The Effects of Steroids on Survival of Mouse and Human Tympanic Membrane Fibroblasts.

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.

Developmental aspects of the tympanic membrane: Shedding light on function and disease.

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.

The Feasibility to Isolate and Expand Tympanic Membrane Squamous Epithelium Stem Cells From Scarred Perforation Margins.

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.

Isolation of Epidermal Progenitor Cells from Rat Tympanic Membrane.

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.

Single origin of the epithelium of the human middle ear.

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.

Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes.

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.

Tympanic Membrane Derived Stem Cell-Like Cultures for Tissue Regeneration.

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 ß1 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.

Cytotoxicity of Ciprofloxacin and Steroids in Mouse Tympanic Membrane Fibroblasts.

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.

Chitosan/PEI patch releasing EGF and the EGFR gene for the regeneration of the tympanic membrane after perforation.

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.

Evidence against the mucosal traction theory in cholesteatoma.

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.

Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

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.

Collagen and α-Tubulin of Mouse Tympanic Membrane Fibroblasts Treated with Quinolones and Aminoglycosides.

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.

A dual wedge microneedle for sampling of perilymph solution via round window membrane.

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.

Secreted biofilm factors adversely affect cellular wound healing responses in vitro.

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.

Morphology and ciliary motion of mucosa in the Eustachian tube of neonatal and adult gerbils.

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.

Establishment of endogenous human tympanic membrane-derived somatic stem cells for stem cell therapy.

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.

Investigation of the human tympanic membrane oscillation ex vivo by Doppler optical coherence tomography.

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.

TGF-α/HA complex promotes tympanic membrane keratinocyte migration and proliferation via ErbB1 receptor.

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.

Langerhans cells in the human tympanic membrane in health and disease: a morphometric analysis.

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.

Utilising silk fibroin membranes as scaffolds for the growth of tympanic membrane keratinocytes, and application to myringoplasty surgery.

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.