curve plot of the TM’sagain resembles an ‘‘S’’ shape (Fig. 5a). The ‘‘S’’shape of the mean curve plot of this group becomessmoother in a similar way as for the control group(Fig. 5b). The largest hysteresis effect was foundaround 50—100 daPa in the positive pressure cycle.The mean curve plot of control and the treatedgroups almost overlapped each other, meaning thatthere was no difference in the stiffness of the TMsbetween the treated and the untreated controls.

4. Discussion

The exact prevalence on chronic tympanic mem-brane perforation for the entire human population isnot available. In developing countries a level wellover one percent is probable. Kamal et al. [14]presented a prevalence >7% in slum dwellers inDhaka City, Biswas et al. [15] reported >12% in

Tympanic membrane perforation and stem cells 1133

Fig. 3 (a) Transmission electron microscopy of a myr-ingotomized control TM after 6 months. Note increasedthickness of lamina propria. Proliferating fibroblast(arrowhead), collagen fibres (arrow), and external audi-tory canal (EAC) are common for figures a—c. Accumula-tion of keratin (K) is evident. The lamina propria shows thecollagen fibre bundles in straight rows. Three clear layersof the TM: the mucosal layer (M), the lamina propria (LP)and the epidermal cell layer (E). (b) Transmission electronmicroscopy of a myringotomized stem cell treated tym-panic membrane after 6 months. Note larger thickness oflamina propria (LP) as compared with control sample in

Fig. 4 Displacement interferogram at an ear canal pres-sure load of +350 daPa of (a) a myringotomized left tym-panic membrane and (b) a myringotomized ES cell treatedright tympanic membrane recorded at 6 months after theintervention. Presence of two fringes in both the anteriorand posterior part of the pars tensa confers the similarchanges in both groups. Orientation: superior rim to theright, posterior rim upward in (a) and downward in (b).

Bangladesh rural areas and Morris et al. 17% inAustralian Aboriginal children [16]. Treating chronicperforation by simple procedures other than con-ventional surgery is still an unresolved problem inthe field of otology. Such a treatment would begreatly beneficial especially for the pediatricpatients. Multiple approaches and graft materialshave been used to reconstruct the lost or damagedTMs to avoid recurrent otitis media and hearing loss.

Recent advances in developmental biology andtissue engineering give the opportunity to repair

(a). Note proliferating fibroblast. (c) Transmission elec-tron microscopy of a myringotomized stem cell treatedtympanic membrane after 6 months in higher magnifica-tion. Note oedema (*), fiber bundle (fb) and dispersedfibers in disorganisation with increased amount of groundsubstance and lots of cells and vessels in lamina propria.

1134 A. Rahman et al.

Fig. 5 (a) Peak displacement vs. pressure plot of a control and a treated tympanic membrane of one animal recorded at6 months after myringotomy. Note that the plotted curves almost coincide in a smooth S-shape. Different readings duringincreasing and decreasing sequences at identical pressures are due to hysteresis. (b) Moire interferometry testing thepressure resistance (daPa) in themyringotomized control and treated group. The two curves are almost identical in the EScell treated group (grey diamond) and control group (black squares). Results were obtained during increasing pressur-ization from 0 to +350 and 0 to �350 daPa.

damaged or lost tissues with cells supplied fromexogenous sources or mobilizing the cells fromendogenous origin. Despite the promising experi-mental results yielded from using adult stem cells,the increasing evidence of their limited plasticitymade them less suitable for transplantation. ES cellsare particularly important because they can beprecommitted towards a specific cell lineage andthey can complete their maturation under in vivosituation. Several studies reported the positive cat-alytic effect of ES cells on tissue repair and regen-eration [17—19]. The potential therapeutic

application of ES cells still depends on addressingsome key prerequisites like cell purity, amplificationand immunogenicity.

Chronic perforation causes distortion of theepithelial cells as well as of the stromal frameworkof the TM. The epithelial cells cannot repair thedamage solely without the mechanical support ofproliferating connective tissue. The healing processof the TM is not similar to that of other cutaneousstructures. The epithelial cell proliferation andinvasion of granulation tissue does not take placeconcomitantly. The epidermis is the first layer to

Tympanic membrane perforation and stem cells 1135

come forward when the TM starts to heal and aproliferation of stratified squamous epitheliummigrates toward the edges and tries to span thegap [20]. These epithelial cells might originate fromthe more vascularized portions of the remnants ofthe TM such as at the umbo and around the handle ofmalleus. This central area of the TM is supposed tohost progenic cells that generate the epithelialmigration known to move in centrifugal direction[21]. This area is at risk during the surgical closingprocedure, so avoiding a surgical procedure in theTMs would be beneficial in this respect. Secondarilyin the healing process, the inner mucosal layercomes forward and finally the lamina propria, whichwill often be incomplete and invade in-betweenthese two epithelial layers [20]. This is not the casein usual wound healing where the fibrous tissuestarts to fill the defect [21]. Another recent, impor-tant finding reported that plasminogen plays animportant role in wound healing especially in theTM perforation [22].

In order to promote healing there should be somesort of mechanical support for the progressingepithelial proliferation. In wound healing, bloodclots may act as a scaffold to epithelial migration,and the extra-cellular matrix proteins provide asubstratum for cells to adhere, migrate and prolif-erate. In the present perforation study we usedgelatin on group A. Gelatin is a heterogeneousmixture of water-soluble protein of high molecularweight which is usually found in collagen. Gelatinwas used to provide the support to the applied stemcells. Another purpose was to prevent the migrationof transplanted cells towards the middle ear cavity.In order to test whether a platform of gelatin couldsupport a faster regeneration, it was applied ingroup A. Such application, however, did not helpto reduce the closing time. On the contrary, group Bnot having gelatin showed faster closure despitethat the perforations were larger. This differencewas found for both the treated and the non-treatedsubgroups. Park et al. [23] reported the effects ofdifferent substances on TM repair and found thesame closer rate of gelatin treated TMs to controlssimilar to the findings of our group A study. Althoughimmunosuppressive drug impairs the wound healing[24], but introduction of cyclosporine in group Brather showed faster healing. So, it cannot be con-cluded that changes of parameters (exclusion ofgelatin, introduction of cyclosporine) from groupA, caused faster healing in group B and the reasonis not known and is not clarified by the presentresults.

The design of the group B study was aimed topromote the possibilities to detect an effect of stemcell treatment with a small number of animals. The

closing time was not shown to reduce with the EScell treatment as compared with control ears. In aprevious study [10], also using mouse ES cells fortreatment an obvious enhancement of the closingtime was found. In that study cells of different originwas used and they were applied on another speciesother than the Sprague—Dawley rat, namely theMongolian gerbil. One may speculate on how differ-ent related species accept xeno-transplantation indifferent ways.

Comparing the healing in groups A and B, signifi-cant difference in the total healing time could notbe found whether the perforation was 0.2 or 0.4 mmor using gelatin or not. When comparing the time forthe closing procedure, a recent study [25] reported9 days in the same animal species, while in thepresent study both the control side and the ES celltreated side closed within 14 days. Earlier studieswith laser myringotomy have shown closing times of9—14 days [11].

Morphologically investigated TMs presently showa dispersed pattern of the lamina propria in themyringotomized area, especially on the ES celltreated side where the thickness of the TM wasincreased by approximately 33%. In the untreatedcontrol TMs the increase is approximately 28%.

The ES cells were originated from mouse andxeno-transplanted into the rat ear. Xeno-transplan-tation has earlier been shown to be successful withthe same type of cells although the host species wasdifferent, i.e. the gerbil [10]. Shortage of donororgans for clinical transplantation and increasingneed for available organs has focused attentionon the possibility of xeno-transplantation. Trans-plantation of xenogenic cells or tissues instead ofcomplex organ provided better therapeutic result.Pig fetal neuronal cells survived and formed dopa-minergic neuron when transplanted into patientswith Parkinson’s disease [26]. Groth et al. [27]reported that the porcine fetal pancreatic cell pro-duce insulin for a limited time after transplantationto diabetic patients. Immune rejection is the great-est challenge for this treatment. The risks ofimmune rejection in the present study may be less,since the TM pars tensa has low metabolism andrestricted vascularization. The ES cells used in thepresent study were not indicative of the origin orlocation at 6 months after treatment. Nevertheless,it cannot be excluded that the implanted ES cellsmight have had indirect effect on healing of the TM,which might have been mediated via secretion ofgrowth factors or other unknown substances fromthe ES cells.

It has been described earlier that the part of parstensa that closed after myringotomy was thickerthan normal up to 1 month later [11]. Presently

1136 A. Rahman et al.

after 6 months the site of the myringotomy wasthicker in the treated TMs than in the controls(36 mm versus 28 mm), which might be due to anintroduction of more proliferating cells. Whetherthis difference is a result of actions of the ES cellscan be argued. Secretion of growth factors from theES cells might be responsible for the larger biologi-cal response in the lamina propria in the treatedears. The elasticity was almost identical with orwithout the treatment, as the displacement bypressure was nearly the same in both groups. Thestrength of the TMs puts a limit to the pressurewhich can be applied, and resulted in rupture duringpressurizations in the untreated group, whereasnone of the treated ears had TM rupture in thissituation.

In the present study investigating the long-termclosing effects of the TM after acute laser perfora-tion, there seems to be no difference between theES cell treated ears and the controls concerningstiffness and pressure tolerance. The large amountof tissue in the lamina propria is however still thereat 6 months after myringotomy. This has been inter-preted, that the loss of orientation of the collagenfibers is compensated by a large amount of groundsubstances and dispersed and disorientated collagenfiber bundles. This way the stiffness and the pres-sure resistance will show the same values as in non-perforated TMs [13].

A possible disadvantage of ES cell treatment isthe risk of teratoma formation in a recipient organ[28]. In the present long-term follow-up study therewas however no such evidence obtained: no tumour-like pathology was encountered in the middle ears.

5. Conclusion

Myringotomy performed with a laser beam showedafter 6 months, a healed TM with the same pressurewithstanding and stiffness, whether the perforationsize varied or ES cells or gelatin was used or not. Themorphology, however, was different. The ES celltreated TMs were thicker than the controls at thesite of the perforation due to disarrangement of thelamina propria that contained a lot of cells, vesselsand ground substances.

Acknowledgements

The authors wish to thank Dr. Gregory Margolin,KarolinskaUniversity hospital, for his kind assistance,Prof John O. Mason, Department of BiomedicalSciences and Center for Developmental Biology,The University of Edinburgh for generously supplying

ES cells for the study. They also wish to thank PaulaMannstrom and Mikael Eriksson, Karolinska Instituteand Jan Van Hecke, University of Antwerp for theirexcellent technical support. The studywas supportedby Centrum for Klinisk Forskning, Landstinget Vast-manland and Helga Hjerpstedts Fond, Sweden andfunding from Karolinska Institute, Sweden, and Uni-versity of Antwerp, Belgium.

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