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Citation for published version (APA):Mozaffari, M., Nash, R., & Tucker, A. (Accepted/In press). Anatomy and development of the mammalian externalauditory canal: Implications for understanding canal disease and deformity. Frontiers in Cell and DevelopmentalBiology.
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Download date: 21. Apr. 2022
Anatomy and development of the mammalian external auditory canal: 1
Implications for understanding canal disease and deformity 2
3
4
Mona Mozaffari1*, Robert Nash2, Abigail S. Tucker1 5
6
1Centre for Craniofacial and Regenerative Research, King’s College London, Floor 27, Guy’s 7
Hospital, London SE1 9RT 8
2Department of Paediatric Otolaryngology, Cochlear Implants, Great Ormond Street Hospital for 9
Children NHS Trust, London, UK 10
11
* Correspondence: 12
Corresponding Author 13
15
16
Key words: Hearing; Deafness; External Ear; Ear Canal; Congenital Aural Atresia 17
18
19
20
21
22
23
24
Anatomy and development of the external auditory canal
2
This is a provisional file, not the final typeset article
Abstract 25
The mammalian ear is made up of three parts (the outer, middle and inner ear), which work together 26
to transmit soundwaves into neuronal signals perceived by our auditory cortex as sound. This review 27
focuses on the often-neglected outer ear, specifically the external auditory meatus (EAM), or ear 28
canal. Within our complex hearing pathway, the ear canal is responsible for funnelling soundwaves 29
toward the tympanic membrane (ear drum) and into the middle ear, and as such is a physical link 30
between the tympanic membrane and the outside world. Unique anatomical adaptations, such as its 31
migrating epithelium and cerumen glands, equip the ear canal for its function as both a conduit and a 32
cul-de-sac. Defects in development, or later blockages in the canal, lead to congenital or acquired 33
conductive hearing loss. Recent studies have built on decades-old knowledge of ear canal 34
development and suggest a novel multi-stage, complex and integrated system of development, 35
helping to explain the mechanisms underlying congenital canal atresia and stenosis. Here we review 36
our current understanding of ear canal development; how this biological lumen is made; what 37
determines its location; and how its structure is maintained throughout life. Together this knowledge 38
allows clinical questions to be approached from a developmental biology perspective. 39
40
41
42
43
44
45
46
47
48
Running Title
3
Introduction 49
Hearing places us within our external environment, allowing us to experience a multi-dimensional 50
world, to listen and to communicate. Sound is essentially a series of pressure waves in our airborne 51
environment. That we can ‘hear’ these waveforms involves a complex array of neurophysiological 52
mechanisms which begin at the outer ear and end at the auditory cortex. The mammalian ear is a 53
crucial and fascinating sensory organ formed from the integration of three parts (Figure 1). The pinna 54
or auricle directs soundwaves into the external auditory Meatus (EAM), which then funnels sound 55
waves towards the ear drum or tympanic membrane (TM), causing it to displace and move the 56
ossicular chain of bones in the air-filled middle ear. The middle ear ossicles connect the TM to the 57
much smaller oval window of the inner ear. This intricate leverage mechanism corrects the 58
impedance mismatch between gas and liquid allowing airborne sound waves to move hair cells in the 59
fluid-filled cochlea, generating neural signals that are transmitted to the auditory cortex via the 60
cochlear nerve. 61
62
Hearing loss is most commonly caused due to pathology in the inner ear, referred to as sensorineural 63
hearing loss, which consequently receives the lion’s share of scientific research (1). However, there 64
are limited options for treating sensorineural hearing loss with many potential treatments in the 65
experimental phase (2). Currently, the clinician’s arsenal is best equipped to treat middle ear disease, 66
which consequently receives the lion’s share of clinical attention (1). This leaves the outer ear, which 67
is also indispensable for hearing, but is comparatively overlooked. Pathologies of the ear canal can 68
cause hearing loss, recurrent infection, and complications including cranial nerve palsy and 69
intracranial sepsis (3). Management of hearing loss in this part of the ear may be particularly 70
challenging, as the interaction between the anatomy and physiology of the canal, and the pathologies 71
Anatomy and development of the external auditory canal
4
This is a provisional file, not the final typeset article
that affect it, may complicate the two most commonly used techniques of hearing rehabilitation – 72
hearing aids and hearing restoration surgery. 73
74
In this review we focus in on the ear canal or EAM. We discuss the anatomy of the canal and the 75
epithelial dynamics involved in canal homeostasis. We then turn to the congenital and acquired 76
defects of the canal, and how they are currently treated, before investigating canal development. As 77
anatomical and molecular details are added to our understanding of ear development, compelling 78
questions are beginning to emerge. What factors determine the development of the ear canal’s 79
specialised epithelium? And how does the canal integrate with the middle ear to form a functioning 80
structure? 81
82
Ear canal anatomy and function 83
With certain exceptions, the EAM is similar across all mammals; a deep-set structure on either side 84
of the skull ending in an ear drum cul-de-sac medially and opening to a pinna laterally (Figure 1). In 85
adult humans, the EAM is typically 25mm in length and 8 mm in diameter (4). Rather than a 86
horizontal tube its shape is that of a soft sigmoid, tilted to face downwards and inwards as it projects 87
medially (5). This curved path varies between individuals (5). Finite element modelling of the ear 88
has highlighted the importance of a patent EAM in hearing higher frequency sound (which is crucial 89
for speech recognition) (4). Lower frequencies with wavelengths larger than the canal itself are not 90
affected by variations in canal diameter, however, as soundwaves concertina at higher frequency, 91
variations in canal dimension have a discernible effect on sound transmission. In particular, the 92
segment of canal closest to the TM plays an important part in high frequency sound transmission (4). 93
Running Title
5
Variation in this pretympanic area, namely a deeper pretympanic recess, therefore can influence 94
acquisition of high frequency sound, and has also been linked to increased susceptibility to chronic 95
otitis externa (5). Such biomechanical modelling at a patient-level would provide valuable 96
information in understanding stenotic canal disease and measuring treatment outcomes. 97
98
In contrast to the deep canal observed in most mammals, in sauropsids (reptiles and birds) and 99
amphibia, the ear canal is typically shallow or non-existent (Figure 2A,B). Indeed, we can consider a 100
deep canal to be a specific mammalian trait. It has been proposed that a cavitated middle ear and 101
tympanic membrane evolved multiple times in land vertebrates (tetrapods), therefore the deep 102
mammalian ear is not homologous to the shallow ear canal of other land vertebrates (6). A deep 103
canal is observed in both therian mammals (placentals and marsupials) and monotremes (egg laying 104
platypus and echidna), however monotremes do not have pinna, suggesting that this was a later 105
therian adaptation as monotremes diverged from other mammals relatively early in evolutionary time 106
(Figure 2C,F) (7). The deep canal means that the mammalian tympanic membrane is protected within 107
the head and therefore potentially at less risk of damage. Despite this, damage to the mammalian 108
tympanic membrane is fairly common, with perforations being caused by loud noises, bangs to the 109
head and from middle ear infections (8). A deep canal also provides a means for funnelling and 110
thereby focusing sound waves, as discussed above. 111
112
Depending on the position of the middle ear with respect to the exit position of the canal, the relative 113
length of the canal can vary across mammals. For example, in the platypus the ear canal exits high on 114
the head, next to the eye, so that both the eye and ear are not submerged while the animal is 115
swimming (7). This results in the development of a very long canal (Figure 2C). How the developing 116
Anatomy and development of the external auditory canal
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This is a provisional file, not the final typeset article
ear canal reaches the middle ear, finding its way to varying exit sites in different species, remains a 117
largely unanswered question. 118
119
A deep-set lumen requires structural support. In humans, the inner two-thirds of the external auditory 120
meatus is bony (typically 16mm in length in the human adult) and the outer third is cartilaginous 121
(around 8mm in length), encasing the canal with a semi-rigid wall (9). How the mammalian ear canal 122
evolved remains an intriguing question. Did pinna cartilage elongate inwards to meet the bony canal 123
or did the cartilaginous canal evolve separately from the pinna? The latter appears more likely as 124
monotremes have no pinna but still have a cartilaginous support that forms around the developing 125
EAM (Figure 2E) (10). Cartilage support for the EAM, therefore, likely predates pinna evolution. 126
127
The newborn human ear canal is soft and rudimentary along its length (11). The osseous canal is not 128
evident during embryonic development so appears to develop postnatally (12). The specifics of its 129
development, however, remain vague. It is not yet known whether the tympanic ring, which is a 130
membranous bone, extends posterolaterally to take part in creation of the osseous canal, or whether 131
the osseous canal forms by endochondral ossification of an initially fibrocartilaginous scaffold. Both 132
scenarios are described in textbooks and articles, however there are as yet no published studies 133
demonstrating the process (9,11). The cartilaginous canal appears prior to and apparently 134
independent of the osseous canal (12). From around 15 gestational weeks, a chain of bar like 135
cartilages form inferiorly between the tragus and helix. The anterior cartilage of the tragus and the 136
posterior cartilage of the helix develop early and are consistent in shape; ring and plate-like 137
respectively (12). Rather than fibrocartilage, glial fibrillary acid protein (GFAP) positive elastic 138
Running Title
7
cartilage makes up the outer cartilaginous canal (12). The cartilages transform morphology via 139
intermediate circular and triangle shapes to become H-shaped cartilage wrapping their arms upwards 140
around the developing EAM epithelium (12). Interestingly, these bars of cartilage appear to develop 141
along a fascial condensation linking Reichert’s cartilage and the tragus (12). No evidence of a 142
corresponding fascial plane extending out from Meckel’s cartilage has been described that would 143
potentially precede a superior wall (Figure 1 insert). Unlike the anterior canal, the posterosuperior 144
canal is usually deficient of cartilage in the adult (9). There is significant individual variation in canal 145
shape, which is perhaps not surprising given the complex of irregular shaped cartilages that interlink 146
to form the cartilaginous ear canal (13). Clinically, this variability appears to be of little consequence. 147
Inevitable gaps between the jigsaw of connecting cartilages does however provide a likely 148
explanation for the aetiology of congenital auricular pseudocysts that can occur in multiples between 149
the perichondrium and the skin of the external ear, requiring drainage and pinna reconstruction (14). 150
151
The lining of the ear canal is continuous with skin covering the pinna laterally and the outer layer of 152
the tympanic membrane medially (15). Being an anatomical cul-de-sac ending at the TM, the ear 153
canal is lined with specialised skin capable of migrating, such that it can self-clean with the 154
epidermal layer sloughing off and moving outwards – as opposed to upwards in skin elsewhere 155
(16,17). A failure of this self-cleaning mechanism leads to the build-up of sloughed off keratinocytes 156
in the canal, causing hearing impairment from blockage and localised tissue damage from chronic 157
inflammation (18). Indeed, failure to remove ear canal skin is also observed in keratosis obturans and 158
ear canal cholesteatoma, which are uncommon but well recognised diseases of the external ear 159
discussed further below (18,19). 160
161
Anatomy and development of the external auditory canal
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This is a provisional file, not the final typeset article
Experiments tracing the movement of ink-tattooed ear canal skin cells in mammals (including 162
humans) show these labelled cells moving laterally, out of the canal over time (16,20). This 163
movement occurs roughly at the rate of fingernail growth with anterior canal skin demonstrating the 164
fastest migration (16). Does this migratory pattern have an epicentre at the tympanic membrane? 165
Dated dye experiments suggest a radial pattern of epidermal cell migration spoking away from the 166
umbo, at the centre of the TM (21). More recent experiments using BrdU to chase and label 167
proliferating cells suggest a more involved and dynamic source of stem and progenitor cells that 168
migrate in different directions in different parts of the TM (22,23). Frumm et al., add further detail to 169
the migratory nature of the keratinocytes in the tympanic membrane’s outer layer. Using a 170
combination of lineage tracing, live imaging and single cell sequencing, they demonstrate that the 171
TM epidermis has distinct stem cell and committed progenitor regions, located close to supporting 172
mesenchyme (the manubrium and annulus respectively). The progeny of these cells migrate out 173
across the TM, maintaining a thin vibratory surface (24). 174
175
There are distinctions in the morphology of the skin that covers the bony canal versus the skin that 176
covers the cartilaginous canal in humans. The former is thin and flush against the canal wall bone, 177
whilst the latter is thickened with a spongey subcutaneous layer possessing modified sebaceous 178
glands called cerumen glands. The bony canal skin, does not possess ceruminous glands or hair 179
follicles (9). Our observations of human cranial skin verses ear canal skin development suggest the 180
latter develops precociously in relation to skin (development paper). This has interesting implications 181
for experimental comparisons drawn between ear canal skin and skin elsewhere, as well as clinical 182
implications, where skin from the limbs is used to reconstruct the canal lumen. If a different 183
Running Title
9
developmental timeline leads to different skin biology, transplanting skin into a reconstructed ear 184
canal may not be the ideal graft tissue. 185
186
The cerumen glands are responsible for the production of ear wax. Serous secretions and sebum from 187
the cerumen glands mix with sloughed keratinised squames. A natural lubricant, ear wax aids the 188
self-cleaning function of the ear canal, and is thought to have antimicrobial properties (25). 189
Interestingly, a little like tongue rolling ability, ear wax type is a dimorphic trait. Whether your wax 190
is wet or dry is determined by one single-nucleotide polymorphism (SNP) of a single gene 191
(ABCC11) (26). Patients with dry wax, which lacks oily components, are at higher risk of suffering 192
from cerumen impaction. Impacted ear wax is the single most common ear canal ailment to affect 193
patients and may require regular removal, especially in those using hearing aids (27). The human 194
ABCC11 gene has no orthologous gene in mammals, except for primates. In mice, cerumen 195
production is from a single large gland, the glandula ceruminosa, which opens into a single duct near 196
the TM (28). Layout of glands and secretion type is likely to be species specific and relate to size and 197
position of the canal. In mice, this gland is present from E15 (28). Human ceruminous glands are 198
noted at around 19 gestational weeks in published literature (29), although we see them even earlier 199
at 13 weeks (unpublished data from Tucker lab). It is likely that ceruminous glands evolved from 200
modified sebaceous glands, as like sebaceous glands these are holocrine glands, were the cells burst 201
to release their content (30). Sebaceous glands fail to develop normally in patients with hypohidrotic 202
ectodermal dysplasia (XLHED), suggesting involvement of the ectodysplasin (EDA) receptor 203
signalling pathway in the development of sebaceous glands (31). Similarly, Eda mutant mice have 204
defects in sebaceous glands, with addition of Eda stimulants able to rescue the defect (32). 205
Meibomian glands, another example of a modified sebaceous gland, also fail to develop in rodent 206
models carrying hypomorphic mutations in the Edar signalling pathway consistent with an XLHED 207
Anatomy and development of the external auditory canal
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This is a provisional file, not the final typeset article
phenotype, with increased Edar signalling leading to larger meibomian glands (33). Recently Edar 208
has been shown to be expressed in developing rat cerumen glands, highlighting a conserved role for 209
this pathway in these holocrine glands (31). In addition to the Eda pathway, Wnt and hedgehog 210
signalling have been shown to play a role in skin sebaceous gland development and therefore may 211
have a role also in cerumen gland development(34,35). Further study into the developmental 212
mechanisms of these unique glands would certainly be revealing. 213
214
Development of the ear canal and how it reaches its target 215
The ear canal develops in two parts, the outer part or primary canal, and the inner part or meatal plug 216
(36) (Figure 3). At around Carnegie stage 17 (6 weeks post conception and equivalent to E12.0 in 217
mice), the primary canal begins to form either by the first cleft deepening or as a new invagination 218
within the 1st arch. Supporting the latter scenario is a recent study unexpectedly suggesting the 219
murine ear canal forms entirely within the first arch (37). This is unexpected because it had 220
previously been assumed that the ear canal forms within the first cleft (38). The meatal plug then 221
forms as a plug of cells adjacent to this cleft. (20). This plug of cells will continue to extend inwards 222
into the surrounding mesenchyme becoming a finger-like projection pointing to the developing 223
middle ear cavity (MEC) (Figure 3B) (39). Temporal and situational differences in cell proliferation 224
appear to influence the extension and directional growth of the meatal plug as it begins to develop 225
(36). Both human and mouse display a distinctive duality in EAM development: the outer, primary 226
canal (which will become the cartilaginous canal) develops differently to the inner, meatal plug 227
(which will become the osseous canal) (36). Initially open, the primary canal closes for some time 228
before opening again along with the meatal plug, to form one long lumen (36) [figure 3C,E]. This 229
closure has been linked to the removal of periderm (36). In mouse embryos, a superficial layer of 230
Running Title
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periderm, denoted by keratin 8 staining, acts as a non-stick Teflon layer to keep the primary canal 231
open in early development (36). 232
233
Programmed cell death, or apoptosis, is a ubiquitous event in development. Its role in ear canal 234
development has been previously alluded to in experiments noting the emergence of TUNEL-positive 235
cells at E15.5 in mice with the surprising lack of them around the time of canal opening, up to 12 236
days postnatally(40). Inhibiting apoptosis in culture and staining K8 positive peridermal cells 237
demonstrated that it is the apoptotic death of the periderm that allows the primary canal to close at 238
around E15.5 in mice (36). Indeed, in Grhl3 null mice with deficient periderm, the primary canal 239
closed prematurely, providing a hopeful avenue for further research into the aetiopathology of CAA 240
(36). 241
242
The canal finally opens by the creation of a central lumen (Figure 3E). This occurs at around 16 243
weeks in human fetal samples, and is preceded by a precocious programme of differentiation, ahead 244
of developing skin elsewhere, and involves the apoptotic cell death of the cornified loricrin-positive 245
layer of squamous epithelium (Figure 3D). The complex and multistage development of the ear canal 246
may account for the variability, both in form and occurrence, of congenital aural atresia. 247
248
As with much of craniofacial development, the EAC is in essence an epithelial structure taking shape 249
in relation to, and influenced by, its surrounding structures. How does the developing ear canal find 250
its path to the middle ear cavity? Current knowledge tells us some detail about these relative relations 251
and signalling pathways, putting the tympanic ring in a lead role. When tympanic ring formation is 252
Anatomy and development of the external auditory canal
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This is a provisional file, not the final typeset article
disrupted using retinoic acid, the EAC also fails to form (41). And when the tympanic ring is 253
duplicated in Hoxa2 null mutant mice, a duplication of the EAC is also noted, as the second arch 254
transforms to a first arch fate (42). In keeping with this phenotype, overexpression of Hoxa2 in the 255
first arch neural crest leads to a duplication of the pinna and loss of the EAM (37). Additionally, 256
Prx1, Gas1, Tshirt (Tsh) and Goosecoid (Gsc) have all been shown to cause EAC defects in the 257
presence of hypoplastic or absent tympanic rings (43–46). Whilst EAC defects are observed in mouse 258
mutants of Prx1 and Gas1, TSH and GSC have been corroborated in human genome wide sequencing 259
studies also (47). Interestingly, mutations in Gsc and Prx1 also cause hypomorphism of the malleal 260
manubrium - an essential link in sound transduction from outside world to cochlea. Experiments in 261
knock out mice as well as associative data corroborating human CT scans and intra-operative 262
findings, indicate an essential role for the EAC in the induction and proper positioning of the 263
manubrium within the eardrum ((15,48)). Not unlike an anatomical love triangle, these findings 264
indicate that formation of the EAM depends on formation of the tympanic ring. In turn formation of 265
the manubrium depends on formation of the EAM. 266
267
Congenital disease of the external auditory meatus 268
Congenital aural atresia (CAA) is a spectrum of defects affecting the EAM at birth. The EAM may 269
fail to form partially, completely or be narrowed. Occurring in around 1 in 10,000 to 20,000 live 270
births, CAA affects males more than females and is typically unilateral, involving the right side more 271
commonly than the left (49). Over the years, a number of systems for classifying CAA have been 272
proposed (50–52). More typically, clinicians group CAA patients into complete atresia (complete 273
obliteration of the canal usually in combination with a bony atretic plate), stenosis (in which there is 274
a continuous lumen to an at least partial tympanic membrane) or partial atresia (variable presence of 275
Running Title
13
a canal lumen lateral to an atretic plate) (Figure 4)(53). Complete atresia and stenosis are much more 276
common than partial atresia, which is rare (54). Severity of canal atresia has been linked with an 277
early arrest in EAM development, although this does not account for the striking difference in the 278
incidence of complete verses partial atresia (11,54). Recent studies described above, examining EAM 279
development at a range of timepoints and using molecular cues suggest a more involved aetiology at 280
play, which may, for example, begin to explain the surprising rarity of partial in comparison to 281
complete atresia (36). 282
283
Genetic basis of CAA 284
Congenital atresia is often associated with pinna and middle ear defects (55). Less frequently inner 285
ear deformities and deformities of the stapes footplate are also present (56). Associations with other 286
craniofacial abnormalities such as facial asymmetry and cleft lip/palate are also common (11). In 287
some patients, these craniofacial defects are part of syndromic conditions such as Treacher-Collins, 288
de Grouchy (chromosome 18q deletion) and Branchio-Oto-Renal syndrome (Figure 4D) (11). 289
Genetic analysis of syndromic patients can provide valuable genetic background to the aetiology of 290
both monogenic and syndromic CAA. For example, Genotype-phenotype mapping of chromosome 291
18q deletions (responsible for de Grouchy syndrome) has consistently shown the critical region for 292
CAA in these patients to be 18q22.3 (47). Further delineating critical regions have highlighted 293
candidate genes to investigate, with a loss of function mutation in the gene TSHZ1, known to be 294
essential for murine middle ear development, being responsible for CAA both in isolated cases and in 295
18q deletion syndrome (45) . A similar approach in a cohort of patients with the syndrome SAMS 296
(short stature, auditory atresia, mandibular hypoplasia, and skeletal abnormalities) has identified 297
mutations in the Goosecoid(GSC) gene as a causative factor (57). GSC is an important regulator of 298
Anatomy and development of the external auditory canal
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This is a provisional file, not the final typeset article
neural crest/mesoderm cell fate and has been shown to modulate tympanic ring development (58). A 299
canal defect in these patients may therefore be secondary to a tympanic ring defect, assigning a key 300
signalling role to the tympanic ring (41). 301
302
Mutations in HOXA2 have been characterised in a number of families with microtia (59). Only one of 303
these families, however, were noted to have additional EAM defects, with shortened and stenotic 304
canals (59). Mouse studies analysing hypomorphic Hoxa2 mutants with 45% the normal level of 305
Hoxa2, and conditional mice, where loss was activated relatively late at E12.5, did not show any 306
defects other than stapes and pinna deformity respectively (37,41). This is consistent with 307
observations that Hoxa2 requirement in craniofacial development is temporal (60). It may be that low 308
Hoxa2 levels only effect EAM development in the early embryonic period by affecting arch fate, 309
converting second arch to a first arch fate. That Alasti et al., report stenotic and shorted canals rather 310
than completely atretic canals is interesting because it contradicts previous thinking that earlier 311
mutations in EAM development lead to more severe anatomical defects (11,54). It is likely that a 312
more complex molecular basis is at play. 313
314
Genes discussed thus far play an indirect role in ear canal development, presumably due to their 315
effect on the tympanic ring. Foxi3, in contrast, is likely involved in the development of the ear canal 316
epithelium itself, making it a particularly interesting gene in the study of outer ear embryology (61). 317
Famously identified in hairless dogs, the Foxi family of genes are expressed in the ectoderm and play 318
important roles in early arch patterning and thus early craniofacial development (62). To date, 319
mutations in Foxi3 have been implicated in ear canal defects in mice, dog and human, suggesting it 320
Running Title
15
plays a role in cases of canal atresia (61). What is currently known about the role of Foxi3 in EAC 321
development is limited and associational. Unfortunately, Foxi3 homozygous null mutants do not 322
survive postnatally, and heterozygous mutants do not show a phenotype. A conditional mouse model 323
would provide an intriguing avenue for further research, in particular into the mechanisms underlying 324
the initiation of a meatal plug in early EAM development. 325
326
Given that CAA is a phenotype observed in multiple syndromes, it is not surprising that multiple 327
genes have been associated with its aetiopathology. The exciting next step will be to harness this 328
information, using conditional knock outs or culture setups, to answer questions such as, what are the 329
signalling pathways allowing the tympanic ring, ear canal and manubrium to communicate? 330
331
Treatment of congenital ear canal disease 332
Dividing canal atresia into complete, partial and stenotic is important to the clinician as it maps 333
patients to key treatment considerations: hearing outcome, likelihood of cholesteatoma (erosive 334
keratin-filled cysts that form when EAM skin cannot migrate out as normal) and the feasibility of 335
achieving a self-cleaning ear should atresiaplasty be considered (63). 336
337
Aesthetics aside, hearing loss is the main problem in complete atresia, as the absence of a lumen 338
precludes the possibility of cholesteatoma forming. Despite significant advances in surgical 339
technique and patient selection, atresiaplasty remains a difficult operation with typically poor 340
outcomes and risk to the facial nerve, which is characteristically laterally placed, and therefore 341
unexpectedly closer to the surgeon’s drill (63). Surgical atresiaplasty predates the use of bone 342
Anatomy and development of the external auditory canal
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This is a provisional file, not the final typeset article
anchored hearing aids (BAHAs), and it is still performed in healthcare settings with limited access to 343
audiological services (64). Complications include restenosis, recurrent keratinous debris build up, 344
poor hearing outcomes (in one study as many as 93% of patients still required a hearing aid post-op) 345
and facial palsy resulting from altered and variable anatomy of the facial nerve in CAA (65). Once 346
the neo-lumen is created, it is covered with skin grafts taken from elsewhere or pedicled from 347
postauricular skin. Restenosis is prevented by performing a wide canalplasty, although this may have 348
cosmetic implications. As there is no native canal skin to use, the neo-lumen has no capacity to clear 349
desquamated debris, requiring regular aural microsuction (66). Hearing outcomes are typically poor 350
despite the opening of the canal and patients may still need a hearing augmentation device as well. 351
Meatoplasty, where the stenotic canal is widened, is more frequently considered in cases of 352
congenital aural stenosis. These patients carry a much greater risk of cholesteatoma to the presence of 353
a narrow canal, which may inhibit epithelial migration(69). Furthermore, native canal skin is often 354
available for canal reconstruction reducing chances of post-operative complications, making surgery 355
a more straightforward decision(69) 356
357
Given the difficulties realting to surgical correction of ear canal atresia, bone conduction devices and 358
auditory implants are often considered instead of atresiaplasty (67). Bone conduction devices bypass 359
the outer and middle ear by transmitting sound vibrations to the cochlea via the skull. Their 360
connection to the skull may be temporary, using methods such as a headband or adhesive; or it may 361
involve the placement of an auditory implant (Figure 4E). Auditory implants can be classified into 362
percutaneous devices, active and passive transcutaneous devices, and middle ear implants. Patient 363
factors, middle ear anatomy and clinician preference determines which devices can be offered (68). 364
Running Title
17
The option of implantable hearing aids is attractive, however, they require near to normal middle ear 365
anatomy and thus are not suitable for many CAA patients.. 366
367
In reality, in Europe and the United States, the majority of patients receive bone anchored hearing 368
aids (64). Whilst these provide good hearing rehab, they are visible, can cause skin irritation and 369
infection and of course do not create a continuous hearing response (they are taken on and off), 370
which is particularly important for spontaneous learning in young patients (64). Therefore, a surgical 371
approach which reconstructs the ear canal with few complications and without the need for further 372
device-assisted hearing, is attractive. Recent research investigating epithelial dynamics in the 373
developing ear canal may provide new avenues to explore the biological basis of atresiaplasty 374
complications (36). For example, surgeons note the greater frequency of restenosis in the lateral 375
reconstructed canal, which is cartilage-lined (70). As the bony and cartilaginous parts of the canal 376
develop in different ways (primary canal verses an extending meatal plate), the development may 377
explain these differences in response after surgery. It would therefore be interesting to study whether 378
there is an inbuilt signalling difference in the outer and inner parts of the EAM that influences their 379
response to injury. A better understanding of EAM development would also further clarify the 380
variable anatomy in CAA, e.g. the route of the facial nerve, easing technical challenges faced by 381
surgeons. 382
383
Acquired disease of the external auditory meatus 384
Acquired disease of the EAM includes of course the breadth of traumatic, infective and rarely 385
neoplastic disease and its comprehensive review is beyond the scope of this review. These can all 386
Anatomy and development of the external auditory canal
18
This is a provisional file, not the final typeset article
lead to a post-inflammatory canal atresia, sometimes referred to as stenosing otitis externa (71). 387
Current treatment involves regular aural hygiene or meatoplasty to expand the canal opening. There 388
is experimental evidence in Guinea pigs for the use of Mitomycin C (an inhibitor of fibroblastic 389
activity) in preventing post-inflammatory canal stenosis (72). Human studies, however, have had 390
contradictory results and further controlled trials are needed to evaluate its potential role in 391
preventing restenosis (73,74). Here, we focus on ear canal diseases that may be further understood 392
through the lens of developmental biology. 393
394
Specific to the specialised biology of ear canal skin and its ability to migrate are two distinct 395
diseases: keratosis obturans (KO) and ear canal cholesteatoma. KO is a rare disease in which 396
squamous debris builds up in the ear canal, often leading to an expansion of canal diameter before 397
presenting with acute pain. Current treatment is regular microsuction to maintain a patent ear 398
canal(19). As with healthy patients, the TM of individuals diagnosed with KO can be labelled with 399
dye to observe the migration of the epidermal layer. One such study suggests that either delayed or 400
disrupted epidermal migration may be the cause of KO (75). External ear canal cholesteatoma 401
(EECC) is also rare and was for a long time grouped together with keratosis obturans as the same 402
disease (76). Whilst ear canal skin in KO is intact with layers of squames building up above it, in 403
EECC there are focal ulcerations in canal skin and the organisation of keratinous squames is random. 404
Importantly there is bony necrosis with focal areas of deep sequestered bone (76). As such, drawing a 405
diagnostic distinction is important, as whilst KO can be managed conservatively, EECC typically 406
requires surgical removal of all the cholesteatoma and reconstruction of the ear canal (76). The 407
aetiopathology of EECC remains uncertain with disordered epithelial migration and/or cerumen 408
gland dysfunction thought to play a role (77). Localised differences in epithelial cell migration in the 409
Running Title
19
tympanic membrane have recently been proposed to underly propensity to middle ear cholesteatoma 410
formation and may provide clues to the aetiology of canal cholesteatoma also (24). The underlying 411
pathology of another category of cholesteatoma, congenital cholesteatoma of the middle ear, also 412
remains controversial. Whilst current consensus rests with an epidermoid formation origin within the 413
middle ear, another proposed theory suggests that an errant developing EAC extending beyond the 414
tympanic ring and into the mastoid cavity may be at fault (78). What determines epithelial behaviour 415
in the ear canal, driving it to focal erosion in EECC rather than circumferential upward layering into 416
the lumen as in KO? Further understanding the epithelial dynamics that bestow EAM skin its 417
migratory potential and its regenerative capacity will certainly begin to answer this question. 418
419
Future Questions 420
Of the three-part mammalian ear, the external auditory canal receives arguably the least attention. 421
Although this has not always been the case. Until the mid-twentieth century and the advent of 422
hearing aids, an exaggerated external ear in the form of an ear trumpet was the most commonly used 423
hearing device, emphasising the valuable role of the external ear in hearing. Congenital disease of 424
the EAM is thankfully rare but crucially important. Infancy is an irretrievable period in which to 425
develop speech and language, which can determine the rest of a child’s life. Clarity and spontaneity 426
of hearing, as well as decibels gained, at this age is critical. Thus, current treatment options, BAHAs 427
or complication-prone canalplasties, leave room for improvement. Better understanding the 428
mechanisms underlying EAM development will provide valuable knowledge towards improving 429
treatment options both for congenital canal atresia and also treatment of acquired canal disease that 430
involves epithelial dysfunction, such as keratosis obturans or canal cholesteatoma. 431
432
Anatomy and development of the external auditory canal
20
This is a provisional file, not the final typeset article
Recent research has pushed forward valuable but dated, largely histology-based, knowledge of EAC 433
development and created new questions. How exactly does the ear canal open along its length, 434
creating innermost a single-cell layer thin tympanic membrane, a thinly lined osseous canal and a 435
spongier outer cartilaginous canal? What determines the different behaviour of epithelium along the 436
canal postnatally? During development, how do the different part of the complex mammalian ear 437
communicate and eventually integrate? Answering these questions will pave the way to answering 438
the clinical quandaries that affect patients’ lives. How to create a self-cleaning ear? How to recreate 439
the external ear epithelium? Which pathways can be modified in the face of congenital ear disease? 440
Looking to developmental biology for the answers will be key. 441
442
Acknowledgements 443
Monotreme images were provided by the zoological museum in Cambridge University and the Hill 444
Collection at the Berlin Museum fur Naturkunde. Thanks to Neal Anthwal for discussions on the 445
monotreme ear. Thanks to Francis Smith for insightful conversations on syndromic ear canal 446
deformity. 447
448
Conflict of Interest 449
The authors declare that the research was conducted in the absence of any commercial or financial 450
relationships that could be construed as a potential conflict of interest. 451
452
Funding 453
Running Title
21
MM is supported through a clinical training fellowship award from the Medical Research Council 454
(MR/P019730/1). AST is funded through a Wellcome Trust Investigator award (102889/Z/13/Z). 455
456
Author Contributions 457
All authors read and contributed to the manuscript and discussion. MM was responsible for 458
conception and writing of the manuscript. AST contributed to the manuscript conception and 459
reviewed the manuscript throughout the writing process. RN provided clinical images and reviewed 460
clinical sections of the manuscript. 461
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649
650
651
652
653
Figure legends 654
655
Figure 1 656
Schematic diagram of the three-part ear; the external ear, which includes the pinna and ear canal; the 657
middle ear which includes the tympanic membrane (TM), middle ear cavity (MEC) and the ossicles. 658
The malleus inserts into the TM at the umbo. The middle ear connects to the nasopharynx via the 659
eustachian tube (ET). The inner ear (IE) includes the cochlea and semi-circular canals. The ear canal 660
Running Title
31
is enclosed in its inner two-thirds by a bony wall and its outer third by a cartilaginous wall, which is 661
lined by hair-bearing skin. The ear canal lumen varies in shape but typically has a pretympanic recess 662
and meatal opening. Inset shows anterior, posterior, superior and inferior axes as referred to in the 663
text. 664
665
Figure 2 666
Variations in the non-mammalian ear canal 667
(A) Bearded dragon (Pogona vivceps) with superficial tympanic membrane outlined by arrow. (B-E) 668
Histology sections (B) Sagittal trichrome section of gecko ear showing a shallow ear canal (star) and 669
superficial tympanic membrane (arrow). (C and D) Developing platypus (Ornithorhynchus anatinus) 670
EAM (C) Frontal section of platypus ear at 2 days post hatching (2 p.h.). The developing ear canal 671
(arrows) is long extending from the malleus to the top of the head. There is no pinna at the canal 672
opening (marked by *). (D) Frontal section of developing ear canal in 10 p.h. day platypus. The 673
EAM develops in a sheath of condensed mesenchyme (arrows). (E and F) Developing echidna 674
(Tachyglossus aculeatus) EAM (E) Horizontal section through the EAM of x p.h. echidna 675
demonstrating cartilage support around the developing EAM (arrows). (F) Horizontal trichrome 676
section through EAM of an echidna at 18 p.h. days showing a tortuous path to the opening (star). P, 677
Pinna; MEC, middle ear cavity; IE, inner ear, IE; EAM, external auditory meatus. 678
679
Figure 3 680
Schematic of mammalian ear canal development 681
Anatomy and development of the external auditory canal
32
This is a provisional file, not the final typeset article
(A) Invagination of the primary canal (marked by *) with a meatal plug at its tip. (B) Extension of the 682
meatus towards the forming middle ear, driven by a potential signal from the tympanic ring. At the 683
same time we see selective loss of periderm in the primary canal caused by apoptosis. (C) Closure of 684
the primary canal (following loss of periderm layer) and further extension of the 685
meatal plug to reach the tympanic ring. (D) Loricrin expression (red dashed line) marks the site of 686
opening of the canal. (E) Following keratinocyte differentiation, the whole external ear canal opens, 687
with the upper/rostral wall of the meatal plate forming the outer surface of the tympanic membrane. 688
Vertical black dashed lines between brown and blue regions represent the two distinct parts of the ear 689
canal. 690
691
Figure 4 692
Patient photograph and scans of congenital aural atresia 693
(A) Congenital aural stenosis. Coronal CT scan shows a normal, patent right ear canal and a stenotic 694
left ear canal marked by *. (B) Complete congenital aural atresia. Coronal CT scan shows a normal 695
ear canal on the right and an atretic plate on the left with no ear canal. (C) Congenital aural stenosis 696
and cholesteatoma. Axial CT scan of a patient with congenital aural stenosis and left-sided 697
cholesteatoma. The bony ear canal has been widened and remodelled by the collecting keratin 698
(marked by *). (D) Photograph of a patient with Treacher-Collins syndrome and complete aural 699
atresia of the left ear canal. The patient has been fitted with an osseointegrated hearing device visible 700
behind the pinna. (E) Example of an osseointegrated device. Computer-generated image of a BAHA 701
Connect (Image courtesy of Cochlear Bone Anchored Solutions AB, ©2020). EAM, external 702
auditory meatus; IE, inner ear; MAC, mastoid air cells. 703