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Objective: Tympanic membrane (TM) fibroblast cytotoxicity of quinolone ear drops is enhanced by dexamethasone and fluocinolone. Hydrocortisone has not been evaluated. We aimed to assess the effects of these 3 steroids on mouse and human TM fibroblast survival., Study Design: In vitro., Setting: Academic laboratory., Subjects and Methods: Mouse and human TM fibroblasts were exposed to hydrocortisone, dexamethasone, or fluocinolone at concentrations in commercial ear drops (1%, 0.1%, or 0.025%, respectively) and at steroid potency equivalents (1%, 0.033%, or 0.0033%, respectively), or dilute ethanol (control), twice within 24 hours or 4 times within 48 hours for 2 hours each time. Cells were observed with phase-contrast microscopy until the cytotoxicity assay was performed., Results: Mouse and human TM fibroblasts treated with any of the steroids had lower survival after 24 and 48 hours compared to control (all P < .0001). After 24 hours, viability of mouse fibroblasts treated with the steroids was not different ( P > .05), while treatment with hydrocortisone decreased human TM fibroblast viability ( P < .0001). After 48 hours, at concentrations found in ear drops and at equivalent steroid potency, dexamethasone and fluocinolone had similar survival in mouse and human fibroblasts (all P > .05), but hydrocortisone had lower survival in both mouse ( P = .02 and P < .0001) and human ( P < .0001) fibroblasts. Phase-contrast images mirrored the cytotoxicity findings., Conclusion: Steroids found in commercial ear drops reduce survival of mouse and human TM fibroblasts. Hydrocortisone appears to be more cytotoxic than the more potent steroids, dexamethasone and fluocinolone. These findings should be considered when assessing clinical outcomes of ototopical preparations.

The ear drum, or tympanic membrane (TM), is a key component in the intricate relay that transmits air-borne sound to our fluid-filled inner ear. Despite early belief that the mammalian ear drum evolved as a transformation of a reptilian drum, newer fossil data suggests a parallel and independent evolution of this structure in mammals. The term "drum" belies what is in fact a complex three-dimensional structure formed from multiple embryonic cell lineages. Intriguingly, disease affects the ear drum differently in its different parts, with the superior and posterior parts being much more frequently affected. This suggests a key role for the developmental details of TM formation in its final form and function, both in homeostasis and regeneration. Here we review recent studies in rodent models and humans that are beginning to address large knowledge gaps in TM cell dynamics from a developmental biologist's point of view. We outline the biological and clinical uncertainties that remain, with a view to guiding the indispensable contribution that developmental biology will be able to make to better understanding the TM., (© 2019 The Authors. Genesis published by Wiley Periodicals, Inc.)

Hypothesis: The scarred rim of chronic tympanic membrane (TM) perforation contains keratinocytes with potential for regeneration while maintaining their morphological and genetic characteristics., Background: The squamous epithelium of the TM has a good regeneration capacity. Successful isolation and expansion of human TM keratinocytes (hTMKR) was reported from a full, en-bloc, healthy TM., Methods: Trimmed margins of the TM perforation (harvested during tympanoplasty) underwent enzymatic digestion (collagenase or trypsin) and were seeded either with serum-containing medium (SCM) or keratinocyte serum-free medium (KSFM) and progenitor cell growth medium (PR) (KSFM:PR, 1:1). Gene expression analysis by real-time qRT-PCR was used to compare between human TM cells derived from scarred perforation margins (hTMKR), normal human skin keratinocytes (NhSKR), and human fibroblasts., Results: Twelve patients were included in the study. In 9 of 12 cases (75%) single-cell isolation with fibroblastic or epithelial cell morphology (or both) was achieved. Cells seeded with KSFM:PR yielded epithelial morphology (hTMKR) while SCM culturing resulted in a fibroblastic morphology (hTMFib). Gene expression analysis revealed significant higher expression of VCAN (p = 0.002) and FOXC2 (p = 0.015) at the mRNA levels (normal hTMKR markers) in hTMKR compared to NhSKR. In addition, a comparison of gene expression between hTMKR and hTMFib revealed significantly higher levels of both VCAN (p = 0.045) and SLC6A14 (p = 0.036) among hTMKR., Conclusion: For the first time, we developed a protocol to isolate hTMKR from scarred TM perforation margins. Furthermore, we succeeded in achieving tissue expansion that preserved the characteristic of healthy TM cells. This study bridges "regenerative medicine" approach with clinical and surgical objectives.

Objective: The epithelium lining the human middle ear and adjacent temporal bone cavity shows a varying morphological appearance throughout these cavities. Its embryologic origin has long been debated and recently got attention in a newly proposed theory of a dual embryologic origin. The epithelial morphology and its differentiating capabilities are of significance in future mucosa-targeted therapeutic agents and could affect surgical approaches of the temporal bone. This study aims to analyze reported murine histological findings that led to the theory of a dual epithelial embryological origin and immunohistochemically investigate whether such an epithelial embryological origin in the human fetal middle ear could be true., Methods: By combining a sagittal sectioning technique and immuno-histochemical staining, a comprehensive immuno-histological overview of the fetal human middle ear during a critical stage of tympanic cavitation was provided. A critical analysis of previously reported findings leading to the theory of a dual epithelial embryological origin and a comparison of these findings to the findings in the human fetal middle ear was performed., Results: The reported findings and critical analysis provide multiple arguments for an entirely endodermal embryonic origin of the epithelium lining the tympanic cavity., Conclusion: Different morphological epithelial appearances throughout the tympanic and temporal bone cavities could be explained by different stages of epithelial differentiation rather than different embryologic origin and endodermal rupture does not seem to be a necessity for these cavities to form., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The eardrum is an important structural component for hearing, but it is delicate and subject to traumatic injury and disease. Healing mechanisms are activated after injury but sometimes healing fails and chronic perforations develop, requiring surgical intervention. To model the wound healing responses we established a simple method for isolating keratinocytes and progenitors from individual eardrums. The central region of the eardrum contains epidermal proliferative centers that produce keratinocytes which migrate to cover the eardrum surface. We dissected out the central region and explanted it to the plastic membrane of a culture well insert. Epidermal cells grew from the explant onto the surface of the insert membrane. The cells could be serially harvested and passaged for continuous culture and characterization. Magnetic immunoseparation methods were used to enrich for epithelial cells with stem cell-like characteristics. Proliferation and migration in vitro was demonstrated, and the cells were shown suitable for tissue engineering applications.

Objectives: To investigate the distribution of ciliated epithelium in the human middle ear and its potential role in the formation of cholesteatoma., Study Design: Comparative human temporal bone study., Methods: We selected temporal bones from 14 donors with a diagnosis of cholesteatoma, 15 with chronic otitis media without retraction pockets, 14 with chronic otitis media with retraction pockets, 14 with cystic fibrosis (CF), and 16 controls. We mapped the distribution of the ciliated cells in the mucosal lining of the middle ear and tympanic membrane using three-dimensional reconstruction analysis, and counted the number of ciliated cells in the middle ear mucosa., Results: Ciliated cells are extremely sparse in the epithelial lining of the lateral surface of the ossicles in the epitympanum and the medial surface of the tympanic membrane. Furthermore, there is a significant decrease in the number of ciliated cells in these areas in temporal bones with cholesteatoma, chronic otitis media, chronic otitis media with retraction pockets, and CF compared to controls. Ciliated cells most commonly are located at the hypotympanum and the Eustachian tube opening but not the tympanic membrane or epitympanum., Conclusion: The paucity of ciliated epithelial cells on the medial side of the tympanic membrane and the lateral surface of the ossicles in the epitympanum in cases with cholesteatoma and/or chronic otitis media do not support the mucosal migration theory of cholesteatoma formation., Level of Evidence: NA. Laryngoscope, 128:1663-1667, 2018., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.

Epidermal cells with stem cell-like characteristics have been identified in the tympanic membrane (TM) and localized specifically to the umbo and annulus regions. While they have been proposed to play a role in the regeneration of both acute and chronic TM perforations, evidence for the mechanism and regulation of their contribution is not yet described. Indeed, the behavior of these putative stem cells is largely unknown, in part due to a lack of refined methods for efficient cell isolation. In this study, we compared different explant techniques using normal and perforated rat TM tissues and investigated their ex vivo characteristics. TM after perforation in vivo showed increased staining for epidermal stem cell markers integrin β1 and cytokeratin (CK) 19, and for proliferation Ki-67, indicating activation of the proliferative centers. A mixed population of fibroblasts and epithelial cells were isolated from explant cultures. Excised TM umbo implanted on a culture well insert was the most effective technique. Explants made from perforated TM produced cells before those from unperforated TM. More importantly, the implanted TM umbo organoid was capable of producing cells in a continuous manner, allowing subsequent harvest using trypsin. Primary rat TM epithelial cell cultures positive for pancytokeratin had colony forming activity and could be enriched for CK 19-positive cells that were capable of culture expansion by proliferation and cell migration when subject to a wound assay. Taken together, trauma to the TM activated the proliferative centers and prompted early cell production from TM umbo organoid cultures, which produced TM stem cell-like cultures that proved suitable for tissue engineering of the TM.

Objective Ciprofloxacin, commonly given as eardrops, has been shown to adversely affect tympanic membrane fibroblasts. Dexamethasone potentiates this effect. A newly available eardrop contains ciprofloxacin and fluocinolone, a more potent steroid. We evaluated the cytotoxic effects of this preparation on mouse tympanic membrane fibroblasts. Study Design Prospective, in vitro. Setting Academic laboratory. Subjects and Methods In experiment 1, fibroblasts were exposed to 1:10 dilutions of commercially available 0.3% ofloxacin, 0.3% ciprofloxacin, 0.3% ciprofloxacin + 0.1% dexamethasone, 0.3% ciprofloxacin + 0.025% fluocinolone, or dilute hydrochloric acid (control), twice within 24 hours. In experiment 2, cells were also treated with the dilutions of the pure form of dexamethasone 0.1% or fluocinolone 0.025%, alone and in combination with ofloxacin or ciprofloxacin. Cells were exposed to the solutions for 2 hours each time and were placed back in growth media after the treatments. Cells were observed with phase-contrast microscope until the cytotoxicity assay was performed. Results Survival of fibroblasts treated with ofloxacin was not different from the control. Fibroblasts treated with ciprofloxacin, ciprofloxacin + dexamethasone, or ciprofloxacin + fluocinolone had much lower survival (all P < .0001). Cells treated with ciprofloxacin + fluocinolone had lower survival than ciprofloxacin ( P < .0001) and ciprofloxacin + dexamethasone ( P = .0001). Steroids alone also decreased fibroblast survival compared to control ( P < .0001). The combination of dexamethasone or fluocinolone with ciprofloxacin, but not ofloxacin, further decreased fibroblast survival ( P < .0001). Phase-contrast images mirrored the cytotoxicity findings. Conclusion Tympanic membrane fibroblast cytotoxicity of ciprofloxacin is potentiated by corticosteroids. This effect may be deleterious when treating a healing perforation but beneficial when treating granulation tissue on the tympanic membrane.

Damage to the eardrum causes acute pain and can lead to chronic otitis media if it develops into chronic tympanic membrane (TM) perforations. Chronic TM perforations are usually treated with surgical methods such as tympanoplasty and myringoplasty. However, these surgeries are not only complicated and difficult but also cost a lot of money. Our research team developed chitosan patches (E-CPs) that release epidermal growth factor (EGF) as a patch therapy to replace surgical methods. However, there was a limitation in the healing ratio of the treatment compared to the surgical methods. In this study, we developed EGF and epidermal growth factor receptor (EGFR) gene-releasing polyethyleneimine (PEI)/chitosan patches (EErP-CPs) to increase the regeneration of TM perforations. The addition of PEI increased the adhesion and migration ability of TM cells on the patches. The simultaneous release of the EGF and the EGFR gene further enhanced TM cell proliferation, adhesion and migratory ability. It was confirmed that the EGF protein and EGFR gene were released for 30 days; however, EGF was released and increased TM cell viability almost immediately after treatment and EGFR took a minimum of 3 days before showing its effect on improved cell viability. It was also shown that EErP-CPs are more hydrophilic and have more positive charge than E-CP because of added amine groups from PEI. In conclusion, the developed EErP-CPs resulted in the improved healing of TM perforations and can potentially be applied to the regeneration of both chronic and acute tympanic membrane perforations.

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

Objective To assess collagen and α-tubulin levels of mouse tympanic membrane fibroblasts treated with quinolone and aminoglycoside antibiotics at concentrations found in eardrops. Study Design Prospective controlled cell culture study. Setting Academic tertiary medical center. Subjects Mouse tympanic membrane fibroblasts. Methods In experiment 1, fibroblasts were treated with the following for 24 or 48 hours: phosphate-buffered saline (negative control), dilute hydrochloric acid (positive control), 0.5% gatifloxacin, or commercially available 0.3% ciprofloxacin, 0.3% ciprofloxacin + 0.1% dexamethasone, 0.3% ofloxacin, 0.5% moxifloxacin, 0.3% gentamicin, or 3.5 mg/mL of neomycin + polymyxin B sulfate + hydrocortisone. In experiment 2, cells were treated with the pure form of gatifloxacin, gentamicin, ofloxacin, or ciprofloxacin. Cells were observed with phase-contrast microscope until harvested. Proteins were extracted for Western blotting with antibodies against collagen α1 type I (collagen 1A1) and α-tubulin, and for densitometry to quantify levels. Results Collagen and tubulin levels in fibroblasts treated with ofloxacin, moxifloxacin, gatifloxacin, or gentamicin for 24 hours were not different from the saline control. Fibroblasts treated with neomycin + polymyxin B + hydrocortisone, ciprofloxacin + dexamethasone, or ciprofloxacin for 24 hours had lower collagen 1A1 and α-tubulin levels (all P < .001) than the negative control. After 48 hours, fibroblasts treated with neomycin + polymyxin B sulfate + hydrocortisone, ciprofloxacin + dexamethasone, ciprofloxacin, or moxifloxacin had lower collagen 1A1 ( P ≤ .007) and α-tubulin ( P < .0001; except ciprofloxacin, P = .033) as compared with control. In experiment 2, only cells treated with ciprofloxacin had lower collagen 1A1 and α-tubulin levels and cell viability (all P < .0001) than control. Cytotoxicity assay and phase-contrast images mirrored the protein findings. Conclusion The adverse impact of topical antibiotic exposure on tympanic membrane collagen and tubulin protein levels is drug specific. This may be important for selection of ototopical therapy.

Precision medicine for inner-ear disease is hampered by the absence of a methodology to sample inner-ear fluid atraumatically. The round window membrane (RWM) is an attractive portal for accessing cochlear fluids as it heals spontaneously. In this study, we report on the development of a microneedle for perilymph sampling that minimizes the size of RWM perforation, facilitates quick aspiration, and provides precise volume control. Here, considering the mechanical anisotropy of the RWM and hydrodynamics through a microneedle, a 31G stainless steel pipe was machined into wedge-shaped design via electrical discharge machining. The sharpness of the needle was evaluated via a surface profilometer. Guinea pig RWM was penetrated in vitro, and 1 μL of perilymph was sampled and analyzed via UV-vis spectroscopy. The prototype wedge shaped needle was successfully fabricated with the tip curvature of 4.5 μm and the surface roughness of 3.66 μm in root mean square. The needle created oval perforation with minor and major diameter of 143 and 344 μm (n = 6). The sampling duration and standard deviation of aspirated volume were 3 s and 6.8 % respectively. The protein concentration was 1.74 mg/mL. The prototype needle facilitated precise perforation of RWMs and rapid aspiration of cochlear fluid with precise volume control. The needle design is promising and requires testing in human cadaveric temporal bone and further optimization to become clinically viable.

Although most chronic wounds possess an underlying pathology, infectious agents also contribute. In many instances, pathogens exist as biofilms forming clusters surrounded by a secreted extracellular substance. We hypothesized that compounds secreted by biofilm bacteria may inhibit normal wound healing events including cell proliferation and migration. Conditioned media from two common bacterial species associated with chronic skin wounds and chronic tympanic membrane perforations, Staphylococcus aureus and Pseudomonas aeruginosa, were evaluated for their capacity to affect keratinocyte proliferation and migration. Additionally, proteomic analysis was performed to identify proteins within the biofilm conditioned media that may contribute to these observed effects. Biofilm conditioned media from both species inhibited proliferation in human tympanic membrane derived keratinocytes, whereas only biofilm conditioned media from S. aureus inhibited migration. Human epidermal keratinocytes were found to be more sensitive to the effects of the conditioned media resulting in high levels of cell death. Heat treatment and microfiltration suggested that S. aureus activity was due to a protein, while P. aeruginosa activity was more likely due to a small molecule. Proteomic analysis identified several proteins with putative links to delayed wound healing. These include alpha hemolysin, alcohol dehydrogenase, fructose-bisphosphate aldolase, lactate dehydrogenase and epidermal cell differentiation inhibitor.

We examined whether somatic stem cells (SSCs) exist in human tympanis membrane (hTM) and whether they could be differentiated into neural lineage cells. The hTM-SSCs could generate neurospheres, which could differentiate into specific neural linage cells under specific differentiation conditions. Also, we conducted another experiment that led to differentiation into neurospheres and neuronal lineage cells, which occurred independent of each other. Independent of each other condition, hTM-SSCs could differentiate into neurospheres, and subsequently, into neuronal lineage cells. However, NS-NR neural differentiation rates are higher than independent of each other culture system.

The Eustachian tube is a small canal that connects the tympanic cavity with the nasal part of the pharynx. The epithelial lining of the Eustachian tube contains a ciliated columnar epithelium at the tympanic cavity and a pseudostratified, ciliated columnar epithelium with goblet cells near the pharynx. The tube serves to equalize air pressure across the eardrum and drains mucus away from the middle ear into the nasopharynx. Blockage of the Eustachian tube is the most common cause of all forms of otitis media, which is common in children. In the present study, we examined the epithelial lining of the Eustachian tube in neonatal and adult gerbils, with a focus on the morphological and functional development of ciliated cells in the mucosa. The length of the tube is ∼8.8 mm in adult gerbils. Scanning electron microscopy showed that the mucosal member near the pharyngeal side contains a higher density of ciliated cells and goblet cells than that near the tympanic side. The cilia beat frequency is 11 Hz. During development, the length of the Eustachian tube increased significantly between postnatal day 1 (P1) and P18. Scanning electron microscopy showed that the mucosa contained a high density of ciliated cells with a few goblet cells at P1. The density of ciliated cells decreased while the density of goblet cells increased during development. At P18, the mucosa appeared to be adult-like. Interestingly, the ciliary beat frequency measured from ciliated cells at P1 was not statistically different from that measured from adult animals. Our study suggests that the Eustachian tube undergoes significant anatomical and histological changes between P1 and P18. The tube is morphologically and functionally mature at P18, when the auditory function (sensitivity and frequency selectivity) is mature in this species.

Investigations of the tympanic membrane (TM) can have an important impact on understanding the sound conduction in the ear and can therefore support the diagnosis and treatment of diseases in the middle ear. High-speed Doppler optical coherence tomography (OCT) has the potential to describe the oscillatory behaviour of the TM surface in a phase-sensitive manner and additionally allows acquiring a three-dimensional image of the underlying structure. With repeated sound stimuli from 0.4 kHz to 6.4 kHz, the whole TM can be set in vibration and the spatially resolved frequency response functions (FRFs) of the tympanic membrane can be recorded. Typical points, such as the umbo or the manubrium of malleus, can be studied separately as well as the TM surface with all stationary and wave-like vibrations. Thus, the OCT methodology can be a promising technique to distinguish between normal and pathological TMs and support the differentiation between ossicular and membrane diseases., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tympanic membrane perforations are common and represent a management challenge to clinicians. Current treatments for chronic perforations involve a graft surgery and require general anaesthesia, including associated costs and morbidities. Bioactive molecules (e.g. growth factors, cytokines) play an important role in promoting TM wound healing following perforation and the use of growth factors as a topical treatment for tympanic membrane perforations has been suggested as an alternative to surgery. However, the choice of bioactive molecules best suited to promote wound healing has yet to be identified. We investigated the effects of hyaluronic acid, vitronectin, TGF-α, IL-24 and their combinations on migration, proliferation and adhesion of cultured human tympanic membrane-derived keratinocytes (hTM), in addition to their possible mechanisms of action. We found that TGF-α, TGF-α/HA and TGF-α/IL-24 promoted wound healing by significantly increasing both migration and proliferation. TGF-α and/or HA treated cells showed comparable cell-cell adhesion whilst maintaining an epithelial cell phenotype. With the use of receptor binding inhibitors for ErbB1 (AG1478) and CD44 (BRIC235), we revealed that the activation of ErbB1 is required for TGF-α/HA-mediated migration and proliferation. These results suggest factors that may be incorporated into a tissue-engineered membrane or directly as topical treatment for tympanic membrane perforations and hence reduce the need for a surgery., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hypothesis: The normal tympanic membrane contains Langerhans dendritic cells, and they play a role in the pathogenesis of chronic suppurative otitis media., Background: The presence of Langerhans dendritic cells in the normal tympanic membrane is disputed. However, they have been identified in tympanic membranes of patients with otitis media. A quantitative analysis of the distribution and morphology of these cells in the types of chronic suppurative otitis media has not been undertaken., Methods: Samples of normal cadaveric tympanic membranes and those from patients with chronic suppurative otitis media of the tubotympanic and atticoantral varieties were stained with the immunohistochemical marker CD1a. The number of cells per unit length of basement membrane, diameters of cells, and number and length of dendritic processes were compared between the groups., Results: CD1a-positive Langerhans dendritic cells were present in the normal tympanic membrane. The number of cells per unit length of basement membrane, diameters of cells, and the length of dendritic processes increased significantly in tubotympanic disease and in atticoantral disease, the difference being more pronounced in the latter form of otitis media., Conclusion: Langerhans cells are present in the normal tympanic membrane, and they probably play differing roles in the pathogenesis of tubotympanic and atticoantral forms of chronic suppurative otitis media.

Background: Chronic tympanic membrane perforations can cause significant morbidity. The term myringoplasty describes the operation used to close such perforations. A variety of graft materials are available for use in myringoplasty, but all have limitations and few studies report post-operative hearing outcomes. Recently, the biomedical applications of silk fibroin protein have been studied. This material's biocompatibility, biodegradability and ability to act as a scaffold to support cell growth prompted an investigation of its interaction with human tympanic membrane keratinocytes., Methods and Materials: Silk fibroin membranes were prepared and human tympanic membrane keratinocytes cultured. Keratinocytes were seeded onto the membranes and immunostained for a number of relevant protein markers relating to cell proliferation, adhesion and specific epithelial differentiation., Results: The silk fibroin scaffolds successfully supported the growth and adhesion of keratinocytes, whilst also maintaining their cell lineage., Conclusion: The properties of silk fibroin make it an attractive option for further research, as a potential alternative graft in myringoplasty.

One current challenge of studying human tympanic membranes (TM) with optical coherence tomography (OCT) is the implementation of optics that avoid direct contact with the inflamed tissue. At the moment, no commercial device is available. We report an optics design for contactless forward imaging endoscopic optical coherence tomography (EOCT) with a large working distance (WD) and a broad field of view (FOV) by restricting the overall diameter of the probe to be small (3.5 mm), ensuring a sufficient numerical aperture. Our system uses a gradient-index (GRIN) relay lens and a GRIN objective lens, and executes a fan-shaped optical scanning pattern. The WD and FOV can be adjusted by manually changing the distance between the triplet and the GRIN relay lens. The measured lateral resolution is ∼28  μm at a WD of 10 mm with a FOV of 10 mm. Additionally, a camera and an illumination beam path were implemented within the probe for image guidance during investigations of the TM. We demonstrated the performance of the EOCT design by 3-D imaging of a human TM ex vivo and in vivo with a k-linear spectral domain OCT system.

Epithelial migration (EM) is a process that serves as a self-cleaning and repair mechanism for the ear canal and tympanic membrane (TM). Epithelial migration has been evaluated in humans and several other species, but not in dogs. The objective of this study was to determine the rate and pattern of EM on the TM in clinically normal laboratory dogs. Eighteen dogs were anaesthetized, and three drops of waterproof drawing ink were placed on two sites of the pars tensa (PT) and one on the pars flaccida (PF). Images were recorded with a video otoscope and digital capture system. Each dog was evaluated and images were recorded every 6-8 days for four evaluations. Migration pattern analysis and EM rate calculation were performed with image-processing software. Descriptive statistics for EM rate (mean, SD, 95% confidence interval) were calculated for all ink-drop locations on the TM (PT1, PT2 and PF) at each time point. No significant differences in the mean EM rates were identified between right and left ears of the fox hounds breeds (beagle or fox hound), or locations PT1 and PT2. The mean overall EM rates (± SD) were 96.4 (± 43.1) and 225.4 (± 128.1) μm/day for the PT and PF, respectively. All ink drops moved outwards, the majority in a radial direction, from the original location to the periphery of the TM. The ink-drop placement method used in this study can be used in future studies to determine the EM rate of the canine TM., (© 2011 The Authors. Veterinary Dermatology. © 2011 ESVD and ACVD.)

The human tympanic membrane (hTM), known more commonly as the eardrum, is a thin, multi-layered membrane that is unique in the body as it is suspended in air. When perforated, the hTM's primary function of sound-pressure transmission is compromised. For the purposes of TM reconstruction, we investigated the phenotype and genotype of cultured primary cells derived from hTM tissue explants, compared to epithelial (HaCaT cells) and mesenchymal (human dermal fibroblasts (HDF)) reference cells. Epithelium-specific ets-1 (ESE-1), E-cadherin, keratinocyte growth factor-1 (KGF-1/FGF-7), keratinocyte growth factor-2 (KGF-2/FGF10), fibroblast growth factor receptor 1 (FGFR1), variants of fibroblast growth factor receptor 2 (FGFR2), fibroblast surface protein (FSP), and vimentin proteins were used to assess the phenotypes of all cultured cells. Wholemount and paraffin-embedded hTM tissues were stained with ESE-1 and E-cadherin proteins to establish normal epithelial-specific expression patterns within the epithelial layers. Immunofluorescent (IF) cell staining of hTM epithelial cells (hTMk) demonstrated co-expression of both epithelial- and mesenchymal-specific proteins. Flow cytometry (FCM) analysis further demonstrated co-expression of these epithelial and mesenchymal-specific proteins, indicating the subcultured hTMk cells possessed a transitional phenotype. Gene transcript analysis of hTMk cells by reverse transcriptase polymerase chain reaction (RT-PCR) revealed a down regulation of ESE-1, E-cadherin, FGFR2, variant 1 and variant 2 (FGFR2v1 and FGFR2v2) between low and high passages, and up-regulation of KGF-1, KGF-2, and FGFR1. All results indicate a gradual shift in cell phenotype of hTMk-derived cells from epithelial to mesenchymal.

Multiharmonic optical microscopy has been widely applied in biomedical research due to its unique capability to perform noninvasive studies of biomaterials. In this study, virtual biopsy based on back-propagating multiple optical harmonics, combining second and third harmonics, is applied in unfixed rat tympanic membrane. We show that third harmonic generation can provide morphologic information on the epithelial layers of rat tympanic membrane as well as radial collagen fibers in middle fibrous layers, and that second harmonic generation can provide information on both radial and circular collagen fibers in middle fibrous layers. Through third harmonic generation, the capillary and red blood cells in the middle fibrous layer are also noted. Additionally, the 3-D relationship to adjacent bony structures and spatial variations in thickness and curvature are obtained. Our study demonstrates the feasibility of using a noninvasive optical imaging system for comprehensive evaluation of the tympanic membrane.

Objective: To explore the effects of rats adipose-derived stem cell (ASC) on the proliferation and migration of fibroblasts from the tympanic membrane, and to discuss the possibility and significance of therapy with ASC for tympanic membrane (TM) healing and regeneration., Methods: Wistar rats were sacrificed, and then the isolation, culture and identification of both ASC and the TM fibroblasts were performed respectively. To verify the effect of ASC on fibroblasts proliferation, transwell co-culture system was used. To examine the effect of ASC on fibroblasts migration, cell migration assay with transwell was also applied. All the data were analyzed under a confocal laser scan microscopy system., Results: Immunofluorescence of cell surface markers indicated that rats ASC were positive for both of CD44 and CD29, but negative for CD34. The rat TM fibroblasts were positive for vimentin. The fibroblasts co-cultured with ASC proliferated faster than the fibroblasts of control group, and the difference of the cell counting number between the two groups was significant (t = 6.75, P = 0.003). Compared with the control group, the fibroblasts cultured with ASC conditioned culture medium migrated significantly faster, and the space between the fibroblasts and the polycarbonate membrane pore was significantly shortened at different time point (1, 2.5 and 4 h, P < 0.05). The cell number of the fibroblasts that had migrated through the polycarbonate membrane had been significantly increased 5 h after migration (P < 0.05)., Conclusion: ASC might promote TM fibroblasts proliferation and migration by paracrine activation, and it will facilitate the regeneration of TM fibrous layer.

Human tympanic membrane cells (hTMCs), harvested from tympanic membrane (TM) explants, were grown in culture and then seeded on membranes prepared from silkworm (Bombyx mori) silk fibroin (BMSF) and on tissue-culture plastic membranes (PET). Fibroin was isolated from silk cast into membranes with a thickness of 10-15 microm. The hTMCs were cultured on both materials for 15 days in a serum-containing culture medium. The cells grown on both substrata were subjected to nuclear staining (DAPI) and counted. Further, the cultures were immunostained for a number of protein markers related to the epithelial/keratinocyte phenotype and cell adhesion complexes. The BMSF membranes supported levels of hTMC growth higher than that observed on the PET membranes. The immunofluorochemical analysis indicated unequivocally that BMSF is a more suitable substratum than PET with respect to the growth patterns, proliferation, and cell-cell contact and adhesion. BMSF appear as a promising substratum in the tissue-engineered constructs for the replacement of TM in case of nonhealing perforations.

The surgical treatment to repair chronic tympanic membrane perforations is myringoplasty. Although multiple autologous grafts, allografts, and synthetic graft materials have been used over the years, no single graft material is superior for repairing all perforation types. Recently, the remarkable properties of silk fibroin protein have been studied, with biomedical and tissue engineering applications in mind, across a number of medical and surgical disciplines. The present study examines the use of silk fibroin for its potential suitability as an alternative graft in myringoplasty surgery by investigating the growth and proliferation of human tympanic membrane keratinocytes on a silk fibroin scaffold in vitro. Light microscopy, immunofluorescent staining, and confocal imaging all reveal promising preliminary results. The biocompatibility, transparency, stability, high tensile strength, and biodegradability of fibroin make this biomaterial an attractive option to study for this utility., (Copyright 2010 American Academy of Otolaryngology-Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.)

Unlabelled: An experimental study was undertaken to investigate the use of a CO(2) laser welding technique in myringoplasty. Albumin solder was used to fix a temporal fascia graft via an overlay transcanal approach. The results of the operative procedure were assessed by microscopic and histopathologic examination over an interval of 1, 2, 3 and 4 weeks postoperatively., Materials and Methods: Forty-eight adult guinea pigs were divided into two groups after permanent perforation of the tympanic membrane was created: laser-assisted myringoplasty group and surgical myringoplasty group. Laser beam power was 0.4 W, pulse duration 0.75 s, pulse interval 0.1 s and spot size 250 microm. Each experimental group was further subdivided into four subgroups of 6 animals each. Histological and microscopic findings of the tympanic membrane for both groups after 1, 2, 3, and 4 weeks were compared., Results: Microscopic examination of the tympanic membrane showed high success rate in the laser-assisted myringoplasty group compared to the control group. Histological evaluation for the tympanic membrane showed complete repair of the tympanic membrane in the laser-assisted myringoplasty group., Conclusion: Laser-assisted myringoplasty using CO(2) might be a promising new method in surgical myringoplasty., (Copyright 2010 S. Karger AG, Basel.)

Ossicular reconstruction is the rebuilding of the damaged middle ear. There are many different prosthesis and techniques used to reconstruct the middle ear ossicles. However, precision in the surgical procedures and prostheses used for ossiculoplasty are still imperfect. The objective of this study was to evaluate the potential of recombinant human bone morphogenetic protein-2 (rhBMP-2)/ atelocollagen composite for ossicular reconstruction implanted in the tympanic cavity of rat. The ossicles were extirpated by perforating the tympanic membranes of rats. rhBMP-2/atelocollagen composite was implanted as substitute of ossicles in intimate contact with the tympanic membrane. Composites were subjected to histological, immunohistochemical, and radiological examination. To evaluate the auditory function, auditory brainstem response (ABR) was measured. rhBMP-2/atelocollagen composites showed good stability and durability without any inflammatory reaction within the tympanic cavity. The process of new bone formation was similar to intramembranous ossification. They also demonstrated that the hearing ability was re-established by ABR threshold shifts. rhBMP-2/atelocollagen composite exhibited excellent potential for ossicular reconstruction, maintaining their vibratory function. This ossicular tissue engineering may be considered as a future therapeutic strategy for ossiculoplasty., (Copyright 2008 Wiley Periodicals, Inc.)

Objective: To reconstruct three different kinds of tympanic membrane in vitro by tissue engineering technique, and to examine their histological structures and mechanical properties., Methods: The skin and dura of pig (weight 30 kg) were processed with high satuated saline and enzymes to make extracellular matrix. Meanwhile, fibroblasts (1 x 10(6)/mL, 0.2 mL) were seeded on the surface of these two scaffolds and collagen. The composite tissues were cultured in vitro for 1 week and examined in histological structure and mechanical properties., Results: Fibroblasts cultured were spindle-shaped and could grow and attach to these scaffolds with a arrangement of sarciniform and parallel. The reconstructed tissue of ECM and collagen appeared to integrate well and had better bio-compatibility. The mean thickness of the collagen, the skin and the dura (all covered with fibroblasts) were 9.4, 10.0 and 10.4 microm respectively. The tension of the collagen was (1.417+/-0.030) N/mm2, of the acellular dermal matrix was (24.500+/-2.040) N/mm2 (being close to the tension of normal tympanic membrane, 26.700 N/mm2), of the acellular dura was (53.300+/-2.600) N/mm2., Conclusion: The results suggest that the tension and the thinkness of acellular dermal matrix is similar to the normal tympanic membrane of guinea pig, it is an ideal material for tympanoplasty.

Hypothesis: Formalin-fixed acellular dermis (AlloDerm; LifeCell Corporation, Branchberg, NJ, USA) is safe and efficacious for tympanoplasty in the chinchilla model as compared with the established technique of nonfixed acellular dermis tympanoplasty., Background: Temporalis fascia autografts have been shown safe and effective in the treatment of near-total tympanic membrane (TM) perforations. The formalin-fixation technique first described by Perkins allows the autologous fascia graft to be shaped to more closely resemble the natural TM and has been successful but somewhat cumbersome. The use of acellular dermis as a fascia substitute has also been shown safe and effective in closing smaller perforations. We sought to determine whether formalin-fixed acellular dermis is as safe and useful as nonfixed acellular dermis for TM perforation repair by examining inflammation and tissue effects in a well-established animal model of perforation., Methods: Fifteen adult chinchillas underwent creation of 2-mm central perforations created using a Coherent CO2 laser. Each chinchilla underwent an immediate formalin-fixed acellular dermis repair in one ear and an unfixed acellular dermis repair in the other ear as a control. The chinchillas were randomized with regard to the repair applied to each ear. After 6 weeks of observation, the animals were killed by using sodium pentobarbital (dose, 150 mg/kg). The TMs were otomicroscopically examined for tissue reaction and repair success by a blinded observer. An estimate of residual perforation, if any, was made at that time. The TMs were then harvested and examined histopathologically by a blinded veterinary pathologist., Results: Thirteen chinchillas survived to the evaluation endpoint. There were no significant differences in inflammation, fibrosis, and cellular ingrowth between the 2 groups, although there was a trend toward less inflammation in the formalin-fixed ears. Effusions were present in 15% of non-formalin-fixed ears. Adhesions were present in 15% of non-formalin-fixed ears and 8% of the formalin-fixed ears. A TM cholesteatoma was observed in one non-formalin-fixed ear. The TMs were completely healed in 11 (85%) of 13 TMs in each group., Conclusion: Formalin-fixed acellular dermis was as safe and efficacious as nonfixed acellular dermis in the repair of TM perforations in a chinchilla model. No adverse tissue effects related to formalin treatment were observed.

Hypothesis: The short-term healing scar that forms after experimental laser myringotomy will revert to a normal lamina propria in the long run. The mechanical stiffness will stay normal., Background: Recent studies have shown severe structural changes in the fibrous layer in the early course after experimental laser myringotomy, whereas the scar quickly restored the strength of the tympanic membrane (TM). A reorganization of the fiber layer is expected to occur., Methods: Potassium titanyl phosphate laser myringotomy was made on one side of the TM in Sprague-Dawley rats. The ear of the other side was untouched and used for control. After half a year of observation, the stiffness and strength of the healed TMs were measured with moiré interferometry and examined with otomicroscopy and light and electron microscopy., Results: The interferometry readings showed a slightly reduced strength in the myringotomized and healed TMs. After half a year, still there were immense structural changes including increased thickness over a wide area of the pars tensa with increased amounts of fibers. An obvious reorganization of the fiber layer was lacking., Conclusion: Laser myringotomy causes profound, long-standing, or permanent structural changes in the lamina propria of the pars tensa, whereas the strength of the TM may become slightly reduced.

Aim: To analyze the effects of PRF (a platelet and immune autologous concentrate) on in vitro proliferation of human keratinocytes and preadipocytes, and to determine if these results may offer an opening on new clinical investigations, particularly in the improvement of tympanoplasties and facial lipostructures (Coleman's technique)., Materials and Methods: Human tympanic keratinocytes and preadipocytes are collected and cultured using the explant technique. 4 series of each type of cells are cultivated either in normal condition (control group) or with PRF (test group). The Petri dishes (of culture) are taken out on the 3rd, 7th, 14th and 21st day, for counting. Evolutions of cells' number are analyzed with a variance test., Results: The number of cells in culture increases of more than 60% on the 7th day, and of almost 150% right from the 14th day when in presence of PRF. The daily proliferation peak occurs around the 14th day. The two cellular tested types react similarly., Conclusion: The PRF, considered as a healing biomaterial, could be used in tympanic and facial lipostructures surgeries, in order to improve the therapeutic result. Other applications in microsurgery and in plastic surgery may be possible, but specific clinical studies need to validate such protocols.

We evaluated the efficiency of transplantation of cultured human allofibroblasts onto tympanic membrane damaged by mine explosion in combination with Tampograss dressing (Paul Hartman). Transplantation of cultured human allofibroblasts was effective in 100% cases. Application of the film with fibroblasts onto perforation occupying from 1/4 to 1/2 of the tympanic membrane was more effective by 15% (by 59% in subtotal perforation) than tympanoplasty with amnion membrane. The mean duration of tympanic membrane restoration after spontaneous healing and amnionoplasty is virtually the same, while transplantation of allofibroblasts accelerated the process in comparison with other groups in perforation of any size; in subtotal defect the duration of tympanic membrane restoration was shorter by 14 +/- 1 days.

Gentamicin is toxic to vestibular hair cells, but its effects on vestibular afferents have not been defined. We treated anesthetized chinchillas with one injection of gentamicin (26.7 mg/ml) into the middle ear and made extracellular recordings from afferents after 5-25 (early) or 90-115 days (late). The relative proportions of regular, intermediate, and irregular afferents did not change after treatment. The spontaneous firing rate of regular afferents was lower (P < 0.001) on the treated side (early: 44.3 +/- 16.3; late: 33.9 +/- 13.2 spikes x s(-1)) than on the untreated side (54.9 +/- 16.8 spikes x s(-1)). Spontaneous rates of irregular and intermediate afferents did not change. The majority of treated afferents did not measurably respond to tilt or rotation (82% in the early group, 76% in the late group). Those that did respond had abnormally low sensitivities (P < 0.001). Treated canal units that responded to rotation had mean sensitivities only 5-7% of the values for untreated canal afferents. Treated otolith afferents had mean sensitivities 23-28% of the values for untreated otolith units. Sensitivity to externally applied galvanic currents was unaffected for all afferents. Intratympanic gentamicin treatment reduced the histological density of all hair cells by 57% (P = 0.04). The density of hair cells with calyx endings was reduced by 99% (P = 0.03), although some remaining hair cells had other features suggestive of type I morphology. Type II hair cell density was not significantly reduced. These findings suggest that a single intratympanic gentamicin injection causes partial damage and loss of vestibular hair cells, particularly type I hair cells or their calyceal afferent endings, does not damage the afferent spike initiation zones, and preserves enough hair cell synaptic activity to drive the spontaneous activity of vestibular afferents.

Objective: To investigate the distributions of epithelial stem cells in the tympanic membrane and the growth characteristics of cultured epithelial cells from different region of tympanic membrane, and to establish culture techniques of stem cells in tympanic membrane., Methods: Four young rats and four adult SD rats were used to observe normal tympanic membrane. The other 28 rats were performed 2 mm size perforations in pars tensa. These animals were sacrificed at different periods after perforation. The tympanic membranes were cut in cryostat sections for immunohistochemistry of cytokeratin 19 and integrin beta1. Thirty tympanic membranes of rats were treated with Mitomycin C to damage the mucosal surface, then divided into two parts: the annulus region and center region of pars tensa, cultured in medium with high amount of epidermal growth factor and low amount of calcium., Results: The immunostaining cells of cytokeratin 19 and integrin beta1 were displayed in both the handle of malleus and annular regions, but there were no staining positive cells in the intermediate region of pars tensa. The positive cells distribution had no significant difference between adult and infancy rats. In the pars flaccid, the positive cells scattered in the basal layer. The positive cells increased after perforation in the annulus and handle of malleus region, but no immunostaining cells were found at the edge of perforation. The redouble time of the culture cells from the annulus region was shorter than the center of pars tensa. The cells adherent within 1 hour formed larger and more colonies, and contained more positive cells., Conclusion: The epithelial stem cells in tympanic membrane were located in both the handle of malleus and annular regions, but no stem cells could be found in the intermediate region of pars tensa. The stem cells of tympanic membrane can be simply purified according to the adherent time.

Objective: The objective was to examine the possible risk of injury to the internal carotid artery during procedures in the middle ear, including myringotomy., Study Design: Histopathological morphometric study of human temporal bones., Methods: One hundred forty-two human temporal bone specimens obtained from 92 individuals without any known ear disease were prepared for light microscopic study. Using 83 bones that were available for measurement, the thickness of the carotid canal wall (CCW), which is the medial wall of the bony portion of the eustachian tube, was measured. Using 15 bones selected for three-dimensional measurement, the closest distance from CCW to the anterior tympanic annulus was measured. Using all 142 temporal bone specimens, the CCW was examined to detect the presence of partial dehiscence. In one case, the images of CCW dehiscence and its surrounding structures were reconstructed by a personal computer., Results: The thickness of the CCW was 0.00 to 0.73 mm (average thickness, 0.24 mm [+/-0.12 mm]). The distance from the CCW to the anterior tympanic annulus was 1.8 to 8.1 mm (average distance, 4.9 [+/-1.7 mm]). Dehiscence of CCW was observed in 7 (4.9%) of 142 temporal bone specimens. The reconstructed image showed that the posterior half of the dehiscence of CCW could be seen from the external ear canal., Conclusions: The CCW was found to be extremely thin or even dehiscent in some cases, rendering the internal carotid artery vulnerable during transtympanic procedures. The study's findings emphasized the need for judicious care when operating in the anterior mesotympanum.

Objective: Recently, attempts have been made to prolong the patency of myringotomy site with topical use of mitomycin C (MMC). It has been shown that MMC inhibits mitosis and proliferation of ocular fibroblasts, however, there are no studies of MMC's effect on tympanic membrane fibroblasts. To investigate the effects of MMC on cultured human tympanic membrane fibroblasts and understand the cellular basis of MMC for maintain myringotomy patency, cultured fibroblasts were exposed to various concentrations of MMC for periods of 5-10 min., Methods: Effect of MMC on cultured fibroblasts was assessed by microscopic observation and cell viability test., Results: Dose-, time- dependent relationship of MMC on cultured fibroblasts was revealed. There was a significant difference between the inhibition effects of MMC at concentrations of 0.4 mg/ml and control following 5 and 10 min exposure intervals. Phase-contrast microscopy showed consistency with the antiproliferative effect of MMC at higher concentration., Conclusions: Therefore, it would appear that intraoperative use of MMC could be effective in delaying the healing of the myringotomy site and extending the period of time for myringotomy patency.

The development of the union between the manubrium of the malleus and the tympanic membrane was studied in human embryo. For that purpose 25 temporal bones of human foetus, aged from 36 days (14 mm) to 29 weeks (270 mm) were analyzed. Samples were fixed in a 10% formaldehyde solution, decalcified with 2% nitric acid, embedded in Paraplast, cut in sections of 7 microns thick and stained with Martin's trichrome method. During the development a pseudojoint between the malleus and the tympanic membrane several stages were seen. In the first stage, the manubrium was adhered to membrane mesenchyme of primitive tympanum, in the second one this mesenchyme was loose and there appeared capillars in it, in the third one there were collagen fibers in a radial disposition, and in the forth stage, the hollow in the meatal plug gave independence to the tympanic membrane for the external acoustic meatus. The distal portion of the manubrium, included in tympanic membrane, has remained with a cartilaginous structure during end of the time it has been studied by us. The collagen fibers at the level of the umbo surround the manubrium, while the rest of it remains in its anterior edge.

The paratympanic organ (PTO) in the middle ear has been described in numerous bird species, but little is known about the distribution of this presumed lateral line remnant in other vertebrate classes. Here we provide evidence for a PTO in juvenile alligators, and make the first detailed description of its location and relation to ligaments in the reptilian middle ear. The alligator PTO measures about 450 micro m in diameter. The alligator PTO contains hair cells whose cilia extend into a mucous substance within the lumen. The PTO connects though a ligament to the ear drum, suggesting that pressure onto the tympanic membrane might induce fluid movement in the PTO. Labeling of innervating nerve fibers with the fluorescent dye, DiI, indicates that the alligator PTO is connected with the vestibular brainstem. Because all bird species examined possess a PTO except for owls and possibly parakeets, we verified the absence of a PTO in parakeets by examination of serial sections combined with GABA immunolabeling for potential hair cells. Bird species with significant upper beak movement lack a PTO, suggesting that PTO function is incompatible with upper beak movement. We also examined the middle ear of an armadillo, a mammal that has a very basal position within the eutherian phylogenetic tree. A small vesicle with ciliated cells was found, but did not label with a hair-cell specific marker, antibodies to myosin VIIa, and thus is not likely to represent a true PTO. Our evidence for a PTO in a non-avian species, the alligator, together with previous reports suggesting the presence of a PTO in some mammals, indicates that ancestral stem amniotes possessed a PTO, and that the PTO was not a de novo invention of birds., (Copyright 2002 S. Karger AG, Basel)

Recent studies using a satellite-linked dive recorder have shown that the hooded seal (Cystophora cristata), a common Arctic pinniped, can dive to a depth of > 1000 m and stay submerged for close to 1 h. At these depths the water pressure reaches 100 atm, entailing obvious risk of serious damage to the hearing apparatus, mainly the tympanic membrane (TM) and middle ear (ME). We dissected and photodocumented the temporal bones of five newborn and three adult hooded seals in order to study the temporal bone structure and reveal its protective mechanisms for extreme pressure changes. Specimens were sectioned and stained for light microscopy. The thicknesses of the pars tensa and pars flaccida were found to average 60 and 180 microm, respectively. The ME cavity hosts a cavernous tissue of thin-walled vessels beneath the modified respiratory epithelium. The ME and external ear canal (EAC) volumes can be altered appreciably by filling/emptying the cavernous tissue with blood. The ossicles were fixed by contracting the tensor tympani and stapedius muscles simultaneously with complete occlusion of the EAC. According to Boyle's law, the volume of the gas-filled ME cavity at a depth of 1000 m is only 1% of its volume at the surface of the sea. Ascent from such a depth allows the gas in the ME cavity to expand, causing the TM to bulge laterally. This movement is counteracted by a reduction in the blood volume inside the cavernous sinuses, action in the tensor tympani and stapedius muscles and discharge of gas through the Eustachian tube. The presence of a firm, broad-based exostosis in the floor of the EAC lateral to the TM helps to obstruct the EAC.

The sterility of the eustachian tube and tympanic cavity of normal individuals is maintained not only by the adaptive immune system, but also by the mucociliary system and the antimicrobial molecules of innate immunity. Mucin production and periciliary fluid homeostasis are essential for normal mucociliary function and dysfunction of this system is an important risk factor for otitis media. The secreted antimicrobial molecules of the tubotympanum include lysozyme, lactoferrin, beta defensins, and the surfactant proteins A and D (SP-A, SP-D). Defects in the expression or regulation of these molecules may also be the major risk factor for otitis media.

This study was done to compare the outcome of porcine ossicular implants in the middle ear and the subcutaneous dorsal region of the guinea-pig to those of allo-implants implanted in parallel in the dorsal region. The implants were heteropic, xenogeneic, demineralized (HCl), lyophilized and sterilized. The evaluation was histological (light microscopy and scanning electron microscopy) and immunological (immunofluorescence staining). Fifty-four guinea-pigs were implanted in the middle ear and 14 of them were also implanted subcutaneously in the dorsal region with xeno-implants and allo-implants. The middle ear implants were found to be constantly reossified and coated with normal mucosa with only a minimal immune reaction. In contrast, the dorsal xeno-implants were found to be the target of mononucleic infiltration, fibrous encapsulation and an influx of immunoglobulins resulting in segregation. The corresponding allo-implants were found to be partially reoccupied and reossified. These findings highlight the value of HCl demineralization in the induction of non-species-specific Bone Morphogenetic Protein and the failure of attempts at immuno-despecification. It appears that the fate of the implant depends less on its antigenic load than on the site of implantation. In this regard the middle ear is apparently very advantageous. The very good short-term tolerance and recovery observed in the middle ear xeno-implant suggest that these implants offer sufficiently good results to warrant clinical testing.

Mongolian gerbil frequently develop spontaneous cholesteatoma. As we reported previously, in the process of gerbiline cholesteatoma formation, effusions inside the pars flaccida are always found in the ears during the early stage, and epidermal growth factor (EGF) is also localized in the pars flaccida, especially in the mucous layer. In this study, to clarify the process of gerbiline cholesteatoma formation, we studied 22 gerbiline temporal bones by using a monoclonal antibody against bromodeoxyuridine (BrdU). BrdU-labeled cells demonstrate a proliferative potential. We also used a carbon dye method to label the micro-vase in 14 gerbiline temporal bones. Cells showing BrdU uptake were more abundant, as demonstrated immunohistochemically, in the pars flaccida of ears with early cholesteatomas than in the pars flaccida of normal ears (p < 0.01). The pars flaccida of ears with early cholesteatomas showed hypertrophy of both epithelial layers, and hyperkeratosis of the epidermal layer. BrdU-labeled cells in the pars flaccida were more localized in the mucous layers than in the epidermal layers. In contrast, in ears with cholesteatomas, BrdU-labeled cells were less abudant than in ears with early cholesteatomas. In addition, BrdU-labeled cells in the pars tensa and external auditory epidermal layers were not increased in ears with any stage of cholesteatoma formation. We used a carbon dye method to detect the micro-vasa in the intermediate layer of the ear drum. Carbon-dye-labeled vasa were more numerous in the pars flaccida with early cholesteatomas than in the pars flaccida of normal ears or ears with cholesteatomas. It is highly suspected that angiogenesis was stimulated in the pars flaccida with early cholesteatomas, because stimulation of angiogenesis by EGF has been reported. The above findings suggest that the mucous layer of the pars flaccida has the greatest proliferative potential in the process of cholesteatoma formation. Angiogenesis in the pars flaccida appears to be a reaction to proliferative changes in the mucous and epidermal layers. These changes are probably stimulated by effusion inside the pars flaccida.

Members of the Rana catesbeiana clade display sexually dimorphic eardrums. In this species assemblage the eardrum of males can be 50% larger than in females of the same body size. There has been, however, no apparent functional explanation for this dimorphism. Measurements of the acoustical coupling (transfer function) of internally generated sound to the enlarged eardrum of male bullfrogs (R. catesbeiana) show distinct energy peaks coincident with those observed in the spectral envelopes of the release and mating calls. Moreover, when the tympanic membranes are artificially damped the spectrum of the release call is drastically altered and the total amount of power radiated decreases substantially. These observations point to a previously unsuspected role for the ears in the sound broadcasting process of the bullfrog and possibly other anurans with similarly modified tympanic membranes.

The external auditory meatus, middle, and inner ear of the deep-diving Weddell seal (Leptonychotes weddelli) were studied with light microscopic, histological, and histochemical techniques in order to contribute to the open discussion on the orientation of this seal in the darkness of the deep Antarctic seas. The external auditory meatus is characterized by a well-developed venous plexus, single apocrine ceruminous, and numerous holocrine sebaceous glands and an incomplete tube of elastic cartilage. The tympanic membrane is comprised of two layers of radially and concentrically arranged collagen fibers and by elastic fibers which are concentrated in the outer part of the ear drum. The tympanic cavity is lined by a pseudostratified prismatic ciliated epithelium with goblet cells; a plexus of wide venous vessels marks the subepithelial lamina propria. The cochlea is about 10 mm high and forms about two and a half turns. The richly pigmented stria vascularis is well vascularized, while the cell-rich prominentia spiralis contains only single small blood vessels. The organ of Corti contains one row of inner and three rows of outer hair cells. Cells of Hensen, Claudius, and Boettcher are present. The basilar membrane is of comparatively uniform simple structure and is composed of abundant glycoproteins, proteoglycans, collagenous fibers, and the loose tissue of the tympanal layer. The spiral ligament is built up by abundant proteoglycans and a complex system of radial and concentric collagen fibers; close to the osseous wall of the bony cochlea it contains fine elastic fibers. The inner zone of the osseous wall of the cochlea strikingly contains hyaline cartilage. The thin lamina spiralis ossea is covered by a limbus spiralis with interdental cells secreting the lamina tectoria, which has a fibrous texture and contains glycoproteins and negatively charged components.

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.

The surgical anatomy of the middle ear was studied in 20 adult gerbils (Meriones unguiculatus) by microdissection and scanning electron microscopy. The most significant finding was a well pneumatized tympanic bulla, which facilitated the identification of useful anatomical landmarks for middle and inner ear dissection. The typical features of middle ear structures in the gerbil are described.

A location of proliferating cells was investigated in eight human normal tympanic membranes (TMs) and external auditory canals (EACs) by an immunohistochemical method using two different types of antibodies for nuclear antigens in proliferating cells: anti-PCNA monoclonal antibody, and anti-Ki-67 polyclonal antibody. Four specimens prepared for cryostat sections were immunostained by both antibodies. Another four were fixed in 4% formaldehyde solution, embedded in paraffin wax and were reacted only with anti-PCNA antibodies. The expression pattern of Ki-67 was basically the same as of PCNA. In the pars tensa (PT), immunoreactivities were expressed in the nuclei of basal layer cells and cells just overlying the basal layer of epidermis both in the handle of the malleus (HM) and annular regions. In the intermediate region of the PT, no immunoreactivity was found basically, apart from a few labelled cells observed in the upper-third of the superior quadrant. In the pars flaccida (PF) and in both the osseous and cartilaginous regions of the EAC, positive cells were also situated in the basal layer and the deeper aspect of the suprabasal layers without any specific distributing pattern. It was certified that the generation centre of epidermal cells (keratinocytes) in the PT was located in both the HM and annular regions, and that stem cells in the PF and the EAC were uniformly scattered in the basal layer and the deeper aspect of the spinous layer. According to these findings, the migratory patterns of auditory epidermal cells in the human TM and EAC were discussed.

The morphology of the tympanic membrane (TM) and external auditory canal (EAC) in mice was examined by transmission electron microscopy, in seven different regions: the handle of the malleus (HM) region, the intermediate and annular regions of the pars tensa (PT), the region of the pars flaccida (PF), the annular and osseous regions of the EAC and the cartilaginous region of the EAC. Each region displayed its own unique findings. In most regions of the TM and EAC-except the intermediate region of the PT-basal layer cells formed cytoplasmic processes (CPs) on their basal surface and adhered to the basement membrane (BM) by means of hemidesmosomes (HDs). This observation may suggest the presence of stem cells. In contrast, basal cells in the intermediate region of the PT did not extend any CPs from the basal surface but attached to the BM by a few characteristic spots of HD-like density but lacking the characteristics of HDs. It is concluded that stem cels are not present there and that basal cells connect loosely with the BM, thus providing the necessary morphological conditions to migrate laterally toward the annular region. The outermost layer of the epidermis in the annular region of the EAC showed numerous cytoplasmic projections (so-called 'finger-like projections'), representing keratinization and desquamation of epidermal cells (keratinocytes). These projections are believed to be the terminal differentiation of epidermal cells originating from the general centre in the HM region of the PT. The epidermis of the PF is an extension of the upper wall of the EAC, but the two regions differ morphologically.