Pathogenesis and aetiology

In principle, a cholesteatoma can present either as a sac that contains keratin with a surrounding keratinising squamous epithelial layer (i.e. matrix) and an adjacent subepithelial connective tissue layer with a bounding mucous cuboidal epithelial layer (i.e. perimatrix), or as a mass-like growth (i.e. haphazard growth with dispersed keratin deposits). Cholesteatoma pathogenesis can be explained on the premises of different triggers (trauma or disease) and different theories (migration, hyperplasia or metaplasia), or a combination thereof, as described in the review article of Louw (2010).Reference Louw¹

The following text briefly summarises excerpts from Louw's (2010) paper. Triggers for cholesteatoma onset are diverse and may involve tympanic membrane trauma (perforation, displacement, retraction and invagination), or disease (chronic inflammation) and/or diseased mucosa of the tympanic cavity (i.e. otitis media with effusion). Taken together, the onset of acquired cholesteatoma can be marked by: epidermal cell migration through tympanic membrane perforations to form mucocutaneous junctions (i.e. pre-cholesteatoma conditions, without inflammation); epidermal cell hyperplasia (i.e. protruding masses through tympanic membrane perforations or from intact tympanic membranes or retraction pockets into the tympanic cavity, triggered by inflammation), with either sac-like growths (i.e. papillary cone and sinus formation patterns that fuse with keratin in the sac) or mass-like growths (i.e. haphazard growth patterns with dispersed keratin); and diseased mucosa of the tympanic cavity (i.e. mucosal metaplasia) with an impact on the tympanic membrane (i.e. cholesteatoma formation with secondary perforation of the tympanic membrane, triggered by inflammation).Reference Louw^1, Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann²

The main aetiological factors in cholesteatoma development are: long-term eustachian tube dysfunction and reduced middle-ear pressure (i.e. poor pneumatisation in the middle ear and/or mastoid process), resulting in tympanic membrane retraction pockets and invaginations (i.e. atelectasis and adhesive otitis media); and inflammatory conditions (i.e. chronic otitis media with effusion), resulting in negative middle-ear pressure and tympanic membrane retraction pockets and invaginations.Reference Louw^1, Reference Seibert and Danner⁴

Chronic inflammation and bacterial biofilms

Chronic inflammation plays an important role in the progression from pre-cholesteatoma conditions (i.e. mucocutaneous junctions, retraction pockets, atelectasis and adhesive otitis media) to cholesteatomas. The role of chronic inflammation in the clinical course of cholesteatoma is summarised in the following text. Bacterial otitis externa can trigger epidermal hyperplasia of the tympanic membrane, with epidermal projections into the tympanic cavity or epidermal ingrowths into the tympanic membrane. Persistent inflammation in tympanic membrane perimatrix and otitis media in the tympanic cavity are considered to be significant factors in cholesteatoma growth, expansion and invasion. Chronic otitis media is the culprit in diseased mucosa, and bouts of chronic infections contribute to cholesteatoma persistence and recurrence.Reference Chole, Sudhoff and Cummings^5–Reference Mustafa, Heta, Kastrai and Dreshaj⁸

Bacterial biofilms have been identified in cholesteatomas. Their actions can be described as either indirect signalling, wherein bacterial adherence to epithelial cells may lead to chronic infection and cell hyperproliferation, or direct signalling of bacterial products (endotoxins) that may elicit host immune responses.Reference Chole and Faddis^9–Reference Macassey and Dawes¹¹Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of forming biofilms, and is the most common organism isolated from infected cholesteatomas.Reference Wang, Jung, Nason, Pashia and Chole¹² Bacterial biofilms can be responsible for inflammatory reactions that may lead to chronic inflammation. This includes biofilms that are: trapped in the external acoustic meatus; present in lacunae, sinuses or sacs within cholesteatomas; or located in hollows of the tympanic cavity.Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann^2, Reference Sudhoff and Tos⁶

Inflammatory mediators are produced by infiltrating immune cells (i.e. neutrophils, monocytes and lymphocytes) and local cells (such as keratinocytes and mast cells) in cholesteatomas.Reference Junh, Jung, Hoffman, Drew, Preciado and Sausen¹³ During initiation of otitis media, the complement part of the immune system (consisting of multiple components that can interact to form enzymes) is activated by bacterial endotoxins; depletion of these endotoxins (such as the chemotactic agent C5a that activates immune cells) is known to cause chronic otitis media.Reference Junh, Jung, Hoffman, Drew, Preciado and Sausen¹³ Research has revealed that bacterially infected cholesteatomas and chronic otitis media eventually disrupt the balance between bone formation (via osteoblasts) and bone resorption (via osteoclasts), leading to bone erosion.Reference Peek, Huisman, Berckmans, Sturk, van Loon and Grote¹⁴ Thus, middle-ear inflammatory mediators and toxins may result in ossicle erosion and hearing loss.Reference Junh, Jung, Hoffman, Drew, Preciado and Sausen¹³

Growth and deterioration

The more severe or chronic the inflammation, the greater the expected impact on cholesteatoma perimatrix–matrix interactions and layer thickness.Reference Dornelles, da Costa, Meurer and Schweiger¹⁵ Thus, chronic and recurrent inflammations determine aggressive growth, and angiogenesis (i.e. microvessel formations) is a prerequisite for tissue maintenance.Reference Nagai, Suganuma, Ide, Shimoda and Kato¹⁶ In addition, chronic inflammatory conditions in the tympanic cavity (and mastoid cells) can lead to the accumulation of thick mucous masses between mucosal folds and within pouches, which can impede ventilation and drainage, with consequent hypopneumatisation and hypoxia.Reference Sudhoff, Dazert, Gonzales, Borkowski, Park and Baird^17, Reference Adunka, Gstoettner, Knecht and Kiener¹⁸ Hypoxic conditions in the tympanic cavity can contribute to tympanic membrane retraction pockets and invaginations that may lead to cholesteatoma onset and growth. Hypoxia eventually leads to microvessel occlusion, with consequent apoptosis and deterioration of cholesteatoma tissue components.Reference Olszewska, Chodynicki and Chyczewski¹⁹ Established cholesteatoma is marked by inflammatory granulation tissue, and fetid exudates and flakes of keratin are present.

Cholesteatoma pathogenesis

Cholesteatoma is a growth disorder, and the epidermal growth factor receptor and keratinocyte growth factor receptor are up-regulated in cholesteatoma pathogenesis,Reference Alves, Pereira, Carvalho M de, Fregnani and Ribeiro^20–Reference Jin, Min, Jeong, Park and Lee²³ as is the ligand for the epidermal growth factor receptor, namely amphiregulin.Reference Macias, Gerkin and Macias²⁴ In addition, increased p63 expression and keratinocyte markers demonstrate uncoordinated hyperproliferation, migration and invasion properties.Reference Kucczkowski, Pawelczyk, Bakowska, Narozny and Mikaszewski^25–Reference Park, Min, Min, Jun, Seo and Kim²⁸ The cell cycle inhibitory protein p27 is down-regulated,Reference Kuczkowski, Bakowska, Pawelczyk, Narozny and Mikaszewski²⁹ and the ErbB-2 protein for accelerated cell proliferation and apoptosis is over-expressed.Reference Sakamoto, Kondo, Yamasodba, Suzuki, Sugasawa and Kaga³⁰ The c-jun protein (associated with proliferation), c-myc protein (associated with differentiation) and p53 tumour suppressor gene (associated with apoptosis) are all up-regulated in cholesteatoma.Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann^2, Reference Osturk, Yildirim, Ascar, Cenik and Keles^31, Reference Motamed, Powe, Kendall, Birchall and Banerjee³² The ras protein (present in keratinocyte membranes) serves as a switch and activates mitogen-activated protein kinases for the transcription of proliferation genes (via c-jun present in keratinocyte nuclei).Reference Huang, Chen, Huang and Shinoda³³ Caspases-3, -8 and -14 signalling pathways play important roles in apoptotic or terminal differentiation of the matrix and accumulation of keratin debris.Reference Miyao, Shinoda and Takahashi^34, Reference Jung, Lee, Cho, Jung, Hwang and Chae³⁵ Survival (anti-apoptotic) signalling pathways, such as those of phosphoinositide 3 kinase-AKT (protein kinase B) and phosphorylated extracellular-regulated kinases 1 and 2, are up-regulated in cholesteatoma perimatrix.Reference Huisman, De Heer and Grote^36, Reference Huisman, De Heer and Grote³⁷ Enhanced vascular endothelial growth factor and fibroblast growth factor 2 expressions can stimulate the production of collagenase and plasminogen activators to enhance fibroblast proliferation and angiogenesis.Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann^2, Reference Sudhoff, Dazert, Gonzales, Borkowski, Park and Baird^17, Reference Niam, Chang, Sadick, Bayerl, Bran and Hormann³⁸ Derailment of the matrix metalloproteinases system plays an active role in cholesteatoma invasion of middle-ear spaces.Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann^2, Reference Morales, Penido N de, da Silva, Stavale, Guilherme and Fukuda³⁹

It is suggested that enhanced human β-defensin 2 and 3 in cholesteatoma may induce an innate immune response.Reference Song, Chae, Woo, Lee, Jung and Hwang⁴⁰ It is also suggested that cell-mediated immunity has an important role in cholesteatoma development and its auto-destructive properties, based on a predominance of T lymphocytes (cluster of differentiation 3+) and histiocytes (cluster of differentiation 68+) in invasive cholesteatomas.Reference Hussein, Sayed and Abu-Dief⁴¹ In addition, the enhanced expression of transforming growth factor β by fibroblasts during cholesteatoma pathogenesis supports the idea that the cholesteatoma activity is similar to that of a chronic wound healing process.Reference Huisman, de Heer, Dijke and Grote⁴² Transforming growth factor β is an important trigger for the production of extracellular matrix components (i.e. fibronectin, collagen, integrins and glycosaminoglycans) that are linked with cell adhesion, migration, growth and differentiation.Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann²

Cholesteatoma complications

The role of osteoclasts in bone erosion has been firmly established.Reference Jung and Chole⁴³ Among molecular mediators implicated in bone resorption are: liposomal enzymes (such as acid hydrolyses) including matrix metalloproteinases and hexosaminidase A;Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann^2, Reference Morales, Penido N de, da Silva, Stavale, Guilherme and Fukuda^39, Reference Olszweska, Borzym-Kluczyk, Olszweska, Rogowski and Zwierz⁴⁴ cytokines (i.e. interleukins (ILs) 1 and 6, and tumour necrosis factor α (TNF-α));Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann^2, Reference Vitale and Ribeiro F de⁴⁵ pro-inflammatory fatty acid metabolites (such as prostaglandin E2);Reference Yuan, Akiyama, Nakahama, Sato, Uematsu and Morita⁴⁶ nuclear transcription factors (such as nuclear factor κβ);Reference Byun, Yune, Lee, Yeo and Park⁴⁷ bacteria (lipopolysaccharide);Reference Iino, Toriyama, Ogawa and Kawakami⁴⁸ and specific pH activities (i.e. demineralisation by acidic cholesteatomas).Reference Olszewska, Wagner, Bernal-Sprekelson, Ebmeyer, Dazert and Hildmann² Previous research indicated that IL-1 and IL-6 can stimulate hexosaminidase (specifically N-acetyl-β-D-hexosaminidase) activity that may lead to bone erosion.Reference Olszweska, Borzym-Kluczyk, Olszweska, Rogowski and Zwierz⁴⁴ More advanced research revealed that TNF-α, together with IL-1 and receptor activator of nuclear factor κβ ligand (ligands for activating nuclear factor κβ receptors), contribute to bone erosion.Reference Nason, Jung and Chole⁴⁹ The above-mentioned research findings correlated with bone erosion in the presence of chronic otitis media, with or without cholesteatoma. With respect to sensorineural hearing loss, it was previously suggested that during chronic suppurative otitis media (with or without cholesteatoma), the passage of bacterial endotoxins through the round window may cause damage to hair cells in the cochlear base.Reference Hellstrom, Eriksson, Yoon and Johansson⁵⁰

Membrane lipids in human cells and bacteria

In the bilipid membrane of a human cell, the phospholipid class and its subclasses are considered the most important lipids. Each class or subclass consists of a fatty acid composition or profile. Fatty acids play critical roles in membrane to nucleus gene regulation and immune responses, and are mediated by a complex array of signalling pathways. Upon stimulation, arachidonic acid is released from membrane phospholipids by phospholipase A2. Arachidonic acid is a source for the production of inflammatory metabolites via cyclo-oxygenase and lipoxygenase activities. Prostaglandin E2 production via cyclo-oxygenase-2 activity plays a crucial role during chronic otitis media. During inflammatory conditions, neutrophils (a major component of phagocytic cells) produce oxygen free radicals (oxidants) via inducible nitric oxide synthases, namely reactive oxygen species and nitric oxide (NO), which cause cell damage. Free radical scavengers (antioxidants), such as superoxide dismutase, glutathione peroxidase and catalase, have a protective effect against oxidative damage.Reference Junh, Jung, Hoffman, Drew, Preciado and Sausen^13, Reference Aggarwal, Shishodia, Sandur, Pandey and Sethi^51, Reference Valko, Leibfritz, Moncol, Cronin, Mazur and Telser⁵² It was demonstrated that superoxide dismutase, glutathione peroxidase and catalase were significantly depleted in cholesteatomas, but there was no correlation with bone erosion.Reference Eskiizmir, Yuceturk, Onur, Var and Temiz⁵³ Arachidonic acid is also a source for lipid peroxidation by free radicals, and the formation of harmful hydroperoxides reduces membrane fluidity and permeability, causing it to collapse. In addition, lipid hydroperoxides can decompose to yield a range of highly cytotoxic aldehydes, which can further perpetuate tissue damage. It was suggested that an increase in oxidative stress occurs in inflammatory conditions as a consequence of elevations in: phospholipase A2, inducible nitric oxide synthase, cyclo-oxygenase-2 and lipoxygenase activities. This leads to increased arachidonic acid release, faster arachidonic acid oxygenation, and increased reactive oxygen species and NO production, which triggers lipid peroxidation and cell damage.Reference Aggarwal, Shishodia, Sandur, Pandey and Sethi^51, Reference Valko, Leibfritz, Moncol, Cronin, Mazur and Telser⁵²

In bacteria, the outer layer of the bilipid membrane predominantly contains lipopolysaccharides, while the inner layer is composed mainly of phospholipids. Lipopolysaccharide essentially comprises a lipid A component, consisting mainly of saturated fatty acids with carbon chain lengths from 10 to 18, and a polysaccharide component that interacts with the environment as a defence mechanism.⁵⁴ Bacterial lipopolysaccharide stimulates the release of arachidonic acid and the production of prostaglandin E2 metabolites and NO radicals during chronic otitis media, and thereby increases mucous secretion and bone erosion. Recently, there has been progression in our understanding of endotoxin lipopolysaccharide as a potent inducer of inflammatory mediators in the pathogenesis of otitis media and sequelae.Reference Junh, Jung, Hoffman, Drew, Preciado and Sausen¹³ Moreover, lipopolysaccharides can stimulate neutrophils for the release of antimicrobial myeloperoxidase under conditions of oxidative stress. Myeloperoxidase reacts with hydrogen peroxide in neutrophil phagolysosomes to form a complex for the oxidation of chloride to hypochlorous acid and other toxic products. In cholesteatoma, excessive secretion of reactive oxygen species and myeloperoxidase has been shown to be correlated with bone erosion.Reference Celebi, Paksoy, Aydin, Sanh, Tasdemir and Gull⁵⁵

Fatty acid role-players during cell growth and apoptosis

Environmental factors (i.e. bacteria, viruses, fungi, free radicals and other foreign particles) can interfere with essential fatty acid metabolism (i.e. metabolism of the omega-6 and omega-3 fatty acid series) with linoleic acid and α-linolenic acid as substrates respectively, by inhibiting Δ6 and Δ5 desaturase activities.Reference Horrobin^56, Reference Das⁵⁷ Essential fatty acid metabolism of the omega-6 and omega-3 fatty acid series is illustrated in Figure 1. Under these circumstances, de novo fatty acid synthase activity is up-regulated during glucose metabolism, with overproduction of palmitic acid as the end product.Reference Shah, Dhir, Golli, Chandran, Lewis and Acquafondata⁵⁸ Based on research evidence, it is apparent that excessive linoleic acid, a ligand for peroxisome proliferator-activated receptor-γ, drives the mitogenetic signalling pathway, and that excessive palmitic acid, a ligand for peroxisome proliferator-activated receptor-δ, drives the apoptotic signalling pathway. However, the eventual over-expression of peroxisome proliferator-activated receptor-δ contributes to apoptotic resistance, which results in conditions of cellular overproduction (e.g. hyperplasia and metaplasia).Reference Martinasso, Oraldi, Trombetta, Maggiora and Bertetto⁵⁹ Notably, peroxisome proliferator-activated receptor-γ is reported to be up-regulated in cholesteatoma.Reference Hwang, Kang, Song, Kang, Woo and Chae⁶⁰ Moreover, linoleic acid peroxidation products, namely 9- and 13-hydroxyoctadecadienoic acid metabolites, which are activators and ligands for peroxisome proliferator-activated receptor-γ, have the capacity to induce apoptosis.Reference Niki, Yoshida, Saito and Noguchi⁶¹ In retrospect, linoleic acid and arachidonic acid are excellent sources for free radical attack, oxidative stress and cell damage. Although the role of arachidonic acid in the clinical course of cholesteatomas has been firmly established, the role of linoleic acid still needs to be confirmed.

Fig. 1 Essential fatty acid metabolism of the omega-6 and 3 fatty acid series. Essential fatty acids include: linoleic acid and α-linolenic acid, which must be consumed by the diet; and γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, which can be consumed by the diet or metabolised in the body. (Fatty acid nomenclature using the example ‘C18:2’: 18 = number of carbon atoms; 2 = number of double bonds.) Elongases are enzymes that elongate the carbon chain by two carbon units, desaturases introduce an additional double bond and β-oxidation refers to the shortening of the carbon chain by two units. Inflammatory metabolites include: prostaglandins and thromboxanes, which are produced via cyclo-oxygeneases; and leukotrienes, produced via lipoxygenases.

Bacterial interference with lipid metabolism may also cause accumulation of very long chain fatty acids (more than 22 carbon atoms), which requires peroxisomal β-oxidation by catalase activity. Peroxisomal β-oxidation is known to be inhibited by endotoxin lipopolysaccharides (i.e. the conversion of hydrogen peroxide into water and oxygen); cells with very long chain fatty acids are rendered toxic and they become apoptotic.Reference Khan, Contreras and Sing⁶² Not surprisingly, cholesteatoma debris was previously associated with bone erosion.Reference Iino, Toriyama, Ogawa and Kawakami⁴⁸ The accumulation of very long chain fatty acids in the epithelial cells of cholesteatoma and their toxicity needs to be confirmed or refuted. Of relevance to cholesteatoma is that newly formed tissue masses are maintained by angiogenesis and a constant supply of systemic dietary lipids. The latter explains why there is no exhaustion of lipid sources and why a cascade of events contributes to cholesteatoma growth and expansion, up to a point where hypoxia and microvessel occlusion lead to cholesteatoma degeneration. It has been revealed that under hypoxic conditions, the hypoxia inducible factor stimulates matrix metalloproteinases that cause cholesteatoma perimatrix degeneration.Reference Knowles, Cleton-Jansen, Korsching and Athanasou⁶³

Fatty acid role-players during chronic inflammation and bone erosion

Central to inflammation are the inflammatory mediators produced by epithelial and endothelial cells, as well as immune infiltrates. These mediators either fight off infection or damage tissues, depending on overproduction, inhibition or an imbalance among family members. It is evident that lipopolysaccharides stimulate tumour necrosis factor α (TNF-α) production, which plays a pivotal role during infection in cholesteatoma and chronic otitis media conditions. Although mainly produced by macrophages, keratinocytes and epithelial cells also have the potential to induce TNF-α activity in cholesteatoma. Tumour necrosis factor α has the capacity to stimulate matrix metalloproteinases involved in cholesteatoma degradation and bone erosion. It can also induce excessive mucous secretion in chronic otitis media (with or without effusion) via mechanisms that stimulate inducible nitric oxide synthases and toxic NO production, which may also lead to cholesteatoma degradation and bone erosion.Reference Junh, Jung, Hoffman, Drew, Preciado and Sausen¹³

Linoleic acid, α-linolenic acid and arachidonic acid are known to be potent natural ligands for peroxisome proliferator-activated receptor-γ. Furthermore, arachidonic acid and γ-linolenic acid metabolites are known to induce receptor activator of nuclear factor κβ ligand and osteoclastogenesis (via cyclo-oxygenase-2 activities).Reference Junh, Jung, Hoffman, Drew, Preciado and Sausen^13, Reference O'Shea, Bassagaya-Riera and Mohede⁶⁴ However, the mechanisms involved still need elucidating.