Preparation of gelatin/genipin nanofibrous membrane for tympanic member repair.

The wound healing for tympanic membrane (TM) perforations is one of the most common clinical treatment in otomicrosurgery. Recently, tissue-engineered composites and grafts, also new designs of biomaterials are applied to the management of TM perforation. In this work, a series of gelatin/genipin nanofibrous films were prepared as a patch for repairing TM perforation. Gelatin, a type of biomaterial with excellent electrospinning performance, has been used for preparing the nanofibers. The genipin, as a crosslinking agent, has been blended into the gelatin nanofibers. The reaction between gelatin and genipin engender suitable tensile strength and water-resistance for TM patch. The survival rate of human umbilical vein endothelial cells and fibroblasts demonstrated that the gelatin/genipin nanofiber scaffolds had good biocompatibility, which indicated the genipin was a kind of effective and nontoxic crosslinking agent for improving the mechanical property and water-resistance of gelatin films. In short, our work provides a novel macromolecular material with good mechanical properties, water-tolerance and excellent biocompatibility which could be used as a potential patch for TM repair.

Gene expression profile of collagen types, osteopontin in the tympanic membrane of patients with tympanosclerosis.

BACKGROUND: Tympanosclerosis is a pathological process involving the middle ear. The hallmark of this disease is the formation of calcium deposits. In the submucosal layer, as well as in the right layer of the tympanic membrane, the calcium deposits result in a significant increase in the activity of fibroblasts and deposition of collagen fibers. OBJECTIVES: The aim of our study was to examine the expression level of genes encoding collagen type I, II, III and IV (COL1A1, COL2A1, COL3A1, COL4A1) and osteopontin (SPP1) in the tympanic membrane of patients with tympanosclerosis. MATERIAL AND METHODS: The total RNA was isolated from middle ear tissues with tympanosclerosis, received from 25 patients and from 19 normal tympanic membranes. The gene expression level was determined by real-time RT-PCR. The gene expression levels were correlated with clinical Tos classification of tympanosclerosis. RESULTS: We observed that in the tympanic membrane of patients with tympanosclerosis, the expression of type I collagen is decreased, while the expression of type II and IV collagen and osteopontin is increased. Moreover, mRNA levels of the investigated genes strongly correlated with the clinical stages of tympanosclerosis. CONCLUSIONS: The strong correlations between the expression of type I, II, IV collagen and osteopontin and the clinical stage of tympanosclerosis indicate the involvement of these proteins in excessive fibrosis and pathological remodeling of the tympanic membrane. In the future, a treatment aiming to modulate these gene expressions and/or regulation of the degradation of their protein products could be used as a new medical approach for patients with tympanosclerosis.

Collagen type distribution in the healthy human tympanic membrane.

BACKGROUND: A weakening of the collagen fibers of the lamina propria of the tympanic membrane is a prerequisite for the formation of a retraction pocket. The various collagen types differ in tensile strength. The distribution of the different collagen types in the lamina propria of the healthy human tympanic membrane has not been reported before. METHODS: Immunohistochemical staining for collagen Types I, II, III, and IV in healthy human tympanic membranes harvested during translabyrinthine surgery for acoustic neuroma. The staining was semiquantified using light microscopy. RESULTS: Collagen Type II was the most abundant collagen of the lamina propria. When subdividing the staining between the 2 fiber layers of the lamina propria, it was observed that the inner layer was enriched with Type III relative to Type II, as reflected in staining patterns. In the outer radial fiber layer of the lamina propria, staining for collagen Type II was predominant.The fibrous annulus could be subdivided into an inner and an outer portion by immunohistochemistry. The inner portion stained strongest for collagen Type II and to a lesser extent for Type I. The outer portion stained strongest for collagen Type III and Type I. CONCLUSION: The differences in distribution of collagen types in the different fiber layers of the lamina propria suggest that the lattice of connective tissue supporting the tympanic membrane is not uniform. Understanding the differences in collagen type distribution and in the physical properties of the individual collagen types themselves may contribute to a comprehensive model of retraction pocket pathogenesis.

Measurement of young's modulus of human tympanic membrane at high strain rates.

The mechanical behavior of human tympanic membrane (TM) has been investigated extensively under quasistatic loading conditions in the past. The results, however, are sparse for the mechanical properties (e.g., Young's modulus) of the TM at high strain rates, which are critical input for modeling the mechanical response under blast wave. The property data at high strain rates can also potentially be converted into complex modulus in frequency domain to model acoustic transmission in the human ear. In this study, we developed a new miniature split Hopkinson tension bar to investigate the mechanical behavior of human TM at high strain rates so that a force of up to half of a newton can be measured accurately under dynamic loading conditions. Young's modulus of a normal human TM is reported as 45.2-58.9 MPa in the radial direction, and 34.1-56.8 MPa in the circumferential direction at strain rates 300-2000 s(-1). The results indicate that Young's modulus has a strong dependence on strain rate at these high strain rates.

Distribution of different collagen types in the rat's tympanic membrane and its suspending structures.

BACKGROUND: The objective of the study was to investigate the histological distribution of collagens in the healthy rat's tympanic membrane. METHODS: Immunohistochemical analysis of collagen type I, II, III, and IV in the tympanic membranes in healthy adult female Sprague-Dawley rats. The staining was semiquantified using light microscopy in a blinded fashion, not knowing what type of collagen the slide had been stained for. RESULTS: The pars tensa of the tympanic membrane was mainly stained for collagen type II and IV. The fibrous annulus could on immunohistochemistry be subdivided into an inner and an outer portion. The inner portion of the fibrous annulus was mainly stained for collagen type II, whereas the outer portion was most strongly stained for collagen type III and collagen type IV. The test-retest reliability of the semiquantative method was 81%. CONCLUSION: Collagen type II and IV are the major collagen constituents of the pars tensa of the tympanic membrane. The outer portion of the fibrous annulus has collagen type III and IV as its major constituents, whereas the inner portion is made up of collagen type II.

Spontaneous development of otitis media in plasminogen-deficient mice.

Inflammatory conditions of the ear, otitis media, are one of the most common disease entities in children. In this study, the role of the plasminogen (plg)/plasmin system for the spontaneous development of chronic otitis media was investigated by the analysis of plg-deficient mice. Whereas essentially all of the wild-type control mice kept a healthy status of the middle ear, all the plg-deficient mice gradually developed chronic otitis media with various degrees of inflammatory changes during an 18-week observation period. Five bacterial strains were identified in materials obtained from the middle ear cavities of six plg-deficient mice. Morphological studies revealed the formation of an amorphous mass tissue and inflammatory changes in the middle ears of plg-deficient mice. Immunohistochemical studies further indicate a mass infiltration of neutrophils and macrophages as well as the presence of T and B cells in the middle ear mucosa of these mice. Extensive fibrin deposition and an abnormal keratin formation were also observed in the tympanic membrane, the middle ear cavity and external ear canal in these mice. These results suggest that plg plays an essential role in protecting against the spontaneous development of chronic otitis media. Our findings also suggest the possibility of using plg for clinical therapy of certain types of otitis media.

The collagen structure of the tympanic membrane: collagen types I, II, and III in the healthy tympanic membrane, during healing of a perforation, and during infection.

OBJECTIVE: To analyze the distribution of 3 collagen types in healthy tympanic membranes, during healing of a perforation, and during infection. DESIGN: Immunohistochemical study of collagen types I, II, and III in the tympanic membranes of healthy rats as well as during healing of a perforation and in the presence of infection with Streptococcus pneumoniae at various time points. SETTING: Laboratory research center at a university hospital. RESULTS: Type II collagen was a main constituent of the lamina propria of the pars tensa, whereas type I collagen was found mainly in the pars flaccida. Collagen types I and III were found at the insertion to the malleus handle and in the loose connective tissue surrounding the main collagen layer of the pars tensa. After myringotomy, collagen types I and III were found at the perforation border and around dilated blood vessels early in the healing phase. During infection, the collagen layer was thickened and stained strongly for type II collagen. Collagen types I and III were found in the edematous connective tissue around the main collagen layer and around dilated blood vessels. Three months after perforation or infection, all 3 collagens were present in the lamina propria of the tympanic membrane. Extensive amounts of all 3 collagen types were present in the scar tissue in the tympanic membranes of rats that had undergone myringotomy during the presence of acute otitis media. CONCLUSIONS: The lamina propria of the pars tensa is mainly made up of type II collagen, whereas type I collagen is found in the pars flaccida. Thus the fibrous structure of the pars tensa and flaccida is composed of diverse collagen types, which reflects the different physiological properties of these tissues. Collagen types I and III are present in the acute healing phase after myringotomy and infection, and the collagen content of the tympanic membrane is modified during the inflammatory and healing process.

Utilization of fresh human tympanic membranes for structural analysis and cytokeratin immunocytochemistry implementing resin techniques.

CONCLUSIONS: The results of this study have demonstrated for the first time that tympanic membrane (TM) structure is preserved following removal of fresh, normal tissue from patients undergoing surgery. Greater clarity has been demonstrated using resin sections than in previous studies on paraffin sections. Of particular note, cytokeratin (CK) immunocytochemistry was successfully performed on resin sections, which has not been previously reported. This may have potential applications for future work involving tissues that express CKs. OBJECTIVES: To analyse the structure of normal, fresh human TM specimens after surgical removal and to evaluate their CK immunocytochemistry using resin techniques, neither of which have been demonstrated previously. MATERIAL AND METHODS: Seven TM specimens were removed during surgery and then preserved in a modified Karnovsky's fixative. Semi-thin and thin sections were examined by means of light and electron microscopy, respectively. For comparison purposes, paraffin block-embedded specimens were also sectioned. CK immunocytochemistry was performed on semi-thin sections using standard immunoperoxidase techniques, with expression being demonstrated using light microscopy. RESULTS: The three-layer architecture of the TM was preserved. The morphology of the TM was vastly superior in the semi-thin resin sections than in the thicker paraffin sections. The outer, middle and inner layers were clearly demonstrated. The integrity of the outer epithelial layer was maintained, with an outer keratinizing stratum corneum and underlying stratum granulosum, stratum spinosum and stratum basale layers resting on the basal lamina. The thin inner mucosal layer was also viable, consisting of simple squamous or cuboidal cells. Preservation of the middle lamina propria was achieved, with demonstration of the outer radial and inner circular fibres. CK immunocytochemistry utilizing resin techniques provided excellent staining of CK 7 and 8 in the inner layer, with positive staining of CK 5 and 10 in the outer layer.

Calcium deposition and expression of bone modelling markers in the tympanic membrane following acute otitis media.

BACKGROUND AND OBJECTIVES: In accordance with clinical findings, myringosclerosis develops after otitis media (OM) and paracentesis in an experimental setting. The pathogenesis of this phenomenon of calcification is poorly understood. As the calcification process and the sclerotic plaques of the drum mimics features of bone tissue, this study explores tympanic membrane calcium deposition in association with the expression of three bone modelling markers: osteopontin (OPN), osteoprotegerin (OPG) and osteonectin (ON). OPN is secreted by osteoblasts and is found at calcification sites, e.g. during pathological calcification in chronic OM. The cytokine OPG is an inhibitor of bone resorption and consequently bone remodelling. ON is a calcium binding glycoprotein necessary for the maintenance of bone mass and remodelling. It is found in bone matrix and synthesized by osteoblasts. METHOD: A rat model of acute otitis media (AOM) caused by non-typeable Haemophilus influenzae was used. Four days following middle ear inoculation, a myringotomy was performed in six animals. Another group of ten animals was inoculated only. The drum was dissected in two animals from each group on day 4, 7, 14 and 28 post-inoculation, and the expression of OPN, OPG and ON was determined by immunohistochemistry. von Kossa staining determined the deposition of calcium and immune staining for CD68 identified macrophages. RESULTS: Calcium depositions were initially accumulated in the cytoplasm of macrophages and dispersed in the connective tissue layers of the pars flaccida and tensa. Late accumulation occurred in the lamina propria of pars tensa, more extensively in myringotomized ears. OPN expression was found early in inflammatory cells including especially macrophages and late in pars tensa fibrocytes. OPG expression was initially located to inflammatory cells and late to pars tensa fibrocytes and the inner basal membrane of pars flaccida. Some ears displayed a marked pars flaccida expression of ON in the connective tissue matrix on early days and at the inner basal membrane on later days. The latter cases were from myringotomized ears. Otherwise, no apparent differences of marker expression occurred between myringotomized and non-myringotomized animals. CONCLUSION: We conclude that osteopontin, osteoprotegerin and osteonectin are expressed by different cell types in the tympanic membrane during calcification in association with AOM, with or without myringotomy. These molecules may accordingly play a role in the pathogenesis of myringosclerosis, in which macrophages and fibrocytes appear as potential major players.

[Microstructure of tympanic membrane in chronic otitis media: immunohistochemical evaluation]. / Mikrostruktura blony bebenkowej w przewleklym zapaleniu ucha srodkowego w badaniach immunohistochemicznych.

Chronic otitis media (COM) is an inflammatory process involving the middle ear mucosa and the tympanic membrane. The healing and epidermization is mostly impaired by immunological response of the host. Investigating the activity and the function of immunological response elements one can learn the immunological mechanisms taking place in chronic otitis media. The ultrastructural investigations of the tympanic membrane were done on its fragments obtained from 19 patients with COM during middle ear surgery, performed at ENT Department of Medical University of Gdansk in the years 1997-1999. Immunohistochemical investigations were performed using monoclonal antibodies against tenascin, S-100 protein, Ki 67, CD 31, F VIII, HLA-DR, TGFbeta1 and EGFR. The control group was 11 healthy tympanic membranes from cadavers. The presence of tenascin was proven in all COM tympanic membranes and in 45.5% of those from control group. S-100 protein was present in 88.9% of the patients with COM and absent in control group. Ki 67 was observed in 44.4% of the patients with COM and in 27.3% of the healthy tympanic membranes. Angiogenesis factors (CD 31 and FVIII) were present in 77.8% of the investigated COM tympanic membranes, in control group in 45.5%. HLA-DR expression was observed in 90% COM patients, in control group in 72.7%. Growth factor TGFbeta1 was present in the all cases in mucous and fibrous layer and in 54.5% of healthy tympanic membranes. EGF receptor was present in 60% of COM patients, mainly in epithelial layer of tympanic membrane and in 54.5% of those from control group. The presented investigations confirm the immunological activity of tympanic membrane in chronic otitis media.

A study of inflammatory mediators in the human tympanosclerotic middle ear.

OBJECTIVE: To analyze immunocompetent cells as well as 2 factors involved in inflammation and also thought to be involved in bone remodeling-interleukin 6 (IL-6) and inducible nitric oxide synthase in the human middle ear, including the tympanic membrane. DESIGN: Biopsy specimens were obtained from the human middle ear and tympanic membrane during surgery. Using an immunohistochemical technique, the expression of macrophages, T cells, B cells, IL-6, and inducible nitric oxide synthase were analyzed. MATERIALS: Nine biopsy specimens from tympanic membranes in children having a transtympanic ventilation tube inserted as a treatment for secretory otitis media and 11 biopsy specimens from tympanosclerotic plaques from patients with chronic otitis media and tympanosclerosis. RESULTS: More positively stained specimens showing macrophages, B cells, and IL-6 were seen in the biopsy specimens from children with secretory otitis media compared with the biopsy specimens from patients with chronic otitis media and tympanosclerosis. The biopsy specimens from patients with chronic otitis media and tympanosclerosis more often showed positive stainings for inducible nitric oxide synthase than the biopsy specimens from children with secretory otitis media. The presence of IL-6 and inducible nitric oxide synthase was shown by staining to be mostly in the surface cells, while macrophages and B cells were stained deeper in the tissues, in connective tissue, or around sclerotic lesions. CONCLUSIONS: The 2 patient groups differed in antigen presentation so that macrophages, B cells, and IL-6 were labeled more frequently in patients with secretory otitis media, that is, an early phase of the disease. Inducible nitric oxide synthase was seen more frequently in the patients with already established tympanosclerosis in a later phase of the disease.

Expression pattern of the plasminogen activator-plasmin system in human cholesteatoma.

The plasminogen activator-plasmin system plays a pivotal role in the delicately regulated process of extracellular matrix remodeling. Recent studies have shown that an imbalance of proteolytic enzymes over specific inhibitors in this system may lead to an aggressive, expanding, and infiltrating cellular phenotype. As cholesteatoma resembles a tumor in many ways, we investigated the pattern of expression for members of the plasminogen activator-plasmin system in 12 human cholesteatomas, using immunohistochemistry. As controls, 3 tympanic membranes and 4 ear canal skin specimens were used. In contrast to the tympanic membranes, all cholesteatoma specimens showed a strong expression of plasminogen at the basal epithelial cell layers. In ear canal skin, only the basal surface of the most basal epithelia stained discretely positive. The urokinase-type plasminogen activator (uPA) could be detected in the basal stratum of the cholesteatoma matrix and in the surrounding granulation tissue, while tissue-type plasminogen activator (tPA) was not detectable at all. Plasminogen activator inhibitor-1 (PAI-1) was expressed in both the granulation tissue and the granular cell layer of the matrix, but not in the basal epithelial cells; PAI-2 showed a pericellular expression pattern in the subbasal and granular cell layers. Neither uPA, tPA, nor the PAIs could be detected in tympanic membrane controls; ear canal skin showed the same staining pattern as cholesteatoma only for PAI-2. Our data demonstrate that there is a clear imbalance in favor of proteolytic activity in the basal epithelial layers of the cholesteatoma matrix, which might at least partly account for the aggressive behavior of this tumorlike lesion. Further, the pattern of expression resembles the pattern described for several epithelial malignancies.

Immunohistochemical study of cell proliferation using BrdU labelling on tympanic membrane, external auditory canal and induced cholesteatoma in Mongolian gerbils.

The Mongolian gerbil is a well-known animal model for induction of aural cholesteatomas. This animal model is useful for studying changes in the keratinizing epithelium. It is not known whether keratin accumulation can increase the proliferative activity of the keratinizing epithelium in tympanic membrane and meatal skin. In this study, we investigated the proliferative activity of the epidermis in induced aural cholesteatoma at various stages and in different areas of the tympanic membrane and meatal skin in normal gerbils. Anti-5-bromo-2- deoxyuridine (BrdU) was injected intraperitoneally to detect the proliferative activity of keratinizing epithelium. Immunohistochemistry with monoclonal BrdU antibody in the normal gerbil showed intense immunolabelled keratinocytes at the handle of malleus, and the superior parts of pars tensa and pars flaccida. Also, mitotic activity in the deep meatal skin was more active than in the lateral meatal skin. The induced aural cholesteatoma showed more active proliferation centre of the epithelial cell than eardrum and external ear canal of the normal gerbil. These observations suggest that the accumulation of the keratin debris might induce changes of the cellular proliferation in the external auditory meatus.

Induction of KGF, basic FGF, and TGFalpha mRNA expression during healing of experimental TM perforations.

KGF (KGF), synthesized and secreted exclusively by stromal cells in epithelialized organs, specifically promotes proliferation of cells of epithelial origin, including keratinocytes. A related peptide, basic fibroblast growth factor (bFGF), has mitogenic properties for fibroblasts and endothelial cells. KGF expression is stimulated markedly in the skin during wound healing. To investigate the physiologic action of KGF in the healing of TM (TM) perforations, we examined KGF and KGF receptor (KGFR) mRNA transcript levels as well as those of bFGF and transforming growth factor-alpha (TGFalpha) in normal and wounded rat TM at varying intervals, using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). We found KGF and TGFalpha mRNA expression to be induced rapidly, peaking 3 days after wounding and then declining. Expression of bFGF was induced gradually and remained increased until 7 days. In contrast, we found KGFR to be expressed in normal TM, remaining unchanged during TM repair. These results indicate that KGF and TGFalpha may mediate migration and proliferation of epithelial cells of the outer layer in the early stage of TM repair while bFGF may mediate the connective tissue reaction in the middle layer in a subsequent stage.

Cholesteatoma: a molecular and cellular puzzle.

HYPOTHESIS: There are at least three possible molecular models of cholesteatoma pathogenesis. Cholesteatoma may arise as a result of 1) the induction of a preneoplastic or neoplastic transformation event; 2) a defective wound-healing process; and/or 3) a pathologic collision of the host inflammatory response, normal middle ear epithelium, and a bacterial infection. BACKGROUND: There have been a number of speculations concerning the factors that foster the development of cholesteatoma. Before resolving the molecular basis for the pathogenesis of cholesteatomas, it is important to present and test plausible models that could explain how a cholesteatoma becomes invasive, migratory, hyperproliferative, aggressive, and recidivistic. METHODS: The authors evaluated by various techniques (e.g., immunohistochemistry, flow cytometry, and image analysis) a large number of cholesteatomas of all types (e.g., primary and secondary acquired, recurrent, and congenital) and a range of normal tissues (tympanic membrane, canal wall skin, and postauricular skin) for the expression of various proteins (e.g., p53, ectopeptidases, tryptase) and for the presence of DNA aneuploidy. RESULTS AND CONCLUSIONS: The authors' published and unpublished studies to date support several suppositions concerning the pathology of cholesteatomas. First, cholesteatoma epithelium behaves more like a wound-healing process than a neoplasm. The available evidence to date does not indicate that cholesteatomas have inherent genetic instability, a critical feature of all malignant lesions. Second, the induction of hyperproliferative cells in all layers of the cholesteatoma epidermis implicates a potential idiopathic response to both internal events as well as external stimuli in the form of cytokines released by infiltrating inflammatory cells. Third, the presence of bacteria may provide a critical link between the cholesteatoma and the host, which prevents the cholesteatoma epithelium from terminating specific differentiation programs and returning to a quiescent state in which it becomes minimally proliferative, nonmigratory, and noninvasive. Fourth, none of our data suggest that there are any obvious molecular or cellular differences among the various types of cholesteatomas (e.g., primary and secondary acquired, recidivistic, and congenital). Continued research should delineate the precise molecular and cellular dysfunction involved in the pathogenesis of cholesteatomas and how this knowledge can be useful in the clinical management of cholesteatomas.

Influence of hyperoxia on in vitro growth of rabbit middle ear epithelium and auditory meatal epithelium.

The oxygen partial pressure of middle ear gas increases more than 3-fold upon insertion of ventilation tubes, while the carbon dioxide partial pressure decreases. Whereas the middle ear gas is normally equilibrated to venous gases and has an oxygen partial pressure of 43 mmHg, 138 mmHg is measured in ventilated ears. The present study was undertaken to compare the effects of these oxygen tensions on in vitro growth and glycoprotein secretion of rabbit middle ear epithelium and for comparison auditory meatal epithelium. Cultures were incubated in atmospheres of 7, 21 or 75% O2 in 5% CO2 and the remnant N2. The cell layer protein mass, [3H]thymidine-incorporation, DNA content and [3H]glucosamine-incorporation was measured in identical subcultures every third day during a 15-day period. In middle ear epithelium the DNA content, DNA synthesis and cell layer protein mass were significantly higher at 7% oxygen compared to 21% and 75%. In conclusion hyperoxia leads to decreased growth of middle ear epithelium in vitro. If applicable to in vivo conditions, this might contribute to the mechanism of action of ventilation tubes. Moreover the proliferation rate of auditory meatal epithelium exceeds that of middle ear epithelium both at 7 and 21% oxygen, an interesting point with regards to cholesteatoma pathogenesis.

Keratinocyte differentiation in acquired cholesteatoma and perforated tympanic membranes.

OBJECTIVE: To evaluate the type of differentiation of keratinocytes of acquired cholesteatoma and its significance for cholesteatoma invasiveness. DESIGN: Forty acquired cholesteatomas and 10 tympanic membranes with persisting perforations were snap frozen and processed for immunohistochemical studies. Cytokeratin antibodies that represented all subgroups and antibodies that were directed against collagen components of the basal lamina were applied. Expression of these constituents was scored by using light microscopy. RESULTS: The phenotype of the matrix was generally characterized by an extension of expression of basal cell cytokeratin 14 and hyperproliferation-associated cytokeratins 6, 16, and 17 into the suprabasal cell layers, while the expression of keratinization marker cytokeratin 10 was down-regulated. These features varied greatly at different sites of the matrix and were most marked at the advancing front of the cholesteatoma. A comparable expression pattern, but less pronounced, was observed at the epidermal front of the mucocutaneous junction of the tympanic membrane perforations. This phenomenon was invariably associated with a mononuclear cell infiltrate in the dermis at both junctions. The basal lamina was always intact. CONCLUSIONS: Acquired cholesteatomas show hyperproliferative features. There is a striking similarity between the pronounced expression of this phenotype and the associated inflammation at the mucocutaneous junctions of cholesteatomas and tympanic membrane perforations and those that are observed after epidermal injury. This indicates that epidermis and middle ear epithelium do not form stable junctions and the front can be considered to be a persisting epidermal defect. This involves the permanent presence of "activated keratinocytes" in the junction area that will lead to proliferation and migration, when additional triggers are present.

Structural characterization of persistent tympanic membrane perforations in man.

In the present structural study the authors investigated the border of permanent tympanic membrane (TM) perforations in patients selected for myringoplasty. Furthermore, a panel of monoclonal antibody markers that recognize different epitopes within glycosaminoglycans as well as antibodies to epidermal growth factor and fibronectin were applied to the sections. In half of the specimens the epithelial junction ended at the inside of the perforation border, whereas in the other half it was located at the perforation border itself. In the junctional area the keratinocytes were covered by a thick keratin layer which protruded as a spur centripetally in order to bridge the perforation. Epidermal cells formed papillae and contained remnants of keratinocyte nuclei that showed similarities to those of the skin in inflammatory conditions. The connective tissue layer was fibrous and showed areas containing sclerotic plaques. The inner epithelium of the TM had abundant ciliae, thus supporting the concept that cells of the mucosal lining of the TM are able to differentiate in inflammatory conditions into ciliated cells and secretory cells. The immunoreactivity of hyaluronan and other glycosaminoglycans, the immunoreactivity of epidermal growth factor, and immunoreactivity of fibronectin, all of which are known to occur in healing wounds, were only scantily demonstrated; this could be one reason for the arrested healing and a reason why the natural drive to complete a mature closure is abandoned.

Cytokeratin expression patterns by one- and two-dimensional electrophoresis in pars flaccida cholesteatoma and pars tensa cholesteatoma.

Expression patterns of cytokeratins (CKs) in normal skin, in pars flaccida type cholesteatoma (PFTC), and in pars tensa type cholesteatoma (PTTC) were examined by means of one- and two-dimensional electrophoretic techniques. Both CKs 14 and 5 pair (CKs 14/5) and CKs 10/1 were found in all materials. Neither CKs 16/6 nor 19 was found in the skin. CKs 16/6 and 19 were both found in 3 out of 5 PFTCs, only CKs 16/6 in 1 out of 5 and neither CKs 16/6 nor 19 in 1 out of 5. CKs 16/6 and 19 were both found in 1 out of 3 PTTCs, only CKs 16/6 in 1 out of 3 and neither CKs 16/6 nor 19 in 1 out of 3. There was no significant difference in the CKs expression patterns between PFTC and PTTC. The expression of CKs 16/6 and 19 suggested that their matrix epithelia were hyperproliferative. However, not all of the cholesteatomas were always hyperproliferative. Patterns of the terminal differentiation of CKs 1, 5, 10 and 14 in the PFTC or the PTTC were basically the same as those in the skin. In the cholesteatoma, eack CK gradually diminished in molecular weight in the cornified layer and debris. Desmosomal proteins were abundant in skin but not in cholesteatomas.

Epidermal growth factor and basic fibroblast growth factor are induced in guinea-pig tympanic membrane following traumatic perforation.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF or FGF-2) have been shown to enhance the healing of traumatic tympanic membrane (TM) perforations. The action of EGF and bFGF in the TM repair process remains unknown. This study was designed to determine the expression of EGF and bFGF in normal and injured TM by immunohistochemistry. EGF was detected in normal TM mainly in the annulus tympani area. After a perforation in the TM posterior/superior quadrant, EGF was detected around the perforated area in polynuclear cells, in pericytes and in basal epithelial cells. EGF was also detected in the antero/superior quadrant in basal epithelial cells and pericytes. The peak of EGF detection was observed 3 days after the perforation. bFGF was not detected in normal TM, but it was expressed 3 days after a traumatic perforation mainly in the perforated area in pericytes and in polynuclear cells. This study suggests that EGF and bFGF are involved in the control of TM acute perforation repair. These findings help to explain the accelerated healing of TM perforations that are seen after application of FGF or EGF, and suggest that antibodies against these growth factors would retard the healing process.