Middle ear mechanics Using temporal bone experiments to improve clinical methods

Anton Rönnblom

Clinical Science, Otorhinolaryngology Umeå 2022

This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN print: 978-91-7855-835-3 ISBN PDF: 978-91-7855-836-0 ISSN: 0346-6612 Series number 2193 Cover photo: Human ossicles cast in bronze by the author Electronic version available at: http://umu.diva-portal.org/ Printed by: Cityprint i Norr AB Umeå, Sweden 2022

“Sometimes the smallest things take up the most room in your heart.” Winnie the Pooh

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Table of Contents

Abstract .................................................................................................................... iii

Abbreviations ............................................................................................................ v

Regarding illustrations and photos .......................................................................... vi

Original Papers ....................................................................................................... vii

Populärvetenskaplig sammanfattning på svenska ................................................. viii Introduction .............................................................................................................. 1

Sound and how it is measured ............................................................................................. 1 Anatomy and physiology of the human ear .......................................................................... 3 The middle ear ................................................................................................................. 4 Muscles of the middle ear ................................................................................................. 6 The mastoid and the Eustachian tube ................................................................................ 8 The cochlea...................................................................................................................... 9 Sensorineural and conductive hearing loss ......................................................................... 10 Middle ear disease ............................................................................................................ 11 Chronic otitis media ....................................................................................................... 12 Cholesteatoma ............................................................................................................... 13 Otosclerosis ................................................................................................................... 14 Trauma and malleus fractures ............................................................................................ 14 Hearing reconstruction ...................................................................................................... 15 PORP, TORP and the challenge in clinical evaluation..................................................... 17 Prosthesis materials ....................................................................................................... 17 Human temporal bone ....................................................................................................... 19 Temporal bone in clinical practice ................................................................................. 19 Temporal bone in research ............................................................................................. 21 Laser Doppler vibrometry .............................................................................................. 22 Vestibulocochlear pressure measurement ........................................................................ 25 Digital holography ......................................................................................................... 25 Muscle analysis and fibre phenotypes ................................................................................ 26 Mathematical models ........................................................................................................ 28

Aims ........................................................................................................................ 29

Materials and Methods ........................................................................................... 30 Papers I – II laser Doppler vibrometry ............................................................................... 30 Paper III ........................................................................................................................... 36 Paper III – IV muscle analysis ........................................................................................... 39

Ethical approval and considerations....................................................................... 41 Results ..................................................................................................................... 42

Paper I .............................................................................................................................. 42 Paper II............................................................................................................................. 43 Paper III ........................................................................................................................... 44 Paper IV ........................................................................................................................... 45

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Discussion ................................................................................................................ 47 Paper I and II .................................................................................................................... 47 Paper III ........................................................................................................................... 51 Paper IV ........................................................................................................................... 53

Conclusion ............................................................................................................... 55 Future perspectives ................................................................................................. 56 Acknowledgement ................................................................................................... 57 References ............................................................................................................... 59

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Abstract

Background

The middle ear transmits and amplifies sound vibrations from the tympanic membrane via three ossicles to the inner ear. Moreover, it contains two muscles, the stapedius muscle (SM) which protects the inner ear from loud noise, and the tensor tympani (TT) whose function is still debated.

The majority of hearing loss caused by disruption of the ossicular chain is a result of chronic otitis media and cholesteatoma. Variations in pathology, surgical skill and individual healing conditions make objective evaluation of ossicular replacement prosthesis in vivo difficult. Prosthesis development and the investigation of trauma mechanisms are affected by the same challenges. With few changes post-mortem, the temporal bone (TB) is suitable for studies of middle ear mechanics and allows a controlled environment. Equally important, it allows theories to be tested without patient risk.

In this thesis we used human TBs to find factors associated with optimal sound transfer in the two types of ossicular replacement prostheses. Furthermore, we investigated the mechanism and forces involved in rare cases of isolated malleus fractures. We also investigated the morphology, fibre phenotype composition and vascularization of the human middle ear muscles in order to better understand their roles.

Materials and Methods

Laser Doppler vibrometry (LDV) is an established method of measuring sound transfer in human TBs. We have further developed a surgical model that allows testing of a wide range of prostheses and their placements. In Paper I beneficial factors in partial ossicular replacement prostheses (PORPs) were tested. In Paper II we evaluated different types of total ossicular replacement prostheses (TORPs) including an experimental prosthesis inspired by the single ossicle system of birds. In Paper III the negative pressure trauma typically associated with isolated malleus fractures, produced by a finger being withdrawn from a wet ear canal after a shower or bath, was simulated in TBs. Based on measurement from control persons the forces involved were calculated and measured in models developed for this purpose. The force of the TT was estimated by comparing its cross-sectional area and fibre composition with those reported in published references. In Paper IV we used immunohistochemical, enzyme histochemical, biochemical and morphometric techniques on TT, SM and human orofacial and limb muscle control samples.

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Results

Of the prostheses, PORPs and TORPs with lateral contact with both the tympanic membrane and the malleus handle performed best, and TORPs with distal malleus contact proved superior. Our experimental bird-type prosthesis was the most stable in such placement and performed equally to or better than other prostheses. In Paper III the application of negative pressure via the ear canal did not fracture the malleus shaft, with only a passive counterforce from support structures, although the force exceeded that required for a malleus shaft fracture. We estimate that when adding calculated counteracting forces from the TT muscle, sufficient force is generated to cause a malleus fracture. Both human middle ear muscles are predominated by fast type 2 fibres, and have rich capillarization and nerve innervation compared with limb muscles. Muscle spindles were found in the TT but not the SM.

Conclusions

Where possible, an ossicular replacement prosthesis should be placed to allow distal contact with both the TM and the malleus handle. The sound transfer capabilities combined with the stable placement of our experimental prostheses suggest room for improvement. The combination of a negative pressure created by a finger being withdrawn from a wet ear canal and a simultaneous counteracting reflexive force by the TT muscle was found to be sufficient to cause an isolated malleus fracture. The finding of muscle spindles in TT, but not in SM, suggests a difference in regulatory control; furthermore, it indicates that the TT can be activated by a sudden stretch reflex as described in the malleus fracture trauma. The human middle ear muscles have a highly specialized muscle morphology, which is more similar to orofacial than to limb muscles. The fibre phenotype composition suggests capability for fine-tuned, fast, strong and relatively sustainable contractions. Based on fibre type patterns the TT is among the fastest muscles in the human body.

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Abbreviations

ABG air–bone gap AC air conduction BC bone conduction CAF number of capillaries around, or related to, each fibre CAFA capillaries related to each individual fibre cross-sectional area CD capillary density CHL conductive hearing loss COM chronic otitis media CT computed tomography CV coefficient of variation dB decibel 2D two-dimensional 3D three-dimensional ET Eustachian tube Hz Hertz IMJ incudomallear joint ISJ incudostapedial joint LDV laser Doppler vibrometry mAb monoclonal antibody MRI magnetic resonance imaging MyHC myosin heavy chain PORP partial ossicular replacement prosthesis SD standard deviation SM stapedius muscle SNHL sensorineural hearing loss SPL sound pressure level TB temporal bone TM tympanic membrane TORP total ossicular replacement prosthesis TT tensor tympani VR virtual reality

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Regarding Illustrations and photos

All illustrations and photographs in this thesis were, unless otherwise specified, made by the author, © Aug. 2022 Anton Rönnblom. For imagery containing patient information, permission to publish de-identified data has been given by the patients. Accurately depicting the complex three-dimensional (3D) anatomy of the human ear in 2D artwork is a challenge. For educational purposes, the illustrations in the thesis have been simplified, and are not to scale, depicting relevant structures with emphasis on their function and relation to each other.

As a complement to these schematic drawings, photos of bronze cast ossicles have been taken. These casts were made by A. Rönnblom, based on enlarged 3D prints of high-resolution computed tomography (CT) scans. They are to scale and fixed at angles representative of specimens in vivo.

In Paper III we had access to a high-resolution camera connected to a rigid 30° angled endoscope allowing overview photos and videos. The access for manipulation and visual confirmation in Papers I–III was similarly generous but in Papers I–II all visual evaluation was done via a surgical microscope. This was decided because the method allows manipulation with both hands; however, one drawback was that it did not give a clear overview for photography of the prosthesis position. In these studies, evaluations were based on inspection from several angles using the microscope. Schematic illustrations were made instead of photos.

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Original papers This thesis is based on the following papers:

I Niklasson A, Gladiné K, Rönnblom A, Von Unge M, Dirckx J, Tano K.

An optimal partial ossicular prosthesis should connect both to the tympanic membrane and malleus. A temporal bone study using laser Doppler vibrometry. Otol Neurotol. 2018;39:333–339

II Rönnblom A, Gladiné K, Niklasson A, Von Unge M, Dirckx J, Tano K.

A new, promising experimental ossicular prosthesis: a human temporal bone study with laser Doppler vibrometry. Otol Neurotol. 2020;41:537–544

III Rönnblom A, Niklasson A, Werner M, Stål P, Tano K.

Forces required for isolated malleus shaft fractures. Otol Neurotol. 2021;42:1515–1520

IV Rönnblom A, Thornell LE, Shah F, Tano K, Stål P.

Unique fibre phenotype composition and metabolic properties of the human stapedius and tensor tympani muscles.

(Manuscript in preparation)

Reprints are made with permission from the publishers.

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Populärvetenskaplig sammanfattning på svenska

Mellanörats funktion är att överföra och förstärka ljud från omgivningen till innerörat, samt att skydda innerörat mot höga ljud och annat trauma. Mellanörat är en hålighet i temporalbenet, en del av skallbasen. Utåt mot hörselgången begränsas det av trumhinnan. Inåt mot det vätskefyllda innerörat begränsas det av ovala och runda fönstret. I mellanörat finns tre sammankopplade hörselben; hammaren, städet och stigbygeln, samt två muskler.

Stapediusmuskeln fäster i stigbygeln som står i det ovala fönstret och drar den åt sidan reflektoriskt vid höga ljud. Tensor tympani-muskeln fäster vid hammarskaftet och drar detta inåt vilket spänner trumhinnan och hörselbenskedjan. Den kan reagera på flertal stimuli men hos människa reagerar den inte på ljud och dess funktion är fortfarande omdebatterad.

Mellanörekirurgi syftar vanligen till att förbättra hörsel och/eller sanera sjukliga förändringar. Det är precisions- och tidskrävande operationer, som utförs under mikroskop och som på grund av anatomiska förhållanden medför risker för allvarliga komplikationer. Resultatet är beroende av både det kirurgiska utförandet och av individuella läkningsförhållanden.

Ett sätt att förbättra resultaten vid framtida operationer är att studera och träna på temporalben från avlidna, som under livet gett sitt medgivande till att donera sin kropp för forskningsändamål. Det sker få förändringar i temporalbenet efter döden, vilket ger oss en bra modell att optimera behandlingar i. Vi kan göra adekvata mätserier med få preparat, optimera vanliga ingrepp i en kontrollerad miljö, utvärdera ovanliga skador och nya tekniker, samt förbättra kunskapsläget om örats egenskaper.

Laserdopplervibrometri (LDV) är en väletablerad metod med vilken man med stor precision kan mäta vibrationer i en specifik punkt utan att mätningen i sig medför en yttre påverkande kontakt. Av detta skäl har denna metod ofta använts för att utvärdera vibrationshastigheten på stigbygelplattan, övergången mellan innerörat och mellanörat. Vibrationshastigheten på stigbygelplattan är nämligen direkt proportionerlig mot ljudupplevelsen hos människa, resultaten kan mätas i decibel och ger oss därmed ett “hörselprov” för temporalbenet. Det traditionella upplägget för LDV-studier på temporalben består av att ljud skickas till trumhinnan varvid hörselbenskedjan sätts i vibration. Laserstrålen som siktats in via ett mikroskop träffar en liten reflekterande lapp, placerad på stigbygelplattans ovansida som reflekteras åter till LDV-maskinen. Då laserstrålen består av enfärgat ljus i samma fas kan man med hjälp av dopplereffekten och en referensstråle mäta vibrationshastighet och riktning i den givna punkten, baserat på frekvensförändringen i det reflekterade ljuset. Åtkomst till stigbygelplattans ovansida är dock begränsad, även om man genomför ganska omfattande kirurgi, eftersom man måste lämna trumhinnan och hörselbenen orörda för att kunna få adekvata mätningar.

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Vi utvecklade en egen variant för LDV-mätning på färskfrysta humana temporalben. En högtalare och kalibreringsmikrofon skruvades fast utanför hörselgången. En öppning gjordes via mellersta skallgropen in till mellanörat för åtkomst till hörselbenskedjan, utan att påverka dess upphängning eller trumhinnans spänst. Via denna öppning kunde sedan olika skador och reparationer i form av hörselbensplastiker utföras. Resultaten mättes inte såsom tidigare från stigbygelns ovansida, utan från stigbygelplattans undersida. Åtkomst till denna skedde genom att vi avlägsnade själva hörselorganet, snäckan. Den största fördelen med vår metod är att den tillåter en större variation av proteser och placeringar av dessa utan att de skymmer laserstrålen.

I delarbete I utvärderades ett antal modeller av hörselproteser som ersätter en del av hörselbenskedjan, så kallade PORP. Dessa placeras på en fungerande stigbygel och skapar kontakt med trumhinnan utan att gå via städet.

Vi fann att PORPar som hade kontakt med både trumhinna och hammarskaft hade bättre resultat i LDV-mätningarna än de med bara trumhinne- eller hammarskaftskontakt.

I delarbete II undersökte vi proteser som ersätter hela hörselbenskedjan, så kallade TORP. Dessa skapar direktkontakt mellan stigbygelplattan och trumhinnan. Även här gav proteser i kontakt med både trumhinna och hammarskaft det bästa resultatet. En viktig iakttagelse var att bästa resultaten uppmättes då protesen var i kontakt med spetsen av hammarskaftet istället för mitten. På grund av den snäva vinkeln mellan stapesplattan och denna punkt är det svårt att få en stabil kontakt med sedvanliga proteser. I denna studie testade vi även en experimentell protes, inspirerad av hörselbenet från en hornuggla. Fåglar har bara ett hörselben, vilket också är vad man får vid en TORP. Fågelprotesen var placerad så att basen stod mot stapesplattan och med tre konstruerade armar omfamnade den ett kvarvarande hammarskaft. Dessa armar gick sedan vidare ut och agerade kontaktpunkter både med hammarskaftets spets och med trumhinnan. Denna protes hörde till dem som gav bäst resultat och den stod stabilt då den var placerad i optimalt funktionsläge.

I delarbete III undersökte vi traumamekanismen och krafterna involverade i de ovanliga, men sannolikt underdiagnostiserade fallen av isolerade hammarskaftsfrakturer. Det finns i litteraturen mindre än 100 publicerade fall, men de senaste 15 åren har vi i Norrbotten hittat i snitt ett fall per år. Det typiska traumat är att man med ett finger försökt “dra ut” vatten från hörselgången i samband med bad eller dusch. I samband med detta har man känt ett kort smärtande hugg, följt av en kvarvarande mild till måttlig hörselnedsättning. Ett hörselprov uppvisar ett ledningshinder orsakat av att hammarskaftet brutits av.

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Vi undersökte mellanörat på temporalben både i vila och under varierande grad av undertryck, levererat via hörselgången. Det visade sig att hammaren och trumhinnan rörde sig relativt fritt utan samtidigt mothåll från tensor tympani-muskeln. Ett antal hammarskaft fakturerades och kraften uppmättes.

På 60 försökspersoner mätte vi hörselgångens längd och hur långt de kunde föra in ett finger i ett torrt öra. Med dessa data gjorde vi matematiska och fysiska modeller där undertryckskraften kunde beräknas och mätas. Vi mätte diametern på tensor tympani-muskeln och analyserade muskelfibersammansättningen av densamma. Med dessa och tidigare publicerade data kunde vi teoretiskt beräkna den maximala kraften hos en human tensor tympani. Vi fann att kraften som genereras om man drar ett finger ur en våt hörselgång är tillräcklig för att orsaka en hammarskaftsfraktur förutsatt att tensor tympani muskeln samtidigt drar i motsatt riktning. Tensor tympani muskeln har förmågan att utgöra en sådan motverkande kraft bland annat genom muskelspolar som reflexmässigt signalerar att muskeln ska dra ihop sig vid en hastig förlängning.

I delarbete IV studerades bland annat mellanöremusklernas muskelfibersammansättning och metabola egenskaper. Tidigare teorier om mellanöremusklernas funktion hos människa bygger till stor del på djurstudier. Syftet med det fjärde delarbetet var därför att studera uppbyggnaden av mellanöremusklerna hos människa. Vi analyserade fibertyper, kapillär- och nervfibertäthet och förekomst av muskelspolar för att därigenom få en uppfattning om funktion, styrka och snabbhet.

Vi fann att dessa muskler är snabba, starka, har gott om nervfibrer för finmotorisk reglering och en god kärlförsörjning vilket antyder att de är uthålliga. Ett antal muskelspolar observerades i tensor tympani-muskeln, medan inga muskelspolar observerades i stapediusmuskeln. Fyndet av receptorer involverade i sträckreflexer i tensor tympani-muskeln stödjer våra teorier i delarbete III om muskelns delaktighet i hammarskaftsfrakturer. Våra fynd utgör en bra grund för framtida forskning gällande musklernas egenskaper och funktion i mellanörat.

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Introduction Sound and how it is measured Sound is defined as vibrations that propagate as pressure waves through a medium, be it gas, liquid or solid, and that can be heard when they reach a person’s or animal’s ear. Generally, in humans, the medium is air and the acoustic wave frequencies that can be perceived lie between 20 and 20 000 Hertz (Hz). Sounds above these levels are called “ultrasound” and those below the levels are called “infrasound”.

In clinical practice, we measure sound levels according to the International Organization for Standarization (ISO) 389 diagnostic standard for pure-tone audiometry in air conduction, which is narrower, ranging from 125 Hz to 8 000 Hz. The most common band width used in telephones is 300–3 400 Hz. This narrow band is a standard based on studies of the requirements of intelligible speech. It does not contain the full width of human speech, but the key core for perceiving it.

Several of the human senses are adapted to notice relative, rather than absolute, changes in stimuli and our hearing is one such example (1). The standard atmospheric pressure (1 atm) is 101.3 kilopascal (kPa) at sea level. The smallest pressure changes in the form of an acoustic wave that an average human can perceive as sound is called the threshold of hearing or p0 (figure 1). p0 = 2x10-5 Pa in air at 1000 Hz. For the same average person, the sound pressure can increase more than 10 billion times before becoming uncomfortable. Because of this wide range, sound pressure is generally presented on the decibel (dB) scale. This scale expresses the ratio of two values on a logarithmic scale. Sound meters are calibrated according to the dB scale, using the sound pressure level (SPL), or level of pressure (Lp):

𝐿𝑝 = 20 × 𝐿𝑜𝑔)* +𝑝𝑝*,

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The human ear does perceive sound logarithmically but the sensitivity is heightened in the mid-range. Sounds of different frequencies that the human ear perceives to be of the same loudness can therefore in fact be of varying intensity (Figure 1). For this reason, audiometers used clinically are calibrated using the dB hearing level scale. This scale is simply the dB SPL scale with a conversion table attached to give a straight line at 0 dB, making deviation from normal hearing easier to depict and explain to a patient.

Figure 1. Depiction of the human hearing threshold on the decibel (dB) sound pressure level (SPL) scale, an average curve based on measurements from a large number of young individuals without prior ear pathology. This curve is equivalent to an audiogram on the dB hearing level scale with a straight line at 0 dB. Note that the y-axis of perceived human hearing in the above dB SPL scale is displayed upside down in comparison with a clinical audiogram, which aims to depict loss of normal function. Copyright-free work by Daniel Callejas Sevilla. (For comparison, a clinical audiogram from a patient can be seen on page 12.)

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Anatomy and physiology of the human ear

Figure 2. Schematic drawing of the human ear and adjacent structures. The tympanic membrane (TM) is coloured light blue. The malleus, incus and stapes are shown in white. The inner ear comprising the cochlea and semicircular canal is depicted in purple. In bright blue along the neck is the internal jugular vein which connects to the sigmoid sinus (pale blue, dashed line). The carotid artery is coloured pink. The nerves are coloured yellow. The audio vestibular nerve runs between the grey brain stem to the right and the base of the cochlea. The facial nerve runs from the brain stem along the audio vestibular nerve, and on, through the middle ear and the base of the mastoid (yellow, dashed line). The gas-filled mastoid process, in this diagram, is depicted as white-contoured cells surrounding the ear canal. Leading downward out of the middle ear is the bony cavity that contains the Eustachian tube (ET).

The ear is divided into three parts: the outer ear, the middle ear and the inner ear. The outer ear consists of the cartilaginous auricle and the ear canal (Figure 2). The auricle aids in vertical localization of sound but provides no amplification in humans (2). The ear canal is about 25 mm long in adults and has a lateral cartilaginous half and a medial bony half (3). The ear canal has its own resonance frequency depending on length, suggested to be the origin of the common noise-induced hearing loss notch at 4 kHz (4, 5).

The middle ear is a gas-filled, bone-encapsulated cavity between the tympanic membrane (TM) and the inner ear. It is connected to the latter via the membranous oval window and the round window. Three ossicles bridge this gap: the malleus which is connected to the TM; followed by the incus; and finally the stapes whose footplate is placed in the oval window (Figure 3). These ossicles are positioned free from the surrounding walls and stabilized by a number of ligaments and two muscles (Figure 4). The middle ear is connected posterosuperiorly to the aerated cells of the mastoid and to the nasopharynx via the Eustachian tube (ET).

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The inner ear is held within a compact bony outer wall, called the “otic capsule”. The inner ear has three main parts: the cochlea, which is the hearing organ, and the semicircular canals with the adjacent vestibule that make up the balance organ. As sound waves hit the TM they are transformed into mechanical displacements that are transmitted via the three ossicles. As the stapes footplate in the oval window vibrates, pressure waves are created in the fluid of the cochlea and stimulate its hair cells. These signals are transmitted via the cochlear nerve to the brain, where the sound is heard.

Other anatomical structures that must be identified and respected during surgery in the temporal bone (TB) are the dura, the outermost layer of the meninges surrounding the brain, the sigmoid sinus, a venous sinus that transports blood from the posterior part of the skull into the internal jugular vein, and the facial nerve. The facial nerve is mostly covered by bone as it extends through the TB; however, partial dehiscence is common in the normal population (6) and this increases with pathology such as cholesteatoma (7). Most of the dehiscence can be found in the tympanic segment adjacent to the oval window (6, 7). The course of the motor branches of the facial nerve through the TB can be seen in Figure 2. The sensory input from the taste buds takes a different path in the form of the chorda tympani nerve which travels across the middle ear, as seen in Figure 4, before it joins the facial nerve.

The middle ear

Figure 3. The tympanic membrane (TM) viewed from above, in a right ear: the double contour lines surrounding the TM represent the limbus. (1) Pars tensa (light blue); (2) pars flaccida (darker blue); (3) anterior mallear fold; (4) posterior mallear fold; (5) lateral process of malleus; (6) handle of malleus; (7) umbo. The bodies of the malleus and incus and (8) the incudomallear joint (IMJ) are presented as a dashed line because these structures are covered by bone. The long process of the (9) incus, (10) stapes, (11) incudostapedial joint (ISJ), and (12) round window can, depending on the angle of the ear canal and the transparency of the TM, sometimes be visualized through the TM, as depicted. (13) The Eustachian tube (ET) passes through the bone to the nasopharynx and is therefore presented as a dashed line. The opening of the ET can sometimes be viewed through a transparent TM.

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The TM constitutes the border between the outer ear and the middle ear and it consists of three layers. The outside, like the ear canal, is covered with skin; the inside, like the middle ear and mastoid, is covered with mucosa; and in between these two there is a fibrous layer. The TM has the shape of a flattened cone with its apex directed inward. Its distal margin, the thicker limbus, attaches the TM to the tympanic sulcus. The TM is attached along the lateral surface of the malleus handle; at the apex of the cone, the tip of the malleus, called “umbo”, is firmly attached in a fibrous sack. (Umbo is Latin for “shield boss”.) The TM is divided by the anterior and posterior mallear folds into the larger pars tensa and the smaller pars flaccida (Figure 3).

Figure 4. The ossicles, and their connecting supports seen from below, in a left ear: (1) malleus; (2) incus, with its long process towards to the right and the short process pointing down in the picture; (3) stapes; (4) tympanic membrane (TM); (5) stapedius tendon; (6) tensor tympani (TT) tendon; (7) incudostapedial joint (ISJ); (8) incudomallear joint (IMJ); (9) posterior incudal ligament; (10) superior incudal ligament; (11) superior mallear ligament; (12) lateral mallear ligament; (13) anterior mallear ligament; and (14) chorda tympani.

From a mechanical point of view, the most important function of the middle ear is to transform the high-volume, low-pressure vibration of the TM into higher-pressure, small-volume vibration at the stapes footplate. The surface area of the TM is 64.3 mm2 (8) and that of the stapes footplate 3.2 mm2 (9). This 20:1 ratio renders a 20-fold increase in pressure and is the most important factor of this transformation (10). Other factors to consider are: the ossicular lever, where the malleus longer limb over the incus shorter limb has been suggested to give an additional ratio of 1.3:1 (10). The curved-membrane mechanism proposes that the greater displacement with less force of the curved TM is translated into less displacement but increased force acting on the malleus. This was suggested to contribute a factor of 2:1 (11).

Combining these factors gives us 20x1.3x2 = 52. Converting the sound pressure to dB, 20log52 = 34 suggests that the middle ear has an amplifying potential of about 34 dB.

6

In reality there will be transmission losses due to friction and resistance in the TM, the ossicles, their ligaments and joints. This amplification has been shown to have a clear frequency dependency with a peak around 1.2 kHz at 23.5 dB (9).

The movement of the TM is frequency-dependent and becomes more complex with higher frequencies (12, 13). How this relates to middle ear sound transmission has been theorized about (12), but is not yet fully understood. The movements of the stapes footplate have been described as primarily piston-like up to 1 kHz and as more complex, with a mix of piston-like and rocking motions around both the long and short axes, at higher frequency, particularly above 4 kHz (14). It has been suggested that both the piston-like movement and the rocking movement of the stapes can generate a travelling wave in the fluid of the cochlea and its basal membrane. In normal conditions when these two motions are combined the piston-like movement has the dominating influence (15). More about this effect can be read under “Materials and Methods”, in the section titled “Papers I and II – laser Doppler vibrometry”.

Another important function of the middle ear is to protect the inner ear from trauma, for example loud noise, barotrauma or direct trauma.

It has been suggested that the incudomallear joint (IMJ), located sideways between the ossicles rather than top to bottom, has a suboptimal construction for sound transfer (Figure 4). The gliding movement of its surfaces supposedly works protectively by decoupling excessive displacements from the TM and malleus to the stapes and cochlea (16). The ossicular chain is mobile, yet stabilized by a number of ligaments, two joints and two muscles. It is also partly covered by bone (Figure 3). This could explain why injury to the ossicular chain is rare and why joint dislocations are more common than fractures or penetration to the inner ear. More on this is presented in “Trauma and malleus fractures” on page 14.

Muscles of the middle ear The exact functions of the middle ear muscles have been under speculation since they were first discovered by Vesalius (1514–1564) (17). In several mammals, both the stapedius muscle (SM) and the tensor tympani (TT) contract in response to auditory stimuli (18-20).

In humans, the stapedius reflex is considered to be the dominant acoustically evoked middle ear muscle reflex pathway (21). The SM is the smallest skeletal muscle in the human body. It is innervated by the facial nerve and its acoustic reflex is triggered by loud sounds (70–100 dB). An SM contraction exerts forces perpendicular to the stapes superstructure, increasing the impedance and decreasing the intensity of the sound transmitted to the cochlea by about 15 dB (21, 22).

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The SM reflex has been suggested to improve speech recognition in low-frequency moderate noise levels, but coexisting pathology in patients with non-existing reflex makes it difficult to find adequate controls (23). A reduced reflex has been associated with degraded speech recognition at high presentation levels (24). Verifying the absence of the stapedius reflex is a cornerstone in otosclerosis diagnostics, even in patients with minimal air–bone gap (ABG) (25). Overall, the SM’s role as protector of the inner ear against loud noises is widely accepted.

The TT muscle is innervated by the trigeminal nerve. The TT exerts force on the handle of the malleus, resulting in an inward movement of the TM. A TT contraction audiometrically results in a low-frequency mixed hearing loss (26). The TT has been reported to be activated by tactile stimulation of facial areas, swallowing, yawning, laughing, coughing, vocalization, and as part of the startle reaction, but not by external sound (21, 27, 28). In one study TT activation during vocalization was considered to be coupled with movement of the pharynx–larynx since the TT response always preceded, or occurred simultaneously with, the onset of sound (28). In contrast to the SM, relatively little is known about the function of the TT, since no valid method of detecting its contraction exists. Much of what is known comes from studies on individuals who are able to voluntarily contract the TT (26, 27, 29).

Even though the function of the human TT is not completely understood, different roles have been attributed to it (21). Hieronymus Fabricius (1533–1619) was the first to introduce structure–function relationships in anatomical research. He suggested that the TT muscle “protects” the TM and contributes to middle ear ventilation (17). More recent speculations suggest that the TT performs a bellows-like function that, combined with ET opening, produces an active middle ear gas exchange (30). A morphological study on human anatomy suggests that the TT and the tensor veli palatini muscles constitute a functional unit (31). The tensor veli palatini is one of the muscles that open the ET (30, 32).

Both middle ear muscles have been suggested to have an alternative function, namely to “exercise” the ossicular joints to maintain mobility and protect them from ankylosis (16). Middle ear myoclonus is a rare condition with repetitive contractions of the middle ear muscles resulting in a form of tinnitus. Whereas SM contraction produces a buzzing sound, TT contraction has been described as a clicking sound (33). No treatment consensus exists.

Activation stimuli of the middle ear muscles (21, 27) and the presence of muscle spindles in both the TT and the SM (34) have been studied in humans, but the strength of these muscles (35) and their muscle fibre composition (36-39) has, to our knowledge, only been studied in animals. For further details on muscle composition in general and middle ear muscles in particular, see “Muscle analysis and fibre phenotypes” on page 26.

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The mastoid and the Eustachian tube Optimal sound transfer requires that the middle ear maintains an atmospheric pressure matching that of the surrounding environment (40). With the exception of the TM and round window membrane the middle ear is rigid and lacks the elasticity required for large volume/pressure changes (40). It is, however, connected via the antrum to the posteriorly located, also rigid, honeycomb-structured, mastoid which functions as a physiological passive pressure buffer (30, 32). The middle ear itself has a volume of about 0.5–0.6 mL and the mastoid pneumatic system varies greatly from <1 mL to about 30 mL (30). Regardless of the cause of this variation, be it hereditary or environmental, there is unanimous agreement that a small mastoid cell system is associated with pathologic conditions (32). A small mastoid does not automatically lead to problems in an otherwise healthy ear, but it may be a disadvantage, as it cannot perform its role as well, resulting in faster deviation from normal pressures and greater forces applied to the TM (32).

The middle ear and mastoid cells are lined with mucosa, through which an ongoing gas exchange takes place that continually generates a net absorption, resulting in negative pressure (30, 32, 40). Persistent inflammation of this mucosa can impair mastoid growth and pneumatization and may further decrease the pressure by altering the gas exchange function of its subepithelial layer (41).

Nature’s solution is ventilation through the ET. The ET exits the middle ear as a mucosa- covered bone funnel. The latter two-thirds of this is a more complex organ with mucosa, cartilage, soft tissue and peritubal muscles. This portion is normally closed in the resting position, but sometimes when we swallow or yawn it is opened. The opening of the ET is attributed to the levator veli palatini and tensor veli palatini muscles (30, 32). A study found that the ET opens on average 1.4 times per minute for 0.4 seconds during daytime (42). During sleep, the frequency is substantially reduced (32). The ET is an essential part of the middle ear pressure regulation system, but other functions are mucociliary clearance into the nasopharynx, protection against reverse transportation of pathogens and vocal sounds (autophony) (32).

The tympanic nerve, a branch of the glossopharyngeal nerve, provides sensation to the middle ear, including the internal surface of the TM, the mastoid cells and ET (and the parotid gland). Whether, and how, this is related to middle ear pressure regulation or other functions is unclear. The importance of well-regulated middle ear pressure outside of sound transferal is made obvious by the pathology that can arise when at least a period of lower than atmospheric pressure endures, including secretory otitis media, chronic otitis media (COM), atelectatic TMs, retraction pockets and cholesteatoma (30). More on this will be discussed under “Middle ear disease” on page 11.

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The cochlea The human cochlea is a snail shell-shaped organ with approximately two and three-quarter turns from base to top. Laid out straight it would measure 42 mm (43). Its first turn forms the centrally located bulge in the middle ear, known as the “promontory”. The cochlea contains two apically narrowing spaces filled with a fluid called “perilymph”. The upper space is called “scala vestibule” and the lower space is the “scala tympani”. They meet at the tip of the cochlea. The scala vestibuli is connected to the middle ear via the oval window and, therefore, the stapes footplate. The scala tympani is connected to the middle ear via the membranous round window which aids in fluid motion and hydraulic pressure equalization in the otherwise rigid otic capsule. Inside the cochlea, the scala vestibuli is separated from the scala media by the extremely thin Reissner’s membrane. This third room, the scala media, is where the organ of Corti (the hearing organ) is located (Figure 5).

Figure 5. (A) Scanning electron microscopy of a cross-section of the human cochlea. The white frame is magnified in (B), showing a higher magnification of the organ of Corti. The supporting inner pillar cell is slightly collapsed, placing the inner hair cells further away from the tectorial membrane than in vivo. (C) Schematic drawing of cochlea with emphasis on the organ of Corti. (B) and (C) have been coloured to match each other. Movements of the basilar membrane (green) create a shearing force between the cilia of the inner (red) and outer (blue) hair cells and the undersurface of the tectorial membrane (lilac). Nerve fibres are presented in yellow. Images (A) and (B) are taken from publications by Helge Rask-Andersen, Anneliese Schrott-Fischer, Rudolf Glueckert and Kristian Pfaller, Uppsala University and Innsbruck Medical University. With permission to reproduce.

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As vibrations propagated via the stapes footplate are transformed into hydrodynamic pressure in the perilymph, this starts a travelling wave in the basilar membrane, creating a shearing force between the hair cell cilia and the tectorial membrane. The 12 500 outer hair cells modify and amplify the movement of the basilar membrane; the 3 400 inner hair cells are the sensory cells of the cochlea, each connected to ten nerve cells responsible for the signal transduction that becomes sound in our brains (44). The basilar membrane, owing to its rigidity gradient, and the inner hair cell nerves, owing to their placement, have a tonotopic arrangement, meaning that the highest frequencies cause basilar membrane movement and neural transmission at the base of the cochlea by the oval window, with gradually decreasing frequencies operating further along the way towards the tip (45).

Sound can reach the cochlea through two different pathways: (a) The first is called “air conduction (AC)” and refers to the normal mode of hearing via the external, middle and inner ear. In normal ears, AC can be subdivided into ossicular coupling (most important), where sound reaches the inner ear via the TM and ossicular chain, and acoustic coupling, which is more relevant when the ossicular chain is disrupted and sound vibrations directly reach the oval or round window (46). (b) The second pathway, “bone conduction (BC)”, involves sound travelling through the skull bone to the cochlea. This pathway involves several mechanisms, such as: cochlear fluid inertia, compression of the cochlear walls, pressure changes via the cerebrospinal fluid, vibrations via the soft tissue and/or a foreign body of the ear canal, and ossicular inertia (47). In everyday life, BC is irrelevant, but BC does have some clinical significance.

Since propagation of the wave in the basilar membrane is identical, regardless of whether the cochlea is stimulated by the AC or the BC pathway (47, 48), BC remains the most reliable way of directly testing cochlear function (47). Air conduction hearing levels are tested via headphones, while BC is tested via a bone vibrator placed on the mastoid. The difference between AC and BC is called the “air–bone gap (ABG)” and it is a measurement of the theoretically possible hearing improvement in reconstructive otosurgery. Bone conduction is also essential for the function of bone-anchored hearing aids that can bypass a non-existent or faulty ossicular chain.

Sensorineural and conductive hearing loss Since p0, the threshold of hearing (see “Sound and how it is measured” in the Introduction) in the dB hearing level scale is based on a population average, there are individuals who can hear <0 dB. Hearing is considered normal at ≤20 dB (49) and references for hearing loss are as follows: mild 21–34 dB, moderate 35–49 dB, moderately severe 50–64 dB, severe 65–79 dB, profound 80–94 dB and complete 95+dB (49).

Sensorineural hearing loss (SNHL) typically originates from pathology in the cochlea or the vestibulocochlear nerve. Hearing loss due to central pathology such as a stroke exists but will not be considered further in this thesis.

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Exposure to loud noise is a well-known cause of SNHL and can be divided into short extreme exposure, where noise >130 dB has been shown to cause damage (4), and prolonged exposure. Prolonged exposure in the work environment is often regulated, since keeping noise levels over 8 hours at 80 dB or below has been shown to minimize the risk (4). Sensorineural hearing loss often increases with age and may be accompanied by tinnitus (44). Other causes of SNHL include infections, ototoxic drugs, genetic factors, and benign or malign tumours in or adjacent to the vestibulocochlear nerve.

In SNHL both the quantity of the perceived signal, as measured by pure-tone audiometry, and the quality of the signal, as measured in speech discrimination tests, can deteriorate. The most common treatment for SNHL is some type of hearing aid. Surgery to improve SNHL is generally performed in severe forms of SNHL, when cochlear implants may be indicated or, in rare cases, brain stem implants (44). However, other types of implantable hearing aids have become more common during the last 20 years.

“Conductive hearing loss (CHL)” refers to pathology that impedes AC. This includes secretory otitis media, foreign bodies or wax in the ear canal, congenital or acquired ear canal stenosis, a ruptured or fixated TM and discontinuation or fixation of the ossicular chain. Because of the BC pathway the CHL cannot exceed 60 dB and in most cases, it can be surgically treated. A combination of SNHL and CHL is referred to as a “mixed hearing loss” (Figure 6).

Middle ear disease In the early 1900s otosurgery mainly consisted of life-saving operations aimed at draining abscesses caused by infection (44). The introduction of the operating microscope (the monocular microscope used for surgery in humans by Nylen in 1921, and the binocular surgical microscope created by Holmgren in 1922 (50)), antibiotics (penicillin, discovered by Fleming in 1928, and in common use since the 1950s) and the electric drill (first used in surgery in the 1950s) had a revolutionary impact on mastoid surgery (51). From the 1960s reconstruction of the TM and ossicular chain with different materials became more common (51). Vaccination programmes and antibiotics have greatly decreased the severity of infections in the high-income countries of the world. Still, mortality due to complications to ear infections occurs and in poorer countries its prevalence is more than tenfold the prevalence in high-income countries (52). Hearing loss that could have been prevented by vaccinations and antibiotics is far more prevalent in countries that lack the means to treat it. Even though hearing loss is largely caused by ageing, it is estimated that its prevalence will increase more in countries with lower access to health care (49).

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Pathological processes, infections and complications after surgical interventions can affect many of the anatomical structures surrounding the ear. Below some examples are listed:

Dura: intracranial bleeding, central spinal fluid leak, meningitis. Sigmoid venous sinus: bleeding, air embolus to the brain, thrombosis. Facial nerve: one-sided partial or complete facial paralysis. Chorda tympani: taste disturbance. Semicircular canal: severe rotational vertigo and balance issues. In one-sided damage this vertigo is often compensated for over time. Cochlea: partial or complete SNHL.

Note that because of their interconnectivity, damage to the inner ear often involves both the cochlea and the semicircular canal, to varying degrees.

Chronic otitis media

Chronic otitis media is a group of syndromes, rather than one illness (30), which have in common a chronic inflammation of the mucosa in the middle ear and mastoid. Permanent TM perforations and/or retraction pockets are seen in COM (53). Risk factors include poor ET function (30, 53), secretory otitis media, recurrent acute otitis media and different craniofacial abnormalities. Acute otitis media is the most common indication for outpatient antibiotic use in children worldwide (54).

Figure 6. Audiogram showing the mixed hearing loss of a patient with left ear cholesteatoma. The right, unaffected ear is marked in red. The blue dotted line shows bone conduction (BC); starting at 2 000 Hz, a sensorineural hearing loss (SNHL) caused by repeated infections can be seen. The air–bone gap (ABG) (area between the blue lines) indicates a conductive hearing loss (CHL). The ABG in this case is not complete even though the ossicular chain was disrupted. This is because the cholesteatoma sack itself bridged the chain and transmitted some sound. dB = decibel; ISO = International Organization for Standardization.

Common COM symptoms are CHL and discharge from the ear. Atelectatic TMs can sometimes erode the bone over the attic area including the ossicles (53). An isolated defect of the long incus process is the most common (55). A perforation of the TM commonly result in an ABG of ≤30 dB while an ABG of >30 dB suggests erosion and disruption of the ossicular chain (53) (Figure 6).

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Cholesteatoma Cholesteatoma, simply put, is skin in the wrong place. It can be congenital, which is mainly seen in children and presents as a white mass underneath an intact TM (56, 57). The more common, acquired cholesteatoma can develop primarily when a retraction pocket loses its ability to self-clean and starts accumulation of keratin debris. This can happen at any site of the TM but is more frequent in the pars flaccida (53) (see Figure 3 on page 4). Implantation due to TM perforation, trauma or surgical intervention has also been reported to cause acquired cholesteatoma (57-59). Cholesteatoma produces an inflammatory reaction that, combined with recurrent infections, causes osteitis and bone resorption in the adjacent area (53, 57). The ossicular chain is often eroded and the most commonly affected ossicle is the incus (55, 60). Cholesteatoma is generally more extensive and destructive in children than in adults (56). Even though the cholesteatoma itself is benign and does not grow invasively into surrounding organs, it can nevertheless be destructive and expansive by resorption of the surrounding bone and ossicles (Figure 7). Moreover, secondary infections can lead to severe or even fatal complications. An example of this was Oscar Wilde who suffered chronic ear infections, temporary loss of balance, and loss of hearing and eventually died on 30 November 1900 from meningoencephalitis despite an attempt of an early form of mastoidectomy. Ultimately his death has been suggested to have been caused by a cholesteatoma (61).

Figure 7. Photographs taken through a surgical microscope during different stages of operation of two left ear cholesteatomas. The nose of each depicted patient is pointing left. (A) Early stages of operation where the tympanic membrane (TM) (1) is still in its natural position. A posterior skin flap of the ear canal has been lifted from the bony ear canal (2) and the mastoid has been opened distally, exposing the cholesteatoma (3). Owing to the size and destructive growth, the bony ear canal (2) eventually had to be removed to safely remove all cholesteatoma, a so-called “canal wall down” procedure. (B) The reconstruction of a less invasive cholesteatoma. What is left of the TM (1) is folded forward. The bony ear canal is left intact (2); this surgery is referred to as “canal wall up”. All cholesteatoma has been removed and the bone underneath carefully polished clean, therefore number (3) is missing in (B). An autologous partial ossicular replacement prosthesis (PORP) (4) has been constructed and placed on the remaining stapes. The lateral semicircular canal (5) as well as the dura (6) can be visualized during the mastoidectomy.

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Otosclerosis Otosclerosis is a disease with localized bone dysplasia, primarily in the otic capsule and stapes footplate (80%), but other parts of the TB including the ossicles can also be affected (62). The disease primarily presents as CHL, but engagement involving the cochlea can cause SNHL and/or mixed hearing loss (62). A typical audiogram shows an ABG with AC hearing loss greater at lower frequencies. Bone conduction hearing is generally normal, with the exception of a distinct notch-like “false” dip around 2 000 Hz (the so-called “Carhart notch”) (25). This notch is not unique to otosclerosis and stapes fixation; it can also be present in incudostapedial joint (ISJ) detachment, malleus or incus fixation (63) and malleus fractures, according to our clinical experience.

A fixated stapes footplate disables the stapes muscle function. Verifying the absence of the stapedial acoustic reflex is a cornerstone in otosclerosis diagnostics even in patients with minimal ABG and CHL (25). The causes of otosclerosis remain unclear (62).

Trauma and malleus fractures Traumatic middle ear lesions are relatively frequent but are rarely reported in scientific publications (64). The most common injury is perforation of the TM followed by incus luxation (64-66). The foremost cause of TM perforation in most countries is assault, often in the form of a slap to the ear (67-69). In second place comes self-inflicted injury due to an attempt to clean the ear with a cotton swab or similar device (66-69). Diving comes third, followed by a mix of motor vehicle accidents, explosive blasts, thermal or caustic burns, etc (67-69).

Traumatic TM perforations spontaneously recover in ca. 90% of cases (68, 69). Even with self-inflicted penetrating injuries to the ear, joint or ossicular luxation is far more common than ossicular fractures (66). Injuries including the malleus are rare and occur in only 2% of cases (64).

Isolated malleus shaft fractures are exceedingly rare, with fewer than 100 published cases (64, 70-79). However, several authors underline that this condition is easily missed, since the TM is intact (70-75, 77-79), thus leading to underdiagnosis (70, 71, 75). The most commonly reported trauma mechanism is a person using a finger to evacuate the wet ear canal following a shower or bath (70).

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Some patients seek medical aid in connection with ear trauma, after an average of 6 days (80). However, many patients wait, for various reasons. The average delay from injury until treatment is 6–7 years (65, 66, 80). In most cases CHL can be treated with good results even when performed long after the trauma, although pathology developing along the way, such as COM, cholesteatoma and subsequently bone corrosion/necrosis, can complicate matters (65, 69, 80). Fractures to the ossicles and injury to the facial nerve and inner ear are more commonly associated with more severe trauma (80).

Temporal bone fractures caused by motor vehicle accidents and other high-energy trauma are connected with relatively high mortality, owing to associated intracranial injuries (81). When the otic capsule is involved, cerebrospinal fluid leaks can increase fourfold, and there is a sevenfold increase in SNHL and a significantly higher risk of facial paralysis (81, 82). Furthermore, there is a high risk of ear canal stenosis and cholesteatoma formation when the ear canal is severely traumatized, as seen with gunshot wounds to the TB (58, 59).

Leakage from the cochlea or vestibule, most commonly through the round or oval window, is called a “perilymphatic fistula”. It can have several causes, for example penetrating trauma, barotrauma and TB fractures. Treatment of these fistulas can be performed by plugging the fistula with fascia, blood or perichondrium in order to prevent future SNHL (83, 84).

Patients with trauma involving the inner ear (for example, luxation of the stapes) often present with severe vertigo, nystagmus and reduced cochlear function and are at risk of severe SNHL or a dead ear (80, 85).

Hearing reconstruction A perforated or defect TM is repaired by using temporalis fascia or cartilage from the tragus/concha, or sometimes with non-autologous grafts (86). Cartilage has been suggested to be beneficial in atelectatic ears when the aim is to avoid new retractions of the TM (87). Data gathered in the Swedish Register of Otosurgery between 2013 and 2020 (including a total of 4 187 myringoplasty operations) show a healing rate of 88%. The reported main goal of the operation was infection prophylaxis in 46% of cases, infection prophylaxis and improved hearing in 48% and improved hearing in 6% (88). This suggests that infections were a significant issue for these patients.

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Figure 8. Schematic drawings of (A) Normal ossicular chain. (1) Circle marking the outline of the tympanic membrane (TM); (2) malleus; (3) incus; (4) stapes. (B) Placement of a partial ossicular replacement prosthesis (PORP) in the presence of an intact and mobile stapes, and (5) the PORP. (C) Placement of a total ossicular replacement prosthesis (TORP), generally used when the superstructures of the stapes are damaged or completely missing, and (6) the TORP. (D) A stapes prosthesis (7), cramped onto the long process of the incus and placed through the stapes footplate that is fixated as a result of otosclerosis. Note that, for ease of visual comprehension, the stapes and its footplate have been rotated approximately 90° counterclockwise from its natural angle.

In otosclerosis, a fixed footplate can be intentionally or unintentionally mobilized during surgery (89, 90); however, recurrent ankylosis is the Achilles heel of this procedure (89). Therefore, stapedectomy was invented, which entails removing the stapes including the footplate or part thereof, and placing a prosthesis between the long process of the incus and a graft covering the oval window. This was first described by Shea in 1956 (91). During the last decades this operation has been widely replaced by stapedotomy (92) which gives a better high-frequency gain, reduced risk for SNHL, and stable long-term results (89, 92). Stapedotomy, entails removing the stapes but keeping the fixed footplate and inserting a 0.4–0.6 mm wide prosthesis through a hole in the footplate that is made with a drill, laser or pick (see Figure 8D). There are many variations in surgical techniques as well as prostheses on the market. Although requiring skill and experience, stapedotomy is a standardized operation with generally reliable short- and long-term results (92). Complications are few, SNHL being the most frequent (92). Deafness is a rare but known complication that increases with revision surgery (90). The most common findings during revision surgery are prosthesis dislocation followed by erosion of incus with fracture (90). More details on this topic can be found under “Future perspectives” on page 56.

The percentage of patients who have their ABG reduced within a given mean is often used as one measurement of success in otosurgical studies. In otosclerosis surgery this mean ABG is generally £10 dB (89, 92, 93). In ossicular replacement prosthesis surgery it is generally £20 dB (94-99). This indicates that a perfect ossicular chain reconstruction, with complete closure of the ABG, is elusive.

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PORP, TORP and the challenge in clinical evaluation Ossicular chain reconstructions evolved during the 1960s and the concepts of partial ossicular replacement prosthesis (PORP) and total ossicular replacement prosthesis (TORP) (Figure 8B and C) were introduced in the early 1970s (51). In more than 80% of patients the cause of ossicular damage is cholesteatoma or COM (95, 100). Trauma or congenital malformation accounts for most of the remaining cases (100). The variations in pathology and individual healing conditions make accurate evaluation after otologic surgery challenging. For this reason, several numeric grading scales, using risk factors to predict hearing outcome, have been developed. Three commonly used scales are: (1) the middle ear risk index (MERI), a scoring system that has evolved from several previous grading scales, and therefore is not based on a database of patients (101). The latest version focuses on otorrhea, ossicular status, revision surgery, middle ear effusion and smoking (102). (2) The Surgical, Prosthetic, Infection, Tissue, Eustachian tube (SPITE) scale is based on twelve factors divided into the five previously named categories associated with significant poor outcomes that were identified in a material of 535 operations performed by the same surgeon. One example from each category is complexity of surgery; other examples are absence of malleus and/or stapes, chronic otorrhea, poor mucosa and middle ear effusion (103). This system, however, does not weigh the risks. (3) Ossiculoplasty outcome parameter staging (OOPS) is based on 200 operations by the same surgeon. This was the first attempt to create a weighted scoring system where fibrotic mucosa, absence of malleus, canal wall down surgery and revision surgery were given higher scores (99).

Using these three scales in a material of 179 cases, one study showed a significant correlation between each of these scales and hearing outcome, suggesting none to be superior, although only a low SPITE score could predict excellent results, of ABG <10 dB (101). One study found the absence of the malleus to be the only significant unfavourable factor (104). Another study surprisingly found that when using a cartilage TM graft, the absence of a malleus did not have a negative effect on the outcome (105).

Prosthesis materials It has been suggested that the main factors affecting long-term hearing results after ossiculoplasty are, firstly, the severity of disease, followed by surgical technique and, to a lesser degree, prosthesis design (99). It is, however, difficult to compare previous studies and compile data because of the variations in these three factors (55). The prosthesis type used in older and newer studies varies, since, for example, alloplastic materials have lost popularity because of high rates of extrusion (94, 100). The optimal prosthesis should be biocompatible, stable, easy to fit and capable of optimal sound transmission (106). Of the other materials that have been used, such as autologous grafts, gold, ceramics, hydroxyapatite, polyethylene and titanium, none have proved superior from a hearing perspective (96).

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Autologous bone and cartilage are biocompatible and do not cause extrusion; however, they may grow fixed to adjacent bone when in contact with it (100). The most commonly used autologous graft is an autologous incus graft, which is typically reshaped as a PORP to fit the stapes head (100). It is often used because of easy retrieval, low cost and biocompatibility but without sufficient cleaning it can pose a risk of residual cholesteatoma (107). Autologous cortical bone is an alternative when the incus or malleus has been too corroded by pathology; however, bone resorption can develop (94).

Foreign materials tend not to grow fixed to adjacent bone, but do at various levels cause extrusion of the prosthesis. Commonly used foreign materials due to their relatively low level of extrusion are hydroxyapatite and titanium, but they are expensive (94). Both hydroxyapatite (108) and titanium prostheses (109) have been used, with and without cartilage placed between the prosthesis and the TM. Using cartilage interposition combined with prostheses made of foreign materials is considered to significantly reduce the risk of extrusion and for this reason such praxis is generally recommended. It has been suggested that hydroxyapatite and most other biomaterials will extrude in the face of recurrent or persistent middle ear disease (110). A meta-analysis found no significant difference in hearing outcome between titanium and non-titanium prostheses; all studies included mentioned the use of cartilage to prevent extrusion (96). A study comparing autologous ossicles, cortical bone, hydroxyapatite and titanium found no significant difference in hearing outcome but a tendency towards a higher success rate for titanium in conditions associated with poor prognosis (94). Titanium has been suggested to have a smaller extrusion rate than hydroxyapatite (98) and a thinner titanium stem can be placed where a thicker hydroxyapatite stem cannot (108). It has been suggested that placing the prosthesis under the malleus stabilizes the implant and may decrease the likelihood that severe TM retraction will lead to prosthesis extrusion. This can also lead to significantly better hearing (108). Cartilage may support the TM and prevent retractions (87) and aid against prosthesis extrusion. On the other hand, it hinders visibility, making visual inspection of for example residual cholesteatoma impossible, and requiring checkup by magnetic resonance imaging (MRI).

Regardless of the choice of material for prostheses and reconstruction, it can be argued that, with the continual improvement of hearing aids and the increasing number of alternative implantable devices such as BC implants, active middle ear implants, Cochlear implants (CI), a long-term stable ear free from infection should be considered as an important outcome besides hearing.

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Figure 9. The choice of material, in percent, of all ossicular replacement prostheses used, as reported in the Swedish Register of Otosurgery between 2013 and 2020. Note that cholesteatoma was included in the register only after this period and is therefore not present in these statistics (88). PORP = partial ossicular replacement prosthesis; TORP = total ossicular replacement prosthesis.

Human temporal bone

Health care professionals and clinical scientists work to cure diseases or lessen their negative effects on our patients. While striving for these goals, we have a responsibility to minimize the risk of side effects or injury. To quote the editor of the Journal of Microsurgery, Donaghy, in 1979, “A first experience has no place in the operating room” and “The laboratory is the place for practice.” (111).

The human TB is well suited for both surgical practice and research as it undergoes few changes post-mortem. The structural integrity of its components and mechanical functions is more or less unchanged if the loss of active muscle contractions of the TT and SM is disregarded. Hence, theories can be tested and skills improved without putting patients at risk.

Temporal bone in clinical practice The complex anatomy and the number of sensitive structures to take into consideration make TB surgery a challenging endeavour fraught with risk of serious complications. Depending on pre-existing pathology the mastoid pneumatization can vary greatly. In cholesteatoma cases, drilling through compact bone in search of landmarks is common. Furthermore, cholesteatoma and infections may weaken or completely remove the protective bone around these sensitive structures, thereby increasing the risk of injury during drilling. Before taking an active role in surgery the clinician needs considerable anatomical knowledge and practical abilities, such as working under magnified vision with adequate hand–eye coordination and fine motor skills.

Previously the only safe way to acquire the surgical knowledge and skills needed was drilling on a TB in a lab with instruction from a surgeon competent in the field. Today, plastic models and virtual reality (VR) simulators exist and are continually being improved. As computer technology advances, VR simulators become more cost-effective. Thanks to integrated tutoring they can save precious time from instructing clinicians.

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Studies show that VR simulation lacks in fidelity but increases the surgical confidence of trainees (112). Virtual reality training before cadaveric dissection has been shown to improve the performance of novices (113). The continuous development of 3D printing machinery and its implementations is improving available options in this field (114). It can provide a variety of anatomical variants for training. The closer the model is to actual surgery, the greater the benefit (114). The present authors, however, all hold TB dissection as gold standard before commencing supervised surgery (112-115). Integration of a standardized assessment format has been suggested for better use of 3D printed TBs and VR simulators (112).

It has been suggested that VR and plastic models may be beneficial in the early stages of training of junior trainees but that fresh frozen cadaveric TB specimens should be utilized once the otologist has reached the autonomous stage (115). This thinking naturally continues into surgery and evaluation of technical skills of residents. Using defined competency milestones for the steps of cortical mastoidectomy has been suggested as a potential way to identify and help remediate weak areas of surgical skills (116). Regardless of the training method, experienced surgeons could probably benefit from practising alternative approaches or new implantable devices before applying them in vivo. In the future, patient-specific models may be beneficial in high-risk operations as they will allow preoperative planning and rehearsal of complicated rare cases.

A UK-based survey, admittedly with only 30% response rate, showed that 43 out of 57 responding trainees attended a hospital with TB facilities. Respondents reported poor availability of human TBs, and only three reported use of these facilities. The availability of supervisors was also poor, which was the reason why 53 trainees were sent on a national course during their first 2 years of training (117). Results were quite different in a European questionnaire study with responses from 113 departments from 23 countries, albeit unevenly distributed. A total of 73% of departments offered in-house training, and of these, 68% utilized TB training only and 21% a combination of TB, VR and physical models. Most reported access to sufficient numbers of TBs. The 11% who exclusively used VR simulation and/or plastic model training stated that these were mainly used because of poor availability of human TBs (113).

Worth noting is that the respondents in the present study were mainly the seniors in charge rather than trainees. However, as a result of legislation, a decrease in donations and autopsies and other factors, some countries have a decline in access to TBs. One such example is the UK (115, 117).

In Sweden a new organ transplant law was introduced in 1996 and, similarly to the UK, it requires consent for donation. The Swedish National Board of Forensic Medicine had previously assisted the health care sector in obtaining donor materials. During 2000 the Board investigated the need for such cooperation and deemed it of humanitarian importance (118). This resulted in a centralized organized process where recovery of

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needed materials is registered and possible donors identified. The process ensures that the organ donor register is controlled, next of kin are contacted and a decision concerning donation is made and documented before the material is delivered. At present, the only other area of use besides donation to patients is that of TBs for surgical practice (118). This shows the importance of collaboration between national agencies when laws change, even when they change for the better.

To summarize, there is development and promise in new technology but most otologists would concur with Duckworth. (119) when stating, “An essential way of learning temporal bone anatomy and surgical technique is through repeated dissections of the temporal bone in a cadaver dissection.”

Temporal bone in research As described under “Hearing reconstruction” on page 15, confounding factors make evaluating otosurgical techniques challenging. It has been acknowledged for many years that trying to draw valid conclusions regarding surgical technique from purely clinical studies is difficult (55, 120). A variable that is rarely considered, but that could be a complicating factor, is our primary method of clinical evaluation, pure-tone audiometry. This is a psychoacoustic method that has a test–retest error in the order of 10–15 dB in adults due to attention fatigue. This variability is largest at very high and low frequencies (121).

The TB has been suggested as a useful model for studies of middle ear function and reconstruction (122). As long as the extracted middle ear is kept moist and at normal air pressure, the input impedance is indistinguishable from in vivo measurements (122). For practical reasons, TBs are often harvested, frozen, transported and thawed by the end user. The effect of this is debated. One study suggested that freezing and thawing made little difference on impedance (122). Another study found that freezing and thawing TBs can change the stapes–cochlear input impedance and that the main factor is air allowed to leak into the inner ear. This can largely be reversed by refilling the inner ear with saline (123). Yet another study found that draining the cochlea made little difference to the stapes motion (124). In our model, removing the cochlea ensured that no ear bubbles or pathology existed within. As our TBs were prepared in Sweden and measured in Belgium, freezing was the only option.

The advantages of TB studies are that it is possible to perform accurate, repeatable and objective measurements. Small changes can be evaluated under the same conditions and reasonable sample sizes can be obtained with various alterations and use of few TBs. This is good for optimization of common procedures in a controlled environment, evaluating rare injuries and new techniques or extending our base of knowledge by studying the mechanisms of the middle ear. Below are some methods used in TB research.

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Laser Doppler vibrometry Laser Doppler vibrometry (LDV) uses changes in the frequency of a reflected low-intensity laser beam to measure vibration velocity at a single point. Since it adds no mass or stiffness to the measured object, it is well suited to measure delicate structures with great accuracy. It has, for example, been used in an excellent TB study to measure the movements of the basilar membrane (48). However, it has primarily been used for many years to assess stapes velocity and thus determine sound transmission in human TBs, which can be translated into hearing, and therefore loss of hearing and level of improvement through reconstruction (125-128).

The Doppler effect was first described by Christian Doppler in 1842. It is the change in frequency of a wave in relation to an observer who is moving relative to the source of the wave. The received frequency, compared with the emitted frequency, increases when the source and the observer move towards each other, as the waves “bunch together”, and decreases when they are moving away from each other as the waves “spread out”. Most people can relate to this phenomenon regarding sound waves. An example is a vehicle with an active siren or a long hoot moving towards you, passing you and then travelling on. The perceived sound will alter because of the Doppler effect. This is true for all kinds of waves even if our senses cannot perceive them. Laser, an acronym for “light amplification by stimulated emission of radiation”, is monochromatic light of the same wavelength in phase. The helium–neon (He-Ne) laser used in Papers I and II is red in colour with a wavelength of 6.33 nm. An LDV machine uses the shift in frequency that occurs when the laser hits a vibrating target and some of the light is reflected back, to determine the speed and direction of movement. If the target is sufficiently reflective it can be directly measured. In TB studies using a “traditional setup”, as well as in our model (described under “Methods”), a light-weight reflective patch is generally placed on the stapes footplate (126, 129).

However, the frequency of the backscattered light is too high to measure. Therefore, a process called “heterodyne interferometry”, used in all LDVs, is implemented. This entails letting the backscattered light interfere with another beam with slightly different wavelength to produce a so-called “beat signal”. The beat signal is lower in frequency but contains the same Doppler shift and can therefore allow detection of speed and direction via a photo detector, as shown in Figure 10. A so-called “Bragg cell” is placed in the path of either the reference beam or the illumination beam. The Bragg cell modulates the frequency by a certain amount, usually 30–40 Hz, thus enabling the beat signal to become measurable in the MHz range.

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Figure 10. Schematic description of the laser Doppler vibrometry (LDV) setup. The LDV detects the frequency shift in backscattered light from a vibrating target. The change in frequency is called the “Doppler effect”. To achieve this, a laser beam with known frequency (f) is split by beam splitter 1 into a reference beam and an illumination beam. The illumination beam passes beam splitter 2 and, when it is reflected from the vibrating target, its frequency is shifted because of the Doppler effect (fd). The reference beam is frequency-modulated (fm) by the Bragg cell. Both beams are recombined to a beat signal after passing beam splitter 3. The beat signal contains the same Doppler shift but since it has a lower frequency, it can be detected by the photo detector. He-Ne = helium–neon.

Laser Doppler vibrometry measurements used to analyse the velocity of the stapes movements are directly proportionate to the hearing sensation. Just like our hearing, the velocity values have a very wide range and are converted to the same logarithmic scale, the dB scale. This is calculated with the formula 20 Log10 (speed/reference speed). The reference speed used is 50 nm/s, which is a particle’s velocity in air at 20 µPa (the threshold pressure of hearing in humans). The ratio of the dB in both audiometry and LDV is comparable, which is why this method has such widespread use in surgical evaluations in TBs.

Footplate motion

Because there is increasing rocking motion with rising frequency, stapes velocity at one site is suggested to be suboptimal at frequencies higher than 2 kHz (130). One study suggests that stapes velocity measurements are representative of footplate volume velocity up to 5 kHz but above this, with a peak at 6.7 kHz, the footplate motion is particularly complex (9). This peak at just over 6 kHz has been explained as the rocking components of the footplate which provide a greater displacement at the edges than the piston-like motion does at this frequency (14). However, with the cochlea drained, the piston-like motion increases relative to the rocking component (14). It has been suggested that because of these variations it is best to measure at the centre of the footplate if only one site is used (14).

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The traditional temporal bone laser Doppler vibrometry setup

In vivo, access to the middle ear is foremost achieved by lifting the TM with an adjacent skin flap; surgery is then performed and the TM and skin put back in place. With time and healing, the TM regains its natural tension. This cannot be done in a TB LDV study as the loss of tension would alter the sound transfer capabilities of the TM.

To measure any displacement in response to sound in TB, a sound delivery system (a speaker and a calibration microphone in an acoustic chamber) is needed, as well as visual access to the intended target of the LDV beam.

The first measurements on TBs using LDV technique measured vibrational velocity at the umbo. The sound delivery system and LDV measurements were therefore all made via the ear canal as long as the acoustic chamber holding the sound delivery system had a transparent glass cover for making the LDV measurements (127).

When accessing the stapes footplate for in vivo LDV measurement while keeping the TM intact, it is logical from an otosurgical point of view to use the alternative access to the middle ear, which is through the mastoid. This is done by performing a mastoidectomy and a posterior tympanotomy. However, this will generally not allow sufficient visual access to the footplate. Therefore, unlike in vivo, the removal of a portion of the facial nerve and its bony canal is usually performed (9, 125, 126) (see Figure 11). Cutting the stapes tendon for further visibility is also common but should not affect measurement since cutting the tendons of the middle ear muscles has been shown to have a negligible effect on middle ear impedance (122).

Figure 11. Schematic drawing of a temporal bone (TB), showing laser Doppler vibrometry (LDV) measurements of the stapes footplate via mastoidectomy. The red arrow represents the laser beam trajectory. X marks the acoustic chamber containing a small speaker and a calibration microphone. Generally, the bony ear canal is drilled open a few mm from the tympanic membrane (TM) and X is fixed and sealed in place, allowing controlled sound stimulus (9, 125, 126).

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An excessive angle diminishes the amount of backscattered light that finds its way back into the LDV device. For the angles in, and purpose of, this type of study, the signal quality is generally sufficient. Since the LDV detects movement in the direction of the measuring laser beam, the measured magnitude will be less than the true magnitude, if measured at an angle. If the angle is known, the true value can be calculated. However, since most studies are concerned with relative results with the same setting, this is generally not done (126). It is, however, important to keep the same angle between measurements and TBs in such a study to ensure inter-study reliability.

Vestibulocochlear pressure measurement A method of opening the inner ear in a TB and inserting a hydrophone into the scala vestibule to measure pressure has been described; these measurements have been compared with stapes velocity measurements and have been found essentially equivalent (131).

A challenge using this method is avoiding introduction of air bubbles and maintaining intracochlear hydrostatic pressure, while the integrity of the inner ear is violated to accommodate the hydrophone (131, 132). Not having the prosthesis placement and the site of measurement competing for the same limited space has been suggested as one benefit of this method. Another suggested benefit is avoiding the effects of the rocking motion at the stapes footplate at higher frequencies (described previously under “Laser Doppler vibrometry”) (132).

Digital holography The method of digital holography has been used in TB studies to visualize the motion of the TM and how it varies due to sound stimulation at different frequencies. How this movement relates to middle ear sound transmission has been theorized (12) but to our knowledge this is not fully understood. These results have, however, been used in the refinement of finite element models (133).

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Muscle analysis and fibre phenotypes Muscle analysis is not typically performed in TB research. To our knowledge, strength assessments (35) and muscle fibre composition (36-39) have previously only been studied in animals. This is likely due to their inaccessibility for testing in vivo. It may be possible to perform tests on the TT and SM during skull base surgery, but this would probably entail unwarranted risks for the patients.

For about a century, it has been recognized that skeletal muscle can be divided by colour and contractile characteristics into slow red and fast white muscles. To date, myosin heavy chain (MyHC) composition is regarded as the best marker of functional heterogeneity of muscle fibres (134). Myosins are a superfamily of motor proteins essential for muscle contraction. They exist in several MyHC isoforms, differently distributed in various fibres. In mammalian skeletal muscles there are four major fibre types with distinct MyHC composition: one slow MyHC isoform, referred to as type 1, and three fast MyHC isoforms, referred to as types 2A, 2X, and 2B, although 2B appears not to exist in humans (134). The different fibre types vary in energy production. Type 1 and 2A fibres primarily use oxidative metabolism, while type 2X and 2B fibres primarily rely on glycolytic metabolism. The slow-contracting type 1 fibres normally have more mitochondria and higher vascularization than fast MyHC fibres. This, combined with low energy demand when activated, gives slow fibres capacity for high endurance. As we progress through the fast fibre types in the abovementioned order, speed, peak power and energy demand all increase, while these fibre types are increasingly prone to fatigue in the same order (134).

In humans, muscle fibres expressing slow MyHC-1 and fast MyHC-2A are the dominant fibre types in skeletal muscle. Muscle fibres expressing two or more isoforms are classified as “hybrid fibres”, i.e. fibres expressing MyHC-1 and MyHC-2A or MyHC-2A and MyHC-2X. Hybrid fibres commonly exist in a relatively low proportion in limb muscles, but increase during fibre transformation, which can occur as a result of significant changes in thyroid hormone levels, or neuronal innervation and physical load (134). However, some specialized human cranial muscles have a relatively high proportion of hybrid fibres as their normal fibre composition (135-138). Interestingly, these muscles also contain fibres expressing various combinations of additional MyHC isoforms that are not present in limb muscles, i.e. embryonic, foetal, alpha-cardiac, extraocular and slow tonic MyHC. Therefore, many of the human cranial muscles can be considered highly specialized, differentiating themselves from other skeletal muscles in terms of their structural and functional characteristics (134).

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Concerning the middle ear muscles, animal studies suggest that the SM has a similar fibre type composition in humans as among several other species, with predominance of fast 2A and 2X MyHC fibres (36-38). Tensor tympani, on the other hand, has been reported to be highly variable in fibre type composition between species (37). Myosin heavy chain-slow tonic has, along with specialized fast fibres, been detected in the TT of several carnivore species (39, 139). Slow tonic fibres are, as the name suggests, slow-contracting and highly resistant to fatigue. In mammals they are usually only found in the extraocular muscles (39, 138). Foetal MyHC isoform has been found in TT slow type 1 and fast 2A fibres in cow and pig and in type-2 fibres in rat (38, 140). This MyHC isoform is typically expressed perinatally and is replaced by adult isoforms after birth. However, after injury and disease, this isoform can be re-expressed and can be a marker of regeneration in limb muscles (134). Foetal MyHC isoform does, however, persist in some specialized human muscles such as the eye, palate and jaw-closing muscles and the intrafusal fibres of muscle spindles (135, 136, 141, 142).

Besides the composition of MyHC fibre types in a muscle, fibre size and vascularization have a central effect on the performance of muscle fibres. The size of the capillary network is a variable indicating the capacity for oxygen supply to the muscle, and thereby a parameter of the muscle’s endurance.

Muscle spindles are sensory stretch receptors within the body of a skeletal muscle. They consist of a continuous capsule containing a number of intrafusal muscle fibres. Their primary function is to detect changes in the length and speed of muscle stretch and convey the information to the central nervous system. Activating motor neurons via the stretch reflex aids in resisting the said stretch. One animal study reported that no muscle spindles were found in the TT or stapedius of any of the species examined (sheep, cat, dog, rabbit, horse, cow, pig) (37). Another study suggested that a specialized fibre pairing might represent some sort of sensory structure, i.e. unencapsulated muscle spindles, in cat (143).

In humans, one study reported muscle spindle and spindle-like structures in middle ear muscles. While the muscle spindles in TT had a normal morphology, with several intrafusal fibres as in limb muscles, the spindle-like structure in the SM differed in morphology from ordinary muscle spindles and had only one intrafusal fibre (34). This is interesting, as the facial muscles in humans, which like the SM are innervated by the facial nerve, lack muscle spindles. More on this and alternative regulation can be read in Paper IV.

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Mathematical models For a long time, attempts have been made to create mathematical representations of the middle ear components to simulate experimental data with reasonable accuracy.

In such models there is always a risk that the relevant acting mechanisms are not correctly accounted for, partly because they have not been well understood. The first type of models that were created to simulate middle ear behavior were electromechanical circuit or network models, such as the one developed by Kringlebotn in 1988 (10).

With increasing quality of experimental data and computing power, alternative methods have been developed to further improve the replication of middle ear mechanics. Finite element models are developed by using micro-computed tomography (CT) data of TBs and experimental data on structural and mechanical properties of the middle ear components. These data can then be validated in an ever-expanding database of experimental middle ear response curves. One such study used micro-CT data from three TBs, so that interindividual differences in the middle ear responses that were due to variations in TB geometries could be accounted for (133).

Some parameters that were expected to be influential turned out to be of minor importance, and different geometries simulated with the same parameters produced significantly different responses, showing the complexity in creating such a model for the middle ear and making it resemble the response of a specific individual.

Other research groups have developed a whole-head human finite element model, based on data from cadaver and live human heads, for simulation of the transmission of bone-conducted sound (144). This model was used to evaluate the performance of different bone-conducting devices (145) and, further, to evaluate how extensive TB surgery affects BC (146). However, it is still important to validate these models by comparing the results with TB studies or with real patients [120]. As these models are being improved and validated, and as computer power increases, it will become possible in the future to develop experimental prostheses and implants from computer optimizations, based on finite element models. At the present date, however, TB experiments play a key role both in improving these models and in aiding our understanding of middle ear mechanics.

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Aims The goals of this thesis, as well as ongoing studies described in “Future perspectives”, are to use and further develop TB models to aid in the understanding of the human middle ear, and to improve clinical practice and guide future research without patient risk.

Paper I. To investigate how to best utilize a PORP for optimal sound transmission.

Paper II. To investigate how to best utilize a TORP for optimal sound transmission, and how a novel TORP design inspired by the bird columella would compare with conventional TORPs.

Paper III. To investigate the mechanism and forces needed to fracture a human malleus shaft, and whether the described trauma could present a risk to a normal ear.

Paper IV. To investigate the fibre composition and other properties of the human middle ear muscles in order to understand their function.

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Materials and Methods Papers I and II – laser Doppler vibrometry In this research we used our LDV setup which had previously been developed for an article on malleus fracture repair (129). It allows good access with minimal invasiveness for a broad variety of injury and/or repair simulations. Freshly frozen human TBs were used in Papers I (6) and II (9). Samples were kept at -20°C, or less, then thawed and used in the experiments. The medial surface of the stapes was exposed using a saw and drill to open the otic capsule and gently, with otosurgical instruments under a microscope, remove the cochlea.

This allowed for near-perfect perpendicular measurements of the lateral footplate surface.

Measurements were made with angles greatly exceeding any potential human error ±10° variation from a perpendicular angle, with no significant effect on the resulting output (data not published).

Removing the cochlea changes the impedance of the stapes footplate. The difference can be calculated but as we were interested in relative numbers between prostheses and this correction would be the exact same for each TB these calculations were not made. The possible change in motion at higher frequencies is of minor importance, since, as previously described, all LDV TB studies have limitations at higher frequencies (9, 14, 130).

The freezing and thawing of the TBs, however, could possibly lead to a perilymphatic fistula or leakage from the cochlea which, if undetected, could cause variations in stapes movement patterns between measurements (123). When we removed the cochlea, we did ensure that no ear bubbles or pathology existed within. In Paper II, two TBs from an initial eleven were excluded owing to pathology, one for obvious and extensive middle ear adhesions; the other had discrete otosclerotic foci at the medial side of the footplate, which without our approach would probably not have been spotted.

In order to access the middle ear in Papers I–III, the epitympanic recess was opened cranially starting at the antrum. Working anteromedially, great care was taken to stay below the bone surrounding the superior incudal, superior mallear, and lateral mallear ligamental attachments to avoid affecting the mobility of the ossicular chain. In some cases, a bone pillar could be left as a second opening was drilled anteriorly to the first. In others, these two were joined into an elongated, slightly bent hole. (The view from such an anterior opening is depicted under “Results” on page 44.)

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Laser Doppler vibrometry measurements were made with and without a bone hatch covering these openings, without significant difference to the results, and therefore they were left open in Papers I and II. These bone hatches have been used in our ongoing radiological studies (see “Future perspectives” on page 56).

A custom-made metal fixture containing an earphone speaker and a condenser microphone was made for each TB and screwed in place and sealed with elastic paste (Otoform AKX; Dreve Otoplastik GmbH, Unna, Germany) at the lateral opening of the bony ear canal of each TB.

Figure 12. Schematic drawing of a temporal bone (TB), with laser Doppler vibrometry (LDV) measurements taken from the medial side of the stapes footplate. The red arrow represents the laser beam trajectory. (A) Speaker delivering controlled sound at different frequencies. (B) Microphone measuring the sound pressure level. (C) Manipulation of the middle ear was done through a separate hole in the epitympanic recess. Note that our access point in this image is different from that in the published illustration in Paper I. The published illustration did not include the facial nerve which was not affected by our approach.

With this setup the output from the earphones was calibrated, with an accuracy better than 1 dB. The sound pressure for all measured frequencies was 90 dB. This level was chosen for its superior signal:noise ratio. The Doppler vibrometer (Polytec model 534 and controller OFV-5000) was mounted on an operating microscope (Zeiss OPMI Sensera, Jena, Germany). Using a joystick, the beam was positioned exactly on a reflective patch (0.04 mg, 0.4x0.4 mm) at the centre of the medial surface of the stapes plate. As long as the stapes footplate was kept moist with saline solution, reliable repeatable LDV measurements could be made without any sign of exhaustion or change over time.

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In Papers I and II all individual TB measurements were made within a number of hours during the same day. As a control, one TB was kept moist and cool in a saline-drenched wrap in a refrigerator for a week, with repeated measurements each day without deterioration (data not published). In this control, as in our other measurements, saline solution was applied to the stapes footplate before measurement. If any injury to the TM or other structures occurred during the manipulations this TB was retired from the study. For this reason, risky manoeuvres such as gluing the PORP to the TM in Paper I and loosening the TM from the malleus in Paper II were done when all other measurements were completed. More on these details can be read in Paper I and Paper II, respectively.

The only alteration in the setup between Paper I and Paper II was that the cartilaginous part of the ear canal was removed in Paper II. This made the sealant connection between the microphone and the bony part of the ear canal more uniform and made its shape resemble the in vivo anatomy. This did not change the distance between the microphone and the TM in the individual bones, which was dictated mainly by the zygomatic and mastoid processes. The rationale was that we noted a small variation in the velocity peak at approximately 4 kHz, which is the average resonance frequency of the human ear canal (4, 5). This peak moved slightly up or down the frequency range depending on the length of the remaining cartilaginous part of the ear canal, which varied between TBs beyond normal anatomical variation because of prior dissection. After adjustment for this variation the baseline recordings showed less interindividual variation, as seen in Figure 13. This alteration is likely insignificant concerning the results of either Paper I or Paper II as the presented data are based on changes within each measured TB; but the adjustment is still suggested in future studies for uniformity.

Figure 13. Mean stapes velocity in temporal bone (TB) prior to manipulation in Paper II, measured using the decibel (dB) sound pressure level (SPL) scale. The shaded area of the graph represents the standard error of the mean. Comparing this image with Figure 1 (on page 2) concerning the human hearing threshold on the dB SPL scale, the similarities suggest that a significant part of the shape of the curve is due to mechanisms in the ear canal and middle ear. Note that one of the curves must be turned upside down for comparison because this curve concerns output levels and Figure 1 shows input thresholds.

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Stapes velocity, in the result part of this thesis, as well as in Paper I and Paper II, are presented in an approximation of the dB hearing level scale and not the dB SPL scale. The results are presented so that the base TB measurement prior to pathology and repair will be a straight line at 0 dB. Deviations from this “normal hearing” due to a disrupted ossicular chain or reconstruction are thus made more visually clear and the results are easier to compare with audiometric results in vivo. See Introduction and Figure 1 for further explanation.

Paper I The effectiveness of different PORP types in restoring stapes vibration was compared. Common clinically used PORPs were chosen. All PORPs were adjusted for best fit in each TB and measured at least twice. Only minor variations occurred, and the best result was saved. The individual measurements of the eight different PORPs showed that they were well suited to be divided into three groups based on their lateral contact (see Figure 14 and Table 1).

Figure 14. Schematic drawing of the three groups of prostheses used in Paper I. The prostheses have been divided into these groups based on their lateral contact: (A) prostheses with only tympanic membrane (TM) contact; (B) prostheses with only malleus contact; and (C) prostheses with both malleus and TM contact.

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Table 1. Partial ossicular replacement prostheses (PORPs) used in Paper I for each temporal bone (TB) and divided into groups A, B or C based on their lateral contact: (A) prostheses with only tympanic membrane (TM) contact; (B) prostheses with only malleus contact; and (C) prostheses with both malleus and TM contact. Two types of prefabricated titanium prostheses were used, with and without a hook. The hook was used to connect to the malleus. All other prostheses were made using autologous bone. MH = pillar-shaped prostheses made from the malleus head. In the Table, all prostheses whose name starts with “PORP” were made from the incus and shaped like a boot. In group A, “PORP posterior” indicates that the “boot tip” pointed posteriorly on the TM while “PORP Umbo” indicates that the tip pointed towards the umbo. In group B, the PORP malleus was placed centrally under the malleus. And as with group A, in “PORP Umbo” in group B the tip was aimed towards the umbo. In group C, NiTa were custom prostheses made from incus. They were shaped so as to accommodate both TM and malleus contact (Figure 14C). *Glued PORPs (n = 4). After gluing, the TB was not used for further measurements.

Paper II The effectiveness of different TORP types and placements in restoring stapes vibration was compared. Common clinically used TORPs as well as an experimental bird-type prosthesis were chosen. All TORPs were adjusted for best fit, and measured and recorded as in Paper I. The different TORPs were subdivided into three main groups and one subgroup according to their lateral contact (see Figure 15 and Table 2 on page 35).

One study has suggested that malleus neck placement is beneficial (132). In our material these prostheses (referred to as “BMN”, for “bone malleus neck”, in Table 2) did not allow any TM contact and preformed inferiorly. Though they were mentioned in Paper II and appear in Table 2, they were therefore not included in any group. Experiments were also performed in one case with a flat and inverted TM, similar to that of a bird. More on this can be read in Paper II.

Group A Group B Group C

Temporal bone

Titanium MH PORP

Posterior PORP Umbo

PORP Malleus

PORP Malleus Umbo

NiTa Titanium

hook

1 1 2* 1 1 1 1 1 0 2 1 1 1 2* 1 1 1 0 3 0 1 1 1 1* 1 2 0 4 1 1 1 1 1 1 1 2 5 1 1 0 1 0 0 1 2 6 1 1 0 1 1 1 1* 1

Total 5 7 4 7 5 5 7 5

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Figure 15. Schematic drawing of the groups of prostheses used in Paper II: (A) lateral contact with the tympanic membrane (TM) only; (B) lateral contact with the TM and malleus; (C) lateral contact with the TM and the malleus, near the umbo (distal malleus); (D) bird-type prosthesis.

Table 2. Total ossicular replacement prostheses (TORPs) used in Paper II for each temporal bone (TB), and groups A, B, C, D based on their lateral contact: (A) prostheses with tympanic membrane (TM) contact only; (B) prostheses with TM and malleus contact; (C) subgroup of prostheses with TM and distal malleus contact; and (D) experimental bird-type prostheses, with TM and extensive malleus contact including distal malleus. Data on subgroup C are not separately presented but are included under group B. In the Table, abbreviations for prefabricated TORPs made of titanium start with “Ti”. All TORPs made of autologous bone are referred to as “bone” and hence start with “B”. These were constructed, either from incus bone or, when this was too short, cortical bone. BMN = bone malleus neck; owing to poor performance these prostheses are only mentioned in Paper II but were not included in any subgroup. TM = TM contact; TMDM = TM and distal malleus contact; TMM = TM and malleus contact; F+I = when all other measurements in this TB were done the malleus was carefully removed. Measurements were made with a TORP under a flat TM without tension and with four different long TORPs in an inverted shape. This is further described in Paper II.

The LDV measurements in Paper II were made during two separate sessions in Antwerp, Belgium. In Sweden the TBs were harvested at a central tissue bank and sent to our lab in Luleå where they and the prostheses were prepared. The different surgeries and LDV controls were performed in Antwerp. During the first session in Antwerp, standard prostheses and placements were tested. Digital holography images were made using a similar setup to the one described by De Greef et al. (147) and the results were in line with previously published data (12, 148).

Group A Group B D Temporal

bone BTM TiTM BMN BTMM

BTMDM (C)

TiTMM Bird F+I

1 1 0 1 1 1 0 0 0 2 1 1 1 1 0 0 0 0 3 1 1 1 0 1 1 0 5 4 1 1 1 1 1 1 0 0 5 1 1 1 1 0 1 1 0 6 1 1 0 1 0 1 1 0 7 1 0 0 0 0 0 1 0 8 0 1 0 0 1 1 1 0 9 0 1 0 0 0 1 1 0

Total 7 7 5 5 4 6 5 5

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High-resolution 3D scans of bird ossicles were examined. These came from parallel work in the Antwerp lab on the mechanics of a single-ossicle ear that Pieter Muyshondt has written his thesis on (149). We wanted to draw inspiration from birds, since using a TORP means creating a single-ossicle ear. Moreover, up to 4 kHz, the hearing ability of birds is comparable to that of humans (150). When designing the bird-type prosthesis, we initially intended to have a scaled-up bird ossicle 3D printed and then to modify it to fit around the malleus shaft and the anatomy of the human ear. Of the high-resolution 3D scans of bird ossicles available to us – chicken, mallard duck, ostrich and long-eared owl – it was the last named that was deemed in need of the least modification to fit our purposes.

We could, however, not find any laboratory at the time capable of 3D printing a prosthesis to this scale and we found no interest in altering the existing production line of titanium middle ear prostheses for a study of this scale. Therefore, we produced five experimental prostheses ourselves at our lab in Luleå. Silver was chosen for its favourable molding properties. The shape was inspired by the aforementioned long-eared owl ossicle, the results from Paper I and our digital holography measurements. During the second session in Antwerp these experimental prostheses were tested along with traditional prostheses.

Paper III Visual inspection of trauma in temporal bone

Ten fresh frozen human TBs, kept at -20°C, were thawed and prepared and, using a pressure application adaptor, screwed and sealed into the bony part of the ear canal. Via the same access point in the epitympanic recess as described in Papers I–II, a rigid, 30° angled endoscope connected to a high-resolution camera was used to obtain overview photos and videos (e.g. see “Results” on page 44). Negative pressure of various levels was applied via the ear canal adaptor to mimic the force of a finger being pulled out of the ear canal. At the same time the effects on the TM and malleus were observed and recorded. The negative pressure was applied either by releasing a built-up vacuum from a surgical pump (Ardo Master; Ardo Medical AG, Unterägeri, Switzerland) or directly via a 100 cl stainless steel syringe filled either with air or water or with a mixture of both. Our access point allowed us to stabilize or pull the TT tendon to visualize structural movements with a fictive muscle reaction.

Force causing a malleus fracture

To establish the force needed to cause a fracture, 30 malleus ossicles were broken in a controlled manner, ten ossicles from fresh frozen TBs and 20 dry-stored ossicles. It has previously been shown that preservation methods have no effect on the structure or mechanical strength of ossicles (151). The head of each individual malleus was fixated. An artificial membrane attached along the shaft was used to mimic the TM. This membrane was pulled via a dynamometer at an angle simulating the intended trauma. The force required to cause a fracture was recorded.

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Ear canal measurements

To calculate and measure the possible negative force exerted by extracting a finger from a wet ear canal both a mathematical and two experimental models were made. The required base data for these models were gathered from 60 adult test subjects, 30 male and 30 female. The length a finger of their choice could be inserted into their ear canal (X in Figure 16A) and the length of said ear canal (L1+X in Figure 16A) were measured.

Calculation of negative pressure force

Using the ideal gas law PV=nRT, where –

P = standard normal air pressure, defined as 101 000 pascal

A = area of the TM, average at 64.3 mm2, according to Pannu et al. (8)

X = length of the external auditory meatus filled with a finger

L1 = distance from the TM to the fingertip

L2 = total length of the ear canal, measured on our test persons to calculate L1 by subtracting X (Figure 16A),

and assuming that the external part of the ear canal forms a perfect seal around the finger during the event, making PV a constant, we can further derive the formula

𝐹 = 𝑃𝐴 × 012301

Using this formula, the force applied via the TM due to air expansion can be calculated. It is also possible to account for several variabilities, such as the likely scenario that the ear canal is not sealed during the entire extraction of the finger, by subtraction from X. Moreover, the amount of air between the finger and the TM could be lessened due to the presence of water (subtraction from L1). Since water expansion can be considered negligible, it was not accounted for in the equation. Hydraulic mechanisms work on the effectiveness of energy transfer in liquid. This mathematical model gives us a theoretical output in a fixed cylinder. It does not account for movement of the TM.

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Figure 16. (A) Schematic illustration of the force generating trauma when a finger is withdrawn the length of X from the ear canal. L1 marks the distance between the tympanic membrane (TM) and the fingertip. The tensor tympani (TT) muscle and its tendon are shown in red. (B) Schematic illustration of our scale model of the trauma with an artificial TM (lilac) with a cast in metallic malleus replica that attaches to the force-measuring dynamometer via a hook marked H. V marks a screw valve for normalization of pressure before the piston that acts as the withdrawn finger is extracted the length of X (drawn here as a hollow arrow to the right). (C) Similar construct as in (B), but when the first piston is drawn the length of X (marked by the hollow arrow to the right), the dynamometer attached to the second piston marked P measures the force required to withdraw P. The friction force needed to withdraw P is separately measured and then subtracted from the result to give the force applied by the negative pressure of withdrawing the first piston. Illustrator: Gustav Andersson.

Negative pressure force measurements in models

To complement our calculations two cylinders resembling the size of an ear canal were constructed. The first cylinder simulated a free-moving TM (Figure 16B) with an artificial TM with a steel wire malleus connected to a dynamometer. Excess air was evacuated via a screw valve (V). A piston was drawn out of the cylinder at the same distance as the mean length that our test subjects could insert their finger into their ear canal (X in Figure 16). The force was measured. The second cylinder was constructed to simulate a non-moving TM resembling a scenario with a counteracting force from the TT muscle (Figure 16C). This model was identical to the previously described cylinder except that the distal end was filled by a second piston with a small sealant lip. The force required to overcome the friction while extracting the second piston without negative pressure in the model was measured. Thereafter negative pressure was established by pulling the first piston to a length representing the measurements from our test subjects. The second piston was then withdrawn. The force required was measured. The previously measured friction was subtracted, resulting in the force generated by the negative pressure from the piston. The cylinder model measurements were performed with only air in the system to give an estimate of the lower end of possible force.

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Calculating the strength of the tensor tympani muscle

The relationship between the maximum force that a muscle can exert is related to its cross-sectional area and to the composition of fibre types that build the muscle. Based on this, the force interval that the TT muscle theoretically develops was calculated by measuring the diameter of the muscle belly and analysing the fibre type composition of the muscle, as described below under “Papers III and IV – muscle analysis”. Without taking fibre type composition into account, the force span in a human muscle has been suggested to be between 35 and 137 N/cm2 for whole muscles (152).

Papers III and IV – muscle analysis Muscle samples and immunohistochemical methods

Nine TTs and eight SMs were obtained post-mortem from previously healthy subjects. In Paper IV, control samples from human masseter, zygomaticus major, palatopharyngeus, biceps brachii and vastus muscles were taken. All specimens were obtained 1–2 days post-mortem, a delay acceptable for obtaining reliable fibre typing (153). The samples were mounted for transverse sectioning in an optimal cutting temperature compound (Tissue-Tek; Miles Inc., Elkhart, IN, USA), rapidly frozen in liquid propane and chilled with liquid nitrogen. The samples were stored at -80ºC until further processing.

Five µm thick serial muscle cross-sections from the middle part of the muscle were cut in a cryostat at -20ºC and mounted on glass slides. The samples were immunochemically stained using a modified standard technique and well-characterized monoclonal antibodies (mAbs). For details on the immunohistochemical multistaining technique and antibodies, see Papers III and IV.

In Paper III, five TT samples were immunochemically stained with an mAb with strong affinity to MyHC-1 and basement membrane laminin α-2 for identification of muscle cell border. In Paper IV, all nine TT and eight SM samples were immunostained with mAbs with strong affinity to MyHC-1, MYHC-2A and MYHC-slow tonic. NCL–MHCneo mAbs were used to target foetal MyHC. Two mAbs against different laminin isoforms were used, α-2 for cell borders and α-5 for capillaries. Double or triple staining was performed using different combinations of mAbs. Bound primary antibodies were visualized by indirect immunofluorescence using affinity-purified antibodies prepared for multiple labelling, and conjugated with fluorochrome with different emission spectra. For staining of myonuclei, 4’,6-diamidino-2-phenylindole (DAPI) was used.

Muscle fibre type classification

The muscle samples stained as previously described were scanned at x40 magnification with a fluorescence microscope equipped with a digital high-speed fluorescence charged-coupled device (CCD) camera.

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In Paper III, the muscle fibres showing immune reactivity with the mAb directed against slow MyHC-1 were classified as “type 1”, while fibres lacking staining were classified as “fast type 2”. In Paper IV, the fibres where classified according to their immunostaining patterns, i.e. muscle fibres exclusively expressing MyHC-1 (type 1), and MYHC-2A (type 2A). Fibres with no MyHC-1 or MyHC-2A staining were classified as “MyHC-2X (type 2X)”. Hybrid fibres expressing both MyHC-1 and MyHC-2A were classified as “type 1/2”, and those expressing both MyHC-2A and MyHC-2X as “type 2A/2X”. (Examples can be found in the “Results” section on page 45.)

Fibre area measurement

The cross-sectional area of muscle fibres, in Papers III and IV, and the capillarization of muscle fibres, in Paper IV, were calculated using a customized morphometric software. Data required for calculations were acquired by manually tracing the basement membrane of each fibre and capillary on a computer image.

Muscle fibre and capillary variables

Fibre types are presented as a proportion of the total sum (%). Variability in fibre area was expressed as the coefficient of variation (CV) for each fibre type. The CV was calculated using the formula standard deviation (SD) x 1,000/mean fibre area. Capillary density (CD) was calculated as the total number of capillaries stained per mm2 of muscle tissue. For the analysis of number of capillaries related to, or around, each fibre (CAF), all capillaries within a distance of 5 µm from an individual muscle fibre were included. The number of capillaries related to each individual fibre, relative to fibre cross-sectional area (CAFA), was calculated according to the formula CAF/fibre cross-sectional area x 103. For further details on these and other variables, see Paper IV. Biochemical methods

Frozen muscle cross-sections (10-40 µm thick) from five TT and five SMs and one control biceps brachii muscle were placed in buffer. The pH in the solution was adjusted to 6.8 before filtering. The sample buffer was thawed and spun in a microcentrifuge, then sonicated in an ultrasonic cleaner, respun and diluted 1:10. The analysis of MyHC isoforms was performed on 5% sodium dodecyl sulphate (SDS)–glycerol gels that were migrated for 26 hours at 130 mV. For immunoblotting, the separated proteins were transferred to nitrocellulose sheets and exposed to mAbs A4.951 and A4.74. For further details, see Paper IV.

Muscle spindles

Muscle spindles were in transverse muscle sections defined as groups of small-sized muscle fibres surrounded by a connective tissue capsule.

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Ethical approval and considerations The TBs used in Papers I–III were donated for research and ethical approval was given by the regional Medical Ethical Committee of Umeå University (dnr 2014-352-31). The measurements on human test subjects in Paper III was approved by the Swedish Ethical Review Authority (No. 2020-03240). Autopsy specimens in Papers III and IV were collected in agreement with the Swedish laws and regulations on autopsy and transplantation, and consent was given by the National Board of Health and Welfare (dnr 5254-17784).

The number of TBs used have been in line with other, similar studies (126, 154). The precise and repeatable methods used were deemed sufficient for exploratory studies. Using larger volumes, for the sake of statistical improvements, was not deemed ethical.

Where possible, the TBs used in these studies were put to further use before destruction in accordance with our local guidelines.

Our method of accessing the middle ear does not require the fairly extensive surgery that a mastoidectomy entails but involves only a small opening that can be sealed up with a bone hatch. This also allowed TBs from Paper II to be used as pilot test bones for the setup of the radiology studies described under “Future perspectives” on page 56.

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Results Figures 17–19 are based on the same data as reported in Papers I and II. The names of the prosthesis groups have been changed to minimize risk of confusion when comparing the two studies.

Paper I The best sound transmission for PORPs was obtained with prostheses with lateral contact with both the malleus handle and the TM, as opposed to either TM only or malleus only contact.

Figure 17. Mean laser Doppler vibrometry (LDV) results of the prostheses grouped according to their lateral contact with the tympanic membrane (TM) and/or malleus handle. The error bars indicate the standard error of the mean. The black line at 0 decibels (dB), labelled “normal”, marks the sound transmission prior to disrupting the ossicular chain. TM = tympanic membrane; TM only = lateral contact with only the TM (blue); Malleus only = lateral contact with the malleus only (red); and TM and malleus = lateral contact with both the TM and the malleus (yellow). The number of prostheses measured for each type is given in parentheses.

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Paper II The best sound transmission for TORPs was obtained with prostheses in contact with both the malleus handle and the TM. The new bird-type prosthesis performed equally well as or better than all other prostheses.

Figure 18. Mean laser Doppler vibrometry (LDV) measurements of the prostheses grouped according to their lateral contact. TM = tympanic membrane; TM only = lateral contact with only the TM (blue); TM and malleus = contact with both malleus and the TM (red); bird-type prosthesis (yellow) = new prosthesis inspired by the ossicle of a long-eared owl. Incus removed (purple) = measurements of a disrupted ossicular chain. Note that the LDV measurements from Paper II are presented in a wider y-axis scale than those from Paper I to allow the air–bone gap (ABG) with incus removed to be included for reference.

Figure 19. Mean laser Doppler vibrometry (LDV) measurements of the prostheses, grouped according to their lateral contact. TM = tympanic membrane; TM only = lateral contact with only the TM (blue); TM and malleus = contact with both malleus and the TM (red). TM and distal malleus = subgroup of “TM and malleus”, with the prostheses angled towards the umbo (yellow). The bird-type prosthesis (purple) = new prosthesis inspired by the ossicle of a long-eared owl. Examples of prosthesis placement are as shown in Figure 15 on page 35.

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Paper III Applying negative pressure equal to or exceeding the force that experimentally fractured the malleus did not fracture the malleus in our TBs. Without fixation of the TT tendon the malleus moved relatively freely along with the TM. Fixation of the TT tendon resulted in reduced movement of both the malleus and the TM. Visual signs of tension in the TM distal to the TT appeared, but no fractures occurred.

Only by pulling the TT muscle, i.e. by imitating a stretch reflex, was the bone broken. Our experimental model did not allow us to apply and measure simultaneous forces in both directions.

The mathematical and physical models, with the exception of the free-moving cylinder simulating the TM, exceeded both the mean and the maximum force required to break a malleus shaft.

Figure 20. Photograph taken via a rigid, 30° angled endoscope through an opening drilled in the epitympanic recess showing the middle ear from its medial side. (A) Anterior ligament; (B) tensor tympani (TT) tendon; (C) malleus shaft; (D) long process of incus; (E) stapes; (F) tympanic membrane (TM).

Table 3. Forces, measured in Newtons, in the respective experiments. The number of measurements the data are based on is given in parentheses. (A) Force required for fracture in all bones tested; (B) freshly sourced bones; and (C) older, stored bones; (D) and (E) calculations based on hypothetical, perfect conditions and therefore only showing the maximum force possible. (D) The unlikely in vivo scenario of an ear canal filled only with air from which a finger is extracted along its full length before the seal between the finger and the outer ear canal is broken. (E) The more likely scenario where one-third of the space between the fingertip and the tympanic membrane (TM) is water-filled and the seal is broken after the finger is withdrawn one-third of the way. (F) and (G) Results of our cylinder model tests: (F) Simulating the forces transferred to the malleus via the TM without any restrictions on its movement (see Figure 16B); and (G) force developed with very little movement of the TM allowed, simulating a simultaneous tensor tympani (TT) muscle pull (see Figure 16C).

Force in Newtons Mean Standard deviation Minimum Maximum Force required for malleus fracture A. All mallei (30) 2.27 0.81 1.0 (3) 4.0 (2) B. Fresh malleus (10) 2.15 0.67 1.0 (1) 3.0 (2) C. Old/dry malleus (20) 2.33 0.88 1.0 (2) 4.0 (2)

Calculated negative pressure force D. Air only, and full pull 13.3

E. 1/3 water-filled and 1/3 seal-held 9.9

Measured negative pressure force F. Free-moving TM (40) 1.00 0.23 0.5 (4) 1.5 (4)

G. Fixed TM (40) 5.03 0.85 3.0 (1) 7.0 (2)

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Calculated tensor tympani muscle strength

The mean diameter of the TT muscle was 2.0 mm, which would give a maximal force in the range of 1.1–4.3 N (152). The muscle was composed primarily of type 2 fibres (86.9%). Since these are generally faster and more powerful than type 1 fibres the maximum force exerted by the TT muscle may be closer to the upper span.

Paper IV

Figure 21. Muscle cross-sections from Musculus tensor tympani (TT) (A) and M. zygomaticus major (B), as well as masseter (C) and biceps brachii muscle (D). Fibres are classified according to their immunostaining patterns, into type 1 (green) and type 2A (red); as well as type 2X (no stain); and hybrid fibres type 1/2A (green and red stain) and type 2A/2X (weak red stain). Examples of typical fibre stain patterns are marked. Note the higher proportion of myosin heavy chain (MyHC)-2A fibres and the smaller fibre area in the TT muscle compared with the facial, jaw and limb muscles. Scale bar = 50 µm.

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General morphology and fibre composition

The muscle fibres of the TT and SM were markedly smaller and displayed a greater variation in size compared with control limb muscles. The middle ear muscles also displayed a more rounded form and were generally less densely packed (see Figure 21). They also had a markedly higher density of small nerve fascicles in the muscle cross-sections compared with limb muscles. Both the TT and the SM were predominated by type 2 fibres in all samples, with a mean of 87% for the TT, and 79% for the SM. A low proportion of hybrid fibres was found in both the TT and the SM. No slow tonic fibres were found in either TT or SM muscles. Muscle fibres co-expressing foetal MyHC were found in all but two of the samples of both middle ear muscles, with an average of 4.3% in the TT and 3.3% in the SM muscles. The mean fibre area was significantly smaller in the SM than in the TT muscles. Fibre type proportion and fibre area are presented in detail in Paper IV.

Myosin heavy chain isoforms as analysed by biochemical methods

The biochemical analysis revealed four different MyHC isoforms in both the TT and the SM muscles, corresponding to slow MyHC-1, fast MyHC-2A, fast MyHC-2X and foetal MyHC. The MyHC-2X isoform was generally present in higher amounts in the gels than revealed by antibody staining.

Capillarization

The middle ear muscles had relatively small fibre size, yet generous capillarization. In other words, the number of capillaries in relation to the individual fibres, and to the CAFA, was high. Of all measured muscles, SM muscles had the highest CAFA value and TT muscles had the second highest value. The differences were significant for both orofacial and limb muscles, with the exception of the zygomaticus major muscle. Compared with the biceps muscle, the CAFA was higher by 51% in SM and by 41% in TT. See Paper IV for more details.

Muscle spindles

Muscle spindles were identified in TT samples but not in SM samples.

Figure 22. Serial cross-section of a muscle spindle in the tensor tympani (TT) muscle. The sections are immunostained for laminin a2 (red), and MyHC-1(green). The spindle capsule, a nerve and intrafusal fibers are marked. Scale bar = 50 µm.

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Discussion Papers I and II Both Papers I and II found that, regardless of the prosthesis material, a superior sound transfer is achieved if the prosthesis is placed in distal contact with both the malleus and the TM. Similar results have been shown in a study on 52 patients using hydroxyapatite PORPs and TORPs, where it was also suggested that “trapping” the prosthesis under the malleus aided in extrusion reduction (155). Use of the malleus to stabilize the prosthesis and prevent displacement has been suggested by other authors (156-158).

Our LDV setup left the bony ear canal in place and therefore did not allow the broad access needed for consecutive digital holography measurements in the same TB. We therefore could not evaluate how the different prostheses affected the TM movements. It has been demonstrated that placing a prosthesis on the surface of the TM leads to significant reductions in the motions of the TM surface adjacent to the prosthesis (13, 159) but has little effect on middle ear sound transmission (13). With the present knowledge of TM movement, digital holography seems to contribute little to the creation of optimal prostheses but this may change as finite element models develop further.

Our LDV setup in Paper II did, however, produce basic stapes vibration patterns with little interindividual variation (see Figure 13 on page 32) and frequency-dependent similarities with human hearing thresholds (see Figure 1 on page 2). This is likely due in part to our attempt to replicate the anatomy of the ear canal and thus standardize the placement of the 4 kHz notch associated with its resonance (4, 5). We have no evidence suggesting that mimicking the resonance effect of the ear canal improved the reliability of our LDV results concerning middle ear impedance or prosthesis evaluation, compared with studies where the speaker was placed next to the TM (122, 126). However, we would argue that there is no reason why a model should not aim to replicate the in vivo mechanics as closely as possible.

Figure 23. Prosthesis used in Paper II. (A) Extra-long bone total ossicular replacement prosthesis (TORP) for lateral malleus placement; (B) silver bird-type prosthesis. The long vertical lines on the horizontal rulers below the prostheses measure 1 mm.

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When it comes to development of future prostheses with superior mechanical function, the LDV results of Paper II suggest that contact with the TM and distal contact with the malleus in combination produced the best results. This could be achieved with both a long autologous bone TORP and with the bird-type prosthesis depicted in Figure 23. The question is, Why is this not regularly performed in in vivo surgery? The answer to this question is that the angle between the stapes footplate and, even more so, the stapes head and the umbo is unfavourable for reconstruction (160). As seen in our experiments, the TORPs with distal contact did perform well but were at an extreme angle, with risk of slipping and fixation in vivo scenarios (Figures 24 and 25). The question, then, is, How do we address these angles?

Figure 24. Bronze statue of the ossicular chain based on three-dimensional (3D) printed, high-resolution scans. Note the angle between the stapes head P and umbo tip X for partial ossicular replacement prosthesis (PORP) placement, and stapes footplate T and umbo tip X for total ossicular replacement prosthesis (TORP) placement.

Elsewhere, malleus relocation has been suggested as a solution to adverse angulation. The main failure reported in that surgery was that, owing to post-surgical movement of the malleus, the prosthesis became too short (158). The same surgical team, led by Vincent, further developed the technique, placing the malleus directly above the stapes and then placing a traditional titanium TORP, stabilized via a silastic band, to an intact stapes. Comparing this method with a PORP with lateral malleus contact, they found the TORP superior (161). In a case with an absent malleus, a titanium malleus replacement prosthesis, placed in a similar fashion by the same team, showed promising results (157). Although these results are promising, moving the malleus correctly to avoid ending up with a short prosthesis requires technique and experience (161); it also requires cutting of the TT tendon (161). Malleus head fixation to adjacent bone if moved into contact is a possible risk. Placing a titanium malleus replacement prosthesis carries a possible extrusion risk. All these techniques are more complex and invasive than regular ossicular replacement surgery and still do not achieve distal contact with the umbo.

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Aiming for the umbo, even disregarding the risk of prosthesis displacement by slipping can be fraught with the risk of lateral bone contact and prosthesis fixation to adjacent bone (see Figure 25).

Figure 25. Schematic drawing of the middle ear showing the anatomical challenges of placing an angled prosthesis on the head or, even more snug, the stapes footplate (below (1)). The risk of lateral contact with a prominent promontory (2) or facial nerve canal (3) can lead to prosthesis fixation. In ears where the space between 2 and 3 is narrow a thin titanium stem prosthesis may be beneficial (108).

Developing an ossicular replacement prosthesis to incorporate features from the bird-type prosthesis could prove successful. Elements we believe to have merit are: a central piston connected with a lateral snug fit to the malleus that extends to its distal end; and two to three sites of contact with the TM. A model built similarly to existing titanium prostheses would result in lighter weight and possibly better sound transmission at the higher frequencies, than seen in Paper II (162). It would also be easy to angle and adapt to individual conditions. The silver prostheses were modified and bent to adapt to the individual ear measured in Paper II but because of their relative thickness this took more effort compared with a typical titanium prosthesis.

Titanium prostheses have been successfully used without cartilage support (109) and the extensive malleus contact could be a further protection against extrusion (156-158), especially with balanced TM contact on either side of the malleus shaft. The benefits of not needing cartilage are: shorter surgery with less trauma; easier prosthesis fit; and ease of post-operative visual inspection. A cartilage TM hinders visibility, making visual inspection, for example of residual cholesteatoma, impossible and requiring the use of MRI for further checkups.

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A titanium PORP similar to those on the market but with a modified distal end could be a viable stable solution; however, because of the angle it is placed in it might not be optimal. A TORP is easier to place advantageously, in both the traditional TORP setting with stapes missing and a setting with intact stapes superstructures present, as described in Paper II. One study suggested that a TORP with a thin malleable shaft cradled by a remaining stapes structure would increase its stability and perform as well as a PORP in the same circumstances (163). As mentioned previously, such a TORP connected to the malleus has shown results superior to a PORP (161). Adding possible support at the footplate in the shape of prefabricated connectors or a cartilage shoe (164) could, in combination with the snug fit of the malleus, increase the stability and reduce the need for more complex surgery, as demonstrated by Vincent et al. (161).

Papers I and II report similar LDV results for reconstructions using prostheses with standard lateral contact with both the TM and the malleus. Only distal malleus prostheses and the bird-type prosthesis give TORPs an advantage. This, as well as the studies referenced above, suggests that both a PORP or a TORP would, where hearing is concerned, function well in similar circumstances, possibly with room for improvement in prosthesis development.

The three-ossicle system in humans may lead to more stability and protection but not necessarily to hearing optimization (16). It has been suggested that the flexibility of the ossicular joints reduces the peak amplitudes of impulsive sounds (165).

When deciding on reconstruction with a PORP or a TORP it is therefore important to consider what, besides mechanical sound transfer levels, we want to alter. The main protective mechanism of the ostrich middle ear was shown to be a buckling movement at the intracolumellar connection between the bone base and cartilage tripod of the single-ossicle system (166). The long-eared owl used for prosthesis inspiration in Paper II has a cartilage tripod. This suggests that a TORP with an elastic element could potentially reduce the risk of inner ear damage (166). Other protective mechanisms to consider in humans are our middle ear muscles. When designing future prostheses, it is important that they should be adaptable to most situations; however, there is no reason why we should not allow the preservation of the TT and SM where possible.

Our laser Doppler vibrometry setup

Measurement of the acoustic properties of middle ear prostheses in an appropriate model prior to insertion in humans should be part of the routine of the prosthesis development and testing process (167).

As seen in Figure 18 on page 43, there is a small gradual decline in response for all prostheses measured above 2 kHz. This can likely be attributed to the previously described complexity of the footplate movement at higher frequency, which cannot be

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picked up by measuring only one site. Another issue that makes the high frequency range more complex to assess is the increase in sound transferred outside the ossicular chain with increasing frequency. The sound transferred through air and bone directly to the footplate is, in Figure 18, labelled “incus removed” and would in the clinical setting be the AC in a case with a complete ABG. In our measurements, likely owing to several factors such as comparable lack of dampening soft tissue in a TB, the direct fixation of the speaker, and the lower impedance due to a removed cochlea that could affect the acoustic coupling, this background noise increases with frequency in a way it does not do in vivo.

The LDV measurements are precise and comparable to each other over the entire frequency range. However, the gap between measurements taken with the incus removed and the different reconstructions is for the above reasons further from the clinical reality at the higher frequencies, and we would concur with a previous study stating that assessments of measurements above 5 kHz are too complex (9).

Our LDV setup uses a tried and tested method and has improved it. It allows footplate displacement to be made in the direction of the assumed velocity vector, thus ensuring reliable signal quality regarding backscattered light and measuring the true size of displacement. Since draining the cochlea has been reported to make little difference to stapes motion (124), our setup should be considered in all TB studies where direct measurements cannot be performed on fresh TBs (as freezing may cause unseen leakage that can affect measurements) (123). The greatest advantage of using the described setup, however, is the increased access to a middle ear that is not both a model and a measuring site. This makes a broader set of experiments possible. Using the traditional LDV method, Paper II would probably have run into the same issues as described by Puria et al. (132). They state that it is nearly impossible to measure the footplate vibration with a large-based TORP in place, because it blocked the laser beam. In their study, vestibulocochlear pressure measurement was used instead (132). We therefore believe the advantages of our setup outweigh the disadvantages.

Paper III In this study, we show that the forces simulating a finger being pulled from a wet ear canal are sufficient to break the malleus shaft if the TT muscle is simultaneously activated. In the described trauma, this muscle activation could theoretically be attributed to a number of stimuli. Among these are local tactile stimulation; the startle reaction to loud sound during the action of inserting and extracting a finger; or simultaneous swallowing/yawning to ease the ear blockage (27). One study has suggested that voluntary contraction of the TT is possible (29). However, the most likely cause, in our opinion, would be a stretch reflex. One study looking for muscle spindles in the human middle ear muscles found them in both the TT and SM (34). In Paper IV we confirm

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finding them in the TT, further supporting this theory. Therefore, when the TM and malleus are rapidly moved outwards the TT with its muscle spindles will stretch. This will immediately increase the alpha motor neuron activity, resulting in muscle contraction to resist the stretching.

A case report presented two cases of malleus fractures caused by a suppressed sneeze (168). These are unique finds, insufficient by themselves to draw conclusions. Purely speculatively they may suggest that the pressure placed on the TM could derive from an open ET, with no stimulus in the ear canal. To cause a fracture, this trauma would, according to our findings in Paper III, require the same counteracting forces from malleus support structures and the TT muscle as in the finger extraction trauma. The malleus itself has good support from six ligaments (169). It has been suggested that the relative fixation of the malleus head in the epitympanum is a sufficient stabilizer of the ossicle for the sudden lateral movement of the shaft to cause fracture (74). Our TB experiments, however, clearly illustrate that there is not enough passive force in these supportive structures to limit the ossicle’s movement sufficiently to cause a fracture. Our mathematical model shows that more than enough force to cause a fracture can be built up under hypothetical conditions. This still applies when the seal around the finger holds for only part of the withdrawal. It is especially evident if accounting for water being present between the fingertip and the TM – which supposedly is the reason for ear prodding after a shower/bath in the first place. Less air left to expand means more force transferred to the TM. Since the mathematical model does not account for the flexibility of the TM our results are likely higher than in vivo. The cylinder models were built as a complement to demonstrate possible forces “in practice”. In vivo the water likely functions both as a sealant between the finger and ear canal, and to increase the force transferred to the TM. To our knowledge, no isolated malleus fractures have been reported from people who have inserted their finger into a dry ear canal. The cylinder models were perfectly circular, unlike an ear canal, and thus an effective seal around the plunger could be created with a rubber ring. With water not necessary for the seal, measurements were made with only air in the system to give an estimate of the lower end of possible forces. The cylinder models showed that the movement of the TM had a significant effect of negative pressure build-up. Taken together, all experiments suggest that there needs to be a TT activation at the same time as the outward movement of the TM to create an isolated malleus shaft fracture. The muscle analysis in Papers III and IV, further discussed in Paper IV, supports that the TT has the capacity needed to cause a fracture. It may in itself be strong enough to generate the 2.3 N mean force required for a fracture. We are convinced that a fracture in an ear with no pre-existing defects can occur if this force is added to the existing passive support of the malleus and the synchronic forces that arise when extracting a finger from the ear canal.

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Both the mathematical and the experimental models of this study represent simplified versions of the trauma mechanisms. The strength of Paper III is that it relies on several methods complementing each other to make a valuable addition to an uncharted field of knowledge. Hopefully this information can aid in the understanding and spreading of knowledge concerning malleus shaft fractures so that fewer fractures occur and those who need help can be properly diagnosed. Paper IV In this study we found that the muscles of the middle ear, i.e. the TT and SM, have a high density of small nerve fascicles, suggesting a more precise regulatory control compared with limb muscles. They are both dominated by type 2 fibres, which makes them among the fastest muscles in the human body. These data suggest that the SM and TT in humans are also among the most type 2-dominated middle ear muscles, compared with other species (37). The higher amount of MyHC-2X in the immunoblotting gels compared to that estimated by the immunohistochemical techniques could be related to the fact that there is no available antibody detecting human MyHC-2X. Estimation of MyHC-2X is based on an absent or weak immunoreaction for MyHC-2A and an absent reaction for MyHC-1. Therefore, muscle fibres classified as MyHC-2A are probably overestimated. Some of these could be type 2X.

We found no trace of MyHC-slow tonic in the SM or TT. This was investigated because MyHC-slow tonic has been found in the TT of several carnivore species (39, 139). Of the two middle ear muscles, the TT has larger fibre areas and is a larger muscle and is therefore stronger than the SM. Its fibre composition also suggests that it is faster. On the other hand, the relatively larger amount of type 1 fibres of SM suggests that the SM could have a higher endurance compared with the faster and stronger TT. Both TT and SM had higher CAFA than the control samples, suggesting a rich oxygen supply, ensuring some endurance even with the extreme fibre composition.

The middle ear muscles of many species reportedly lack muscle spindles (37). Small but morphologically classic muscle spindles have been found in human TT and single fibre spindles in SM (34). In Paper IV we could verify muscle spindles in the TT muscle but found none in the SM. Concerning hybrid and foetal MyHC, the reason for their presence is unclear.

A study on SM fibre composition in rats found fibres with more than one subclass of myosin. Its authors hypothesized that the MyHC hybridism reflects functional demands of the muscle (36). Suggested benefits were finer gradation of contraction speed per fibre and effective aerobic as well as anaerobic metabolic capabilities to generate adenosine triphosphate (ATP), thus specializing the muscles for their specific demands of fast fine-tuned contraction and fatigue resistance (36). Foetal MyHC does exist in some specialized human muscles (135, 136, 141, 142) as well as in the middle ear muscles of pigs and cows

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(140). The cause of such an arrested development (if this is what it is) was unknown to the authors of the study on pigs and cows, and is likewise unknown to us concerning similar findings in Paper IV.

The MyHC composition of the SM supports its widely acknowledged function as a fast, reactive muscle that responds to loud noise. The fibre hybridization, rich nerve innervation and capillarization suggest that the SM is a fine-tuned muscle with some fatigue resistance. This could support the more advanced role suggested in speech recognition modulation in noise. The high variability in fibre type of the TT in different mammals suggests that it plays different roles in different species (37). Our data neither confirm nor refute the theory that the TT could be an active part of middle ear ventilation (30). There is evidence proposing that the TT in humans, unlike several other mammals (18-20), has non-acoustic functions (28). There are, however, protective mechanisms to consider that are not associated with sudden or loud noise.

The barotrauma via an open ET during a sneeze, cough or Valsalva manoeuvre can be significant. A suppressed sneeze has been suggested to be enough to cause an isolated malleus fracture (168). The middle ear in humans and the IMJ in particular has been suggested to be designed more for protection than for sound transfer optimization. With the ossicles located side by side rather than top to bottom, this allows the decoupling of excessive TM displacements (16). Hüttenbrink (16) suggests that the malleo–cochleariform ligament enveloping the TT tendon is the most important of the six described ligaments connected to the malleus, and that it protects the malleus against outward displacement (169). In Paper III we found that the passive supportive structures of the malleus had a limited effect on malleus movement. Without tension in the TT, simulating muscle contraction, the malleus was relatively free to move with the TM. As reported in Paper III, malleus fractures are rare although they likely are underdiagnosed; as discussed, the typical cause is a finger pulled out of a wet ear canal after a bath or shower. From an evolutionary perspective, regular bathing and consequently putting a finger in a wet ear canal are relatively new concepts (170). However, coughing, sneezing and Valsalva-like actions to remedy ear discomfort have most likely been with us since the dawn of humankind. The quick and strong fibres of the TT and its muscle spindles make it suited to respond and protect the inner ear from excessive movement of the TM. Further findings, and theories on what these may mean concerning TT and SM function, are covered in Paper IV. The findings of Paper IV support the conclusion of Paper III, that the TT has the capacity to generate the counteracting force needed to cause an isolated malleus fracture. Paper IV is also a relevant contribution to the body of academic knowledge and is hoped to contribute to future attempts to better understand the functions of the human middle ear muscles.

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Conclusion

Paper I

The best sound transmission was found in PORPs with lateral contact with the TM and the handle of the malleus.

Paper II

The best sound transmission was found for TORPs with lateral contact with the TM and the distal parts of the handle of the malleus. Our experimental bird-type prosthesis provided a stable contact of this kind, suggesting that there is room for prosthesis design development to optimize hearing outcome after surgery.

Paper III

Negative pressure, created by a finger extracted from a wet ear canal, is sufficient to cause an isolated malleus fracture with intact TM, but only if there is a simultaneous force in the opposite direction. The TT muscle has the capacity to generate this counteracting force.

Paper IV

The human TT and SM are highly specialized. Their fibre-type pattern, rich capillarization and nerve innervation suggest that they are adapted for fine-tuning, high speed and peak power performance and modified for some fatigue resistance. Based on fibre-type patterns, the TT is among the fastest muscles in the human body. These findings and the verification of TT muscle spindles furthermore support the conclusion in Paper III, that the TT can react to a stretch reflex and has the capacity to generate a sufficient force to cause a fracture in the described trauma.

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Future perspectives In collaboration with, among others, radiologist Malin Vestin Fredriksson and physicist Love Kull, a modular anthropomorphic phantom based on a human dry skull, tissue-equivalent materials and space for interchangeable fresh temporal bones (TBs) has been created to produce relevant findings with minimal alterations to the anatomy.

Using the model, the middle ear is accessed in the same way as in Papers I–III, after which the epitympanic opening is sealed with a bone hatch. This model is being used in ongoing studies regarding optimization of clinical protocols on CT and cone-beam computed tomography (CBCT) modalities aiming to compare the ability of different radiological methods to detect pathology in the TB. To our knowledge, no other such model currently exists.

Figure 26. An anthropomorphic phantom based on a human skull, using tissue-equivalent materials and including space for interchangeable fresh TBs, for radiological research on the human middle ear.

It is hoped that the findings from Papers I and II, recommending distal connection of prosthesis with the malleus shaft and simultaneous contact with the TM, may inspire prospective clinical studies aiming to verify these results in vivo.

The bird-type prosthesis of Paper II suggests that there is room for prosthesis development, concerning both stability and sound transfer. Further LDV studies and/or clinical trials on an adaptable titanium prosthesis using its key features would be desirable.

In stapedotomy the prosthesis fixation around the long process of incus can be made with crimping plyers or by applying heat to a prosthesis made of shape-memory metal (nitinol). Erosion of the long process is a common finding in revision surgery (90). With our LDV method it would be possible to fixate the stapes footplate and study different crimping methods and materials and analyse which method gives the best sound transmission with the least amount of crimping force applied.

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Acknowledgements

There are many friends, family and coworkers, who have inspired, guided and supported me through the years and without whom I would not be the person I am today. I sincerely thank you all. Below I would like to specially mention those who have been crucial to this specific adventure.

Krister Tano, as my former boss you convinced me that ENT and otosurgery was the best path for me and recruited me to our clinic. As a senior colleague you have supported my practical education. Finally, as my main supervisor you have guided me through this work. No one else has had such an impact on my professional choices. Thank you.

Magnus von Unge, my first co-supervisor in this work. You introduced me to LDV and the wider world of international research collaborations and the great benefits they can bring.

Mimmi Werner, my second co-supervisor in this work. You have given me much sound advice and shown me how to find the gems in local collaboration.

Anders Niklasson, we started off as colleagues, collaborated in research sparked by our common curiosity, and grew to become good friends.

Our Antwerpen collaborators: Joris Dirckx, Kilian Gladiné and Pieter Muyshondt. It has been a joy to match our clinical knowledge and questions with your expertise in the biophysics of sound and hearing in both humans and birds.

Our Umeå collaborators: Per Stål and Farhan Shah, your enthusiasm and skill in muscle fibre analysis have strengthened my, and our, work, as well as the world’s knowledge about the composition of the middle ear muscles. Gustav Andersson, your work on the illustrations in Paper III has enhanced the thesis both artistically and pedagogically.

I would like to thank Diana Berggren, Per Olof Eriksson, Michael Gaihede, Ann Hermansson, Fredrik Karlsson and Eva Westman for reviewing my work and giving me valuable feedback.

All the staff at the ENT clinic in region Norrbotten.

Ulf Mercke, with never-ending patience and generous support you stood by as I made my first steps into otosurgery.

Lennart Edfeldt, you taught me how to drill deeper, besides many other important life lessons.

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Per Olof Eriksson, thank you for continuing to support me as I strive to improve as a surgeon.

Marie Bunne, Karin Strömbäck, Niklas Danckwardt-Lillieström, many thanks for guidance and support in otosurgical matters through the years and over the extreme distances that working in a remote location entails.

Helge Rask-Andersen, you have generously shared your knowledge and given me free access to your lab during my internship in Uppsala, which gave me my first insight into temporal bone research.

My dear wife Elisabeth. You were my proofreader and my inspiration to earn a degree. What won’t a man do to impress the woman he loves?

My children Astrid and Edvard, for whom I try to improve myself.

Thomas and Ingrid Hedlund, my parents-in-law, for all your support through the years and for always having a spare bedroom ready for me on all those Umeå research visits.

Financial disclosure

I would like to express my gratitude to the Research Council of Norrbotten, Sweden, for their support, especially in financing time for research (Akademisk miljö, Krister Tano, NLL-939827, NLL-930215 and NLL-968473).

Furthermore, I also want to thank the Department of Clinical Science/Otorhinolaryngology, Umeå University, for support regarding equipment.

Last but not least, I would like to send a respectful thought to all those who donate their body to research. It is a valuable gift and we have tried to make the most of it.

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