In-vitro perforation of the round window membrane via direct 3-D printed microneedles.

The cochlea, or inner ear, is a space fully enclosed within the temporal bone of the skull, except for two membrane-covered portals connecting it to the middle ear space. One of these portals is the round window, which is covered by the Round Window Membrane (RWM). A longstanding clinical goal is to reliably and precisely deliver therapeutics into the cochlea to treat a plethora of auditory and vestibular disorders. Standard of care for several difficult-to-treat diseases calls for injection of a therapeutic substance through the tympanic membrane into the middle ear space, after which a portion of the substance diffuses across the RWM into the cochlea. The efficacy of this technique is limited by an inconsistent rate of molecular transport across the RWM. A solution to this problem involves the introduction of one or more microscopic perforations through the RWM to enhance the rate and reliability of diffusive transport. This paper reports the use of direct 3D printing via Two-Photon Polymerization (2PP) lithography to fabricate ultra-sharp polymer microneedles specifically designed to perforate the RWM. The microneedle has tip radius of 500 nm and shank radius of 50 µ m, and perforates the guinea pig RWM with a mean force of 1.19 mN. The resulting perforations performed in vitro are lens-shaped with major axis equal to the microneedle shank diameter and minor axis about 25% of the major axis, with mean area 1670 µ m2. The major axis is aligned with the direction of the connective fibers within the RWM. The fibers were separated along their axes without ripping or tearing of the RWM suggesting the main failure mechanism to be fiber-to-fiber decohesion. The small perforation area along with fiber-to-fiber decohesion are promising indicators that the perforations would heal readily following in vivo experiments. These results establish a foundation for the use of Two-Photon Polymerization lithography as a means to fabricate microneedles to perforate the RWM and other similar membranes.

A dual wedge microneedle for sampling of perilymph solution via round window membrane.

Precision medicine for inner-ear disease is hampered by the absence of a methodology to sample inner-ear fluid atraumatically. The round window membrane (RWM) is an attractive portal for accessing cochlear fluids as it heals spontaneously. In this study, we report on the development of a microneedle for perilymph sampling that minimizes the size of RWM perforation, facilitates quick aspiration, and provides precise volume control. Here, considering the mechanical anisotropy of the RWM and hydrodynamics through a microneedle, a 31G stainless steel pipe was machined into wedge-shaped design via electrical discharge machining. The sharpness of the needle was evaluated via a surface profilometer. Guinea pig RWM was penetrated in vitro, and 1 µL of perilymph was sampled and analyzed via UV-vis spectroscopy. The prototype wedge shaped needle was successfully fabricated with the tip curvature of 4.5 µm and the surface roughness of 3.66 µm in root mean square. The needle created oval perforation with minor and major diameter of 143 and 344 µm (n = 6). The sampling duration and standard deviation of aspirated volume were 3 s and 6.8 % respectively. The protein concentration was 1.74 mg/mL. The prototype needle facilitated precise perforation of RWMs and rapid aspiration of cochlear fluid with precise volume control. The needle design is promising and requires testing in human cadaveric temporal bone and further optimization to become clinically viable.

Silver/silver chloride microneedles can detect penetration through the round window membrane.

HYPOTHESIS: Silver-plated microneedles can be used to confirm penetration of semi-permeable membranes such as the round window membrane (RWM) by detection of voltage change at the moment of perforation. BACKGROUND: The introduction of microperforations in the RWM can significantly enhance intracochlear delivery of therapeutics. However, the moment of needle penetration through the RWM cannot be reliably detected by visualization or sensation alone. We explore the ability of electrochemical detection of penetration in defining the precise instant a microneedle enters the inner ear. METHODS: 0.2 mm diameter stainless steel Minutien pins were electroplated with copper, then silver. Pins were then soaked in bleach for 24 h to complete Ag/AgCl plating. Experiments were performed using a 3 mL Franz cell diffusion system with 1%, 2%, 3%, 4%, and 5% saline solution in the donor chamber and artificial perilymph solution in the receptor chamber separated by 5-µm pore synthetic membrane. Continuous voltage measurements were made throughout the process of membrane penetration by the microneedle (N = 6 for each saline concentration). RESULTS: Silver-plated needles were able to detect an instantaneous change in voltage when traversing the membrane from saline solution into artificial perilymph. As calculated, the magnitude of the change in voltage upon penetration increased with increasing saline concentration and was stable across trials. CONCLUSION: Ag/AgCl coated microneedles are effective in detecting the moment of penetration across semi-permeable membranes. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 307-311, 2017.

Human cochlear hydrodynamics: A high-resolution µCT-based finite element study.

Measurements of perilymph hydrodynamics in the human cochlea are scarce, being mostly limited to the fluid pressure at the basal or apical turn of the scalae vestibuli and tympani. Indeed, measurements of fluid pressure or volumetric flow rate have only been reported in animal models. In this study we imaged the human ear at 6.7 and 3-µm resolution using µCT scanning to produce highly accurate 3D models of the entire ear and particularly the cochlea scalae. We used a contrast agent to better distinguish soft from hard tissues, including the auditory canal, tympanic membrane, malleus, incus, stapes, ligaments, oval and round window, scalae vestibule and tympani. Using a Computational Fluid Dynamics (CFD) approach and this anatomically correct 3D model of the human cochlea, we examined the pressure and perilymph flow velocity as a function of location, time and frequency within the auditory range. Perimeter, surface, hydraulic diameter, Womersley and Reynolds numbers were computed every 45° of rotation around the central axis of the cochlear spiral. CFD results showed both spatial and temporal pressure gradients along the cochlea. Small Reynolds number and large Womersley values indicate that the perilymph fluid flow at auditory frequencies is laminar and its velocity profile is plug-like. The pressure was found 102-106° out of phase with the fluid flow velocity at the scalae vestibule and tympani, respectively. The average flow velocity was found in the sub-µm/s to nm/s range at 20-100Hz, and below the nm/s range at 1-20kHz.

Serrated needle design facilitates precise round window membrane perforation.

The round window membrane (RWM) has become the preferred route, over cochleostomy, for the introduction of cochlear implant electrodes as it minimizes inner ear trauma. However, in the absence of a tool designed for creating precise perforation, current practices lead to tearing of the RWM and significant intracochlear pressure fluctuations. On the basis of RWM mechanical properties, we have designed a multi-serrated needle to create consistent holes without membrane tearing or damaging inner ear structures. Four and eight-serrated needles were designed and produced with wire electrical discharge machining (EDM). The needle's ability to create RWM perforations was tested in deidentified, commercially acquired temporal bones with the assistance of a micromanipulator. Subsequently, specimens were imaged under light and scanning electron microscopy (SEM). The needles created consistent, appropriately sized holes in the membrane with minimal tearing. While a four-serrated crown needle made rectangular/trapezoid perforations, the octagonal crown formed smooth oval holes within the membrane. Though designed for single use, the needle tolerated repeated use without significant damage. The serrated needles formed precise perforations in the RWM while minimizing damage during cochlear implantation. The octagonal needle design created the preferred oval perforation better than the quad needle. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1633-1637, 2016.

Microperforations significantly enhance diffusion across round window membrane.

HYPOTHESIS: Introduction of microperforations in round window membrane (RWM) will allow reliable and predictable intracochlear delivery of pharmaceutical, molecular, or cellular therapeutic agents. BACKGROUND: Reliable delivery of medications into the inner ear remains a formidable challenge. The RWM is an attractive target for intracochlear delivery. However, simple diffusion across intact RWM is limited by what material can be delivered, size of material to be delivered, difficulty with precise dosing, timing, and precision of delivery over time. Further, absence of reliable methods for measuring diffusion across RWM in vitro is a significant experimental impediment. METHODS: A novel model for measuring diffusion across guinea pig RWM, with and without microperforation, was developed and tested: cochleae, sparing the RWM, were embedded in 3D-printed acrylic holders using hybrid dental composite and light cured to adapt the round window niche to 3 ml Franz diffusion cells. Perforations were created with 12.5-µm-diameter needles and examined with light microscopy. Diffusion of 1 mM Rhodamine B across RWM in static diffusion cells was measured via fluorescence microscopy. RESULTS: The diffusion cell apparatus provided reliable and replicable measurements of diffusion across RWM. The permeability of Rhodamine B across intact RWM was 5.1 × 10(9-) m/s. Manual application of microperforation with a 12.5-µm-diameter tip produced an elliptical tear removing 0.22 ± 0.07% of the membrane and was associated with a 35× enhancement in diffusion (P < 0.05). CONCLUSION: Diffusion cells can be applied to the study of RWM permeability in vitro. Microperforation in RWM is an effective means of increasing diffusion across the RWM.

Microanatomic analysis of the round window membrane by white light interferometry and microcomputed tomography for mechanical amplification.

OBJECTIVE: The round window membrane (RWM) is increasingly becoming a target for amplification using active middle ear implants. However, the current strategy of using available transducer tips may have negative consequences for the RWM. We investigated the microanatomy of the RWM to establish a basis for the design of the transducer tip for the RWM driver. STUDY DESIGN: Using the guinea pig as an animal model, microcomputed tomography (µCT) and white light interferometry were used to study the topography of the RWM and RW niche (RWN). The curvatures of the RWM surface were calculated using the topography data. MAIN OUTCOME MEASURES: The 3-dimensional structure of the scala tympani terminal, saddle-shaped surface topography, and surface curvature were determined. RESULTS: The size of the scala terminal was approximated as an ellipse for which the major and minor axes were 1.29 and 0.95 mm. The average minimum and maximum radii of curvature around the center of RWM were -0.44 and +0.70 mm along the minor and major axis. CONCLUSION: The microanatomies of the RWM and RWN have important implications for the design of the transducer tip to maximize energy transfer while minimizing its distortion and permanent disruption. Our results suggest that the size of the transducer tip should be smaller than the minor axis of the scala terminal to avoid collision with the RWN. The driver should be designed to conform to the topography and radius of curvature of the center portion of the RWM, which for a guinea pig is 0.44 mm.

Intravascular Neural Interface with Nanowire Electrode.

A minimally invasive electrical recording and stimulating technique capable of simultaneously monitoring the activity of a significant number (e.g., 10(3) to 10(4)) of neurons is an absolute prerequisite in developing an effective brain-machine interface. Although there are many excellent methodologies for recording single or multiple neurons, there has been no methodology for accessing large numbers of cells in a behaving experimental animal or human individual. Brain vascular parenchyma is a promising candidate for addressing this problem. It has been proposed [1, 2] that a multitude of nanowire electrodes introduced into the central nervous system through the vascular system to address any brain area may be a possible solution. In this study we implement a design for such microcatheter for ex vivo experiments. Using Wollaston platinum wire, we design a submicron-scale electrode and develop a fabrication method. We then evaluate the mechanical properties of the electrode in a flow when passing through the intricacies of the capillary bed in ex vivo Xenopus laevis experiments. Furthermore, we demonstrate the feasibility of intravascular recording in the spinal cord of Xenopus laevis.