International Journal of Pediatric Otorhinolaryngology (2007) 71, 1129—1137 www.elsevier.com/locate/ijporl Healing time, long-term result and effects of stem cell treatment in acute tympanic membrane perforation Anisur Rahman a,*, Magnus von Unge a, Petri Olivius a, Joris Dirckx b, Malou Hultcrantz a a Center for Hearing and Communication Research and Department of Otorhinolaryngology, Karolinska University Hospital and Institute, 17176 Stockholm, Sweden b Biomedical Physics Group, University of Antwerp, Belgium Received 8 February 2007; received in revised form 4 April 2007; accepted 5 April 2007 KEYWORDS Laser; Moiré interferometry; Morphology; Myringotomy; Rat; Stem cell Summary Objective: The incidence of otitis media in children between the age of 2 and 6 years is well documented. Repeated attacks may cause acute and chronic perforations. The surgical treatment for repairing chronic perforation is quite uncomfortable for the patients of this age group because of the invasiveness of this treatment. The aim of this study was to determine the long-term influence of embryonic stem cells on acute perforations and the effect of gelatin as a vehicle for applied stem cells. The possibility of teratogenic effects of the stem cells was also observed. Methods: Bilateral laser myringotomy was performed in 17 adult Sprague—Dawley rats, divided into two groups. Gelatin, a substance suitable as vehicle for bioactive material was used bilaterally around the perforation in group A, to serve as a scaffold for repairing tissue. The stem cells were used in the right tympanic membrane perforation leaving the left tympanic membrane as a control. The animals in group B received the same treatment except for the use of gelatin and in addition received an immuno-suppressive agent. After half a year of observation the mechanical stiffness of the tympanic membrane was measured by moiré interferometry for group B and the morphological study was performed by light microscopy for both groups A and B and electron microscopy for group A. Results: Stem cell treated ears did not show any enhanced healing of the perforation although a marked thickening of the lamina propria was observed compared with control group. After half a year the strength and the stiffness of the tympanic membrane 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 of connective tissue and fibers in the lamina propria, possibly mediated by secreted substances, although the stiffness properties do not seem to be altered. The use of gelatin does not seem to enhance the healing process of the tympanic membrane perforation. # 2007 Published by Elsevier Ireland Ltd. 1. Introduction 2. Material and methods Perforation of the tympanic membrane (TM) is an important clinical problem worldwide, especially in children. It can cause a conductive hearing loss and repeated infections. It is often the result of repeated otitis media or trauma or as sequel after treatment with ventilating tubes [1]. Traumatic acute perforations heal spontaneously in a relatively large number of cases whereas chronic perforation generally requires surgical repair with a tissue graft. Surgical treatment (myringoplasty) is successful in 88—95% of the cases but can cause operative risks, causes discomfort for the patient and costs for society [2]. Millions of people from developing and under-developed countries have not got access to such treatment and are left with having the problem life-long. Repairing a perforation by office techniques has been successful for a few cases for small traumatic perforations [3]. These attempts have however been disappointing in case of nontraumatic or large perforations. The healing process of TM perforations has been subjected to several investigations with the purpose of finding better treatment of chronic TM perforations [4]. Because of a broad clinical potential, it is important to test the healing enhancing capability of various substances that can be applied to the perforation, so that conventional surgery might become unnecessary. Studies have been reported on enhancing the healing with the use of hyaluronic acid [5— 7], blood [8] and growth factors [9]. So far, no final solution for a simple out-patient procedure of closing chronic TM perforations has been established. Since the first successful derivation of embryonic stem (ES) cells from the blastocyst-stage, these cells are in the center of tremendous interest over the potential application in the newly emerging field of regenerative medicine. ES cells are pluripotent and they can proliferate infinitely in an undifferentiated state in vitro. In a previous study the application of mouse ES cells in a gerbil acute TM perforation model appeared to enhance the healing [10]. The purpose of the present study is to test and compare the capability of applied mouse ES cells to enhance the closure of acute perforations with and without the use of gelatin. Twenty adult female Sprague—Dawley rats, weighing between 250 and 300 g, were used. Three rats died during anesthesia and the 17 remaining rats were divided in two different settings, 10 rats in group A and seven rats in group B. The animals were bred at Biomedicinskt Centrum, Uppsala, and were kept in the animal department during the experiments. The animals were kept in a single-species, temperature-controlled, 12 h light/dark cycle facility. Food and water were provided ad libitum. The research protocol was approved by Stockholms Norra Djurförsöksetiska Nämnd (N344/02). The Tau-GFP-labeled mouse ES cells were collected from the laboratory of Prof. John O. Mason, department of biomedical sciences and center for developmental biology, Edinburgh, United Kingdom. The cells were dispended in physiological saline giving a solution of approximately 1  104 cells/mL in each application. Myringotomy was performed bilaterally on all 17 rats, under general anesthesia of 100 mg ketamine hydrochloride (Ketalar, Pfizer AB, Täby, Sweden) and 10 mg xylazine hydrochloride (Rompun, Bayer Health Care AG, Germany) intraperitoneally. The myringotomy was done under an operating microscope using a KTP laser beam, directed through a 0.2 mm fiber in group A and a 0.4 mm in group B that were entered via the external auditory canal. 0.5 s single pulses of 1 W were given repeatedly. The myringotomy was made in the postero-superior quadrant of the pars tensa. It was verified under a scaled ocular microscope that the fiber diameter coincided with the created perforation size (Fig. 1). In group A, a droplet of gelatin was applied bilaterally on the TM around the perforation. The purpose of putting gelatin over the perforation was to produce a platform for the ES cells to migrate or proliferate upon. Ten minutes after the application, the gelatin has formed a layer which spans the perforation. Thereafter the solution of ES cells was applied to the perforation site on the right TMs and the same solution without ES cells was used in the left, control side. The seven animals in group B were treated in the same way as described above, except for the gelatin Tympanic membrane perforation and stem cells Fig. 1 0.4 mm perforation observed by otomicroscopy immediately after laser myringotomy. Margin of perforation (arrow) and handle of malleus (M) are indicated. application, and a larger diameter of laser fiber of 0.4 mm was used. In order to minimize the immunological rejection, the transplanted host animals received injections of cyclosporine (Novartis, Sweden, 0.56 mg/100 gm body weight) at every alternate day from the day of transplantation up to 2 weeks. Postoperatively, all TMs in groups A and B were monitored with otomicroscopic examination daily for three consecutive days followed by every second day until the perforations were closed, and at the study end after 6 months. Observation was made regarding the presence of a perforation, blood clot, infection, myringosclerosis and thickened TM. The animals in group A were euthanized under CO2 anesthesia after 6 months. Two ES cell treated TMs were dissected out along with the annulus, placed on a glass slide and photographed using fluorescence light under a Zeiss Axioplan. The remaining ES cell treated right ears, as well as the non-treated, left ears were fixed in 2.5% glutaraldehyde for 24 h. The tympanic bulla was opened, the external ear canal and the cochlea were 1131 removed and the temporal bone was trimmed. Post-fixation was done in osmium tetroxide for 1 h. The specimens were then embedded according to the standard methods in agar-resin for light- and transmission electron microscopy. Thickness measurements were made at 10 different places in TM sections based on electron micrograph pictures. In group B the animals were sacrificed at 6 months after myringotomy and the fresh TMs were prepared according to the protocol for moiré interferometry [11,12] to assess the mechanical strength and stiffness. The displacement of the TM was measured during sequences of static pressures applied to the middle ear in the range between 350 to +350 daPa. At first a positive pressure cycle was run, starting from 0 to +350 daPa and then in reverse sequence from +350 back to 0 daPa. A negative pressure cycle from 0 to 350 daPa and back to 0 was performed in analogy with the positive one. After measurements the TMs were prepared for light microscopy as described above. 3. Results 3.1. Otomicroscopy Two rats from group A died during the experiment from the effects of anesthesia. Closure time of the TM perforations was recorded in all other ears (see Table 1). In both groups the control side closed earlier than the ES cell treated side, indifferent of whether the immunosuppressive agent was administered or not. These differences, however, were minute and insignificant. It was obvious that the TMs of group B, larger perforation (0.4 mm) without gelatin, tended to close earlier than those of group A. The 16 TMs of group A and the 14 of group B showed neither signs of blood clot, nor infection or thickening of the TM. At 1 month after myringotomy most of the TMs of both groups showed a visible scar, as an opalescent ring in the postero-superior quadrant of the pars tensa. Visible scars were no longer found at the end of the study where all TMs appeared normal. Table 1 Accumulated number of closed TMs at various days after myringotomy Group A (gelatin) Group B (no gelatin, immuno-suppressive agent) Day Control Treated Control Treated 6 8 10 12 14 0 1 4 6 8 0 0 1 6 8 1 7 7 7 7 0 6 6 6 7 1132 3.2. Fluorescence microscopy Two treated TMs of group A were investigated for fluorescent labeled cells and a faint staining of unspecific origin could be found. It was not possible to correlate the staining to any specific cell or structure in the TM. 3.3. Light and electron microscopy For both groups A and B, light microscopy have showed a similar thickening of the TM at and around the site where the myringotomy was done. This thickened area covered almost the entire posterosuperior quadrant (Fig. 2a and b). The anterior quadrants appeared completely unaffected. Electron microscopy performed in group A revealed that the thickening mostly consists of changes in the collagen layers of the TM lamina propria. The thickening was more pronounced in the ES cell-treated TMs than in the controls. The mean TM thickness as measured at different places in a few treated and a few control ears were approximately 36 and 28 mm, respectively. The normal thickness is approximately 5 mm [11]. The thickening was in both groups characterized by edema with dispersed and disconnected fiber bundles running in diverse directions (Fig. 3a and b). There was also an abundance of cells, mainly fibroblast and vessels in the fibrous layer (Fig. 3c). In some TMs the keratin layer was A. Rahman et al. found unevenly thickened. Myringosclerosis was not found in any of the TMs. 3.4. Moiré interferometry 3.4.1. Displacement of control ears Moiré interferometry allows to measure TM deformation over the entire surface, and was described in our previous paper [11]. Successful moiré interferometry measurements were obtained from six out of the seven TMs in group B. One specimen leaked before starting the pressure cycle. One TM ruptured at 160 daPa while the others went through the complete positive and negative pressure sequences of 350 daPa. The appearance of the moiré fringes was normal (Fig. 4a) [13]. In Fig. 5a the peak displacement values calculated from the interferograms of a control ear are plotted versus the applied pressures, and the curve shows an ‘‘S’’ shape which is typical for visco-ealstic material. The displacement is more extensive in the negative pressure zone as compared to the corresponding positive pressure zone. In a mean peak displacement curve plot for the entire group of control ears the ‘‘S’’ shape becomes even smoother (Fig. 5b). The mean peak displacement for the control group at +350 daPa pressure was 2.65  10 4 m with a S.D. of 0.68  10 4 m and at 350 daPa was 3.45  10 4 m with a S.D. of 0.10  10 4 m. 3.4.2. Displacement of treated ears All seven treated TMs were successfully measured during the pressure sequences between 0 and 350 daPa. Moiré fringes showed similar patterns as compared with the control ears (Fig. 4b). The displacement versus pressure curve plot of the TM’s again resembles an ‘‘S’’ shape (Fig. 5a). The ‘‘S’’ shape of the mean curve plot of this group becomes smoother in a similar way as for the control group (Fig. 5b). The largest hysteresis effect was found around 50—100 daPa in the positive pressure cycle. The mean curve plot of control and the treated groups almost overlapped each other, meaning that there was no difference in the stiffness of the TMs between the treated and the untreated controls. 4. Discussion Fig. 2 Light microscopy photograph of healed (a) a control and (b) a stem cell treated tympanic membrane at 6 months after myringotomy. The myringotomy site (arrow), handle of malleus (h), middle ear cavity (ME), external auditory canal (EAC) are indicated. Note that the thickness increase is largest in the treated tympanic membrane. The exact prevalence on chronic tympanic membrane perforation for the entire human population is not available. In developing countries a level well over one percent is probable. Kamal et al. [14] presented a prevalence >7% in slum dwellers in Dhaka City, Biswas et al. [15] reported >12% in Tympanic membrane perforation and stem cells 1133 Fig. 4 Displacement interferogram at an ear canal pressure load of +350 daPa of (a) a myringotomized left tympanic membrane and (b) a myringotomized ES cell treated right tympanic membrane recorded at 6 months after the intervention. Presence of two fringes in both the anterior and posterior part of the pars tensa confers the similar changes in both groups. Orientation: superior rim to the right, posterior rim upward in (a) and downward in (b). Fig. 3 (a) Transmission electron microscopy of a myringotomized control TM after 6 months. Note increased thickness of lamina propria. Proliferating fibroblast (arrowhead), collagen fibres (arrow), and external auditory canal (EAC) are common for figures a—c. Accumulation of keratin (K) is evident. The lamina propria shows the collagen fibre bundles in straight rows. Three clear layers of the TM: the mucosal layer (M), the lamina propria (LP) and the epidermal cell layer (E). (b) Transmission electron microscopy of a myringotomized stem cell treated tympanic membrane after 6 months. Note larger thickness of lamina propria (LP) as compared with control sample in Bangladesh rural areas and Morris et al. 17% in Australian Aboriginal children [16]. Treating chronic perforation by simple procedures other than conventional surgery is still an unresolved problem in the field of otology. Such a treatment would be greatly beneficial especially for the pediatric patients. Multiple approaches and graft materials have been used to reconstruct the lost or damaged TMs to avoid recurrent otitis media and hearing loss. Recent advances in developmental biology and tissue engineering give the opportunity to repair (a). Note proliferating fibroblast. (c) Transmission electron microscopy of a myringotomized stem cell treated tympanic membrane after 6 months in higher magnification. Note oedema (*), fiber bundle (fb) and dispersed fibers in disorganisation with increased amount of ground substance 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 at 6 months after myringotomy. Note that the plotted curves almost coincide in a smooth S-shape. Different readings during increasing and decreasing sequences at identical pressures are due to hysteresis. (b) Moiré interferometry testing the pressure resistance (daPa) in the myringotomized control and treated group. The two curves are almost identical in the ES cell treated group (grey diamond) and control group (black squares). Results were obtained during increasing pressurization from 0 to +350 and 0 to 350 daPa. damaged or lost tissues with cells supplied from exogenous sources or mobilizing the cells from endogenous origin. Despite the promising experimental results yielded from using adult stem cells, the increasing evidence of their limited plasticity made them less suitable for transplantation. ES cells are particularly important because they can be precommitted towards a specific cell lineage and they can complete their maturation under in vivo situation. Several studies reported the positive catalytic effect of ES cells on tissue repair and regeneration [17—19]. The potential therapeutic application of ES cells still depends on addressing some key prerequisites like cell purity, amplification and immunogenicity. Chronic perforation causes distortion of the epithelial cells as well as of the stromal framework of the TM. The epithelial cells cannot repair the damage solely without the mechanical support of proliferating connective tissue. The healing process of the TM is not similar to that of other cutaneous structures. The epithelial cell proliferation and invasion of granulation tissue does not take place concomitantly. The epidermis is the first layer to Tympanic membrane perforation and stem cells come forward when the TM starts to heal and a proliferation of stratified squamous epithelium migrates toward the edges and tries to span the gap [20]. These epithelial cells might originate from the more vascularized portions of the remnants of the TM such as at the umbo and around the handle of malleus. This central area of the TM is supposed to host progenic cells that generate the epithelial migration known to move in centrifugal direction [21]. This area is at risk during the surgical closing procedure, so avoiding a surgical procedure in the TMs would be beneficial in this respect. Secondarily in the healing process, the inner mucosal layer comes forward and finally the lamina propria, which will often be incomplete and invade in-between these two epithelial layers [20]. This is not the case in usual wound healing where the fibrous tissue starts to fill the defect [21]. Another recent, important finding reported that plasminogen plays an important role in wound healing especially in the TM perforation [22]. In order to promote healing there should be some sort of mechanical support for the progressing epithelial proliferation. In wound healing, blood clots may act as a scaffold to epithelial migration, and the extra-cellular matrix proteins provide a substratum for cells to adhere, migrate and proliferate. In the present perforation study we used gelatin on group A. Gelatin is a heterogeneous mixture of water-soluble protein of high molecular weight which is usually found in collagen. Gelatin was used to provide the support to the applied stem cells. Another purpose was to prevent the migration of transplanted cells towards the middle ear cavity. In order to test whether a platform of gelatin could support a faster regeneration, it was applied in group A. Such application, however, did not help to reduce the closing time. On the contrary, group B not having gelatin showed faster closure despite that the perforations were larger. This difference was found for both the treated and the non-treated subgroups. Park et al. [23] reported the effects of different substances on TM repair and found the same closer rate of gelatin treated TMs to controls similar to the findings of our group A study. Although immunosuppressive drug impairs the wound healing [24], but introduction of cyclosporine in group B rather showed faster healing. So, it cannot be concluded that changes of parameters (exclusion of gelatin, introduction of cyclosporine) from group A, caused faster healing in group B and the reason is not known and is not clarified by the present results. The design of the group B study was aimed to promote the possibilities to detect an effect of stem cell treatment with a small number of animals. The 1135 closing time was not shown to reduce with the ES cell treatment as compared with control ears. In a previous study [10], also using mouse ES cells for treatment an obvious enhancement of the closing time was found. In that study cells of different origin was used and they were applied on another species other than the Sprague—Dawley rat, namely the Mongolian gerbil. One may speculate on how different related species accept xeno-transplantation in different ways. Comparing the healing in groups A and B, significant difference in the total healing time could not be found whether the perforation was 0.2 or 0.4 mm or using gelatin or not. When comparing the time for the closing procedure, a recent study [25] reported 9 days in the same animal species, while in the present study both the control side and the ES cell treated side closed within 14 days. Earlier studies with laser myringotomy have shown closing times of 9—14 days [11]. Morphologically investigated TMs presently show a dispersed pattern of the lamina propria in the myringotomized area, especially on the ES cell treated side where the thickness of the TM was increased by approximately 33%. In the untreated control TMs the increase is approximately 28%. The ES cells were originated from mouse and xeno-transplanted into the rat ear. Xeno-transplantation has earlier been shown to be successful with the same type of cells although the host species was different, i.e. the gerbil [10]. Shortage of donor organs for clinical transplantation and increasing need for available organs has focused attention on the possibility of xeno-transplantation. Transplantation of xenogenic cells or tissues instead of complex organ provided better therapeutic result. Pig fetal neuronal cells survived and formed dopaminergic neuron when transplanted into patients with Parkinson’s disease [26]. Groth et al. [27] reported that the porcine fetal pancreatic cell produce insulin for a limited time after transplantation to diabetic patients. Immune rejection is the greatest challenge for this treatment. The risks of immune rejection in the present study may be less, since the TM pars tensa has low metabolism and restricted vascularization. The ES cells used in the present study were not indicative of the origin or location at 6 months after treatment. Nevertheless, it cannot be excluded that the implanted ES cells might have had indirect effect on healing of the TM, which might have been mediated via secretion of growth factors or other unknown substances from the ES cells. It has been described earlier that the part of pars tensa that closed after myringotomy was thicker than normal up to 1 month later [11]. Presently 1136 after 6 months the site of the myringotomy was thicker in the treated TMs than in the controls (36 mm versus 28 mm), which might be due to an introduction of more proliferating cells. Whether this difference is a result of actions of the ES cells can be argued. Secretion of growth factors from the ES cells might be responsible for the larger biological response in the lamina propria in the treated ears. The elasticity was almost identical with or without the treatment, as the displacement by pressure was nearly the same in both groups. The strength of the TMs puts a limit to the pressure which can be applied, and resulted in rupture during pressurizations in the untreated group, whereas none of the treated ears had TM rupture in this situation. In the present study investigating the long-term closing effects of the TM after acute laser perforation, there seems to be no difference between the ES cell treated ears and the controls concerning stiffness and pressure tolerance. The large amount of tissue in the lamina propria is however still there at 6 months after myringotomy. This has been interpreted, that the loss of orientation of the collagen fibers is compensated by a large amount of ground substances and dispersed and disorientated collagen fiber bundles. This way the stiffness and the pressure resistance will show the same values as in nonperforated TMs [13]. A possible disadvantage of ES cell treatment is the risk of teratoma formation in a recipient organ [28]. In the present long-term follow-up study there was however no such evidence obtained: no tumourlike pathology was encountered in the middle ears. 5. Conclusion Myringotomy performed with a laser beam showed after 6 months, a healed TM with the same pressure withstanding and stiffness, whether the perforation size varied or ES cells or gelatin was used or not. The morphology, however, was different. The ES cell treated TMs were thicker than the controls at the site of the perforation due to disarrangement of the lamina propria that contained a lot of cells, vessels and ground substances. Acknowledgements The authors wish to thank Dr. Gregory Margolin, Karolinska University hospital, for his kind assistance, Prof John O. Mason, Department of Biomedical Sciences and Center for Developmental Biology, The University of Edinburgh for generously supplying A. Rahman et al. ES cells for the study. 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