The importance of mechanical and biological cues of tympanic membrane grafts to ensure optimal regeneration.

Chronic suppurative otitis media (CSOM) is often associated with permanent tympanic membrane (TM) perforation and conductive hearing loss. The current clinical gold standard, using autografts and allografts, suffers from several drawbacks. Artificial replacement materials can help to overcome these drawbacks. Therefore, scaffolds fabricated through digital light processing (DLP) were herein created to support TM regeneration. Various UV-curable printing inks, including gelatin methacryloyl (GelMA), gelatin-norbornene-norbornene (GelNBNB) (crosslinked with thiolated gelatin (GelSH)) and alkene-functionalized poly-ε-caprolactone (E-PCL) (crosslinked with pentaerythritol tetrakis(3-mercaptopropionate) (PETA4SH)) were optimized regarding photo-initiator (PI) and photo-absorber (PA) concentrations through viscosity characterization, photo-rheology and the establishment of working curves for DLP. Our material platform enabled the development of constructs with a range of mechanical properties (plateau storage modulus varying between 15 and 119 kPa). Excellent network connectivity for the GelNBNB and E-PCL constructs was demonstrated (gel fractions >95 %) whereas a post-crosslinking step was required for the GelMA constructs. All samples showed excellent biocompatibility (viability >93 % and metabolic activity >88 %). Finally, in vivo and ex vivo assessments, including histology, vibration and deformation responses measured through laser doppler vibrometry and digital image correlation respectively, were performed to investigate the effects of the scaffolds on the anatomical and physiological regeneration of acute TM perforations in rabbits. The data showed that the most efficient healing with the best functional quality was obtained when both mechanical (obtained with the PCL-based resin) and biological (obtained with the gelatin-based resins) material properties were taken into account.

Prestrain in the eardrum investigated using laser-ablation perforation: A proof of principle study on the New Zealand white rabbit.

While the presence of residual stress (also called prestress) in the tympanic membrane (TM) was hypothesized more than 150 years ago by von Helmholtz (1869), little experimental data exists to date. In this paper, a novel approach to study residual stress is presented. Using a pulsed laser, the New Zealand white rabbit TM is perforated at seven predefined locations. The subsequent retraction of the membrane around the holes is computed using digital image correlation (DIC). The amount of retraction is the so-called prestrain, which is caused by the release of prestress due to the perforation. By measuring the prestrain using DIC, we show that residual stress is clearly present over the entire rabbit TM surface. In total, fourteen TMs have been measured in this work. An automated approach allows tracking the holes' deformation during the measurement process and enables a more robust analysis than was previously possible. We find similar strains (around 5%) as reported in previous work, in which slits were created manually using flattened surgical needles. However, the new approach greatly reduces measurement time, which minimizes dehydration artifacts. To investigate the effect of perforation location on the TM, the spatial decrease of the prestrain (α) around the perforation was quantified. Perforations inferior to the umbo showed the least negative α values, i.e., the most gradual decrease around the hole, and were the most consistent. Perforations on other locations showed more negative α values, i.e., steeper decrease in strain, but were less consistent across samples. We also investigated the effect of the holes' creation sequence but did not observe a significant change in the results. Overall, the presented method allows for consistent residual stress measurements over the TM surface. The findings contribute to our fundamental knowledge of the mechanics of the rabbit TM and provide a basis for future work on human TMs.

Rabbit tympanic membrane thickness distribution obtained via optical coherence tomography.

Knowing the precise tympanic membrane (TM) thickness variation is crucial in understanding the functional properties of the TM and has a significant effect on the accuracy of computational models. Using optical coherence tomography, we imaged five left and five right TMs of domestic New Zealand rabbits. From these data, ten thickness distribution maps were computed. Although inter-specimen variability is present, similar features could be observed in all samples: The rabbit TM is thickest around the umbo, with values of 150 ± 32 µm. From the umbo towards the TM annulus, the thickness gradually decreases down to 38 ± 7 µm around the midway location, but increases up to 54 ± 19 µm at the TM annulus. The thickness values at the umbo are comparable to literature data for humans, but the rabbit TM is thinner at the TM annulus and in-between the umbo and annulus. Moreover, the rabbit TM thickness distribution is highly symmetrical, which is not the case for the human TM. The results improve our general understanding of TM structure in rabbits and may improve numerical models of TM dynamical behavior.

Material Identification on Thin Shells Using the Virtual Fields Method, Demonstrated on the Human Eardrum.

Characterization of material parameters from experimental data remains challenging, especially on biological structures. One of such techniques allowing for the inverse determination of material parameters from measurement data is the virtual fields method (VFM). However, application of the VFM on general structures of complicated shape has not yet been extensively investigated. In this paper, we extend the framework of the VFM method to thin curved solids in three-dimensional, commonly denoted shells. Our method is then used to estimate the Young's modulus and hysteretic damping of the human eardrum. By utilizing Kirchhoff plate theory, we assume that the behavior of the shell varies linearly through the thickness. The total strain of the shell can then be separated in a bending and membrane strain. This in turn allowed for an application of the VFM based only on data of the outer surface of the shell. We validated our method on simulated and experimental data of a human eardrum made to vibrate at certain frequencies. It was shown that the identified material properties were accurately determined based only on data from the outer surface and are in agreement with literature. Additionally, we observed that neither the bending nor the membrane strain in an human eardrum can be neglected and both contribute significantly to the total strain found experimentally.

Prestrain in the rabbit eardrum measured by digital image correlation and micro-incisions.

Prestrain in the absence of external loads can have an important effect on the vibrational behavior of mechanical systems such as the middle ear. Studies that measure tympanic membrane (TM) prestrain are scarce, however, and provide no conclusive answer on the existence and nature of the prestrain. In this study, prestrain is measured in the TM of cadaveric rabbit ears by stereo digital image correlation. To release the prestrain, straight incisions of 0.33 mm are made on different locations in the TM with a direction parallel to either the radial or circular fibers in the membrane. The effect of sample dehydration during different stages in the experimental procedure is assessed and eliminated by rehydrating the samples directly before each measurement. The measurements demonstrate average prestrain values around the incisions between 3.52±2.34% and 13.62±7.92% for the different locations, with a noise floor of 0.07%. No clear differences were found between the prestrain values obtained for radial and circular incisions. Observed local variations in TM prestrain could not be clearly related to specific locations on the TM. The results suggest that TM prestrain may need to be considered in future studies of middle-ear function if the findings can be confirmed in human ears.

Eardrum displacement and strain in the Tokay gecko (Gekko gecko) under quasi-static pressure loads.

The eardrum is the primary component of the middle ear and has been extensively investigated in humans. Measuring the displacement and deformation of the eardrum under different quasi-static loading conditions gives insight in its mechanical behavior and is fundamental in determining the material properties of the eardrum. Currently, little is known about the behavior and material properties of eardrums in non-mammals. To explore the mechanical properties of the eardrum in non-mammalian ears, we investigated the quasi-static response of the eardrum of a common lizard: the Tokay gecko (Gekko gecko). The middle ear cavity was pressurized using repetitive linear pressure cycles ranging from -1.5 to 1.5 kPa with pressure change rates of 0.05, 0.1 and 0.2 kPa/s. The resulting shape, displacement and in-plane strain of the eardrum surface were measured using 3D digital image correlation. When middle-ear pressure is negative, the medial displacement of the eardrum is much larger than the displacement observed in mammals; when middle-ear pressure is positive, the lateral displacement is much larger than in mammals, which is not observed in bird single-ossicle ears. Peak-to-peak displacements are about 2.8 mm, which is larger than in any other species measured up to date. The peak-to-peak displacements are at least five times larger than observed in mammals. The pressure-displacement curves show hysteresis, and the energy loss within one pressure cycle increases with increasing pressure rate, contrary to what is observed in rabbit eardrums. The energy lost during a pressure cycle is not constant over the eardrum. Most energy is lost at the region where the eardrum connects to the hearing ossicle. Around this eardrum-ossicle region, a 5% increase in energy loss was observed when pressure change rate was increased from 0.05 kPa/s to 0.2 kPa/s. Other parts of the eardrum showed little increase in the energy loss. The orientation of the in-plane strain on the eardrum was mainly circumferential with strain amplitudes of about +1.5%. The periphery of the measured eardrum surface showed compression instead of stretching and had a different strain orientation. The TM of Gekko gecko shows the highest displacements of all species measured up till now. Our data show the first shape, displacement and deformation measurements on the surface of the eardrum of the gecko and indicate that there could exist a different hysteresis behavior in different species.

Sound localization in the lizard using internally coupled ears: A finite-element approach.

A number of interesting differences become apparent when comparing the hearing systems of terrestrial vertebrates, especially between mammals and non-mammals. Almost all non-mammals possess only a single ossicle, enabling impedance matching and hearing below 10 kHz. The middle ear (ME) evolved as a chain of three ossicles in mammals, enabling sound transmission up to higher frequencies than in similar-sized non-mammals. The relatively low-frequency hearing in non-mammals is associated with audible wavelengths that are significantly larger than the head. Therefore, it is unlikely that localization of the sound source can be obtained by using external cues between the ears (intensity and time difference between both sides), especially when compared to similarly sized mammals. The heads of many non-mammals contain large air-filled cavities, which acoustically couple both MEs. This article studies acoustic responses and sound-source localization capabilities of the coupled MEs of the brown anole (Anolis sagrei), using finite-element modeling. Based on high-resolution µCT data, 3D finite-element models of the ME and interaural cavity were constructed. The parameter values in the ME model were determined such that the response of the isolated ME matches experimental data of literature and the velocity ratio between the tympanic membrane (apex) and footplate reflects the anatomical arrangement of the columellar lever in the anole. It was found from simulation of the coupled ME model that the interaural connection amplifies intensity differences between both sides and thus enhances the capability of sound-source localization. In addition, the interaural canal doubles the phase differences of the incident external sound waves between the eardrums. In isolated ears, generating such phase differences would require head sizes twice as large. Effects of the inner-ear loading on the sound-source localization of the coupled MEs were investigated as well. The inner-ear load lowered the peak velocity ratios between the ears, but created broader plateaus of useful directionality, indicating that inner-ear loading not only influences sound perception but also sound localization in internally connected ears.