Accessing the inner ear presents a significant challenge for the diagnosis and treatment of inner ear diseases. Many existing techniques to access the inner ear are invasive and can cause permanent damage to the cochlea. Recently, a novel microneedle has been fabricated to perforate the round window membrane (RWM) – a membrane sealing one of the two openings in the cochlea. These perforations enhance drug delivery into the inner ear, potentially improving the efficacy of therapeutics. Furthermore, they allow for the aspiration of perilymph samples, which is essential for diagnosing inner ear diseases. However, owing to limited knowledge about the mechanical properties of the RWM, certain technical aspects remain unexplored. Specifically, the interaction between the RWM and the microneedle during perforation is yet to be examined. This investigation is pivotal for the optimal design of microneedles — those robust enough to perforate RWMs yet delicate enough to minimize damage. In this thesis, we conduct a thorough examination of the guinea pig RWM, encompassing its geometry and its mechanical responses to pressures from the middle ear and inner ear. Additionally, we also formulate a comprehensive constitutive law for the guinea pig RWM. Our exploration begins with the creation of a U-Net model tailored to automatically segment the RWM. Despite the presence of other structures in the same image—such as bone, the basilar membrane, and ambient noise—the model proved invaluable for efficiently and automatically segmenting the RWM. To enhance accuracy, post-processing techniques like connected component analysis and majority voting were incorporated. Using this 3D model, we proceeded to study the RWM’s geometry. Recognizing the shrinkage observed in fixed RWMs, we integrated fresh RWM data to estimate the shrinkage ratio. Subsequently, we analyzed both the overall RWM thickness and that of the middle connective tissue layer—crucial metrics for future RWM modeling. Next, we proposed a method to evaluate the in-plane deformation of the RWM due to applied pressure. This involved using a bulge test system to pressurize and deform the RWM, combined with confocal microscopy to track stained nuclei or pre-introduced fluorescent beads on the RWM. We then utilized the coherent point drift (CPD) algorithm to measure the displacement of beads and nuclei. Results indicated that both markers could be successfully used to measure the RWM’s displacement. Further analysis revealed the in-plane Lagrangian strain of the RWM, with a significant observation being that the direction of maximum in-plane Lagrangian strain is perpendicular to the fiber direction. This underscores the crucial role of collagen fibers in determining the RWM’s mechanical properties. To conclude our study, we devised a constitutive law for the RWM, conceptualizing it as a combination of the ground substance and a family of dispersed fibers. This model was integrated into a FEBioStudio plugin, facilitating simulations of the RWM’s mechanical reactions to different pressures. Although our simulations closely aligned with experimental findings, some discrepancies were noted, likely stemming from an incomplete understanding of fiber dispersions. Nevertheless, our constitutive law reinforces the notion that fibers primarily govern the RWM’s mechanical characteristics.

Indiana University-Purdue University Indianapolis (IUPUI) / The inner ear contains specialized sensory epithelia that detect head movements, gravity and sound. Hearing loss and imbalance are primarily caused by degeneration of the mechanosensitive hair cells in sensory epithelia or the sensory neurons that connect the inner ear to the brain. The controlled derivation of inner ear sensory epithelia and neurons from pluripotent stem cells will be essential for generating in vitro models of inner ear disorders or developing cell-based therapies. Despite some recent success in deriving hair cells from mouse embryonic stem (ES) cells, it is currently unclear how to derive inner ear sensory cells in a fully defined and reproducible manner. Progress has likely been hindered by what is known about induction of the nonneural and preplacodal ectoderm, two critical precursors during inner ear development. The studies presented here report the step-wise differentiation of inner ear sensory epithelia from mouse ES cells in three-dimensional culture. We show that nonneural, preplacodal and pre-otic epithelia can be generated from ES cell aggregates by precise temporal control of BMP, TGFβ and FGF signaling, mimicking in vivo development. Later, in a self-guided process, vesicles containing supporting cells emerge from the presumptive otic epithelium and give rise to hair cells with stereocilia bundles and kinocilium. Remarkably, the vesicles developed into large cysts with sensory epithelia reminiscent of vestibular sense organs (i.e. the utricle, saccule and crista), which sense head movements and gravity in the animal. We have designated these stem cell-derived structures inner ear organoids. In addition, we discovered that sensory-like neurons develop alongside the organoids and form putative synapses with hair cells in a similar fashion to the hair cell-to-neuron circuit that forms in the developing embryo. Our data thus establish a novel in vitro model of inner ear organogenesis that can be used to gain deeper insight into inner ear development and disorder.