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 m. 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.

Hypothesis: Microneedles can create microperforations in the round window membrane (RWM) without causing anatomic or physiologic damage.Background: Reliable delivery of agents into the inner ear for therapeutic and diagnostic purposes remains a challenge. Our novel approach employs microneedles to facilitate intracochlear access via the RWM. This study investigates the anatomical and functional consequences of microneedle perforations in guinea pig RWMs in vivo.Methods: Single 3D-printed, 100 μm diameter microneedles were used to perforate the guinea pig RWM via the postauricular sulcus. Hearing was assessed both before and after microneedle perforation using compound action potential and distortion product otoacoustic emissions. Confocal microscopy was used ex vivo to examine harvested RWMs, measuring the size, shape, and location of perforations and documenting healing at 0 hours (n = 7), 24 hours (n = 6), 48 hours (n = 6), and 1 week (n = 6).Results: Microneedles create precise and accurate perforations measuring 93.1 ± 29.0 μm by 34.5 ± 16.8 μm and produce a high frequency threshold shift that disappears after 24 hours. Examination of perforations over time demonstrates healing progression over 24-48 hours and complete perforation closure by one week. Conclusion:Microneedles can create a temporary microperforation in the RWM without causing significant anatomic or physiologic dysfunction. Microneedles have the potential to mediate safe and effective intracochlear access for diagnosis and treatment of inner ear disease.

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