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Immunology of human tympanic membrane in otitis media
International Congress Series 1240 (2003) 1095 – 1100
Immunology of human tympanic membrane in otitis media Immunohistochemical study of epidermal cytokines Jerzy Kuczkowski*, Waldemar Naroz˙ny, BoguslCaw Mikaszewski Department of Otolaryngology and Department of Immunopathology, Medical University of Gdan´sk, 81-211 Gdan´sk, ul. De˛binki Str.7, Poland
Abstract The immune response plays a main role in disregulation of epithelial growth in chronic otitis media (COM). Immunohistochemical study was performed on 11 specimens of human tympanic membranes (TMs) with COM correlated with 11 normal human TMs from cadavers. The specimens of TM near perforation expressed high intensity of TGFh1 and HLA-DR antigen. TGFh1 expression was observed in the lamina propria of perforated TM. Expression of EGF-R was various, more intensive in the margin tissue of TM perforation. The fragments of TM showed the highest staining of tenascin- and S-100-positive cells. The expression of S-100-positive cells in the TMs and granulation tissue indicated the presence of dendritic cells, which are the part of immunity. These data support the hypothesis, that epithelial cells in the COM are activated through the growth factors. D 2003 International Federation of Otorhinolaryngological Societies (IFOS). All rights reserved. Keywords: Chronic otitis media; Tympanic membrane; Epidermal cytokines; Immunohistochemistry
1. Introduction Chronic otitis media (COM) is an acquired inflammation of mucous lining of the middle ear, associated with the inflammatory reaction and one of its symptoms is tympanic membrane (TM) perforation and effusion. Although the clinical diagnosis is easy, the treatment may be difficult. Important in the treatment of this disease is to obtain a dry ear and restored status of TM. Epidermization process of the operated ear is a very slow process, sometimes undergone with difficulties or even unfinished. The middle ear
* Corresponding author. Fax: +48-58-346-11-97. E-mail address: [email protected] (J. Kuczkowski). 0531-5131/ D 2003 International Federation of Otorhinolaryngological Societies (IFOS). All rights reserved. doi:10.1016/S0531-5131(03)00906-3
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reconstruction is performed by auto- or allotransplantation and by support of epithelialization processes [1,7,10,13]. Chronic tympanic perforation can be of infectious or traumatic origin. At cellular level, the epithelialization of the wound’s edge is often considered as the responsible factor, which prevents the spontaneous healing of the TM [6]. Growth factors play the main role in this reaction. As a part of COM, an increase of immunological reactions is considered [2,9]. Infection rapidly increases the rate of exfoliation of keratin and granulation tissue [12]. The cytokines take an important part in cellular interaction modulating cell growth and differentiation in COM [2,5,8,11]. EGF stimulates the proliferation of epithelial cells, fibroblasts, endothelial cells and migration of keratinocytes [11]. Strong staining for receptor EGF in perforated TM was observed mainly in the squamous epithelium and in the basal layer [3,8,9]. Strong positive immunoreactivity for TGFh1 was found in the subepithelial and fibrous layer [2]. The deposition of TGFh1 is prominent in the fibrous layer, at the border of the tympanic perforation. The greatest difference between perforated membrane and the normal one was found for TGFh1. There was strong staining in the basal epithelial layer of the TM [2,5,11]. This suggests, that TGFh1 plays an important role in the pathogenesis of TM healing [2,5]. The aim of this study was to characterize the functional status of TM in COM. In relation to the above cytokines, we have studied immunohistochemistry of others markers, using different kind of antibodies.
2. Materials and methods The investigated materials were parts of TM. A total of 22 TM (11 perforated and 11 normal TM) were studied. The material was obtained from 11 patients with COM treated in Department of Otolaryngology Medical University of Gdan˜sk in the years 1997– 2000. All perforations of TM were of infectious and cholesteatomas origin and had persisted for longer than 1 year. These samples were taken immediately after tympanomastoid surgery. A part of samples was rapidly frozen in liquid nitrogen. Cryosections of 4 Am were cut and prepared for the peroxidase – antiperoxidase (PAP) methods. Other samples were fixed in formalin and embedded in paraffin. The sections of 4 Am from each case were cut and prepared for immunostaining. All specimens were stained with routine hematoxylin and eosin staining. Eleven intact TMs were dissected from cadavers (adults, 10 –16 h postmortem). The following antibodies were used: 1. Monoclonal anti-human LAP (TGFh1) neutralizing antibody (RD System Europe, UK, at dilution of 1:10) 2. Monoclonal anti-epidermal growth factor receptor (clone EGFRI1) RPN 513 (Amersham, England, at dilution of 1:50) 3. Monoclonal mouse anti-human HLA-DR antigen, Beta Chain, clone DK 22 (DAKO, Denmark, at dilution of 1:200) 4. Rabbit anti-cow S-100, LOT 129 (DAKO, at dilution of 1:200) 5. Monoclonal mouse anti-human tenascin (DAKO tenascin TN2) LOT 063 (DAKO, at dilution of 1:80)
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Table 1 Distribution of growth factors in TMs from cadavers Antibody
TM Epithelium
EGFR TGFh1
Pars fibrosa
Endothelium
+
+
+
is absent, + is mild, ++ is extensive.
6. Monoclonal mouse anti-human von Willebrand Factor (clone F8/86), LOT 125 (DAKO, at dilution of 1:25) 7. Monoclonal mouse anti-human endothelial cell, CD31, LOT 110 (DAKO, at dilution of 1:20) 8. Anti-human Ki67 Antigen/HRP, LOT 023 (DAKO). Bound antibody was detected using commercial Kit (DAKO, LSA B2 Peroxidase 675). The results were compared to the control group. Additionally as positive control, reactions from skin were used. For negative control, sections were treated with mouse nonimmune sera.
3. Results The 11 normal TM showed no particular histological features. The 11 perforated TMs specimens came from 6 male and 5 female patients (mean age 36.2). Ten specimens from perforated TM showed histological anomalies. Inflammatory infiltration and fibrosis in the fibrous layer were observed in all cases. Tympanosclerosis was present in eight cases. The epithelial layer showed diffuse hyperplasia. The medial, mucosal layer consisted of cuboidal and pseudostratified columnar cells with ciliated and goblet cells. Strong positive staining was found for receptor of EGF in perforated as well as in nonperforated TM specimens mainly in the stratified squamous epithelium. Immunoreactivity was more intense at the based membrane and in the basal layer. In the specimens of perforated TM, the seemingly more prominent staining in hyperplastic areas was due to the overall increased epithelial thickness and not to greater deposition. In the fibrous layer specimens, chronic TM demonstrates no or minimal staining of EGFR. There was also a slight staining of the vascular walls mainly at the basal membrane. The deposition of TGFh1 was particularly prominent in the fibrous layer at the border of the perforation. The Table 2 Distribution of growth factors in TMs COM Antibody
TM Epithelium
EGFR TGFh1
Pars fibrosa
Endothelium
+
++
++
is absent, + is mild, ++ is extensive.
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Table 3 Reactivity to the monoclonal antibodies in the TMs at COM and normal TMs from cadavers Antibody
TGFh1 EGFR HLA-DR Tenascin S-100 Ki67 CD31 FVIII
Substrate TMs normal
TMs COM
6/11 6/11 8/11 5/11 0/11 2/11 7/11 5/11
11/11 7/11 9/11 11/11 8/11 3/11 9/11 9/11
Number of positive results/number of investigated cases.
most striking difference was seen at the level of the subepithelial layer and pars fibrosa. In normal TM, hardly any or no immunoreactivity could be shown in these layers, whereas in perforated TMs, areas of strong positive staining were revealed in the subepithelial layer and in the lamina propria (Tables 1 and 2). Some specimens of TMs showed high activity of this factor. Intensity of reaction for the presence of EGFR in any case of COM was small, much smaller than for TGFh1. Anti-HLA-DR antigen reacted with all DR loci in Langerhans cells. In specimens from TM, there was a strong reaction with HLA-DR, suggesting the presence of Langerhans cells. The analysis of fixed in formalin tissue from TMs showed presence of many positive immunohistochemical reactions. Expression of tenascin was observed in all cases of investigated TMs tissue samples. The tenascin expression was often found in granulation tissue and margin tissue of TM perforation. The cells containing S-100 protein proved to be present most often in the tissue from TM perforation margin and granulation tissue. Single cases of positive reactions were of poor intensity. Ki67 antigen expression was present in 3 of 11 cases. The endothelium cells were identified using monoclonal antibodies CD31. Their presence was common in the inner layer of TM. Positive reaction for CD31 presence was observed in 9 of 11 of investigated cases. Factor von Willebrand (F VIII) was observed in 9 of 11 cases. It was most commonly present in endothelium cells of granulation tissue. Immunohistochemical investigation of intact cadaver TM showed low expression of tenascin, endothelial cells with CD31 antigen and von Willebrand factor. The presence of S-100-positive cells was not proven (Table 3).
4. Discussion In COM, there are active immunological processes present, with a particularly important role of growth factors. Cytokines play a central role in leukocyte migration through peripheral blood and tissue, and they induce and maintain the proliferation of both lymphocytes and keratinocytes [2 –4,13]. As it was shown, TGFh1 is frequently observed in tissue of TM perforation, which could prove its important role in repairing processes of TM.
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TGFh1 in general inhibits the growth of epithelium cells and stimulates the growth of mesenchymal cells [2,5,11]. In consequence, it may alter the architecture of stromal and/or epithelial elements. Inflammatory cells present in the earliest lesions may release TGFh1 possibly resulting in chemotactic recruitment of additional chronic inflammatory cells. If so, it would confirm the expression of TGFh1 observed in our study. TGFh1 expression was observed in lamina propria of TM with chronic perforation. This presence at the perforation border seemed to be related to the degree of fibroplasia. EGFR is not so common in COM. Mondain and Ryan [8] and Ramsay et al. [9] proved that the presence of EGF, known to be important in wound healing, can be demonstrated only minimally in the rims of chronic perforations by immunohistochemical means. This cytokine probably is of less importance in COM process. The positive HLA-DR reactions showed the presence of dendritic cells [4]. Their activity in tissue and margin tissue of TM is important for understanding the immune status of middle ear. The presence of Ki67 antigen is typical for rapid proliferating cells, especially in malignant neoplasms. The assessment of cell proliferation by Ki67 antigen in chronic inflammation cell population has been shown to be of a little prognostic value. That fact, that CD31 and F VIII determine vascular involvement in repair process in investigated cases, is obvious.
5. Conclusions 1. The cellular elements of chronic inflammation are present in the TM s in chronic otitis media. 2. TGFh1 is present in the TM perforation margin and endothelium. 3. EGFR is present in epithelium of TM but absent in pars fibrosa and endothelium. 4. The positive HLA-DR reactions and presence of S-100 protein showed the presence of Langerhans cells in COM.
References [1] D. Boedts, Tympanic membrane perforations, Acta Otorhinolaryngol. Belg. 49 (1995) 149 – 158. [2] W. Border, E. Ruoslahti, Transforming growth factor-beta in disease: the dark side of tissue repair, J. Clin. Invest. 90 (1992) 1 – 7. [3] D. Dvorak, G. Abbas, T. Ali, S. Stevenson, B. Welling, Repair of chronic membrane perforations with longterm epidermal growth factor, Laryngoscope 105 (1995) 1300 – 1304. [4] B. Hussl, G. Egg, W. Kong, N. Romani, A. Schrott-Fischer, Dendritic cells in the normal human tympanic membrane, Ann. Otol. Rhinol. Laryngol. 104 (1995) 803 – 807. [5] R. Koba, I. Kawabata, Immunohistochemical study of transforming growth factor-a expression in normal and perforated tympanic membrane, Ann. Otol. Rhinol. Laryngol. 104 (1995) 793 – 797. [6] D.J. Lim, Structure and function of the tympanic membrane: a review, Acta Otorhinolaryngol. Belg. 49 (1995) 101 – 115. [7] W. Meyerrhoff, Ch. Kim, M. Paparelle, Pathology of chronic otitis media, Ann. Otol. Rhinol. Laryngol. 87 (1978) 749 – 760. [8] M. Mondain, A. Ryan, Epidermal growth factor and basic fibroblast growth factor are induced in guinea-pig tympanic membrane following traumatic perforation, Acta Oto-laryngol. (Stockh.) 115 (1995) 50 – 54.
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[9] H. Ramsay, E. Heikkonen, P. Laurila, Effect of epidermal growth factor on tympanic membranes with chronic perforations: a clinical trial, Otolaryngol. Head Neck Surg. 113 (1995) 37379 – 37775. [10] C. Ruah, R. Penha, P. Schachern, M. Paparella, Tympanic membrane and otitis media, Acta-rhino-laryngol. Belg. 49 (1995) 173 – 180. [11] H.H. Steenfos, Growth factor and wound healing, Scand. J. Plast. Reconstr. Hand Surg. 28 (1994) 95 – 105. [12] H. Sudhoff, J. Bujia, A. Holly, C. Kim, A. Fisseler-Eckhoff, Functional characterisation of middle ear mucosa residues in cholesteatoma samples, Am. J. Otol. 15 (1994) 217 – 221. [13] T. Yamashita, Histology of the tympanic perforation and the replacement membrane, Acta Oto-laryngol. (Stockh.) 100 (1985) 66 – 71.
Immunology of human tympanic membrane in otitis media Immunohistochemical study of epidermal cytokines Jerzy Kuczkowski*, Waldemar Naroz˙ny, BoguslCaw Mikaszewski Department of Otolaryngology and Department of Immunopathology, Medical University of Gdan´sk, 81-211 Gdan´sk, ul. De˛binki Str.7, Poland
Abstract The immune response plays a main role in disregulation of epithelial growth in chronic otitis media (COM). Immunohistochemical study was performed on 11 specimens of human tympanic membranes (TMs) with COM correlated with 11 normal human TMs from cadavers. The specimens of TM near perforation expressed high intensity of TGFh1 and HLA-DR antigen. TGFh1 expression was observed in the lamina propria of perforated TM. Expression of EGF-R was various, more intensive in the margin tissue of TM perforation. The fragments of TM showed the highest staining of tenascin- and S-100-positive cells. The expression of S-100-positive cells in the TMs and granulation tissue indicated the presence of dendritic cells, which are the part of immunity. These data support the hypothesis, that epithelial cells in the COM are activated through the growth factors. D 2003 International Federation of Otorhinolaryngological Societies (IFOS). All rights reserved. Keywords: Chronic otitis media; Tympanic membrane; Epidermal cytokines; Immunohistochemistry
1. Introduction Chronic otitis media (COM) is an acquired inflammation of mucous lining of the middle ear, associated with the inflammatory reaction and one of its symptoms is tympanic membrane (TM) perforation and effusion. Although the clinical diagnosis is easy, the treatment may be difficult. Important in the treatment of this disease is to obtain a dry ear and restored status of TM. Epidermization process of the operated ear is a very slow process, sometimes undergone with difficulties or even unfinished. The middle ear
* Corresponding author. Fax: +48-58-346-11-97. E-mail address: [email protected] (J. Kuczkowski). 0531-5131/ D 2003 International Federation of Otorhinolaryngological Societies (IFOS). All rights reserved. doi:10.1016/S0531-5131(03)00906-3
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reconstruction is performed by auto- or allotransplantation and by support of epithelialization processes [1,7,10,13]. Chronic tympanic perforation can be of infectious or traumatic origin. At cellular level, the epithelialization of the wound’s edge is often considered as the responsible factor, which prevents the spontaneous healing of the TM [6]. Growth factors play the main role in this reaction. As a part of COM, an increase of immunological reactions is considered [2,9]. Infection rapidly increases the rate of exfoliation of keratin and granulation tissue [12]. The cytokines take an important part in cellular interaction modulating cell growth and differentiation in COM [2,5,8,11]. EGF stimulates the proliferation of epithelial cells, fibroblasts, endothelial cells and migration of keratinocytes [11]. Strong staining for receptor EGF in perforated TM was observed mainly in the squamous epithelium and in the basal layer [3,8,9]. Strong positive immunoreactivity for TGFh1 was found in the subepithelial and fibrous layer [2]. The deposition of TGFh1 is prominent in the fibrous layer, at the border of the tympanic perforation. The greatest difference between perforated membrane and the normal one was found for TGFh1. There was strong staining in the basal epithelial layer of the TM [2,5,11]. This suggests, that TGFh1 plays an important role in the pathogenesis of TM healing [2,5]. The aim of this study was to characterize the functional status of TM in COM. In relation to the above cytokines, we have studied immunohistochemistry of others markers, using different kind of antibodies.
2. Materials and methods The investigated materials were parts of TM. A total of 22 TM (11 perforated and 11 normal TM) were studied. The material was obtained from 11 patients with COM treated in Department of Otolaryngology Medical University of Gdan˜sk in the years 1997– 2000. All perforations of TM were of infectious and cholesteatomas origin and had persisted for longer than 1 year. These samples were taken immediately after tympanomastoid surgery. A part of samples was rapidly frozen in liquid nitrogen. Cryosections of 4 Am were cut and prepared for the peroxidase – antiperoxidase (PAP) methods. Other samples were fixed in formalin and embedded in paraffin. The sections of 4 Am from each case were cut and prepared for immunostaining. All specimens were stained with routine hematoxylin and eosin staining. Eleven intact TMs were dissected from cadavers (adults, 10 –16 h postmortem). The following antibodies were used: 1. Monoclonal anti-human LAP (TGFh1) neutralizing antibody (RD System Europe, UK, at dilution of 1:10) 2. Monoclonal anti-epidermal growth factor receptor (clone EGFRI1) RPN 513 (Amersham, England, at dilution of 1:50) 3. Monoclonal mouse anti-human HLA-DR antigen, Beta Chain, clone DK 22 (DAKO, Denmark, at dilution of 1:200) 4. Rabbit anti-cow S-100, LOT 129 (DAKO, at dilution of 1:200) 5. Monoclonal mouse anti-human tenascin (DAKO tenascin TN2) LOT 063 (DAKO, at dilution of 1:80)
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Table 1 Distribution of growth factors in TMs from cadavers Antibody
TM Epithelium
EGFR TGFh1
Pars fibrosa
Endothelium
+
+
+
is absent, + is mild, ++ is extensive.
6. Monoclonal mouse anti-human von Willebrand Factor (clone F8/86), LOT 125 (DAKO, at dilution of 1:25) 7. Monoclonal mouse anti-human endothelial cell, CD31, LOT 110 (DAKO, at dilution of 1:20) 8. Anti-human Ki67 Antigen/HRP, LOT 023 (DAKO). Bound antibody was detected using commercial Kit (DAKO, LSA B2 Peroxidase 675). The results were compared to the control group. Additionally as positive control, reactions from skin were used. For negative control, sections were treated with mouse nonimmune sera.
3. Results The 11 normal TM showed no particular histological features. The 11 perforated TMs specimens came from 6 male and 5 female patients (mean age 36.2). Ten specimens from perforated TM showed histological anomalies. Inflammatory infiltration and fibrosis in the fibrous layer were observed in all cases. Tympanosclerosis was present in eight cases. The epithelial layer showed diffuse hyperplasia. The medial, mucosal layer consisted of cuboidal and pseudostratified columnar cells with ciliated and goblet cells. Strong positive staining was found for receptor of EGF in perforated as well as in nonperforated TM specimens mainly in the stratified squamous epithelium. Immunoreactivity was more intense at the based membrane and in the basal layer. In the specimens of perforated TM, the seemingly more prominent staining in hyperplastic areas was due to the overall increased epithelial thickness and not to greater deposition. In the fibrous layer specimens, chronic TM demonstrates no or minimal staining of EGFR. There was also a slight staining of the vascular walls mainly at the basal membrane. The deposition of TGFh1 was particularly prominent in the fibrous layer at the border of the perforation. The Table 2 Distribution of growth factors in TMs COM Antibody
TM Epithelium
EGFR TGFh1
Pars fibrosa
Endothelium
+
++
++
is absent, + is mild, ++ is extensive.
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Table 3 Reactivity to the monoclonal antibodies in the TMs at COM and normal TMs from cadavers Antibody
TGFh1 EGFR HLA-DR Tenascin S-100 Ki67 CD31 FVIII
Substrate TMs normal
TMs COM
6/11 6/11 8/11 5/11 0/11 2/11 7/11 5/11
11/11 7/11 9/11 11/11 8/11 3/11 9/11 9/11
Number of positive results/number of investigated cases.
most striking difference was seen at the level of the subepithelial layer and pars fibrosa. In normal TM, hardly any or no immunoreactivity could be shown in these layers, whereas in perforated TMs, areas of strong positive staining were revealed in the subepithelial layer and in the lamina propria (Tables 1 and 2). Some specimens of TMs showed high activity of this factor. Intensity of reaction for the presence of EGFR in any case of COM was small, much smaller than for TGFh1. Anti-HLA-DR antigen reacted with all DR loci in Langerhans cells. In specimens from TM, there was a strong reaction with HLA-DR, suggesting the presence of Langerhans cells. The analysis of fixed in formalin tissue from TMs showed presence of many positive immunohistochemical reactions. Expression of tenascin was observed in all cases of investigated TMs tissue samples. The tenascin expression was often found in granulation tissue and margin tissue of TM perforation. The cells containing S-100 protein proved to be present most often in the tissue from TM perforation margin and granulation tissue. Single cases of positive reactions were of poor intensity. Ki67 antigen expression was present in 3 of 11 cases. The endothelium cells were identified using monoclonal antibodies CD31. Their presence was common in the inner layer of TM. Positive reaction for CD31 presence was observed in 9 of 11 of investigated cases. Factor von Willebrand (F VIII) was observed in 9 of 11 cases. It was most commonly present in endothelium cells of granulation tissue. Immunohistochemical investigation of intact cadaver TM showed low expression of tenascin, endothelial cells with CD31 antigen and von Willebrand factor. The presence of S-100-positive cells was not proven (Table 3).
4. Discussion In COM, there are active immunological processes present, with a particularly important role of growth factors. Cytokines play a central role in leukocyte migration through peripheral blood and tissue, and they induce and maintain the proliferation of both lymphocytes and keratinocytes [2 –4,13]. As it was shown, TGFh1 is frequently observed in tissue of TM perforation, which could prove its important role in repairing processes of TM.
J. Kuczkowski et al. / International Congress Series 1240 (2003) 1095–1100
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TGFh1 in general inhibits the growth of epithelium cells and stimulates the growth of mesenchymal cells [2,5,11]. In consequence, it may alter the architecture of stromal and/or epithelial elements. Inflammatory cells present in the earliest lesions may release TGFh1 possibly resulting in chemotactic recruitment of additional chronic inflammatory cells. If so, it would confirm the expression of TGFh1 observed in our study. TGFh1 expression was observed in lamina propria of TM with chronic perforation. This presence at the perforation border seemed to be related to the degree of fibroplasia. EGFR is not so common in COM. Mondain and Ryan [8] and Ramsay et al. [9] proved that the presence of EGF, known to be important in wound healing, can be demonstrated only minimally in the rims of chronic perforations by immunohistochemical means. This cytokine probably is of less importance in COM process. The positive HLA-DR reactions showed the presence of dendritic cells [4]. Their activity in tissue and margin tissue of TM is important for understanding the immune status of middle ear. The presence of Ki67 antigen is typical for rapid proliferating cells, especially in malignant neoplasms. The assessment of cell proliferation by Ki67 antigen in chronic inflammation cell population has been shown to be of a little prognostic value. That fact, that CD31 and F VIII determine vascular involvement in repair process in investigated cases, is obvious.
5. Conclusions 1. The cellular elements of chronic inflammation are present in the TM s in chronic otitis media. 2. TGFh1 is present in the TM perforation margin and endothelium. 3. EGFR is present in epithelium of TM but absent in pars fibrosa and endothelium. 4. The positive HLA-DR reactions and presence of S-100 protein showed the presence of Langerhans cells in COM.
References [1] D. Boedts, Tympanic membrane perforations, Acta Otorhinolaryngol. Belg. 49 (1995) 149 – 158. [2] W. Border, E. Ruoslahti, Transforming growth factor-beta in disease: the dark side of tissue repair, J. Clin. Invest. 90 (1992) 1 – 7. [3] D. Dvorak, G. Abbas, T. Ali, S. Stevenson, B. Welling, Repair of chronic membrane perforations with longterm epidermal growth factor, Laryngoscope 105 (1995) 1300 – 1304. [4] B. Hussl, G. Egg, W. Kong, N. Romani, A. Schrott-Fischer, Dendritic cells in the normal human tympanic membrane, Ann. Otol. Rhinol. Laryngol. 104 (1995) 803 – 807. [5] R. Koba, I. Kawabata, Immunohistochemical study of transforming growth factor-a expression in normal and perforated tympanic membrane, Ann. Otol. Rhinol. Laryngol. 104 (1995) 793 – 797. [6] D.J. Lim, Structure and function of the tympanic membrane: a review, Acta Otorhinolaryngol. Belg. 49 (1995) 101 – 115. [7] W. Meyerrhoff, Ch. Kim, M. Paparelle, Pathology of chronic otitis media, Ann. Otol. Rhinol. Laryngol. 87 (1978) 749 – 760. [8] M. Mondain, A. Ryan, Epidermal growth factor and basic fibroblast growth factor are induced in guinea-pig tympanic membrane following traumatic perforation, Acta Oto-laryngol. (Stockh.) 115 (1995) 50 – 54.
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[9] H. Ramsay, E. Heikkonen, P. Laurila, Effect of epidermal growth factor on tympanic membranes with chronic perforations: a clinical trial, Otolaryngol. Head Neck Surg. 113 (1995) 37379 – 37775. [10] C. Ruah, R. Penha, P. Schachern, M. Paparella, Tympanic membrane and otitis media, Acta-rhino-laryngol. Belg. 49 (1995) 173 – 180. [11] H.H. Steenfos, Growth factor and wound healing, Scand. J. Plast. Reconstr. Hand Surg. 28 (1994) 95 – 105. [12] H. Sudhoff, J. Bujia, A. Holly, C. Kim, A. Fisseler-Eckhoff, Functional characterisation of middle ear mucosa residues in cholesteatoma samples, Am. J. Otol. 15 (1994) 217 – 221. [13] T. Yamashita, Histology of the tympanic perforation and the replacement membrane, Acta Oto-laryngol. (Stockh.) 100 (1985) 66 – 71.