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 rests
on a stroma of varying thickness, the perimatrix. The resulting hyperkerato-
sis and shedding of keratin debris usually results in a cystic mass with a
surrounding inflammatory reaction. It may present extradurally and intra-
durally. Extradurally, cholesteatoma most commonly involves the middle
ear cleft but can occur in all portions of the petrous bone including the
mastoid, petrous apex, and external auditory canal. Intradurally, cholestea-
toma, also known as epidermoid, have been described in a variety of
anatomic 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 what
might correspond to a cholesteatoma. He described an abscess of the bone
originating from the auditory canal that opened behind the auricle, forming
a fistula above the mastoid process, shedding the small sheets composed of
what he describes as scales. The abscess described was accompanied by
a bad odor and gave rise to what was described as grave accidents. He
also mentioned that the same process easily enters the middle ear cleft
through 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 an
avascular tumor originating from the cells of the subarachnoid space. Inde-
pendently, Müller [4] in 1838 used the term cholesteatoma as he became
aware 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 the
epidermal origin of these lesions. In 1855, Virchow [5] classified

* Corresponding author.
E-mail address: Cliff.Megerian@uhhs.com (C.A. Megerian).

0030-6665/06/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.otc.2006.08.003 oto.theclinics.com
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 that
undergo dedifferentiation and then redifferentiation into epithelial cells.
This postulation represents the first theory suggesting that cholesteatoma
arise 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 external
meatus are able to cause pressure-induced osteolysis of the bony wall of the
meatus and thus invasion of the mastoid and the middle ear with extension
if unchecked into the transverse sinus and brain. Gruber [8], Wendt [9] and
Rokitansky [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 squamae
elimination became narrower. The theory of metaplasia became well ac-
cepted among otologists in the 19th century. At the end of the century, by
studying two different pathologic entities, Bezold [11] and Habermann
[12] proved that cholesteatoma could originate from the skin of the external
auditory meatus, which migrates into the middle ear under the influence of
chronic 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 studying
patients with marginal tympanic membrane perforation after acute necrotiz-
ing otitis; Bezold, however, studied cholesteatoma formation in patients
with attic or posterosuperior retraction pockets secondary to eustachian
tube dysfunction.
Middle ear cholesteatoma occurs as two principle different entities that
share many pathological resemblances: congenital and acquired. The latter
is divided further into the more common primary acquired or attic retrac-
tion pocket cholesteatoma and the secondary acquired cholesteatoma as it
occurs secondary to epithelial migration into the middle ear at the site of
a tympanic membrane perforation or iatrogenically implanted during an
otologic procedure.
In this review, we limit our discussion to middle ear cholesteatoma and
provide an updated literature review on the pathophysiology of congenital
and acquired cholesteatoma. Emphasis will be placed on the pathophysiol-
ogy of congenital and primary acquired cholesteatoma, cytokine-mediated
inflammation and bony destruction.

Congenital cholesteatoma
The first published description of a congenital cholesteatoma appeared in
1885, by Lucae [13]. Körner’s initial criteria [14] to distinguish acquired
from congenital cholesteatoma were revived half a century later by Derlacki
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1145

and Clemis [15] who reintroduced the concept of congenital cholesteatoma

in 1965. They proposed that congenital cholesteatoma be defined as a pearly
white mass behind an intact tympanic membrane in the absence of history of
otitis or otorrhea, tympanic membrane perforation, or previous otologic
procedures. In 1986, Levenson and coworkers [16] suggested that the pres-
ence of prior bouts of otitis media does not necessarily exclude the presence
of congenital cholesteatoma, because this inflammatory condition is very
common among children.
The incidence of congenital cholesteatoma is 0.12 per 100,000 [17]. There
has 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 otologic
examination (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 presence
of epithelial rests in fetal temporal bones that disappeared by 33 weeks of
gestation. He postulated that the persistence of these cells leads to formation
of congenital cholesteatoma. These rests were localized in the lateral wall of
the eustachian tube in proximity of the tympanic ring in the anterosuperior
quadrant of the middle ear. These findings were confirmed later by Michaels
in 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. This
was the first description of these epithelial rests persisting beyond 33 weeks
of 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 small
mass of squamous epithelium was seen embedded in the mucosa of the ante-
rosuperior quadrant of the middle ear at the junction of the columnar and
cuboidal epithelia. In their clinical study of a series of 160 congenital choles-
teatoma, Potsic and coauthors [21] found that in cases of isolated quadrant
involvement, 77% were anterosuperior and 22% were posterosuperior. The
number of quadrants involved increased with age. The incidence of isolated
posterosuperior quadrant involvement appears to be higher than initially
thought.
Many theories have been proposed to explain the origin of congenital
cholesteatoma. The Teed-Michaels’ epithelial rest theory has been well ac-
cepted among otologists. Rüedi [22,23] speculated that inflammatory injury
to an intact tympanic membrane results in microperforations in the basal
layer that lead to invasion of the squamous epithelium by proliferating ep-
ithelial cones through a macroscopically intact but microscopically injured
tympanic membrane. These epithelial cones fuse and expand forming a mid-
dle ear cholesteatoma. Tos [17] recently questioned the epithelial rest theory
and proposed a different explanation for the pathogenesis of this disease. He
observed that anterosuperior cholesteatoma had a frequent attachment to
1146 SEMAAN & MEGERIAN

the anterior aspect of the malleus handle or neck and that posterosuperior
cholesteatoma had an attachment to the posterior aspect of the malleus han-
dle and to the incudostapedial joint. This location was far from the anterior
tympanic annulus and the lateral wall of the eustachian tube where epithelial
rests are usually found. Furthermore, he speculated that if the site of origin
was the lateral eustachian tube wall and the area anterior to the tympanic
annulus, cholesteatoma would block the eustachian tube before extending
into the tympanic cavity and the area of the malleus handle, a finding
that 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 that
keratinized squamous epithelium may be implanted or included into the
tympanic 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.
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1147

keratinized epithelial cells of the retracted and adherent tympanic mem-

brane to the malleus handle, malleus neck, or the long process of the incus
are 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 epithelial
cells 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 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.
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 inclusion cholesteatoma.
3. Microperforations of the traumatized retracted tympanic membrane
result in invasion of the basal membrane by epithelial cones. As the
ear drum is suddenly loosened these cones are left behind and included
in the tympanic cavity.
4. Similar to the previous mechanism, repeated inflammation of the tym-
panic membrane results in proliferating 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.
In response to Tos’ observations, Liang and coauthors [24] performed an
immunohistochemical 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 8
months postpartum at least one epidermoid formation with a total of 116.
The majority were found in the middle ear epithelium in the anterosuperior
annular region of the tympanic cavity with a small number of epidermoid
formations seen in the posterosuperior, anteroinferior, and posteroinferior
region 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 cuboidal
epithelium used to characterize the epidermoid formation seen in temporal
bones 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 previously
described by Michaels [19,25]. All epidermoid formations seen in their study
stained positive for epidermal cytokeratin. The epidermoid formation pre-
cursor found in both temporal bones of an embryo aged 6 gestational weeks
1148 SEMAAN & MEGERIAN

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 the
tubotympanic 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 by
gestational week 16, epidermal cells become encroached onto the fibroblasts
of 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 pertain
to the biological factors that predict aggressiveness, growth, and recidivism
of 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 cholesteatoma
has been debated for more than two centuries. Four predominant theories
have 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 primary
mechanism 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.
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1149

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 structural
weakening of the tympanic membrane and development of retraction
pockets. The pars flaccida, having the weaker structural support, is the
most common site of formation of a retraction pocket. Sade [26] and
Sade and Halevy [27] described four stages of tympanic membrane retrac-
tion: stage I, retracted membrane; stage II, retraction onto the incus; stage
III middle ear atelectasis; and stage IV, adhesive otitis media. The geomet-
rical changes attributed to progressive retraction lead to narrowing of the
anatomic passages and impairment of the epithelial migration and cleaning
of the keratin debris. As the pocket deepens and insinuates between mucosal
folds and crevices, it becomes non–self cleaning and leads to accumulation
of keratin debris (Fig. 3) Bacterial proliferation and super-infection of the
accumulated debris form a biofilm that leads to chronic infection and
epithelial 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 induced
Mongolian gerbils and 24 human specimens. The investigators detected the
amorphous polysaccharide matrix suggestive of biofilm formation in 21 of 22
animals 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.
1150 SEMAAN & MEGERIAN

capable of producing biofilm and become highly resistant to antimicrobial

therapy. These findings strongly suggest a role of bacterial biofilm in the
pathogenesis of cholesteatoma.
The experimental model illustrating the implication of eustachian tube
dysfunction in the formation of retraction pockets and later cholesteatoma
was described by Kim and Chole [30]. By ligating the eustachian tube of
Mongolian 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 active
cholesteatoma in some patients with an attic retraction pocket while others
continue to have a quiescent and self-cleaning pocket remain unclear. It has
been shown recently that the combination of tympanic membrane retraction
and basal cell proliferation is the hallmark for cholesteatoma formation and
development.
In a cohort of healthy children age 5 to 16 years, the prevalence of attic
retractions was between 14% and 25% of ears [31]. In a separate cohort of
children treated for secretory otitis with pressure equalization tube insertion
with or without adenoidectomy and followed up to 18 years, the incidence of
severe retractions (behind the scutum with some bone resorption) was 5% to
6% and attic cholesteatoma was 0.2% to 1.7%. Sudhoff and Tos [31] per-
formed immunohistochemical analysis of surgical specimens obtained
from 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 canal
skin. In addition, the investigators analyzed the integrity of the basement
membrane by using avidin biotin complex peroxidase to stain collagen
type 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.
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1151

continuity was seen at the cholesteatoma–lamina propria interface, whereas

the integrity was preserved in the adjacent normal auditory canal skin. They
also showed an increased expression of MIB-1 in the keratinocytic popula-
tion of the basal cell layer. This increased expressivity was consistent with
proliferating keratinocytes localized primarily in small epithelial cones or
pseudopods growing into the subepithelial stroma through interruptions
of 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 and
the 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 created
breaks in the basement membrane allowing the formation of epithelial cones
that grow toward the stroma (papillary ingrowth theory). The combination
of subepithelial invasion and keratinocytic proliferation in the form of
microcholesteatoma is the hallmark of the precholesteatomatous stage of
cholesteatoma.
As the microcones expand and fuse together, an attic cholesteatoma is
formed. Using the normal postauricular skin as control, Albino and co-
workers [32] found a nine- to 20-fold increase in the expression of p53 in
cholesteatoma tissue, trough all epithelial layers. The p53 proteins by acti-
vating downstream products (p21/WAF1, GADD45, and mdm2) appear
to have a role in the down-regulation of cellular proliferation and promo-
tion of apoptosis [33], a checkpoint control mechanism to protect the cell
from genetic alterations. Similarly, they noted a two-fold increase in the ex-
pression of Ki-67, a marker of cellular proliferation, in cholesteatoma tissue
compared with control normal postauricular skin. According to Albino
and coauthors [34], the increased p53 expression was a feedback negative
response to control an increased proliferative state as witnessed by the
increase 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 of
the cholesteatoma and to a lesser degree an increase in the expression of CK
5/6 and CK 1/10, markers of epithelial migration. They concluded that cel-
lular migration (or invasion) and proliferation play a role in the expansion
of 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
1152 SEMAAN & MEGERIAN

resulted in destruction of the tympanic membrane, erosion of the ossicular

chain, 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 surrounding
middle ear soft tissue structures and bone. It remains unclear to what factors
predict the biologic behavior of these lesions, such as recidivism and a more
aggressive clinical course.
Mallet and colleagues [37] found a correlation between the aggressiveness
of the clinical behavior of cholesteatoma and the index of proliferation. In
their analysis of surgical specimen from 91 ears with cholesteatoma, MIB-1
was detected in 23% of the ears with moderate bony destruction (single os-
sicle affected) versus 56% of ears with severe bony destruction (two or more
ossicles, meningeal exposure, denudation of the facial nerve or sigmoid
sinus, and erosion of the lateral semicircular canal). These findings were
statistically 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. They
compared the expression pattern of eight cholesteatoma to normal postaur-
icular skin samples. They found an upregulation or induction in genes
involved in cellular proliferation and differentiation (calgranulin A, calgra-
nulin B, psoriasin, thymosin b-10) and cell invasion (cathepsin C, cathepsin
D, cathepsin H, and matrix metalloproteinase 9 [MMP-9]). These results
were 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 of
the 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 the
S100 protein family, has been shown to be increased in hyperproliferative
and inflammatory skin conditions and are believed to play a role in kerati-
nocytic differentiation [40]. Upregulation and induction of these genes may
reflect an alteration in keratinocyte differentiation and migration leading to
keratin overproduction and accumulation as seen in cholesteatoma. The ca-
thepsin family is a group of lysosomal proteases that play a key role in the
degradation of intracellular and extracellular proteins in the epidermis and
have 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 cell
death resulting in the production of larger amount of keratin debris respon-
sible for the expansion and keratin accumulation seen in cholesteatoma. The
implication of apoptotic cell death has been demonstrated recently [43]. Cas-
pase-8 activation, a known effector of the extrinsic pathway of apoptosis, is
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1153

triggered by activation of the cell surface death receptors (tumor necrosis

factor [TNF] family, Fas-L/Fas-R). This activation results in the activation
of an end product of apoptosis, caspase-3, that induces the nuclear translo-
cation of effector molecules that result in apoptosis and programmed cell
death. 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 inactivation
of IkB-a activates NF-kB and results in nuclear translocation of the tran-
scription factor. The activation of NF-kB suppresses apoptosis induced by
TNF-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 granular
layer. The retroauricular skin was used as a control. The NF-kB proteins
were localized in the perinuclear region suggesting that the mechanism of
negatively controlling apoptosis was inactivated leading to keratinocyte
cell death and keratin accumulation.
These findings strongly suggest differential properties inherent to choles-
teatoma compared with normal epidermal keratinocytes that may explain
their 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 and
factors responsible for the genesis and progression of middle ear cholestea-
toma remain unclear, and further research is warranted to help elucidate
these missing links.

Secondary acquired cholesteatoma

Secondary acquired cholesteatoma has been described to occur as the
result of the migration of tympanic membrane epidermis into the middle
ear at the site of a marginal perforation or as the result of the implan-
tation of viable keratinocytes into the middle ear cleft. The implantation
occurs 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 latter
have 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 from
147 soldier-patients that sustained blast injuries with perforation of tym-
panic membrane localized to the pars tensa. These investigators reported
an incidence of 4.8% of invasive cholesteatoma. Freeman [45] reported three
cases of cholesteatoma secondary to temporal bone fracture. The keratino-
cytes appear to have invaded into the middle ear cleft through the fracture
sites.
1154 SEMAAN & MEGERIAN

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 cholesteatoma
attributed to the insertion of the pressure equalization tube. The presence of
cholesteatoma around the tube site was a prerequisite to incriminate the
procedure as a cause of the cholesteatoma. They also noted a higher inci-
dence in children aged less than 5 years, those with placement of Goode
T-tubes, children with frequent reinsertions, patients with duration of place-
ment exceeding 12 months, and ears with history of frequent postoperative
otorrhea. Ferguson and coworkers [47] described the reasons for cholestea-
toma formation after a stapedectomy. The investigators described four
mechanisms: prosthesis extrusion independent of eustachian tube dysfunc-
tion, inadvertent implantation of keratinocytes with the oval window fat
graft, malpositioned inverted tympanomeatal flap, and migration at the
site of a marginal tympanic membrane perforation.
Eavey and coworkers [48], and Camacho and colleagues [49] were able to
produce 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 keratin
was observed up to 9 months postimplantation. Various histopathologic
changes ranging from granulation tissue to cholesteatoma formation were
described. The investigators concluded that neonatal aspiration of lanugo
and viable keratinocytes can result in middle ear inflammation that in the
chronic stage can lead to cholesteatoma formation. Bernal-Sprekelsen and
coworkers [50], argued against this model of implantation of keratinocytes
as an etiology for cholesteatoma and failed to find keratinizing epithelial
cells in 31 temporal bones of infants who died before 1 year of age and
27 temporal bones of preterm fetuses that succumbed to various conditions.
Despite the fact that the neonatal aspiration of viable keratinocytes may not
fully account for the development of congenital cholesteatoma, it provides
a valuable experimental platform that the implantation of viable keratino-
cytes can lead to formation of middle ear or mastoid cholesteatoma. This
is observed frequently in revision middle ear surgery and described as
a ‘‘cholesteatomatous pearl’’ formation that is the result of a trapped viable
keratinocytic 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 at
the edges of a tympanic membrane perforation as a possible cause for cho-
lesteatoma development. By creating a tympanic membrane perforation and
latex with 50% propylene glycol, the investigators succeeded in producing
cholesteatoma in 90% and 80% of their animals, respectively. They con-
cluded that latex provides a biomembrane that favors neoangiogenesis
and forms a bridge for epithelial migration. This environment is enhanced
further by a cytokine-producing acute or chronic inflammatory milieu cre-
ated by the inciting material. This model may provide evidence that
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1155

epithelial migration of keratinizing epithelium at the site of a tympanic

membrane 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 by
the ongoing research to help elucidate the mechanism of expansion, bone
destruction and invasion seen in middle ear cholesteatoma. Two predomi-
nant mechanisms are believed to account for the osteolysis seen in middle
ear cholesteatoma: pressure-induced bone resorption and enzymatic dissolu-
tion of bone by cytokine-mediated inflammation. Pressure necrosis initially
described by Steinbrügge in 1879 and Walsh in 1951, and direct bone resorp-
tion as described by Chole and coworkers [52] in 1985 have been proposed
as possible mechanisms of bone destruction. Chole and colleagues im-
planted silicone sheets in the middle ear of gerbils without cholesteatoma
and 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 biomechanical
factors 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-induced
and cytokine-induced bone destruction has been studied in the last two de-
cades. Matrix metalloproteinases (MMP), a family of zinc metalloenzymes
that degrades unmineralized extracellular matrix, have been shown to be
present in the cholesteatoma [55]. MMP-2 (72 kD collagenase) and MMP-9
(92 kD collagenase) were expressed in suprabasal epithelial layers of
cholesteatoma.
Other investigators found the increased expression of MMP-9 but not
MMP-2 in cholesteatoma cells [56]. Schmidt and coworkers [56] analyzed
the 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 37
cholesteatoma and nine external ear skin specimens. IL-1a production was
found to be significantly elevated; however, no correlation was found between
MMP-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 compared with 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 activity
that produced acid phosphatase-induced bone demineralization. The
amount of osteolysis was increased by adding osteoblasts to the TNF-a–
treated osteoclasts containing medium, suggesting a cell to cell interaction
mediated by TNF-a. In addition, the latter enhanced the production of
1156 SEMAAN & MEGERIAN

collagenases by macrophages and osteoblasts. However, by performing en-

zyme-linked immunosorbent assay on tissue samples from 23 patients with
cholesteatoma 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 of
two patients with ruptured cholesteatoma sac resulting in local inflamma-
tion and osteolysis [59]. These changes were associated with a small abscess
formation at the site of the rupture. They noted a marked inflammatory
cellular infiltrate surrounding the rupture site with evidence of epithelial
proliferation 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 vivo
analysis of a murine model of cholesteatoma-induced bone resorption and
in vitro analysis of osteoclast culture, the investigators studied the gene ex-
pression of nitric oxide synthase (NOS) and the effect of aminoguanidine (an
inhibitor of cytokine mediated nitrite production). They showed a selective
upregulation of the inducible NOS or NOS II compared with NOS I and III
and a dose-dependent stimulation of osteoclastic activity (not proliferation)
using low concentration of nitric oxide donors (sodium nitroprusside and
S-nitro-N-acetyl-D, L-penicillamine). In vitro, only interferon (IFN)-g (not
IL-1b or TNF-a) was able to generate nitrite. This nitrite production was
blocked in vitro by the addition of aminoguanidine (but not in vivo) and
was synergistically enhanced in the presence of IFN-g, IL-1b, and TNF-a.
These findings indicate a role for nitric oxide in the osteoclastic-mediated
bone resorption in cholesteatoma and suggest the implication of additional
cytokines in the in vivo osteoclastogenesis and bone resorption. In contrast
to 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 in
the perimatrix of 21 cholesteatoma surgically obtained. These studies high-
light the importance of osteolysis and its regulatory mechanisms in the bone
destruction 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 appear
to 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 between
anatomic, inflammatory, and regulatory factors of cellular proliferation
and differentiation, the exact mechanism responsible for the invasion, recid-
ivism, and destruction seen in this disease remains unknown.
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1157

References
[1] Soldati D, Mudry A. Knowledge about cholesteatoma, from the first description to the mod-
ern histopathology. Otol Neurotol 2001;22(6):723–30.
[2] Duverney J. Traite de l’Organe de l’Ouie. Paris: E.Michaillet; 1683.
[3] Cruveilhier J. Anatomie Pathologique du Corps Humain. Paris: Bailliere; 1829.
[4] Müller J. Ueber den feineren Bau und die formen der krankhaften Geschwulste. Berlin:
G.Reimer; 1838.
[5] Virchow R. Ueber Perlgeschwulste. Arch Anat Physiol Klin Med 1855;8:371–418.
[6] VonTroeltsch A. Lehrbuch der Ohrenheilkundle. Leipzig Germany: FCW Vogel; 1873.
[7] VonTroeltsch A. Die Anatomie des Ohres in ihrer Anwendung auf die Praxis und die Krans-
kheiten des Gehorogans. Leipzig Germany: FCW Vogel; 1861.
[8] Gruber J. Das Cholesteatome (Perlgeschwulst). Berlin: Cart Gerald’s Sohn; 1888.
[9] Wendt H. Desquamativd Entzundung des Mittelohres. Arch Phys Heilkdt Wagner 1873;14:
428–46.
[10] Rokitansky C. Neubildung von ausserer haut, Schleim und seroser Haut. Vienna: W Brau-
muller; 1855.
[11] Bezold F. Cholesteatom, Perforation der Membrana Flaccida Schrapnelli und Tubenver-
schulus: eime atiologische studie. Vol 20: Z. Ohrenheilkd; 1889.
[12] Habermann J. Cholesteatom des Mittelohres, seine Entstehung. Z. Ohrenheilkdt. 1889;
19:348.
[13] Lucae. Politzer’s Textbook of the disease of the ear for students and practitioners. Philadel-
phia: Lea and Febiger; 1926.
[14] Körner O. Die eitrigen Erkrankungen des Schlafenneins. Weisbaden Germany: JF Berg-
mann; 1899.
[15] Derlacki EL, Clemis JD. Congenital cholesteatoma of the middle ear and mastoid. Ann Otol
Rhinol Laryngol 1965;74(3):706–27.
[16] Levenson MJ, Parisier SC, Chute P, et al. A review of twenty congenital cholesteatomas of
the middle ear in children. Otolaryngol Head Neck Surg 1986;94(5):560–7.
[17] Tos M. A new pathogenesis of mesotympanic (congenital) cholesteatoma. Laryngoscope
2000;110(11):1890–7.
[18] Teed RW. Cholesteatoma verum tympani (its relationship to the first epibranchial placode).
Arch Otolaryngol 1936;24:455–74.
[19] Michaels L. An epidermoid formation in the developing middle ear: possible source of cho-
lesteatoma. J Otolaryngol 1986;15(3):169–74.
[20] Karmody CS, Byahatti SV, Blevins N, et al. The origin of congenital cholesteatoma. Am
J Otol 1998;19(3):292–7.
[21] Potsic WP, Korman SB, Samadi DS, et al. Congenital cholesteatoma: 20 years’ experience at
The Children’s Hospital of Philadelphia. Otolaryngol Head Neck Surg 2002;126(4):409–14.
[22] Rüedi L. Cholesteatoma formation in the middle ear in animal experiments. Acta Otolaryngol
1959;50(3–4):233–40 [discussion: 240–2].
[23] Rüedi L. Pathogenesis and treatment of cholesteatoma in chronic suppuration of the tempo-
ral bone. Ann Otol Rhinol Laryngol 1957;66(2):283–305.
[24] Liang J, Michaels L, Wright A. Immunohistochemical characterization of the epidermoid
formation in the middle ear. Laryngoscope 2003;113(6):1007–14.
[25] Michaels L. Origin of congenital cholesteatoma from a normally occurring epidermoid rest
in the developing middle ear. Int J Pediatr Otorhinolaryngol 1988;15(1):51–65.
[26] Sade J. Retraction pockets and attic cholesteatomas. Acta Otorhinolaryngol Belg 1980;
34(1):62–84.
[27] Sade J, Halevy A. The natural history of chronic otitis media. J Laryngol Otol 1976;90(8):
743–51.
[28] Chole RA, Faddis BT. Evidence for microbial biofilms in cholesteatomas. Arch Otolaryngol
Head Neck Surg 2002;128(10):1129–33.
1158 SEMAAN & MEGERIAN

[29] Wang EW, Jung JY, Pashia ME, et al. Otopathogenic pseudomonas aeruginosa strains as
competent biofilm formers. Arch Otolaryngol Head Neck Surg 2005;131(11):983–9.
[30] Kim HJ, Chole RA. Experimental models of aural cholesteatomas in Mongolian gerbils.
Ann Otol Rhinol Laryngol 1998;107(2):129–34.
[31] Sudhoff H, Tos M. Pathogenesis of attic cholesteatoma: clinical and immunohistochemical
support for combination of retraction theory and proliferation theory. Am J Otol 2000;21(6):
786–92.
[32] Albino AP, Reed JA, Bogdany JK, et al. Expression of p53 protein in human middle ear cho-
lesteatomas: pathogenetic implications. Am J Otol 1998;19(1):30–6.
[33] Perry ME, Levine AJ. Tumor-suppressor p53 and the cell cycle. Curr Opin Genet Dev 1993;
3(1):50–4.
[34] Albino AP, Kimmelman CP, Parisier SC. Cholesteatoma: a molecular and cellular puzzle.
Am J Otol 1998;19(1):7–19.
[35] Kim HJ, Tinling SP, Chole RA. Expression patterns of cytokeratins in cholesteatomas:
evidence of increased migration and proliferation. J Korean Med Sci 2002;17(3):381–8.
[36] Olszewska E, Lautermann J, Koc C, et al. Cytokeratin expression pattern in congenital and
acquired pediatric cholesteatoma. Eur Arch Otorhinolaryngol 2005;262(9):731–6.
[37] Mallet Y, Nouwen J, Lecomte-Houcke M, et al. Aggressiveness and quantification of epithe-
lial proliferation of middle ear cholesteatoma by MIB1. Laryngoscope 2003;113(2):328–31.
[38] Tokuriki M, Noda I, Saito T, et al. Gene expression analysis of human middle ear cholestea-
toma using complementary DNA arrays. Laryngoscope 2003;113(5):808–14.
[39] Goebeler M, Roth J, van den Bos C, et al. Increase of calcium levels in epithelial cells induces
translocation of calcium-binding proteins migration inhibitory factor-related protein 8
(MRP8) and MRP14 to keratin intermediate filaments. Biochem J 1995;309(Pt 2):419–24.
[40] Algermissen B, Sitzmann J, LeMotte P, et al. Differential expression of CRABP II, psoriasin
and cytokeratin 1 mRNA in human skin diseases. Arch Dermatol Res 1996;288(8):426–30.
[41] Ravdin PM. Evaluation of cathepsin D as a prognostic factor in breast cancer. Breast Cancer
Res Treat 1993;24(3):219–26.
[42] Amar MS, Wishahi HF, Zakhary MM. Clinical and biochemical studies of bone destruction
in cholesteatoma. J Laryngol Otol 1996;110(6):534–9.
[43] Miyao M, Shinoda H, Takahashi S. Caspase-3, caspase-8, and nuclear factor-kappaB
expression in human cholesteatoma. Otol Neurotol 2006;27(1):8–13.
[44] Wolf M, Kronenberg J, Ben-Shoshan J, et al. Blast injury of the ear. Mil Med 1991;156(12):
651–3.
[45] Freeman J. Temporal bone fractures and cholesteatoma. Annals of Otology, Rhinology and
Laryngology 1983;92(6 Pt 1):558–60.
[46] Golz A, Goldenberg D, Netzer A, et al. Cholesteatomas associated with ventilation tube
insertion. Arch Otolaryngol Head Neck Surg 1999;125(7):754–7.
[47] Ferguson BJ, Gillespie CA, Kenan PD, et al. Mechanisms of cholesteatoma formation
following stapedectomy. Am J Otol 1986;7(6):420–4.
[48] Eavey RD, Camacho A, Northrop CC. Chronic ear pathology in a model of neonatal
amniotic fluid ear inoculation. Arch Otolaryngol Head Neck Surg 1992;118(11):1198–203.
[49] Camacho AE, Eavey RD, Northrop C. Juvenile keratin inoculation induces chronic ear
pathology. Am J Otol 1997;18(6):773–9.
[50] Bernal-Sprekelsen M, Sudhoff H, Hildmann H. Evidence against neonatal aspiration of ker-
atinizing epithelium as a cause of congenital cholesteatoma. Laryngoscope 2003;113(3):
449–51.
[51] Massuda ET, Oliveira JA. A new experimental model of acquired cholesteatoma. Laryngo-
scope 2005;115(3):481–5.
[52] Chole RA, McGinn MD, Tinling SP. Pressure-induced bone resorption in the middle ear.
Ann Otol Rhinol Laryngol 1985;94(2 Pt 1):165–70.
[53] Burger EH, Klein-Nulen J. Responses of bone cells to biomechanical forces in vitro. Adv
Dent Res 1999;13:93–8.
 PATHOPHYSIOLOGY OF CHOLESTEATOMA 1159

[54] Klein-Nulend J, van der Plas A, Semeins CM, et al. Sensitivity of osteocytes to biomechan-
ical stress in vitro. FASEB J 1995;9(5):441–5.
[55] Schonermark M, Mester B, Kempf HG, et al. Expression of matrix-metalloproteinases and
their inhibitors in human cholesteatomas. Acta Otolaryngol 1996;116(3):451–6.
[56] Schmidt M, Grunsfelder P, Hoppe F. Up-regulation of matrix metalloprotease-9 in middle
ear cholesteatoma–correlations with growth factor expression in vivo? Eur Arch Otorhino-
laryngol 2001;258(9):472–6.
[57] Yan SD, Huang CC. The role of tumor necrosis factor-alpha in bone resorption of choles-
teatoma. Am J Otolaryngol 1991;12(2):83–9.
[58] Yetiser S, Satar B, Aydin N. Expression of epidermal growth factor, tumor necrosis factor-
alpha, and interleukin-1alpha in chronic otitis media with or without cholesteatoma. Otol
Neurotol 2002;23(5):647–52.
[59] Suzuki C, Ohtani I. Bone destruction resulting from rupture of a cholesteatoma sac: tempo-
ral bone pathology. Otol Neurotol 2004;25(5):674–7.
[60] Jung JY, Pashia ME, Nishimoto SY, et al. A possible role for nitric oxide in osteoclastogen-
esis associated with cholesteatoma. Otol Neurotol 2004;25(5):661–8.
[61] Hamzei M, Ventriglia G, Hagnia M, et al. Osteoclast stimulating and differentiating factors
in human cholesteatoma. Laryngoscope 2003;113(3):436–42.