applied sciences

Article Round Window Reinforcement-Induced Changes in Intracochlear Sound Pressure

Nuwan Liyanage 1, Lukas Prochazka 1, Julian Grosse 1, Adrian Dalbert 1, Sonia Tabibi 1, Michail Chatzimichalis 2, Ivo Dobrev 1 , Tobias Kleinjung 1 , Alexander Huber 1 and Flurin Pfiffner 1,*

1 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Zurich, University of Zurich, 8091 Zurich, Switzerland; [email protected] (N.L.); [email protected] (L.P.); [email protected] (J.G.); [email protected] (A.D.); [email protected] (S.T.); [email protected] (I.D.); [email protected] (T.K.); [email protected] (A.H.) 2 Dorset County Hospital, Dorchester DT1 2JY, UK; [email protected] * Correspondence: flurin.pfi[email protected]; Tel.: +41-44-255-1111

Abstract: Introduction: The round window membrane (RWM) acts as a pressure-relieving membrane for incompressible cochlear fluid. The reinforcement of the RWM has been used as a surgical intervention for the treatment of superior semicircular canal dehiscence and hyperacusis. The aim of this study was to investigate how RWM reinforcement affects sound pressure variations in the cochlea. Methods: The intracochlear sound pressure (ICSP) was simultaneously measured in the scala tympani (ST) and scala vestibuli (SV) of cadaveric human temporal bones (HTBs) in response to acoustic stimulation for three RWM reinforcement materials (soft tissue, cartilage, and medical-grade



 silicone). Results: The ICSP in the ST was significantly increased after RWM reinforcement for frequencies below 2 kHz. Between 400 and 600 Hz, all three materials demonstrated the highest Citation: Liyanage, N.; Prochazka, L.; Grosse, J.; Dalbert, A.; Tabibi, S.; median pressure increase. The higher the RWM stiffness, the larger the pressure increase: silicone Chatzimichalis, M.; Dobrev, I.; (7 dB) < soft tissue (10 dB) < cartilage (13 dB). The ICSP in the SV was less affected by reinforcement. Kleinjung, T.; Huber, A.; Pfiffner, F. The highest median pressure increase was 3 dB. The experimental findings can be explained with Round Window Reinforcement- numerical models of cochlear mechanics. Discussion and conclusions: RWM reinforcement increases Induced Changes in Intracochlear the sound pressure in ST at lower frequencies but only has a minor influence on the SV pressure. Sound Pressure. Appl. Sci. 2021, 11, 5062. https://doi.org/10.3390/ Keywords: cochlea; round window membrane; intracochlear sound pressure; numerical models app11115062

Academic Editor: Ilaria Cacciotti 1. Introduction Received: 13 April 2021 The biomechanical hearing process relies on sound transmission from the outer ear Accepted: 26 May 2021 Published: 30 May 2021 via the middle ear ossicles toward the inner ear (cochlea). The mechanical vibration of the stapes footplate at the end of the middle ear drives sound energy into the cochlea.

Publisher’s Note: MDPI stays neutral This transmitted sound creates intracochlear sound pressure (ICSP) in the cochlear fluid, with regard to jurisdictional claims in particularly in the scala vestibuli (SV) and scala tympani (ST), which are interconnected at published maps and institutional affil- the apex. The ICSP difference (PDIFF) between the SV and ST triggers a passive traveling iations. wave in the basilar membrane and leads to the excitation of sensorial structures [1]. The round window membrane (RWM) mechanically seals the fluid-filled cochlea from the middle ear cavity at the base of the ST. The RWM is an approximately 70 µm-thick, elliptically shaped, compliant biological structure that is anatomically located within the round window niche in the medial wall of the middle ear [2]. The RWM with its compliant Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. characteristics serves as a pressure release mechanism for the incompressible fluid in the This article is an open access article cochlea, enabling the PDIFF at the basal part of the cochlea. distributed under the terms and Increasing the stiffness of the RWM by manipulating its intrinsic properties or by any conditions of the Creative Commons artificial means is termed the “reinforcement of the RWM”. RWM reinforcement techniques Attribution (CC BY) license (https:// for clinical applications have received attention for more than two decades [3]. Artificially creativecommons.org/licenses/by/ reinforced RWMs are claimed to provide better hearing for patients suffering from dis- 4.0/). eases such as superior semicircular canal dehiscence, hyperacusis [4], and perilymphatic

Appl. Sci. 2021, 11, 5062. https://doi.org/10.3390/app11115062 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 5062 2 of 19

fistula [5]. The main materials used in clinical applications for RWM reinforcement are perichondrium, cartilage, and fascia [5,6]. After cochlear implantation surgery through the RWM, several clinical studies claimed post-operative low-frequency (<1 kHz) hearing loss in the range of 20–25 dB [7–10]. Among several causes (i.e., insertion trauma), the change in ICSP due to the stiffness increase in the RWM (artificially reinforced RWM) after the CI surgery is also regarded as a possible reason for this auditory defect. Thus far, sufficient data are missing to fully understand the cause of the potential loss in residual hearing after CI surgery. To understand the influence of RWM reinforcement on hearing, Wegner et al. (2016) examined the changes in the middle ear sound transfer function (METF) after RWM reinforcement. The study involved cadaveric human temporal bones (HTBs) and RWM re- inforcement with perichondrium and cartilage. A drop in METF with RWM reinforcement was attributed to the dominance of the cochlear input impedance in RWM stiffness at lower frequencies [5,11,12]. However, the authors concluded that the reinforcement of the RWM had no statistically significant effect on the METF. Similarly, Guan et al. (2018) claimed for one HTB sample that the effect on the METF after RWM reinforcement was negligible even after maximum reinforcement using perichondrium, cartilage and Jeltrate [13]. Beyond understanding the effects of RWM reinforcement on METF, sound pressure measurement in the SV (PSV) and ST (PST) provides insight into the ICSP changes caused by RWM reinforcement. Guan et al. (2018) reported an increased ICSP gain (PSV and PST relative to the ear canal pressure (PEC)) with RWM reinforcement in both scalae. The gain in ST and SV showed a maximum increase of 18 dB between 50 and 800 Hz and 10 dB below 500 Hz, respectively. In the lower-frequency spectrum (200–1000 Hz), the authors observed a reduced PDIFF of approximately 5 dB but no significant influence on PDIFF at higher frequencies. However, to verify the significance of these experimental claims, a study with a larger sample size is crucial. Numerical models of the cochlea based on approximated physiological parameters are useful for understanding the experimental results and estimating values that are not directly measurable. A lumped element model (LEM) of the cochlea was introduced by Nakajima et al. (2009) to estimate the RWM impedance (ZRWM) behavior on the cochlear mechanics. Later, Elliot et al. (2016) carried out a study based on a finite element model (FEM) with an improved LEM of the cochlea. Elliot’s model could predict the above- mentioned post-operative hearing loss after cochlear implantation (CI) due to the stiffening of the RWM. Their numerical simulations predicted that while a 10-fold increase in RWM stiffness would significantly influence residual hearing loss, a 100-fold stiffness increase might cause a residual hearing loss of up to 20 dB [8]. In this study, we investigate the effects of the ICSP changes (PST, PSV, and PDIFF) caused by RWM reinforcement with three clinically used materials on HTBs. In addition, the experimental findings are compared with the simulation output of the numerical models developed by Nakajima et al. (2009) and Elliot et al. (2016). Up to date, this is the first study to report data of ICSP changes due to RWM reinforcement on a series of HTBs, and to compare experimental data with data from numerical models.

2. Materials and Methods 2.1. Human Temporal Bone Samples and Experimental Protocol For this study, cadaveric HTBs (Science Care, Phoenix, AZ, USA) harvested within 48 h and immediately frozen after the death of the donors were used. The HTBs were stored at −19 ◦C in a temperature-regulated freezer until they were used in the experiments. The HTBs were thawed under atmospheric conditions for four to five hours prior to surgical preparation. Experiments with the HTBs were approved by the local ethics committee (KEK-ZH-Nr. 2014-0544) and were in accordance with the Helsinki Agreement of 1975 revised in 2013. A total of 20 HTBs were used for the experiments. Three HTBs were used in pre-trial experiments to develop the experimental methods. Of the remaining 17 HTBs, 11 were Appl. Sci. 2021, 11, 5062 3 of 19

excluded due to nonconformity during the experiment preparation or anomalous results. The age range of the final 6 samples (3 female and 3 male) was a mean of 74 years with a standard deviation (SD) of ±16 years. The experimental protocol consisted of surgical preparation (cp. 2.2) and functionality verification tests (cp. 2.4) of the HTBs before ICSP measurements were performed under several RWM reinforcement conditions (cp. 2.5).

2.2. Surgical Preparation When preparing an HTB sample, first the excess soft tissue was removed. Then, the bony ear canal was carefully drilled until there was an unobstructed view of the tympanic membrane (TM). The upper end of the bony ear canal was kept approximately even and 1–2 mm above the TM. The middle ear cavity was opened wide from the facial recess structures and the facial nerve was removed to access the middle and inner ear. The surgical approach was optimized to gain a broader view of the stapes footplate and the RWM. The bony overhang in the round window niche was drilled to ensure an orthogonal view on the RWM [14]. The cochlear promontory near the oval and round window was thinned during preparation to perform a swift cochleostomy procedure for the insertion of hydrophone probes. Furthermore, the soft tissue membrane on the stapes footplate and the outer epithe- lium of the RWM (pseudo-membrane) were carefully removed to prevent water retention and to provide optimal conditions for laser Doppler velocity measurement on the stapes and RWM [15]. As an artificial ear canal (AEC), a solid plastic tube with a volume of 2 mL was fixed above the TM with a strong adhesive product (BLUFIXX GmbH, Wesseling, Germany) and Play-Doh. Throughout the experiment, the HTB sample was kept moist by spraying water at five- to ten-minute time intervals.

2.3. Measurement Setup The HTB experiments were carried out on a passive damped optical table with pneu- matic isolation to eliminate vibration artifacts from the environment. The prepared HTB was attached on the z-stage of a micromanipulator customized for ICSP measurement. A loudspeaker and a reference microphone probe tip (ER-2 and ER-7C, Etymotic Research Inc., Elk Grove Village, IL, USA) fixed through a foam earplug were inserted into the AEC and placed 2–3 mm above the tympanic membrane. Acoustic stimulation was generated by an audio analyzer (APx585, Audio Precision Inc., Beaverton, OR, USA) and delivered to the loudspeaker through an audio amplifier (RMX 850, QSC Audio Products LLC, Costa Mesa, CA, USA) as a stepped sine stimulation signal consisting of at least 21 frequencies logarithmically distributed within the frequency range of 200–8000 Hz. The sound pressure levels in the ear canal were recorded by the reference microphone. All experiments were performed with sound pressure levels between 80 and 120 dB SPL at the TM. A single-point laser Doppler vibrometer (LDV) (CLV-2534-3, Polytec GmbH, Germany) mounted on a surgical microscope was used to measure the velocities of the stapes footplate (VSTAP) and the RWM (VRWM). To maximize the intensity of the reflected laser beam, retro-reflective beads (aluminum-coated solid barium titanate glass microspheres, 50 µm diameter; Cospheric LLC, Goleta, CA, USA) were placed on the stapes footplate and the RWM. ICSP was measured in the ST and SV in accordance with a previously described method by our group [16,17]. The probes for the ICSP recording (miniature hydrophones) were built on a commercial micro-electromechanical system (MEMS) condenser micro- phone (ADMP504, Analog Devices Inc., Norwood, MA, USA), whose pressure port was customized with a tiny micro-tube terminated with a polyimide diaphragm as the pressure sensing element (cf. Figure1A). The sensors were calibrated before the experiments using a vibrating water column method, as described in [17]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 20

were built on a commercial micro-electromechanical system (MEMS) condenser micro- phone (ADMP504, Analog Devices Inc., Norwood, MA, USA), whose pressure port was Appl. Sci. 2021, 11, 5062 customized with a tiny micro-tube terminated with a polyimide diaphragm as the pres- 4 of 19 sure sensing element (cf. Figure 1A). The sensors were calibrated before the experiments using a vibrating water column method, as described in [17].

Figure 1.Figure (A) Custom-made 1. (A) Custom-made intracochlear intracochlear acoustic acoustic receiver receiver(ICAR) sensor (ICAR) with sensor a zoomed with a zoomedview on view on the soundthe receptor sound receptor and micro-electromechani and micro-electromechanicalcal system system (MEMS) (MEMS) condenser condenser microphone. microphone. (B) Ex- (B) Experi- perimentalmental setup setup with with a human a human temporal temporal bone bone (HTB) (HTB) sample, sample, artifi artificialcial ear ear canal canal (AEC), (AEC), and and two two ICARs ICARs for simultaneous pressure measurement in SV and ST. For a precise sensor insertion into for simultaneous pressure measurement in SV and ST. For a precise sensor insertion into the cochlea, the cochlea, both hydrophones are positioned using multi-axis micromanipulator units. both hydrophones are positioned using multi-axis micromanipulator units.

The ICSPThe in ICSP the inST theand ST SV and and SV the and AEC the sound AEC sound pressure pressure level levelwere wererecorded recorded simul- simulta- taneouslyneously using using the Audio the Audio Analyzer Analyzer APx585 APx585 (Audio (Audio Precision Precision Inc., Beaverton, Inc., Beaverton, OR, USA). OR, USA). The rawThe data raw were data post-processed were post-processed by a bandpass by a bandpass filter based filter on a based digital on third-order a digital third-orderBut- terworthButterworth filter. An average filter. An of averageover 3 subsequent of over 3 subsequent measurements measurements was performed was for performed each for step ofeach the applied step of thestepped applied acoustic stepped stimul acoustication stimulationfrequency sweep. frequency The sweep.measurement The measurement data obtaineddata from obtained a LabVIEW from 2017 a LabVIEW (NI, Austin, 2017 TX, (NI, USA) Austin, virtual TX, interface USA) virtual for data interface acquisition for data ac- and signalquisition processing and signal were processing further anal wereyzed further and plotted analyzed using and MATLAB plotted using 2019a MATLAB (Math- 2019a Works,(MathWorks, Natick, MA, Natick,USA). MA, USA).

2.4. HTB2.4. Functionality HTB Functionality and ICSP and Measurements ICSP Measurements After theAfter surgical the surgical preparation, preparation, the METF the METFand the and inner the ear inner hydrodynamic ear hydrodynamic condition condition of the cochleaof the cochlea were assessed were assessed to confirm to confirm proper proper sound soundtransmission transmission from the from ear the canal ear to canal to the cochlea.the cochlea. The METFThe was METF assessed was assessed as the VSTAP asthe in responseVSTAP in to response acoustic tostimulation. acoustic stimulation. It was calcu- It was lated andcalculated compared and with compared the standard with the reference standard values reference published values by published the American by the Soci- American ety forSociety Testing for and Testing Materials and (ASTM) Materials [18]. (ASTM) [18]. To verifyTo verifythat no that air bubbles no air bubbles that could that modify could modify the hydrodynamics the hydrodynamics of the ICSP of the were ICSP were presentpresent in the cochlear in the cochlear fluid, the fluid, phase the difference phase difference between the between VSTAP and the VRWMSTAP wasand calcu-VRWM was ◦ ◦ lated. Acalculated. phase difference A phase of difference180° (SD ± of 7.2°) 180 between(SD ± 7.2the )V betweenSTAP and V theRWMV inSTAP theand frequencyVRWM in the range betweenfrequency 100 range and between500 Hz is 100 an andindication 500 Hz that is an no indication air bubbles that are no present air bubbles in the are coch- present in lear ductsthe cochlear[19]. ducts [19]. For sensorFor sensorinsertion, insertion, a cochleostomy a cochleostomy was performed was performed by an experienced by an experienced surgeon surgeon (A.D, (A.D., M.C.) inM.C.) the exposed in the exposed bony basal bony turn basal of turnthe cochlea. of the cochlea. The first The cochleostomy first cochleostomy was performed was performed in the STin theand ST the and second the secondin the SV, in theapprox SV,imately approximately 1–2 mm 1–2medial mm to medial the ventral to the edge ventral of edge of the RWM. A skeeter drill (Medtronic Xomed, Jacksonville, FL, USA) with a diameter (0.6 mm) slightly larger than that of the sensor head (0.5 mm) was used. The drilling process was carried out under saline water to prevent air infiltration to the cochlea through the cochleostomies. The surgeon confirmed that no damages occurred to the anatomical structures during the cochleostomy procedure. Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 20

the RWM. A skeeter drill (Medtronic Xomed, Jacksonville, FL, USA) with a diameter (0.6 mm) slightly larger than that of the sensor head (0.5 mm) was used. The drilling process Appl. Sci. 2021, 11, 5062 5 of 19 was carried out under saline water to prevent air infiltration to the cochlea through the cochleostomies. The surgeon confirmed that no damages occurred to the anatomical struc- tures during the cochleostomy procedure. The HTBs were fixedfixed withwith thethe cochleostomiescochleostomies locatedlocated approximatelyapproximately atat thethe highesthighest point of the the cochlea, cochlea, as as depicted depicted in in Figure Figure 1B.1B. The The sensor sensor used used for forST STmeasurements measurements was wasnavigated navigated using using a micromanipulator a micromanipulator with seven with sevendegrees degrees of freedom of freedom and a 10 and μm a adjust- 10 µm adjustmentment precision. precision. The second The second sensor sensor (SV meas (SVurement) measurement) was mounted was mounted on an on articulated an articulated arm armwith witha 2-axis a 2-axis micromanipulator micromanipulator at the at thebase base and and an anadditional additional micro-positioning micro-positioning stage stage at atthe the front front end end to to control control sensor sensor insertion. insertion. A A surgical surgical microscope microscope was was used used to insert thethe sensors into the cochlea. The insertion depth of the sensors was adjusted betweenbetween 0.50.5 andand 1.0 mmmm forfor allall samples.samples. The liquidliquid levellevel ofof thethe cochleacochlea waswas maintainedmaintained constantconstant duringduring sensor insertion to avoid airair beingbeing entrainedentrained inin thethe cochleacochlea fluid.fluid. The gapgap betweenbetween thethe outer rim of the sound receptor tube and the bony cochlear surfacesurface waswas sealedsealed withwith aa thinthin layer of soft tissuetissue toppedtopped withwith aa layerlayer ofof JeltrateJeltrate toto reinforcereinforce thethe sealseal (cf.(cf. FigureFigure2 2).).

Figure 2. Schematic drawing of the RWM and the ICARs inserted into ST and SV (left) and a Figure 2. Schematic drawing of the RWM and the ICARs inserted into ST and SV (left) and a zoomed zoomed microscope image of the basal region of the cochlea (right). The spaces between the ICAR microscope image of the basal region of the cochlea (right). The spaces between the ICAR sound sound receptor tube and the bone were first sealed with a thin layer of soft tissue and then rein- receptorforced with tube Jeltrate. and the bone were first sealed with a thin layer of soft tissue and then reinforced with Jeltrate. The first ICSP measurement after sensor fixation was used as the baseline measure- The first ICSP measurement after sensor fixation was used as the baseline measure- ment for comparison with the ICSP data reported in the literature. The noise floor and ment for comparison with the ICSP data reported in the literature. The noise floor and signal-to-noise ratio (SNR) were determined while the acoustic stimulation was switched signal-to-noise ratio (SNR) were determined while the acoustic stimulation was switched off. Only data with an SNR higher 7 dB were considered as results in the present study. off. Only data with an SNR higher 7 dB were considered as results in the present study.

2.5. Reinforcement of RWM For the reinforcement of thethe RWM,RWM, threethree clinicallyclinically usedused materialsmaterials werewere applied:applied: softsoft tissue, cartilage, and medical-grade silicone (Invotec(Invotec International, Inc., Jacksonville, FL, USA). Soft tissue and cartilage samples were harvested during the surgical preparation ofof the HTBHTB samplesample andand keptkept inin aa salinesaline solutionsolution untiluntil use.use. TheThe reinforcementreinforcement materialmaterial waswas approximately shapedshaped toto the size of the RWMRWM to uniformly cover the whole RWM surface area. TheThe reinforcementreinforcement material material was was applied applied during during the the experiment experiment in a in specific a specific order order that wasthat definedwas defined by an by increasing an increasing stiffness stiffness of the of material: the material: soft tissue,soft tissue, cartilage, cartilage, and silicone.and sili- Thecone. order The inorder which in thewhich material the material was applied was wasapplied defined was to defined minimize to minimize the risk of the damaging risk of thedamaging RWM duringthe RWM reinforcement. during reinforcement. Remnants of water on the RWM or in the middle ear cavity were removed care- fully by suction before and after each experiment. The structural integrity of the RWM was inspected with a surgical microscope and verified for any damage that might have occurred during the manipulation. The ICSP was recorded after the removal of each ma- terial to exclude RWM impedance changes caused by the manipulation and as a baseline measurement for the next material reinforcement step. Appl. Sci. 2021, 11, 5062 6 of 19

In general, only the effect of the RWM reinforcement on the ICSP was investigated in the present study. However, in samples HTB-11, HTB-12, and HTB-20, VSTAP was additionally recorded after RWM reinforcement to verify its effect on the METF and for comparison with Wegner et al. (2016) research findings (AppendixA).

2.6. Numerical Approach to Assess the Effect of RWM Reinforcement on Cochlear Hydrodynamics Numerical simulations using two models of the cochlea [8,11] were conducted to support our experimental data from the ICSP measurements after the RWM reinforcement. The simulations enabled us to estimate the RWM impedance after reinforcement and its components, including inertia and stiffness. Quantifying the RWM impedance by the simul- taneous measurement of PST and the RWM volume velocity (URWM) is challenging, since the RWM motion modes differ from simple piston movement across frequencies [20,21]. Additionally, after the reinforcement process, the RWM is occluded and the reliable mea- surement of the URWM is not possible. Both numerical models considered in the present study are LEMs that rely on the analogy between electrical, mechanical, fluidic, and acoustic domains to describe the behavior of a specific physical system as a circuit of lumped elements. The analogy only holds if the wavelength of the sound wave in the given system’s fluidic or solid medium is much larger than the largest geometrical dimension of the system [22]. A LEM of the cochlea transforms its fluid dynamic behavior into an electrical system and represents the fluidic characteristics by electrical components: inductors (L), capacitors (C), and resistors (R). The sound pressure (P) and volume velocity (U) are equivalent to the voltage (V) and current (I), respectively, of an electrical system. The model published by Nakajima et al. [11] used the stapes volume velocity (USTAP) to calculate the ZRWM of a normal RWM using the assumption that USTAP = URWM [20]. Due to the RWM reinforcement, it is doubtful whether this assumption is still valid, as other anatomical channels (vestibular aqueduct (VA) and cochlear aqueduct (CA)) can be activated to relieve the pressure. Considering this model limitation, Elliot et al. (2016) [8] presented an extended model of the cochlea by including VA and CA leakage pathways into their model to act as high-impedance pressure-relieving channels when the RWM is stiffened. Despite the mentioned weakness of the Nakajima model if applied on a cochlea with reinforced RWM, we still considered it for comparison with the experimental data due to its simplicity and the need for fewer modeling assumptions compared to the Elliot model. A detailed description of both models, the corresponding model parameters, and a discussion of the results from the numerical simulations and the experiments are added to Section 4.3.

3. Results 3.1. Validation of the Experimental Method 3.1.1. Assessment of the Middle Ear Function and the Hydrodynamic Condition of the Cochlea The METF of each HTB and the corresponding median of all samples are shown in Figure3A in comparison to the ASTM mean and SD [ 18]. The median METF follows the lower end of the ASTM standard range, with a peak at approximately 1.1 kHz. Although not every HTB showed an METF within the ASTM standard, the corresponding samples were still considered for the current study, as the main aim was to investigate the relative ICSP changes caused by RWM reinforcement. The phase difference between the stapes and the RWM in the low-frequency spectrum (100–500 Hz) is shown in Figure3B. Except for HTB-20, all the other HTBs are within a range defined as a ±7.2◦ phase deviation from half a cycle phase difference. The cochlear duct system of these HTB samples was therefore considered as free of air bubbles. In HTB-20, the phase difference decays with increasing frequency and deviates by 20◦ at 500 Hz. Therefore, HTB-20 was not considered for further investigations with reinforced RWM. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 20

Appl. Sci. 2021, 11, 5062 7 of 19

were still considered for the current study, as the main aim was to investigate the relative ICSP changes caused by RWM reinforcement.

Figure 3. 3. (A()A METF) METF data data of 6 HTBs of 6 HTBs compared compared with the with ASTM the F2504e05 ASTM standard. F2504e05 The standard. dotted black The dotted black line represents the mean ASTM F2504e05 standard and the 95% confidence interval is in blue. The thickline representsblack line corresponds the mean to ASTM the median F2504e05 of 6 HTBs. standard (B) Phase and difference the 95% between confidence stapes interval and is in blue. The RWMthick blackfor six lineHTBs corresponds in the lower frequency to the median range (100–500 of 6 HTBs. Hz). ( BThe) Phase dotted difference black lines betweenrepresent stapesthe and RWM ±7.2°for six deviation HTBs inband the from lower a 180° frequency phase difference range (100–500 similar to Hz).the study The of dotted Frear et black al. (2018) lines [19]. represent the ±7.2◦ Each HTB sample is represented by a specific color. deviation band from a 180◦ phase difference similar to the study of Frear et al. (2018) [19]. Each HTB sampleThe is phase represented difference by abetween specific the color. stapes and the RWM in the low-frequency spec- trum (100–500 Hz) is shown in Figure 3B. Except for HTB-20, all the other HTBs are within a3.1.2. range Intracochlear defined as a ±7.2° Pressure phase deviation Measurements from halfin a cycle Scala phase Tympani difference. and The Scala cochlear Vestibuli duct systemFigure of4A these (magnitude) HTB samples and was Figure theref4oreB (phase) considered display as free the of individualair bubbles. In experimental HTB- ICSP 20,data the for phase ST, difference while Figure decays4C with (magnitude) increasing and frequency Figure and4D deviates (phase) by display 20° at the500 Hz. corresponding Therefore, HTB-20 was not considered for further investigations with reinforced RWM. data for SV. The ICSP data are normalized with respect to the ear canal pressure PEC and denoted as the ICSP gain. For comparison, the minimum/maximum range of the ICSP gain 3.1.2. Intracochlear Pressure Measurements in Scala Tympani and Scala Vestibuli data reported by Nakajima et al. (2009) was plotted in the same graphs as a shaded area. Figures 4A (magnitude) and Figure 4B (phase) display the individual experimental In general, the ICSP gain values of both scalae were lower compared to the reference data. ICSP data for ST, while Figure 4C (magnitude) and Figure 4D (phase) display the corre- spondingFor ST, at data approximately for SV. The ICSP 500 data Hz are the norm medianalized ICSPwith respect gain drops to the ear below canal the pressure lower limit of the PreferenceEC and denoted data as range the ICSP by up gain. to For 12 dB.comparison, At frequencies the minimum/maximum above 800 Hz, range the median of the ICSP gain ICSPgenerally gain data coincides reported with by Nakajima the lower et limit al. (2009) of the was reference plotted in data the range.same graphs For SV, as a the deviation shadedfrom the area. reference In general, data the follows ICSP gain a similarvalues of trend both asscalae for were ST, except lower compared that the comparatively to the low referenceICSP gain data. values For ST, within at approximately the low-frequency 500 Hz rangethe median are seenICSP atgain up drops to 1.1 below kHz. the The phase data Appl. Sci. 2021, 11, x FOR PEER REVIEWlower limit of the reference data range by up to 12 dB. At frequencies above 8008 of Hz, 20 the for ST and SV can be considered in fair agreement with the reference data. median ICSP gain generally coincides with the lower limit of the reference data range. For SV, the deviation from the reference data follows a similar trend as for ST, except that the comparatively low ICSP gain values within the low-frequency range are seen at up to 1.1 kHz. The phase data for ST and SV can be considered in fair agreement with the reference data.

Figure 4. Simultaneous ICSP measurements in ST and SV: magnitude of the pressure gain in ST Figure(PST/PEC) 4. (ASimultaneous) and in SV (PSV/PEC ICSP) (B); respective measurements phase data in for ST ST and (C) and SV: SV magnitude (D). of the pressure gain in ST (PST/PEC)(A) and in SV (PSV/PEC)(B); respective phase data for ST (C) and SV (D). 3.1.3. Effect of RWM Reinforcement on ICSP Measurement The typical change in ICSP gain due to RWM reinforcement that was observed in the present study is illustrated in the data from four simultaneous ICSP measurements on HTB-10 and HTB-11 using cartilage and soft tissue as reinforcement material (cf. Figure 5). The individual figures show the ICSP gain for both scalae before (baseline) and after the RWM reinforcement. In general, a considerable increase in ICSP after RWM reinforce- ment was identified in ST at frequencies below 1 kHz. In SV, the corresponding pressure increase is moderate or even negligible. Appl. Sci. 2021, 11, 5062 8 of 19

3.1.3. Effect of RWM Reinforcement on ICSP Measurement The typical change in ICSP gain due to RWM reinforcement that was observed in the present study is illustrated in the data from four simultaneous ICSP measurements on HTB-10 and HTB-11 using cartilage and soft tissue as reinforcement material (cf. Figure5). The individual figures show the ICSP gain for both scalae before (baseline) and after the RWM reinforcement. In general, a considerable increase in ICSP after RWM reinforcement was identified in ST at frequencies below 1 kHz. In SV, the corresponding pressure increase Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 20 is moderate or even negligible.

Figure 5. Two exemplary cases for the ICSP gain changes in ST and SV after the reinforcement of Figure 5. Two exemplary cases for the ICSP gain changes in ST and SV after the reinforcement of the the RWM with soft tissue and cartilage compared to the respective baselines for HTB-10 (A,B) and RWMHTB-11 with (C, softD). The tissue bold and lines cartilage represent compared the ICSPto gain the af respectiveter reinforcement baselines in each for HTB-10 scenario ( Afor,B ST) and HTB-11 (Cand,D ).SV The (red bold and linesblue, representrespectively) the and ICSP the gain dotted after line reinforcement pattern for the inbaselines each scenario in the same for STlight and SV (red andcolor blue, shades. respectively) and the dotted line pattern for the baselines in the same light color shades.

TheThe samplesample HTB-10 experienced experienced the the largest largest increase increase in ST in STpressure pressure by more by more than than15 15 dB afterdB after adding adding soft soft tissue tissue to to the the RWM RWM (cf. (cf. Figure Figure5 A).5A). The The cartilagecartilage showed similar similar be- behavior tohavior the soft to the tissue, soft tissue, despite despite its higher its higher material material stiffness stiffness (cf. (cf. Figure 5B).5B). It It was was expected expected that softthat tissue soft tissue could could be better be better adapted adapted to the to surfacethe surface geometry geometry of theof the RWM RWM and and hence hence allow for allow for a more repeatable and efficient means of RWM reinforcement than using stiffer a more repeatable and efficient means of RWM reinforcement than using stiffer cartilage cartilage material. However, using HTB-11 cartilage led to a higher pressure in ST than material.using soft However,tissue (13 dB using versus HTB-11 5 dB, cf. cartilage Figure 5C,D), led tobut a overall, higher the pressure change inin ST pressure than using soft tissuewas less (13 pronounced dB versus 5compared dB, cf. Figure to that5 forC,D), HTB-10, but overall, especially the if changethe effect in of STreinforcement pressure was less pronouncedon the SV pressure compared was considered. to that for The HTB-10, investigations especially on HTB-10 if the effect nicely of demonstrate reinforcement that on the SVthe pressure pressure difference was considered. across the The partition investigations can be considerably on HTB-10 reduced nicely or demonstrate can even van- that the pressureish after RWM difference reinforcement across the and partition at frequencies can be below considerably 500 Hz. Consequently, reduced or a can reduced even vanish afterhearing RWM sensation reinforcement within that and frequency at frequencies range might below result 500from Hz. RWM Consequently, manipulation. a reduced hearingTo sensationhighlight the within effect that of RWM frequency reinforcemen ranget might on the result ICSP in from ST and RWM SV manipulation.for all con- sideredTohighlight HTBs, the the ratio effect between of RWM the reinforcement ICSP after (indicated on the ICSP with in an ST apostrophe) and SV for all and considered before HTBs, the(indicated ratio between as the thebaseline) ICSP after reinforcement (indicated withwas calculated an apostrophe) and denoted and before as the (indicated relative as pres- the baseline) reinforcementsure gain. was calculated and denoted as the relative pressure gain. The corresponding data are shown in Figure 6. Figure 6A,B quantify the effect of The corresponding data are shown in Figure6. Figure6A,B quantify the effect of cartilage reinforcement in ST and SV for all considered HTBs together with the respective cartilagemedians, reinforcementand Figure 6C,D in show ST and the SVsame for bu allt for considered silicone used HTBs as togethera reinforcement with the mate- respective medians,rial. Finally, and the Figure median6C,D curves show for the all the same reinforcement but for silicone materials used are as summarized a reinforcement in Fig- material. ure 6E,F. The individual data for soft tissue are not shown, since they do not provide more insight into the effect of RWM reinforcement on the inner ear sound pressure. The data confirm the aforementioned considerably higher increase in sound pressure in ST com- pared to SV after RWM reinforcement and within the low-frequency range (<2 kHz), Appl. Sci. 2021, 11, 5062 9 of 19

Finally, the median curves for all the reinforcement materials are summarized in Figure 6E,F. The individual data for soft tissue are not shown, since they do not provide more insight into the effect of RWM reinforcement on the inner ear sound pressure. The data confirm the aforementioned considerably higher increase in sound pressure in ST compared to SV after RWM reinforcement and within the low-frequency range (<2 kHz), where pressure Appl. Sci. 2021, 11, x FOR PEER REVIEW 10 of 20 changes are attributed to RWM reinforcement. The large variation in relative pressure gain between the individual samples is noticeable. With cartilage reinforcement, the gain in ST can wherereach pressure a maximum changes gain are attributed of up to to 25 RWM dB (HTB-12), reinforcement. but inThe another large variation trial a in maximum rel- gain as small ativeas 5 pressure dB was gain identified between the (HTB-9). individual The samples other is reinforcementnoticeable. With cartilage materials reinforce- show similar variations ment,between the gain the in samples. ST can reach Considering a maximum the gain median of up to values,25 dB (HTB-12), cartilage but provides in another the highest increase trialin STa maximum pressure, gainfollowed as small byas 5 soft dB was tissue. identified Although (HTB-9). silicone The other is reinforcement regarded as the material with materials show similar variations between the samples. Considering the median values, cartilagethe highest provides stiffness the highest of theincrease three in ST materials pressure, consideredfollowed by soft in tissue. the present Although study, it shows the siliconelowest is regarded efficiency as the for material RWM with reinforcement. the highest stiffness We of believe the three that materials the properconsid- placement of the eredreinforcement in the present study, materials it shows that the are lowest in goodefficiency contact for RWM with reinforcement. the RWM We is crucial be- for a high and lieverepeatable that the proper reinforcement placement of effect. the reinfo Thercement relatively materials stiff that silicone are in materialgood contact makes it challenging with the RWM is crucial for a high and repeatable reinforcement effect. The relatively stiff siliconeto achieve material this makes requirement. it challenging Furthermore, to achieve this larger requirement. anatomical Furthermore, variations larger in the RWM niche anatomicalbetween variations samples in and the RWM the lack niche of between ability samples to objectively and the lack assess of ability the placementto objec- of the material tivelyon the assess RWM the placement make it of difficult the material to achieve on the RWM a better make it repeatability difficult to achieve between a better individual samples. repeatability between individual samples.

Figure 6. Relative pressure gain in ST and SV observed after the RWM reinforcement with cartilage and silicone (A–D). Median values are represented as a solid black line. (E,F) represent the median relative pressure gain values for all three materials, (E) ST, and (F) SV. Appl. Sci. 2021, 11, 5062 10 of 19

4. Discussion 4.1. Validation of the HTB Functionality The functionality of the HTB samples was assessed by using the METF against the standard ASTM and verifying the phase difference between the stapes and the RWM at low frequencies (cf. Figure3). The METF of several samples was below the ASTM standard range across the whole considered frequency range (200 Hz–8 kHz). We assume that the lower METF is mostly the result of age-related effects, such as a less compliant tympanic membrane that reduces the sound energy transmitted to the middle ear [23]. The mean age of the sample was 74 years. In addition, the ossicles and soft tissue structures in the middle ear are subjected to common diseases that can affect the METF [24]. The freezing of the HTBs after extraction might affect the membranous structures such as the RWM and TM. Therefore, using fresh samples might lower the variability of the RWM reinforcement effect on the ICSP between individual samples. During the experiment, the samples were kept from drying with assisted moisturization. However, the drying of the sample during the time taken for preparations and measurements cannot be completely ignored [25]. After the cochleostomy in ST and SV were drilled and the sensors were inserted the surgeon visually confirmed that no damages occurred to the middle or inner ear structures. Additional confirmation of the HTB functionality after sensor insertion by measuring the stapes and RWM motion was not feasible due to the obstructed visual access to the stapes by the two hydrophones. The measured ICSP data were compared with the corresponding data reported by Nakajima et al. [11]. A trend towards lower values than the reference data was also observed during the ICSP measurements. Mainly at frequencies below 2 kHz, where the effect of RWM reinforcement on the ICSP is significant, the measured ICSP data were well below the lower limit of the reference data. While in the present study we are more interested in changes in the ICSP due to RWM reinforcement than in absolute pressure values, it is important to assess the hydrodynamic condition of the cochlea under investigation and thus confirm that the comparatively low METF and ICSP data are not related to the inner ear mechanics. This was accomplished by calculating the input impedance (ZC) of the cochlea, which is defined as the ratio between the vestibuli pressure 2 PSV and the volume velocity of the stapes (VSTAP * A), where A (3.2 mm ) indicates the area of the stapes footplate [11,23]. The VSTAP was measured prior hydrophone insertion, as simultaneous measurements of ICSP and the VSTAP were not possible due to space restrictions in the opened middle ear cavity. The data were then compared with several reference data from the literature [11,23,26]. The behavior of the ZC closely resembles resistive behavior, as the phase data show values close to zero across the considered frequency range and similar to the reference data (cf. Figure7). The wider SD in the phase data above 1 kHz suggests that the complex motion of the stapes gains significance. This could have been alleviated by using a 3D LDV for stapes velocity measurements instead of a single-point LDV, as available for the present study [11]. The median magnitude of ZC is in good agreement with Aibara et al. (2001) and slightly below the data of Nakajima et al. (2009) until 6 kHz. Above 6 kHz, a larger deviation can be identified between our data and the reference data. Since the effect of RWM reinforcement on the ICSP is limited to frequencies below 2 kHz, the HTBs used in the present study can be classified as being valid for this study. Appl. Sci. 2021, 11, 5062 11 of 19 Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 20

FigureFigure 7. Input 7. Input cochlea cochlea impedance impedance (Zc) (comparedZc) compared to the to reference the reference data: data: (A) (magnitudeA) magnitude of the of theZc Zc and and (B) respective phasephase data.data. The bold black line andand the shaded area representrepresent thethe studystudy datadata median medianand and the SD,the SD, respectively. respectively. The The literature literatur datae data are indicatedare indicated in individual in individual colors. colors.

4.2. 4.2.Effect Effect of RWM of RWM Reinforcement Reinforcement on the on ICSP the ICSP

UsingUsing cartilage cartilage as a asreinforcement a reinforcement material, material, a maximum a maximum median median (of (ofall samples) all samples) PST PST changechange of 13 of dB 13 was dB was identified identified after after the theRWM RWM reinforcement reinforcement (cf. (cf.Figure Figure 6). 6In). general, In general, significantsignificant RWM RWM reinforcement-induced reinforcement-induced changes changes in P inST PwereST were observed observed only only at frequencies at frequencies belowbelow 2 kHz. 2 kHz. These These results results are in are good in good agreement agreement with with the thecorresponding corresponding data data reported reported previouslypreviously by Guan by Guan et al. et(2018) al. (2018)for one forHTB one [13]. HTB The [ 13sound]. The pressure sound in pressure the basal in part the of basal the partscala ofvestibule the scala (P vestibuleSV) did change (PSV) considerably did change considerably less (<3 dB) than less (<3in the dB) scala than tympani. in the scala Thistympani. behavior Thisindicates behavior that indicatespressure-relieving that pressure-relieving mechanisms such mechanisms as the cochlear such as and the cochlearves- and vestibular aqueducts prevent a strong increase in Z due to the stiffened RWM. The tibular aqueducts prevent a strong increase in ZC due to the Cstiffened RWM. The fact that RWMfact reinforcement that RWM reinforcement did not have dida significant not have influence a significant on the influence stapes velocity on the stapes (cf. Figure velocity A1, (cf.Appendix Figure A1A) ,further Appendix confirmsA) further the releva confirmsnce theof the relevance aqueducts of the as aqueducts soon the RWM as soon is the RWM is reinforced. However, Guan et al. (2018) reported an increase in P of up to 10 dB reinforced. However, Guan et al. (2018) reported an increase in PSV of up toSV 10 dB after stiffeningafter stiffeningthe RWM thewith RWM a combination with a combination of perichondrium, of perichondrium, cartilage, and cartilage, Jeltrate, andwhereas Jeltrate, in ourwhereas study, in we our could study, only we observe could only a pressure observe increase a pressure below increase 3 dB below using 3 cartilage. dB using Nev- cartilage. Nevertheless, it is important to note that the results of Guan et al. (2018) stem from only ertheless, it is important to note that the results of Guan et al. (2018) stem from only one one HTB sample. HTB sample. Considering the individual samples instead of the averaged data, RWM reinforcement- Considering the individual samples instead of the averaged data, RWM reinforce- induced changes in PST of up to 25 dB but also below 5 dB were observed. We expect that ment-induced changes in PST of up to 25 dB but also below 5 dB were observed. We expect the large variation in the reinforcement effect between individual samples was mainly that the large variation in the reinforcement effect between individual samples was mainly due to the manipulation of the material during the reinforcement of the RWM. The com- due to the manipulation of the material during the reinforcement of the RWM. The com- paratively small effect of the reinforcement on the ICSP using silicone with the highest paratively small effect of the reinforcement on the ICSP using silicone with the highest material stiffness of the three considered materials confirms our assumption. Using a material stiffness of the three considered materials confirms our assumption. Using a stiff stiff reinforcement material makes it challenging to firmly attach the material to the 3D reinforcement material makes it challenging to firmly attach the material to the 3D curva- curvature of the RWM. In addition, the placement of the material must be conducted with turegreat of the care RWM. to minimize In addition, the the risk placement of RWMdamage of the material and, hence, must loss be conducted of the sample. with great care to minimizeTo assess the the risk effectiveness of RWM damage of the reinforcement and, hence, procedure,loss of the sample. one would need to quantify theTo RWMassess the impedance effectiveness after of reinforcement the reinforcement from procedure, the simultaneous one would measurement need to quan- of the tify the RWM impedance after reinforcement from the simultaneous measurement of the RWM volume velocity and PST (ZRWM = PST/URWM). This is difficult to accomplish, RWMmainly volume due velocity to the opaque and PST nature (ZRWMof = thePST/ reinforcementURWM). This is material,difficult to which accomplish, prevents mainly the direct due interrogationto the opaque of nature the RWM of the motion reinforcemen using thet material, LDV. Estimating which prevents the RWM the motion direct inter- from the rogationvelocity of the measurements RWM motion on using the surface the LDV. of the Estimating reinforcement the RWM material motion is inappropriate from the veloc- because ity measurementsof the relatively on thick the surface reinforcement of the reinfo materialrcement and itsmaterial uncontrolled is inappropriate interface withbecause the RWM.of the relativelyA betteralternative thick reinforcement to the reinforcement material and material its uncontrolled used in theinterface present with study the RWM. would be A bettera transparent alternative material to the reinforcement that also provides mate arial good used binding in the withpresent the study RWM. would A transparent be a transparenthydrogel material compound that also developed provides by a Weigood et binding al. [27] with which the directly RWM. reactsA transparent with biological hy- drogeltissues compound might be developed an option by for Wei further et al. research. [27] which Besides directly the reacts advantages with biological of a well-controlled tissues mightinterface be an option between for further the hydrogel research. and Besides the RWM the advantages and direct of optical a well-controlled access to the interface RWM, the betweenhydrogel the hydrogel might also and allow the RWM for a stepwiseand direct increase optical access in RWM to the stiffness RWM, during the hydrogel the curing might also allow for a stepwise increase in RWM stiffness during the curing process. A Appl.Appl. Sci. Sci.2021 2021, 11, 11, 5062, x FOR PEER REVIEW 1213 of of 19 20

process.reinforcement A reinforcement material with material such with properties such properties would allow would forallow more forrepeatable more repeatable and con- andtrollable controllable measurement measurement conditions, conditions, which whichwe see we as seecrucial as crucial in order in orderto gain to a gain more a morequan- quantitativetitative knowledge knowledge of the of RWM the RWM reinforcemen reinforcementt effect effect on the on thecochlear cochlear hydrodynamics. hydrodynamics.

4.3.4.3. Comparison Comparison with with Numerical Numerical Calculations Calculations and and RWM RWM Impedance Impedance Estimation Estimation InIn the the present present study, study, we we were were not not able able to estimate to estimate the RWMthe RWM impedance impedance after reinforce-after rein- mentforcement by experimental by experimental means. means. Instead, Instead, two numerical two numerical LEM models LEM of models the cochlea of the proposed cochlea byproposed Nakajima by etNakajima al. (2009) et and al. (2009) Elliot etand al. Elliot (2016) et wereal. (2016) used were to approximate used to approximate the missing the quantitymissing fromquantity the from best fitthe between best fit between the model theand model the and experiment. the experiment. Nakajima Nakajima proposes pro- aposes simple a simple model ofmodel the cochleaof the cochlea that assumes that assumes an equal an volumeequal volume velocity velocity between between the oval the andoval round and round window, window, an assumption an assumption that is notthat compatible is not compatible with a reinforced with a reinforced RWM leading RWM toleading a reduced to a reduced vibrational vibrational motion motion of the RWM of the butRWM a similar but a similar stapes stapes velocity velocity as before as before the reinforcement.the reinforcement. Elliot’s Elliot’s model model tries tries to account to account for this for effect this effect by considering by considering cochlear cochlear and vestibularand vestibular aqueducts aqueducts that act that as act pressure as pressure relief elementsrelief elements in parallel in parallel withthe with RWM. the RWM.

4.3.1.4.3.1. LEM LEM of of the the Cochlea Cochlea Proposed Proposed by by Nakajima Nakajima et et al. al. (2009) (2009) Nakajima et al. proposed a simple LEM of the cochlea (cf. Figure8) that describes Nakajima et al. proposed a simple LEM of the cochlea (cf. Figure 8) that describes the the cochlea input impedance ZC as a serial circuit containing two lumped parameters, the cochlea input impedance ZC as a serial circuit containing two lumped parameters, the im- impedance of the cochlear partition (ZDIFF) and ZRWM (Equation (1)): pedance of the cochlear partition (ZDIFF) and ZRWM (Equation (1)): Z = Z + Z C𝑍 = DIFF𝑍 + 𝑍RWM (1)

FigureFigure 8. 8. LEM circuit circuit of of the the cochlea cochlea proposed proposed by byNaka Nakajimajima et al. et (2009). al. (2009). Ground Ground connection connection repre- sents the sound pressure at the surgically opened middle ear cavity that is assumed to be zero. represents the sound pressure at the surgically opened middle ear cavity that is assumed to be zero.

ZDIFF was experimentally determined in several studies [8,11,19,23]. The magnitude ZDIFF was experimentally determined in several studies [8,11,19,23]. The magnitude of ZDIFF represents a resistive behavior independent of the frequency, and the phase is of ZDIFF represents a resistive behavior independent of the frequency, and the phase is DIFF approximatelyapproximately close close to to zero zero cycles cycles for for most most frequencies. frequencies. Hence, Hence,Z DIFFZ can can be be approximated approximated withwith one one lumped lumped parameter parameter describing describing the the resistive resistive behavior behavior (R DIFF(RDIFF).). The TheZ DIFFZDIFFvalue value from from AibaraAibara et et al. al. (2001) (2001) was was used used in in both both models. models.Z RWMZRWMwas was calculated calculated using using the the proposed proposed iterativeiterative Foster Foster form form network network model model with with predetermined predetermined parameter parameter values values (Table (Table1). 1). An An adaptedadapted figure figure of of the the RWM RWM LEM LEM from from Nakajima Nakajima et et al. al. (2009) (2009) is is added added for for clarification clarification in in TableTable1[ 111 [11].].

Table 1. Parameter values used in Nakajima et al.’s lumped element model (LEM).

Lumped Element Inclusive Elements SI Unit Value Reference ZDIFF RDIFF Nsm−5 2.0 × 1010 [23] C N−1m5 9.0 × 10−14 ZRWM L kgm−4 4.62 × 107 [11] R Nsm−5 2.34 × 108 Appl. Sci. 2021, 11, 5062 13 of 19

Table 1. Parameter values used in Nakajima et al.’s lumped element model (LEM). Appl. Sci. 2021, 11, x FOR PEER REVIEW 14 of 20 Lumped Element Inclusive Elements SI Unit Value Reference −5 10 ZDIFF RDIFF Nsm 2.0 × 10 [23] Z −1 5 × −14 RWM X (spacing)C N m 9.0 √1010 L kgm−4 4.62 × 107 [11] R Nsm−5 2.34 × 108 √ X (spacing) 10 N (number of branches) 6 N (number of 6 branches)

The USTAP is considered as the input to the cochlea. According to Stenfelt et al. (2004)’s findings,The U theSTAP magnitude is considered of UasSTAP the inputapproximately to the cochlea. equals According the magnitude to Stenfelt of etU RWMal. (2004)’s[20]. findings, the magnitude of USTAP approximately equals the magnitude of URWM [20]. USTAP = URWM (2) 𝑈 = 𝑈 (2) ZDIFF = (PSV − PST)/USTAP (3) 𝑍 =(𝑃 −𝑃 )/𝑈 (3) As we have confirmed by experiments (cf. Appendix A), and as is also reported in the literatureAs we [5 have,13], theconfirmed input to by the expe cochleariments can (cf. be Appendix assumed A), to notand be as affectedis also reported by the RWM in reinforcement—i.e.,the literature [5,13], theU STAPinput= toU’ theSTAP cochlea, where can the be assumed apostrophe to not denotes be affected the conditions by the RWM after RWMreinforcement—i.e., reinforcement. USTAP The = modelU’STAP, furtherwhere the assumes apostrophe equal RWMdenotes motion the conditions before and after after reinforcementRWM reinforcement. (URWM The= U’ modelRWM). further Considering assumes these equal assumptions RWM motion and before using and the after equations re- RWM RWM forinforcementPST (P’ST ()U ((4) and = U’ (5))). and ConsideringPSV (P’SV )these ((7) andassumptions (8)), respectively, and using the the relative equations pressure for PST (P’PST0ST) ((4) andP0SV (5)) and PSV (P’SV) ((7) and (8)), respectively, the relative pressure gain gain ( P and P ) caused by the RWM reinforcement can be derived as the ratio between ( andST ) causedSV by the RWM reinforcement can be derived as the ratio between the the RWM impedance after and before reinforcement for ST (6) and the ratio between the cochleaRWM impedance input impedance after and after beforeZc reinforcement0 and before reinforcement for ST (6) and Zcthefor ratio SV between (9). the coch- lea inputBefore impedance reinforcement after Zc’ for and ST: before reinforcement Zc for SV (9). Before reinforcement for ST: PST = URWM ∗ ZRWM (4) 𝑃 = 𝑈 ∗ 𝑍 (4) After reinforcement for for ST: ST:

P𝑃′0ST = U𝑈′0RWM ∗∗ 𝑍′Z0RWM (5) (5)

From Equations (4) and (5) and the assumption URWM = U’RWM0 : From Equations (4) and (5) and the assumption URWM = U RWM: 𝑃′ 𝑍′ P0 = Z0 (6) 𝑃ST = 𝑍RWM (6) PST ZRWM Similarly, before reinforcement for SV: Similarly, before reinforcement for SV: 𝑃 = 𝑈 ∗ (𝑍 + 𝑍) (7)

After reinforcement (forP SV):SV = USTAP ∗ (ZDIFF + ZRWM) (7) After reinforcement (for𝑃′ SV): = 𝑈′ ∗ (𝑍 +𝑍 ) (8)

Since USTAP = U’STAP: 0 P0SV = U0STAP ∗ (ZDIFF + Z RWM) (8) 𝑃′ 𝑍 +𝑍′ 0 = (9) Since USTAP = U STAP: 𝑃 𝑍 +𝑍 P0 Z + Z0 SV = DIFF RWM (9) + 4.3.2. LEM of the Cochlea ProposedPSV by ElliotZDIFF et al. (2016)ZRWM 4.3.2.In LEM contrast of the to Cochlea the LEM Proposed of Nakajima by Elliotet al., etthe al. model (2016) proposed by Elliot et al. (2016) includes high-impedance leakage pathways. These are the CA connected to ST and the In contrast to the LEM of Nakajima et al., the model proposed by Elliot et al. (2016) VA connected to SV. The circuitry describing the model is shown in Figure 9. As a conse- includes high-impedance leakage pathways. These are the CA connected to ST and the VA quence of the alternative pressure-relieving paths that are considered in the model, the connected to SV. The circuitry describing the model is shown in Figure9. As a consequence URWM and U’RWM (after reinforcement) can be of different magnitudes. However, USTAP (the input to the cochlea), ZVA, and ZCA are assumed to not be affected by the reinforcement of Appl. Sci. 2021, 11, 5062 14 of 19

of the alternative pressure-relieving paths that are considered in the model, the URWM and 0 U RWM (after reinforcement) can be of different magnitudes. However, USTAP (the input to the cochlea), ZVA, and ZCA are assumed to not be affected by the reinforcement of the Appl. Sci. 2021, 11, x FOR PEER REVIEWRWM. Based on these assumptions, the relative pressure gain can be calculated for the15 STof 20

and SV using Equations (10) and (11).    ZRWM  (ZDIFF + ZVA) 1 + + ZRWM P0ST Z0RWM 𝑍 ZCA =  (𝑍 + 𝑍 )1+ + 𝑍  (10) P 𝑃′ Z 𝑍′ Z𝑍0RWM ST RWM(ZDIFF + Z ) 1 + + Z0 = VA ZCA RWM (10) 𝑃 𝑍 𝑍′ (𝑍 + 𝑍)1+ 𝑍 + 𝑍′       0   ( + ) + ZRWM + + Z RWM + 0 P0 ZDIFF ZVA 1 Z ZRWM ZDIFF 1 Z Z RWM SV CA CA = (11)  ( )      P Z0RWM ZRWM SV (ZDIFF + ZVA=) 1 + + Z0RWM ZDIFF 1 + + ZRWM ZCA ZCA (11) ( )

Figure 9. Adapted LEM circuit of the cochlea from Elliot et al. (2016). For descriptive purposes, the Figure 9. Adapted LEM circuit of the cochlea from Elliot et al. (2016). For descriptive purposes, the individual circuit components describing the four indicated equivalent impedance are not shown. individual circuit components describing the four indicated equivalent impedance are not shown.

TheThe parameters parameters and and their their respective respective values values are are summarized summarized in in Table Table2. 2.

Table 2. Parameters and respective values used in Elliot et al.’s LEM. Table 2. Parameters and respective values used in Elliot et al.’s LEM. Lumped Element Inclusive Elements SI Units Value Reference Lumped Element Inclusive Elements SI Units Value Reference ZDIFF RDIFF Nsm−5 2.0 × 1010 [23] −5 10 ZDIFF RDIFF Nsm 2.0 × 10 [23] LRWM Ns2m−5 1.0 × 106 L Ns2m−5 1.0 × 106 ZRWM RRWM RWMNsm−5 2.5 × 109 ZRWM −5 9 CRWM RRWMN−1m5 Nsm 1.0 × 10−132.5 × 10 2 −5 −1 5 7 −13 LVA CRWMNs m N m 5.1 × 101.0 × 10 [8] ZVA 2 −5 7 [8] L −5 × RVA VANsm Ns 1.1m × 1010 × 5.1 Hz10 ZVA −5 q f RVA 2 −5 Nsm 8 10 LCA Ns m 5.61.1 × ×1010 × 1000 Hz CA 2 −5 8 Z LCA Ns m 5.6 × 10 Z RCA Nsm−5 3.5 × 1011 × Hz CA −5 q f RCA Nsm 11 3.5 × 10 × 1000 Hz 4.3.3. Results Considering cartilage as a reinforcement material, the best fit between the two mod- els and the experiment (median data) is achieved if a compliance value that is five times smaller than the initial RWM compliance (C0) and an inertance (added mass) that is twice the initial inertance (L0) are considered for the lumped parameter representing the rein- forced RWM. Cartilage reinforcement was chosen for the comparison, as it produced, on average, the highest increase in inner ear pressure (cf. Figure 6E). As a comparison, Elliot Appl. Sci. 2021, 11, 5062 15 of 19

4.3.3. Results Considering cartilage as a reinforcement material, the best fit between the two models and the experiment (median data) is achieved if a compliance value that is five times smaller than the initial RWM compliance (C ) and an inertance (added mass) that is twice the initial Appl. Sci. 2021, 11, x FOR PEER REVIEW 0 16 of 20 inertance (L0) are considered for the lumped parameter representing the reinforced RWM. Cartilage reinforcement was chosen for the comparison, as it produced, on average, the highest increase in inner ear pressure (cf. Figure6E). As a comparison, Elliot et al. (2016) etreported al. (2016) a reductionreported a in reduction the RWM in compliance the RWM comp by a factorliance of by 10 a tofactor 100 afterof 10 theto 100 implantation after the implantationof a cochlear of implant. a cochlear implant. AsAs illustrated illustrated in in Figure Figure 10A, 10A, Nakajima’s Nakajima’s model model with with twice twice the the initial initial RWM RWM inertance inertance (2L(2L0)0 )and and one one fifth fifth of of the the initial initial RWM RWM compliance compliance (0.2 (0.2 C C0)0 )is is in in fair fair agreement agreement with with the the experimentallyexperimentally determined determined relati relativeve pressurepressure gaingain in in ST ST and and SV SV at at frequencies frequencies above above 500 500 Hz. Hz.Below Below 500 500 Hz, Hz, the the model model overestimates overestimates the the relative relative pressure pressure gaingain byby up to 6 6 dB dB in in ST ST andand 3 3dB dB in in SV. SV. In In comparison, comparison, Elliot’s Elliot’s model model overestimates overestimates the the reinforcement reinforcement effect effect at at lowlow frequencies frequencies even even more more but but predicts predicts lower lower values values in in ST ST than than the the experiment experiment between between 500500 Hz Hz and and 1 1kHz. kHz. The The relative relative pressure pressure gain gain curve curve peaks peaks below below 200 200 Hz Hz in in the the positive positive directiondirection and and shows shows a anegative negative peak peak at at 800 800 Hz. Hz. If If the the initial initial RWM RWM compliance compliance is is reduced reduced byby a afactor factor of of 100 100 (the (the highest highest stiffening stiffening of of the the RWM RWM Elliot Elliot observed observed after after CI CI implantation), implantation), bothboth peaks peaks become become more more pronounced, pronounced, rise rise in in magnitude, magnitude, and and are are shifted shifted towards towards a ahigher higher frequencyfrequency (700 (700 Hz Hz and and 3.6 3.6 kHz). kHz). This This curve curve characteristic characteristic stems stems from from the the cochlear cochlear and and vestibularvestibular aqueducts aqueducts ZZCACA andand ZZVAVA. The. The peak peak magnitude magnitude and and frequency frequency of of occurrence occurrence are are dependentdependent on on the the RWM RWM compliance. compliance. The The experime experimentalntal data data representing representing the the ST STshow show a cleara clear indication indication of a ofpositive a positive peak peak in the in rela thetive relative pressure pressure gain curve, gain curve, but a negative but a negative peak cannotpeak cannotbe identified be identified in the averaged in the averaged data. Looking data. Lookingat the data at theof the data individual of the individual HTBs, a negativeHTBs, a peak negative is obvious peak is in obvious the relative in the relativepressure pressure gain curve gain for curve certain for certainHTBs and HTBs rein- and forcementreinforcement materials materials (cf. Figure (cf. Figure 6A,C).6A,C). The reason The reason why whythe negative the negative peak peak is damped is damped out forout certain for certain samples samples has not has yet not been yet been clarified. clarified. To obtain To obtain a better a better agreement agreement (especially (especially in thein thefrequency) frequency) between between Elliot’s Elliot’s model model and andthe experimental the experimental data, data, a closer a closer parameter parameter op- timizationoptimization of the of lumped the lumped elemen elementsts representing representing the aqueducts the aqueducts or even or even an adaption an adaption of the of wholethe whole model model seems seems to be to necessary. be necessary. Such Such model model optimization optimization was wasbeyond beyond the thescope scope of theof thepresent present study. study. The The limitation limitation of ofNakaji Nakajima’sma’s model model becomes becomes obvious obvious at at high high RWM RWM reinforcements,reinforcements, where where the the assumption assumption of of equa equall volume volume velocities velocities at at the the oval oval and and round round window (U ≈ U ) breaks down. For a 0.01 C RWM impedance, the model predicts window (USTAPSTAP ≈ URWMRWM) breaks down. For a 0.01 C0 RWM0 impedance, the model predicts aa high high relative relative pressure pressure gain gain above above 40 40 dB dB that that we we were were not not able able to to reproduce reproduce with with our our experiments and that does not appear very realistic. experiments and that does not appear very realistic.

Figure 10. Experimental relative pressure gain values of reinforced RWM with simulated data. Figure 10. Experimental relative pressure gain values of reinforced RWM with simulated data. Ex- Experimental data with cartilage reinforcement (median and SD) and simulated values from two perimental data with cartilage reinforcement (median and SD) and simulated values from two models models (Nakajima et al. and Elliot et al.) are represented for (A) ST and (B) SV. After reinforce- A B ment,(Nakajima the RWM et al. inertance and Elliot is set et al.)to twice are representedthe initial value for (to )mimic ST and the ( added) SV. After mass reinforcement,effect with ma- the terialsRWM during inertance reinforcement. is set to twice RWM the initial compliance value to after mimic reinforcement the added mass was set effect to an with initial materials compli- during ancereinforcement. (C0) of 0.2 and RWM 0.01. compliance after reinforcement was set to an initial compliance (C0) of 0.2 and 0.01.

4.4. Effect of RWM Reinforcement on the Pressure Difference across the Partition

The pressure difference PDIFF across the basal part of the cochlear partition triggers the propagation of the traveling wave on the basilar membrane and, hence, is an im- portant measure to assess the influence of RWM reinforcement on the hearing sensation. The PDIFF of each HTB sample was calculated in the complex domain as the difference in pressure in the SV and ST normalized with the ear canal pressure PEC. To illustrate the Appl. Sci. 2021, 11, 5062 16 of 19

4.4. Effect of RWM Reinforcement on the Pressure Difference across the Partition

The pressure difference PDIFF across the basal part of the cochlear partition triggers the propagation of the traveling wave on the basilar membrane and, hence, is an important measure to assess the influence of RWM reinforcement on the hearing sensation. The PDIFF Appl. Sci. 2021, 11, x FOR PEER REVIEW 17 of 20 of each HTB sample was calculated in the complex domain as the difference in pressure in the SV and ST normalized with the ear canal pressure PEC. To illustrate the RWM reinforcement-induced change in PDIFF, the ratio between PDIFF after and before RWM reinforcementRWM reinforcement-induced was calculated change and depicted in PDIFF, in the dB. ratio Figure between 11A showsPDIFF after the correspondingand before ratioRWM for reinforcement cartilage and was Figure calculated 11B shows and depic it forted soft in dB. tissue Figure reinforcement. 11A shows the Only corre- data at frequenciessponding ratio below for 2.2 cartilage kHz are and considered Figure 11B where shows changes it for soft in ISCP tissue can reinforcement. be attributed Only to RWM manipulations.data at frequencies For below certain 2.2 HTBkHz are samples, consider reinforcemented where changes materials, in ISCP andcan be frequencies, attributed the pressureto RWM inmanipulations. SV and ST showed For certain almost HTB the samp sameles, magnitude reinforcement or anmaterials, even higher and frequen- pressure in STcies, than the in pressure SV (cf. Figurein SV 5and). Such ST showed pressure almo conditionsst the same are notmagnitude physical or and an areeven attributed higher to thepressure measurement in ST than uncertainty in SV (cf. Figure of the 5). sensor Such systempressure and conditions other interfering are not physical effects. and However, are theseattributed invalid to the data measurement were removed uncertainty and not of considered the sensor forsystem further and analysis other interfering (certain curvesef- fects. However, these invalid data were removed and not considered for further analysis not continuous). Except for HTB-19, where the PDIFF was reduced by up to 5 dB using (certain curves not continuous). Except for HTB-19, where the PDIFF was reduced by up to cartilage and up to 10 dB using soft tissue, the change in PDIFF was small or even negligible. 5 dB using cartilage and up to 10 dB using soft tissue, the change in PDIFF was small or The reason for this is that the PDIFF is mainly pretended by the vestibuli pressure, which is significantlyeven negligible. larger The than reason the for pressure this is inthat ST. the Despite PDIFF is themainly clear pretended increase inby ST the pressure vestibuli after pressure, which is significantly larger than the pressure in ST. Despite the clear increase reinforcement that is obvious throughout the samples, it has a mostly negligible effect on in ST pressure after reinforcement that is obvious throughout the samples, it has a mostly PDIFF or is compensated by an increased vestibuli pressure. negligible effect on PDIFF or is compensated by an increased vestibuli pressure.

FigureFigure 11.11. RatioRatio betweenbetween thethe pressurepressure differencedifference acrossacross thethe cochleacochlea partitionpartition normalizednormalized to the ear canal canal pressurePSV −P (ST ) after and prior RWM reinforcement with cartilage (A,C) and soft tissue pressure ( ) after and prior RWM reinforcement with cartilage (A,C) and soft tissue (B,D). In PEC (B,D). In (A,B), PDIFF was calculated in the complex space considering the phase shift between PSV (A,B), PDIFF was calculated in the complex space considering the phase shift between PSV and PST. In and PST. In (C,D), zero phase shift was assumed for the PDIFF calculation. Invalid data (discontinu- (C,D), zero phase shift was assumed for the P calculation. Invalid data (discontinuous curves) arise ous curves) arise due to the uncertainties in theDIFF sensor measurements, and other interfering effects duewere to neglected. the uncertainties in the sensor measurements, and other interfering effects were neglected.

Another reason for this that might explain the small effect of RWM reinforcement on the PDIFF is the phase shift between the pressure in SV and ST, which deviates significantly from zero for certain HTB samples (cf. Figure 4 C,D) and, hence, reduces the effective PDIFF. An existing phase shift between the two pressure readings at low frequencies contradicts Appl. Sci. 2021, 11, 5062 17 of 19

Another reason for this that might explain the small effect of RWM reinforcement on the PDIFF is the phase shift between the pressure in SV and ST, which deviates significantly from zero for certain HTB samples (cf. Figure4C,D) and, hence, reduces the effective PDIFF. An existing phase shift between the two pressure readings at low frequencies contradicts the theory of a fast wave propagating down the perilymph-filled cochlea duct [28]. Considering an approximately 70 mm-long channel between the oval and round window [29] and a propagation speed similar to the speed of sound in water, a phase shift of less than 18◦ at 1 kHz should arise between the pressure in the basal part of the SV and ST. Moreover, the phase shift of 180◦ that was measured in the present study between the motion of the oval and round window at low frequencies (cf. Figure3B) stipulates a vanishing phase shift in the simultaneous ICSP readings at low frequencies. Thus far, it is not clear why for certain HTBs the phase shift deviates from the expected range. We do not assume a measurement (system)-related problem, mainly because a similar phase shift in the pressure readings can also be observed in the study of Nakajima et al. (2009), where another pressure measurement system was used. We believe, rather, that the hydrophone signals might be distorted by mechanical vibrations of the sample, which are triggered by the acoustic stimulation and inducing pressure fluctuations in the cochlear fluid. However, Figure 11C,D illustrate the effect of RWM reinforcement on the PDIFF if no phase shift in the pressure readings is assumed. In this case, HTB-10, HTB-12, and HTB-19 show an RWM reinforcement-induced change in the PDIFF of up to 20 dB for cartilage and even more than 20 dB with soft tissue as the reinforcement material. The other samples still show a negligible influence of RWM reinforcement on PDIFF that can be attributed to a less effective reinforcement due to the difficultly in handling the material. A change in PDIFF of 20 dB or more might be an indication that the clinically observed loss in residual hearing at low frequencies after CI implantation through the RWM might be attributed to an RWM that is stiffened by the CI electrode.

5. Conclusions In the present study, we performed simultaneous ICSP measurements on HTBs to investigate the influence of RWM reinforcement on the sound pressure in the basal part of the ST and SV. The resulting pressure difference induces the traveling wave on the basilar membrane and, hence, is regarded as an important measure for hearing sensation. The experiments revealed RWM reinforcement-induced changes in ICSP within the low-frequency range (<2.2 kHz), with the largest effect in ST below 1 kHz. In ST, a sound pressure increase of up to 25 dB was observed after RWM reinforcement. In contrast to the literature, we could identify a considerably smaller pressure rise in SV (<10 dB). The effect of the RWM reinforcement on ICSP varied largely between the individual HTB samples. The poor repeatability was mainly attributed to the difficult handling of the reinforcement material during RWM reinforcement, together with the lack of a direct measurement of the RWM impedance after reinforcement. Suggestions for a more appropriate reinforcement material that could considerably improve the repeatability of the experiment were made. The question raised in the literature—whether an artificially or pathologically reinforced RWM may cause conductive hearing loss at low frequencies—cannot be conclusively answered by this study. However, some data indicate a drop in the pressure difference across the partition of almost 10 dB. If the phase shift between the pressure readings in SV and ST is neglected, the resulting PDIFF can even drop by more than 20 dB after RWM reinforcement. This is in the range of the clinically observed low-frequency hearing loss that can occur after CI surgeries. To provide better evidence in this context, the methodology of RWM reinforcement and ICSP measurement would require further improvement to increase the reinforcement effectiveness and the repeatability of the experiment. Appl. Sci. 2021, 11, 5062 18 of 19

Author Contributions: Conceptualization, F.P., L.P., N.L., and I.D.; methodology, N.L., L.P., A.D., M.C., and F.P.; software, L.P. and N.L.; formal analysis, N.L., J.G., and L.P.; resources, A.H., T.K., and F.P.; data curation, N.L., J.G., and S.T.; writing—original draft preparation, N.L.; writing—review Appl. Sci. 2021, 11, x FOR PEER REVIEWand editing, L.P., F.P., S.T., A.D., M.C., and I.D.; visualization, N.L.; supervision, F.P., A.H., and19 of T.K.; 20

funding acquisition, T.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the European Union’s Horizon 2020 research and innovation programFunding: under This research the Marie was Sklodowska-Curie funded by the European grant agreement Union’s Horizon number 2020 722046. research and innovation program under the Marie Sklodowska-Curie grant agreement number 722046. Institutional Review Board Statement: The study was conducted according to the guidelines of the DeclarationInstitutional of Review Helsinki Board and approved Statement: by The the study local ethicswas conducted committee, according Canton to of the Zurich, guidelines Switzerland of the (KEK-ZH-Nr.Declaration of 2014-0544, Helsinki and approved approved on 25by Februarythe local ethics 2015). committee, Canton of Zurich, Switzerland (KEK-ZH-Nr. 2014-0544, approved on 25 February 2015). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The authors would like to thank Jae Hoon Sim for his contributions in the modelAcknowledgments: simulation validation The authors and resultwould interpretation like to thank andJae valuableHoon Sim insights for hisinto contributions the experimental in the model simulation validation and result interpretation and valuable insights into the experimental results, as well as all the members of the “Otology and Biomechanics of Hearing” research group for results, as well as all the members of the “Otology and Biomechanics of Hearing” research group their support. for their support. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or design of the study; in the collection, analysis, or interpretation of data; in the writing of the manu- in the decision to publish the results. script; or in the decision to publish the results.

AppendixAppendix A. A. StapesStapes VelocityVelocity MeasurementsMeasurements withwith RWMRWM ReinforcementReinforcement The effect of RWM reinforcement on the METF (V normalized to P ) was deter- The effect of RWM reinforcement on the METF (VSTAPSTAP normalized to PECEC) was deter- minedmined in in three three HTBs.HTBs. ItIt isis plottedplotted inin FigureFigure A1 as the difference between the the METF METF in in dB dB afterafter and and before before reinforcement.reinforcement. BesideBeside the data from the the individual individual HTBs, HTBs, the the correspond- correspond- inging median median andand thethe referencereference datadata fromfrom Wegner et al. (2016) (2016) are are also also added added to to the the plots. plots. TheThe left left figure figure represents represents thethe casecase withwith softsoft tissuetissue and the right one with cartilage as as the the reinforcementreinforcement material. material.

Figure A1. Effect of RWM reinforcement on the METF expressed as the difference between the Figure A1. Effect of RWM reinforcement on the METF expressed as the difference between the METF in dB before and after the reinforcement with (A) soft tissue and (B) cartilage. The thick METF in dB before and after the reinforcement with (A) soft tissue and (B) cartilage. The thick dashed-dotted lines represent the corresponding median values. The solid lines with circular dashed-dottedmarkers indicate lines the represent reference the data corresponding extracted from median Wegner values. et al. The for solidperichondrium lines with circular(n = 9) and markers indicatecartilage the (n = reference 4), respectively. data extracted from Wegner et al. for perichondrium (n = 9) and cartilage (n = 4), respectively. Within the low-frequency range (<2 kHz), where piston-like motion pretends the vi- brationWithin behavior the low-frequency of the stapes rangefootplate, (<2 a kHz), RWM where reinforcement-induced piston-like motion change pretends in the the vibrationMETF of behaviorless than 1 of dB the was stapes identified. footplate, This aco RWMnfirms reinforcement-induced the work of Wegner et al. change (2016). in and the METFGuan ofet lessal. (2018) than 1claiming dB was identified.a negligible This effect confirms on the the METF work due of Wegnerto RWM et reinforcement al. (2016) and Guan[5,13]. et al. (2018) claiming a negligible effect on the METF due to RWM reinforcement [5,13].

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