The Pathophysiology of Cholesteatoma

Otolaryngol Clin N Am

39 (2006) 1143–1159

The Pathophysiology of Cholesteatoma

Maroun T. Semaan, MD, Cliff A. Megerian, MD*Department of Otolaryngology and Head and Neck Surgery, University Hospitals

of Cleveland, Case Western Reserve University, LKS 4500, 11100 Euclid Avenue,

Cleveland, OH 44106, USA

Cholesteatoma is a cystic lesion formed from keratinizing stratified squa-mous epithelium, the matrix of which is composed of epithelium that restson a stroma of varying thickness, the perimatrix. The resulting hyperkerato-sis and shedding of keratin debris usually results in a cystic mass with asurrounding inflammatory reaction. It may present extradurally and intra-durally. Extradurally, cholesteatoma most commonly involves the middleear cleft but can occur in all portions of the petrous bone including themastoid, petrous apex, and external auditory canal. Intradurally, cholestea-toma, also known as epidermoid, have been described in a variety ofanatomic locations, the most common being the cerebellopontine angle.

The history of cholesteatoma has been reviewed recently [1] and is sum-marized briefly. In 1683, Duverney [2] published the first description of whatmight correspond to a cholesteatoma. He described an abscess of the boneoriginating from the auditory canal that opened behind the auricle, forminga fistula above the mastoid process, shedding the small sheets composed ofwhat he describes as scales. The abscess described was accompanied bya bad odor and gave rise to what was described as grave accidents. Healso mentioned that the same process easily enters the middle ear cleftthrough the auditory canal, destroying its contents and resulting in deafness.Nearly a century and a half after Duverney’s original description, Cruveilh-ier [3] provided in 1829 a detailed description of what he thought was anavascular tumor originating from the cells of the subarachnoid space. Inde-pendently, Muller [4] in 1838 used the term cholesteatoma as he becameaware of the presence of cholesterin and fat in what he believed to be a tu-mor. Although, he noted the resemblance between the squamae of choles-teatoma and the cells of the stratum corneum he did not postulate theepidermal origin of these lesions. In 1855, Virchow [5] classified

* Corresponding author.

E-mail address: [email protected] (C.A. Megerian).

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1144 SEMAAN & MEGERIAN

cholesteatoma among squamous cell carcinomas and atheromas. However,because these lesions grew in bone, where epidermis does not exist, he con-sidered them as heteroplastic tumors arising from mesenchymal cells thatundergo dedifferentiation and then redifferentiation into epithelial cells.This postulation represents the first theory suggesting that cholesteatomaarise from mesenchymal cells undergoing metaplasia. Despite being a misno-mer, the term cholesteatoma is still used today.

Von Troeltsch [6,7] was the first to consider the epidermal origin of cho-lesteatoma. He theorized that epidermal debris accumulating in the externalmeatus are able to cause pressure-induced osteolysis of the bony wall of themeatus and thus invasion of the mastoid and the middle ear with extensionif unchecked into the transverse sinus and brain. Gruber [8], Wendt [9] andRokitansky [10] considered that middle ear mucosa rather than bone under-went malpighian metaplasia in response to chronic inflammation. The des-quamated cells developed into cholesteatoma as the passage for squamaeelimination became narrower. The theory of metaplasia became well ac-cepted among otologists in the 19th century. At the end of the century, bystudying two different pathologic entities, Bezold [11] and Habermann[12] proved that cholesteatoma could originate from the skin of the externalauditory meatus, which migrates into the middle ear under the influence ofchronic inflammation. Similar to normal skin, the migrated skin desqua-mated, and as the drainage passages became too narrow to enable migra-tion, cholesteatoma forms. Habermann based his findings on the studyingpatients with marginal tympanic membrane perforation after acute necrotiz-ing otitis; Bezold, however, studied cholesteatoma formation in patientswith attic or posterosuperior retraction pockets secondary to eustachiantube dysfunction.

Middle ear cholesteatoma occurs as two principle different entities thatshare many pathological resemblances: congenital and acquired. The latteris divided further into the more common primary acquired or attic retrac-tion pocket cholesteatoma and the secondary acquired cholesteatoma as itoccurs secondary to epithelial migration into the middle ear at the site ofa tympanic membrane perforation or iatrogenically implanted during anotologic procedure.

In this review, we limit our discussion to middle ear cholesteatoma andprovide an updated literature review on the pathophysiology of congenitaland acquired cholesteatoma. Emphasis will be placed on the pathophysiol-ogy of congenital and primary acquired cholesteatoma, cytokine-mediatedinflammation and bony destruction.

Congenital cholesteatoma

The first published description of a congenital cholesteatoma appeared in1885, by Lucae [13]. Korner’s initial criteria [14] to distinguish acquiredfrom congenital cholesteatoma were revived half a century later by Derlacki

1145PATHOPHYSIOLOGY OF CHOLESTEATOMA

and Clemis [15] who reintroduced the concept of congenital cholesteatomain 1965. They proposed that congenital cholesteatoma be defined as a pearlywhite mass behind an intact tympanic membrane in the absence of history ofotitis or otorrhea, tympanic membrane perforation, or previous otologicprocedures. In 1986, Levenson and coworkers [16] suggested that the pres-ence of prior bouts of otitis media does not necessarily exclude the presenceof congenital cholesteatoma, because this inflammatory condition is verycommon among children.

The incidence of congenital cholesteatoma is 0.12 per 100,000 [17]. Therehas been a recent increase in the reported incidence of this disease likely sec-ondary to an increased awareness among pediatricians and otolaryngolo-gists along with improvement in office based tools used for otologicexamination (ie, otomicroscopy, halogen lightening, and photodocumenta-tion). The pathogenesis of congenital cholesteatoma sparked an active de-bate that continues to this day. In 1936 Teed [18] described the presenceof epithelial rests in fetal temporal bones that disappeared by 33 weeks ofgestation. He postulated that the persistence of these cells leads to formationof congenital cholesteatoma. These rests were localized in the lateral wall ofthe eustachian tube in proximity of the tympanic ring in the anterosuperiorquadrant of the middle ear. These findings were confirmed later by Michaelsin 1986 [19] but failed to prove their persistence after 33 weeks of gestation.In 1998, Karmody and colleagues [20] described histologic findings of squa-mous epithelial rest in the temporal bones of two postpartum patients. Thiswas the first description of these epithelial rests persisting beyond 33 weeksof gestation. In their first patient, they described the presence of a cup-shaped elevation of squamous epithelium with a keratin cap noted in the an-terosuperior quadrant of the middle ear. In their second patient, a smallmass of squamous epithelium was seen embedded in the mucosa of the ante-rosuperior quadrant of the middle ear at the junction of the columnar andcuboidal epithelia. In their clinical study of a series of 160 congenital choles-teatoma, Potsic and coauthors [21] found that in cases of isolated quadrantinvolvement, 77% were anterosuperior and 22% were posterosuperior. Thenumber of quadrants involved increased with age. The incidence of isolatedposterosuperior quadrant involvement appears to be higher than initiallythought.

Many theories have been proposed to explain the origin of congenitalcholesteatoma. The Teed-Michaels’ epithelial rest theory has been well ac-cepted among otologists. Ruedi [22,23] speculated that inflammatory injuryto an intact tympanic membrane results in microperforations in the basallayer that lead to invasion of the squamous epithelium by proliferating ep-ithelial cones through a macroscopically intact but microscopically injuredtympanic membrane. These epithelial cones fuse and expand forming a mid-dle ear cholesteatoma. Tos [17] recently questioned the epithelial rest theoryand proposed a different explanation for the pathogenesis of this disease. Heobserved that anterosuperior cholesteatoma had a frequent attachment to


the anterior aspect of the malleus handle or neck and that posterosuperiorcholesteatoma had an attachment to the posterior aspect of the malleus han-dle and to the incudostapedial joint. This location was far from the anteriortympanic annulus and the lateral wall of the eustachian tube where epithelialrests are usually found. Furthermore, he speculated that if the site of originwas the lateral eustachian tube wall and the area anterior to the tympanicannulus, cholesteatoma would block the eustachian tube before extendinginto the tympanic cavity and the area of the malleus handle, a findingthat has not been described previously. Therefore, he argued against the ep-ithelial rest theory and explained the pathogenesis of congenital cholestea-toma by the acquired inclusion theory (Fig. 1). This theory speculates thatkeratinized squamous epithelium may be implanted or included into thetympanic cavity during one of many pathological events affecting the tym-panic membrane and middle ear in childhood. According to Tos, viable

Fig. 1. ‘‘Acquired’’ inclusion theory suggested by Tos. (A1, 2) The tympanic membrane re-

tracted and adherent to the malleus handle, malleus neck, or long process of the incus is loos-

ened and torn leaving a small cuff of viable keratinized epithelium adherent to the ossicles with

a small residual tear in the tympanic membrane. As the tear heals, the included epithelium leads

to formation of an inclusion cholesteatoma. (B1, 2) A tangential tear is created as the retracted

and adherent tympanic membrane is loosened from the underlying structure resulting in a rem-

nant of epithelial cells without a perforation of the tympanic membrane that results in an inclu-

sion cholesteatoma. (C1, 2) Microperforations of the traumatized retracted tympanic

membrane result in invasion of the basal membrane by epithelial cones. As the ear drum is sud-

denly loosened, these cones are left behind and included in the tympanic cavity. (D1, 2) Similar

to the previous mechanism, repeated inflammation of the tympanic membrane result in prolif-

erating epithelial cones that penetrate the basal membrane and proliferate into the subepithelial

space. These cones are included in the tympanic cavity as the drum is loosened and detached

from the underlying bony structures.


keratinized epithelial cells of the retracted and adherent tympanic mem-brane to the malleus handle, malleus neck, or the long process of the incusare left behind after loosening of the drum and are included into the tym-panic cavity.

Four mechanisms are thought to account for the inclusion of epithelialcells into the tympanic cavity.

1. The tympanic membrane retracted and adherent to the malleus handle,malleus neck, or long process of the incus is loosened and torn leavinga small cuff of viable keratinized epithelium adherent to the ossicles witha small residual tear in the tympanic membrane. As the tear heals, theincluded epithelium leads to formation of an inclusion cholesteatoma.

2. A tangential tear is created as the retracted and adherent tympanicmembrane is loosened from the underlying structure resulting in a rem-nant of epithelial cells without a perforation of the tympanic membranethat results in an inclusion cholesteatoma.

3. Microperforations of the traumatized retracted tympanic membraneresult in invasion of the basal membrane by epithelial cones. As theear drum is suddenly loosened these cones are left behind and includedin the tympanic cavity.

4. Similar to the previous mechanism, repeated inflammation of the tym-panic membrane results in proliferating epithelial cones that penetratethe basal membrane and proliferate into the subepithelial space. Thesecones are included in the tympanic cavity as the drum is loosened anddetached from the underlying bony structures.

In response to Tos’ observations, Liang and coauthors [24] performed animmunohistochemical analysis of 36 temporal bones of 19 fetuses aged be-tween 6 gestational weeks to 15 months postpartum. The investigators ob-served in each of the 22 temporal bones aged 16 gestational weeks to 8months postpartum at least one epidermoid formation with a total of 116.The majority were found in the middle ear epithelium in the anterosuperiorannular region of the tympanic cavity with a small number of epidermoidformations seen in the posterosuperior, anteroinferior, and posteroinferiorregion of the lateral wall in the vicinity of the annular zone. In addition,Liang and colleagues [24] examined the differential expression of 34bE12,a cytokeratin antigen expressed by the external ear epidermis and the pseu-dostratified columnar epithelium at all gestational ages, and 35bH11, a cyto-keratin antigen expressed by pseudostratified columnar and simple cuboidalepithelium used to characterize the epidermoid formation seen in temporalbones histological sections. In addition, they used antibodies to antilym-phoid enhancing factor-1 (LEF-1), a marker expressed by embryonic epi-dermis, to analyze the epidermoid formation precursor previouslydescribed by Michaels [19,25]. All epidermoid formations seen in their studystained positive for epidermal cytokeratin. The epidermoid formation pre-cursor found in both temporal bones of an embryo aged 6 gestational weeks


did not stain for LEF-1. Thus, they concluded that the epidermoid forma-tion precursor initially reported is likely the result of a tangential cut arti-fact of a thickened actively growing epithelial bud from the tip of thetubotympanic recess. Microscopically, they observed that as the anterosu-perior tip of the meatal plate (precursor of the pars tensa) develops, by ges-tational week 12, the epidermal interface becomes jagged, and bygestational week 16, epidermal cells become encroached onto the fibroblastsof the bilaminar collagen layer. As the fibroblasts become more condensed,small clumps of epidermal cell become trapped within the condensed bila-minar collagen layer.

Despite improvements in our understanding, the pathophysiology of con-genital cholesteatoma continues to be controversial and actively debated.Furthermore, many questions remain unanswered. These questions pertainto the biological factors that predict aggressiveness, growth, and recidivismof middle ear congenital cholesteatoma (Fig. 2).

Acquired cholesteatoma

Primary acquired cholesteatoma

The pathophysiology of acquired cholesteatoma is similarly controver-sial. As previously eluded to, the precise pathogenesis of cholesteatomahas been debated for more than two centuries. Four predominant theorieshave fueled the debate: (1) invagination, (2) basal cell hyperplasia or papil-lary ingrowth, (3) metaplasia, and (4) epithelial invasion.

The invagination theory is currently regarded as one of the primarymechanism of the formation of primary acquired attic cholesteatoma.Anatomic or pathological conditions that predispose to eustachian tube

Fig. 2. Site of origin and patterns of spread of congenital cholesteatoma according to (A) Tos

‘‘acquired’’ inclusion theory and (B) Teed-Michael’s epidermal rest theory.


dysfunction result in barometric perturbation of the middle ear space. Im-paired ventilation secondary to a dysfunctional eustachian tube leads to neg-ative middle ear pressure. The negative pressure is the culprit for structuralweakening of the tympanic membrane and development of retractionpockets. The pars flaccida, having the weaker structural support, is themost common site of formation of a retraction pocket. Sade [26] andSade and Halevy [27] described four stages of tympanic membrane retrac-tion: stage I, retracted membrane; stage II, retraction onto the incus; stageIII middle ear atelectasis; and stage IV, adhesive otitis media. The geomet-rical changes attributed to progressive retraction lead to narrowing of theanatomic passages and impairment of the epithelial migration and cleaningof the keratin debris. As the pocket deepens and insinuates between mucosalfolds and crevices, it becomes non–self cleaning and leads to accumulationof keratin debris (Fig. 3) Bacterial proliferation and super-infection of theaccumulated debris form a biofilm that leads to chronic infection andepithelial proliferation. The latter appears to be influenced by the cytokine-mediated inflammatory response. Chole and Faddis [28], analyzed the pres-ence of biofilm matrix in cholesteatoma debris of 22 surgically inducedMongolian gerbils and 24 human specimens. The investigators detected theamorphous polysaccharide matrix suggestive of biofilm formation in 21 of 22animals and 16 of 24 human cholesteatoma. Recently, Wang and coworkers[29] found that otopathogenic strains of pseudomonas aeruginosa are

Fig. 3. Mucosal compartmentalization of the middle ear. The mucosal folds of the middle ear

cleft define the spaces that limit the boundaries of the retraction pockets. Knowledge of their

anatomy helps understand the formation and extension of primary acquired cholesteatoma

(black arrows). (1) superior incudal fold, (2) superior malleolar fold, (3) lateral incudal fold,

(4) anterior malleolar fold, (5) lateral malleolar fold, (6) posterior malleolar fold. ET, eustachian

tube orifice; HAC, hypotympanic air cells; RW, round window niche. Eustachian tube dysfunc-

tion results in formation of a retraction pocket. Often, a pars flaccida retraction pocket is

formed (star). As the pocket deepens and insinuates between folds, the self-cleaning mechanism

is altered and keratin accumulates.


capable of producing biofilm and become highly resistant to antimicrobialtherapy. These findings strongly suggest a role of bacterial biofilm in thepathogenesis of cholesteatoma.

The experimental model illustrating the implication of eustachian tubedysfunction in the formation of retraction pockets and later cholesteatomawas described by Kim and Chole [30]. By ligating the eustachian tube ofMongolian gerbils, the investigators succeeded in creating an induced, sur-gical model of primary acquired cholesteatoma (Fig. 4).

The exact mechanism and triggers that lead to development of an activecholesteatoma in some patients with an attic retraction pocket while otherscontinue to have a quiescent and self-cleaning pocket remain unclear. It hasbeen shown recently that the combination of tympanic membrane retractionand basal cell proliferation is the hallmark for cholesteatoma formation anddevelopment.

In a cohort of healthy children age 5 to 16 years, the prevalence of atticretractions was between 14% and 25% of ears [31]. In a separate cohort ofchildren treated for secretory otitis with pressure equalization tube insertionwith or without adenoidectomy and followed up to 18 years, the incidence ofsevere retractions (behind the scutum with some bone resorption) was 5% to6% and attic cholesteatoma was 0.2% to 1.7%. Sudhoff and Tos [31] per-formed immunohistochemical analysis of surgical specimens obtainedfrom 14 patients with middle ear cholesteatoma. In their clinical study,they compared the expression of MIB-1, a marker of cellular proliferation,between the cholesteatoma content and the normal external auditory canalskin. In addition, the investigators analyzed the integrity of the basementmembrane by using avidin biotin complex peroxidase to stain collagentype IV. At the level of the basement membrane, interruption in the

Fig. 4. Patterns of spread of primary acquired cholesteatoma from an attic retraction pocket

(D). (A) Antrum, most common; (B) posterior mesotympanum, second most common; and

(C) anterior mesotympanum, least common.


continuity was seen at the cholesteatoma–lamina propria interface, whereasthe integrity was preserved in the adjacent normal auditory canal skin. Theyalso showed an increased expression of MIB-1 in the keratinocytic popula-tion of the basal cell layer. This increased expressivity was consistent withproliferating keratinocytes localized primarily in small epithelial cones orpseudopods growing into the subepithelial stroma through interruptionsof the basement membrane. Their observation provides experimental evi-dence that support the implication of both the retraction and basal cell hy-perplasia theories. They postulated that in the initial retraction pocket stage,the epithelial migratory pattern is maintained until the pockets deepen andthe drainage pathways become small leading to keratin debris accumulation.As the debris becomes infected, the bacterial proliferation and resultant in-flammation leads to an influx of inflammatory cells and production of cyto-kines. This progression along with local release of collagenases createdbreaks in the basement membrane allowing the formation of epithelial conesthat grow toward the stroma (papillary ingrowth theory). The combinationof subepithelial invasion and keratinocytic proliferation in the form ofmicrocholesteatoma is the hallmark of the precholesteatomatous stage ofcholesteatoma.

As the microcones expand and fuse together, an attic cholesteatoma isformed. Using the normal postauricular skin as control, Albino and co-workers [32] found a nine- to 20-fold increase in the expression of p53 incholesteatoma tissue, trough all epithelial layers. The p53 proteins by acti-vating downstream products (p21/WAF1, GADD45, and mdm2) appearto have a role in the down-regulation of cellular proliferation and promo-tion of apoptosis [33], a checkpoint control mechanism to protect the cellfrom genetic alterations. Similarly, they noted a two-fold increase in the ex-pression of Ki-67, a marker of cellular proliferation, in cholesteatoma tissuecompared with control normal postauricular skin. According to Albinoand coauthors [34], the increased p53 expression was a feedback negativeresponse to control an increased proliferative state as witnessed by theincrease expression of Ki-67.

Using immunohistochemistry, Kim and coworkers [35], analyzed the pat-tern of cellular proliferation and epithelial migration in the Mongolian ger-bil animal model. They showed an increase in the expression of cytokeratin(CK) 13/16, markers of epidermal cell proliferation, in the expanding part ofthe cholesteatoma and to a lesser degree an increase in the expression of CK5/6 and CK 1/10, markers of epithelial migration. They concluded that cel-lular migration (or invasion) and proliferation play a role in the expansionof cholesteatoma.

On the other hand, Olszewska and coauthors [36], by studying the expres-sion of five different cytokeratin (CK 10, CK 14, CK 18, CK 19 and 34bE12)concluded that congenital and acquired cholesteatoma exhibit a similar ex-pression pattern. These findings suggested that the so-called ‘‘acquired’’ cho-lesteatoma in children may be an advanced congenital cholesteatoma that


resulted in destruction of the tympanic membrane, erosion of the ossicularchain, and invasion of the mastoid cavity.

Epithelial invasion by cholesteatoma appears to be an important charac-teristic of this disease. Cholesteatoma expand by invading into surroundingmiddle ear soft tissue structures and bone. It remains unclear to what factorspredict the biologic behavior of these lesions, such as recidivism and a moreaggressive clinical course.

Mallet and colleagues [37] found a correlation between the aggressivenessof the clinical behavior of cholesteatoma and the index of proliferation. Intheir analysis of surgical specimen from 91 ears with cholesteatoma, MIB-1was detected in 23% of the ears with moderate bony destruction (single os-sicle affected) versus 56% of ears with severe bony destruction (two or moreossicles, meningeal exposure, denudation of the facial nerve or sigmoidsinus, and erosion of the lateral semicircular canal). These findings werestatistically significant. Young age was found to be a predictor of aggressive-ness as witnessed by a higher proliferative index in children.

Tokuriki and coworkers [38] performed gene expression analysis on hu-man middle ear cholesteatoma using complementary DNA arrays. Theycompared the expression pattern of eight cholesteatoma to normal postaur-icular skin samples. They found an upregulation or induction in genesinvolved in cellular proliferation and differentiation (calgranulin A, calgra-nulin B, psoriasin, thymosin b-10) and cell invasion (cathepsin C, cathepsinD, cathepsin H, and matrix metalloproteinase 9 [MMP-9]). These resultswere confirmed using reverse transcriptase-polymerase chain reaction(RT-PCR) analysis.

Immunohistochemical analysis showed increased expression of calgranu-lin A, calgranulin B, and calgranulin D in the cytoplasm of all cell layers ofthe cholesteatoma epithelium. Calgranulin proteins belong to the S100 pro-tein family. In epithelial cells they may be involved in Ca2þ- dependent re-organization of cytoskeletal filaments [39]. Psoriasin, also a member of theS100 protein family, has been shown to be increased in hyperproliferativeand inflammatory skin conditions and are believed to play a role in kerati-nocytic differentiation [40]. Upregulation and induction of these genes mayreflect an alteration in keratinocyte differentiation and migration leading tokeratin overproduction and accumulation as seen in cholesteatoma. The ca-thepsin family is a group of lysosomal proteases that play a key role in thedegradation of intracellular and extracellular proteins in the epidermis andhave been shown to contribute to the invasive properties of some neoplasms[41]. Cathepsin B has been shown to play a role in the osteolysis seen in cho-lesteatoma [42].

The increased keratinocyte proliferation is coupled with an increased celldeath resulting in the production of larger amount of keratin debris respon-sible for the expansion and keratin accumulation seen in cholesteatoma. Theimplication of apoptotic cell death has been demonstrated recently [43]. Cas-pase-8 activation, a known effector of the extrinsic pathway of apoptosis, is


triggered by activation of the cell surface death receptors (tumor necrosisfactor [TNF] family, Fas-L/Fas-R). This activation results in the activationof an end product of apoptosis, caspase-3, that induces the nuclear translo-cation of effector molecules that result in apoptosis and programmed celldeath. The transcription factor nuclear factor (NF)-kB is a known key me-diator of the TNF-mediated cellular response. NF-kB proteins are intracy-tosolic and are inactivated by IkB-a (an inhibitory protein). The inactivationof IkB-a activates NF-kB and results in nuclear translocation of the tran-scription factor. The activation of NF-kB suppresses apoptosis induced byTNF-a. Miyao and coauthors [43] found an increased expression of cas-pase-3 localized to the granular and spinous layers of the cholesteatoma ep-ithelium and an increased expression of caspase-8 confined to the granularlayer. The retroauricular skin was used as a control. The NF-kB proteinswere localized in the perinuclear region suggesting that the mechanism ofnegatively controlling apoptosis was inactivated leading to keratinocytecell death and keratin accumulation.

These findings strongly suggest differential properties inherent to choles-teatoma compared with normal epidermal keratinocytes that may explaintheir clinical aggressiveness and behavior responsible for the expansion,bony destruction and recidivism. Numerous studies have confirmed the im-plication of invagination, basal cell hyperplasia, and invasion in the patho-genesis of primary acquired cholesteatoma. The exact inciting events andfactors responsible for the genesis and progression of middle ear cholestea-toma remain unclear, and further research is warranted to help elucidatethese missing links.

Secondary acquired cholesteatoma

Secondary acquired cholesteatoma has been described to occur as theresult of the migration of tympanic membrane epidermis into the middleear at the site of a marginal perforation or as the result of the implan-tation of viable keratinocytes into the middle ear cleft. The implantationoccurs during a blast injury to the tympanic membrane leaving keratino-cytes behind a healed perforation, at the site of a temporal bone fracture,or as the result of an iatrogenic introduction of these cells. The latterhave been described to occur in various otologic surgeries such as stape-dectomy, tympanoplasty, pressure equalization tube placement, and mid-dle ear exploration.

Wolf and coauthors [44] described the otologic findings in 210 ears from147 soldier-patients that sustained blast injuries with perforation of tym-panic membrane localized to the pars tensa. These investigators reportedan incidence of 4.8% of invasive cholesteatoma. Freeman [45] reported threecases of cholesteatoma secondary to temporal bone fracture. The keratino-cytes appear to have invaded into the middle ear cleft through the fracturesites.


Golz and coauthors [46] performed a retrospective analysis of 2829 chil-dren who underwent a ventilation tube placement between 1978 and 1997.These investigators noted an incidence of 1.1% of middle ear cholesteatomaattributed to the insertion of the pressure equalization tube. The presence ofcholesteatoma around the tube site was a prerequisite to incriminate theprocedure as a cause of the cholesteatoma. They also noted a higher inci-dence in children aged less than 5 years, those with placement of GoodeT-tubes, children with frequent reinsertions, patients with duration of place-ment exceeding 12 months, and ears with history of frequent postoperativeotorrhea. Ferguson and coworkers [47] described the reasons for cholestea-toma formation after a stapedectomy. The investigators described fourmechanisms: prosthesis extrusion independent of eustachian tube dysfunc-tion, inadvertent implantation of keratinocytes with the oval window fatgraft, malpositioned inverted tympanomeatal flap, and migration at thesite of a marginal tympanic membrane perforation.

Eavey and coworkers [48], and Camacho and colleagues [49] were able toproduce viable keratinocytes in the bulla of gerbils and chinchilla, respec-tively, by implanting the mastoid space with autogenous keratinocytes ob-tained from the conchal surface of the pinna. Production of new keratinwas observed up to 9 months postimplantation. Various histopathologicchanges ranging from granulation tissue to cholesteatoma formation weredescribed. The investigators concluded that neonatal aspiration of lanugoand viable keratinocytes can result in middle ear inflammation that in thechronic stage can lead to cholesteatoma formation. Bernal-Sprekelsen andcoworkers [50], argued against this model of implantation of keratinocytesas an etiology for cholesteatoma and failed to find keratinizing epithelialcells in 31 temporal bones of infants who died before 1 year of age and27 temporal bones of preterm fetuses that succumbed to various conditions.Despite the fact that the neonatal aspiration of viable keratinocytes may notfully account for the development of congenital cholesteatoma, it providesa valuable experimental platform that the implantation of viable keratino-cytes can lead to formation of middle ear or mastoid cholesteatoma. Thisis observed frequently in revision middle ear surgery and described asa ‘‘cholesteatomatous pearl’’ formation that is the result of a trapped viablekeratinocytic formation that leads to a small localized cholesteatoma.

Another experimental model recently described by Massuda and Oliveira[51] provides physiopathologic evidence that supports epithelial migration atthe edges of a tympanic membrane perforation as a possible cause for cho-lesteatoma development. By creating a tympanic membrane perforation andlatex with 50% propylene glycol, the investigators succeeded in producingcholesteatoma in 90% and 80% of their animals, respectively. They con-cluded that latex provides a biomembrane that favors neoangiogenesisand forms a bridge for epithelial migration. This environment is enhancedfurther by a cytokine-producing acute or chronic inflammatory milieu cre-ated by the inciting material. This model may provide evidence that


epithelial migration of keratinizing epithelium at the site of a tympanicmembrane perforation, in the setting of recurrent inflammatory events,may be the culprit for cholesteatoma formation.

Mechanism of bone destruction

The ongoing debate on the pathogenesis of cholesteatoma is paralleled bythe ongoing research to help elucidate the mechanism of expansion, bonedestruction and invasion seen in middle ear cholesteatoma. Two predomi-nant mechanisms are believed to account for the osteolysis seen in middleear cholesteatoma: pressure-induced bone resorption and enzymatic dissolu-tion of bone by cytokine-mediated inflammation. Pressure necrosis initiallydescribed by Steinbrugge in 1879 and Walsh in 1951, and direct bone resorp-tion as described by Chole and coworkers [52] in 1985 have been proposedas possible mechanisms of bone destruction. Chole and colleagues im-planted silicone sheets in the middle ear of gerbils without cholesteatomaand noted bone resorption at the pressure sites. They estimated that pres-sures of 50 to 120 mm Hg resulted in osteoclastic-induced bone resorption.The interaction of osteoclasts and osteoblasts to extrinsic biomechanicalfactors is a well-documented biological response [53,54].

It is uncertain to what degree the pressure-induced activation of osteo-clasts play a role in the osteolysis seen in cholesteatoma. Enzymatic-inducedand cytokine-induced bone destruction has been studied in the last two de-cades. Matrix metalloproteinases (MMP), a family of zinc metalloenzymesthat degrades unmineralized extracellular matrix, have been shown to bepresent in the cholesteatoma [55]. MMP-2 (72 kD collagenase) and MMP-9(92 kD collagenase) were expressed in suprabasal epithelial layers ofcholesteatoma.

Other investigators found the increased expression of MMP-9 but notMMP-2 in cholesteatoma cells [56]. Schmidt and coworkers [56] analyzedthe in vivo significance of MMP-9 activity in relation to the production of cy-tokines interleukin (IL)-1a, IL-1b, TNF-a, transforming growth factor(TGF)-b, and epidermal growth factor (EGF) in tissue homogenates of 37cholesteatoma and nine external ear skin specimens. IL-1a production wasfound to be significantly elevated; however, no correlation was found betweenMMP-9 activity and cytokine production. IL-1 and IL-8, important intercel-lular mediators of osteoclastic activities have been shown to increase in cul-tured cholesteatoma cells comparedwith normal external auditory canal skin.

The role of another important cytokine, TNF-a, has also been found.Yan and coauthors [57] found that by in vitro stimulating monocytes,they were able to produce multinucleated cells with osteoclastlike activitythat produced acid phosphatase-induced bone demineralization. Theamount of osteolysis was increased by adding osteoblasts to the TNF-a–treated osteoclasts containing medium, suggesting a cell to cell interactionmediated by TNF-a. In addition, the latter enhanced the production of


collagenases by macrophages and osteoblasts. However, by performing en-zyme-linked immunosorbent assay on tissue samples from 23 patients withcholesteatoma and 16 patients with chronic otitis without cholesteatoma,the detection of IL-1a, TNF-a, and EGF was significantly higher in the cho-lesteatoma samples [58].

Recent histopathologic evidence was obtained from the temporal bone oftwo patients with ruptured cholesteatoma sac resulting in local inflamma-tion and osteolysis [59]. These changes were associated with a small abscessformation at the site of the rupture. They noted a marked inflammatorycellular infiltrate surrounding the rupture site with evidence of epithelialproliferation at the lining of the perforation site.

Recent work by Jung and coworkers [60] showed the possible role of ni-tric oxide as an important mediator of osteoclast function. Using in vivoanalysis of a murine model of cholesteatoma-induced bone resorption andin vitro analysis of osteoclast culture, the investigators studied the gene ex-pression of nitric oxide synthase (NOS) and the effect of aminoguanidine (aninhibitor of cytokine mediated nitrite production). They showed a selectiveupregulation of the inducible NOS or NOS II compared with NOS I and IIIand a dose-dependent stimulation of osteoclastic activity (not proliferation)using low concentration of nitric oxide donors (sodium nitroprusside andS-nitro-N-acetyl-D, L-penicillamine). In vitro, only interferon (IFN)-g (notIL-1b or TNF-a) was able to generate nitrite. This nitrite production wasblocked in vitro by the addition of aminoguanidine (but not in vivo) andwas synergistically enhanced in the presence of IFN-g, IL-1b, and TNF-a.These findings indicate a role for nitric oxide in the osteoclastic-mediatedbone resorption in cholesteatoma and suggest the implication of additionalcytokines in the in vivo osteoclastogenesis and bone resorption. In contrastto the increased osteoclastic activity without increase in the number of oste-oclasts seen by Jung and colleagues [60], in a separate study, Hamzei and co-authors [61] found an increase in the number of the osteoclast precursor cells inthe perimatrix of 21 cholesteatoma surgically obtained. These studies high-light the importance of osteolysis and its regulatory mechanisms in the bonedestruction seen in middle ear cholesteatoma that results in significant mor-bidity and mortality.

Summary

The pathophysiology of cholesteatoma continues to be debated widely.Cholesteatoma is classified as congenital or acquired. Recent studies appearto favor a possible common origin and overlap in the pathophysiology be-tween both entities. Despite the growing evidence that the genesis, expan-sion, and progression of cholesteatoma is a complex interaction betweenanatomic, inflammatory, and regulatory factors of cellular proliferationand differentiation, the exact mechanism responsible for the invasion, recid-ivism, and destruction seen in this disease remains unknown.


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The Pathophysiology of Cholesteatoma

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