Adeno-Associated Virus-Encapsulated Alginate Microspheres Loaded in Collagen Gel Carriers for Localized Gene Transfer.

This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.

Mechanical effect of reconstructed shapes of autologous ossicles on middle ear acoustic transmission.

Conductive hearing loss is caused by a variety of defects, such as chronic otitis media, osteosclerosis, and malformation of the ossicles. In such cases, the defective bones of the middle ear are often surgically reconstructed using artificial ossicles to increase the hearing ability. However, in some cases, the surgical procedure does not result in increased hearing, especially in a difficult case, for example, when only the footplate of the stapes remains and all of the other bones are destroyed. Herein, the appropriate shapes of the reconstructed autologous ossicles, which are suitable for various types of middle-ear defects, can be determined by adopting an updating calculation based on a method that combines numerical prediction of the vibroacoustic transmission and optimization. In this study, the vibroacoustic transmission characteristics were calculated for bone models of the human middle ear by using the finite element method (FEM), after which Bayesian optimization (BO) was applied. The effect of the shape of artificial autologous ossicles on the acoustic transmission characteristics of the middle ear was investigated with the combined FEM and BO method. The results suggested that the volume of the artificial autologous ossicles especially has a great influence on the numerically obtained hearing levels.

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound.

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l-lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.

Radiological and audiological predictors of stapes destruction in adherent pars tensa.

PURPOSE: Progressive adherent pars tensa occasionally induces ossicular erosion. Specifically, stapes discontinuity adversely affects postoperative hearing. However, this irretrievable sequela is challenging to prove preoperatively, partly because perimatrix inflammation on the pars tensa can obscure the visibility of the ossicles or the partial volume effect of computed tomography (CT) imaging can hamper detailed ossicular visualization. Therefore, there is no consensus regarding the ideal timing for switching from a wait-and-see approach to a surgical one. Herein, we aimed to explore the potential predictors of stapes superstructure destruction in adherent pars tensa. METHODS: This retrospective cohort study enrolled consecutive patients who underwent primary tympanoplasty for adherent pars tensa categorized as grade IV on Sadé's grading scale between April 2016 and September 2021. The impact of features on otoscopy and CT and air-bone gap (ABG) on stapes superstructure destruction was assessed using uni- and multivariable logistic regression analyses. RESULTS: Sixty-four ears were included. Multivariate analysis revealed the presence of debris on the adherent pars tensa (odds ratio [OR] [95% confidence interval {CI}]): 4.799 [1.063-21.668], p = 0.0415), presence of soft-tissue density occupying the oval window (OR [95% CI]: 13.876 [3.084-62.437], p = 0.0006), and a ≥ 20-dB preoperative ABG at 3 kHz (OR [95% CI]: 7.595 [1.596-36.132], p = 0.0108) as independent predictors for stapes superstructure destruction. CONCLUSION: High preoperative awareness of the possibility of destruction of the stapes superstructure would enable the surgeon to make a timely decision to provide surgical intervention before progression to severe stapes destruction, thereby maintaining long-term satisfactory hearing.

Epigenetic Regulation as a New Therapeutic Target for Middle Ear Cholesteatoma.

HYPOTHESIS: To evaluate the effectiveness of the menin-MLL inhibitor, MI503, as a conservative treatment of middle ear cholesteatoma (cholesteatoma) in a mouse model and to confirm its safety profile regarding auditory function in vivo. BACKGROUND: Cholesteatoma is a mass formed by the keratinizing squamous epithelium in the tympanic cavity and/or mastoid and subepithelial connective tissue and by the progressive accumulation of keratin debris with/without a surrounding inflammatory reaction. Although the main treatment is surgical therapy, the techniques to prevent recurrence remain a critical area of research. Recently, the use of MI503 in experiments resulted in the inhibition of the growth of cholesteatoma in vivo under histone modification. METHODS: After cholesteatoma was induced in ICR mice (n = 7) by keratinocyte growth factor expression vector transfection, MI503 (50 µM) or phosphate-buffered saline was topically injected for 14 days. The effects of MI503 against cholesteatoma were analyzed by micro-computed tomography images. For the in vivo ototoxicity study, a single intratympanic injection of MI503 (50 or 500 µM) or phosphate-buffered saline (n = 4 each) was done in the ICR mice. An auditory brainstem response was performed at days 0, 1, and 14. For morphological analysis, immunostaining for Phalloidin/F-actin and Myo7a was performed. RESULTS: MI503 reduced keratinocyte growth factor-induced cholesteatoma in vivo (4 of 4 [100%]). No difference was found in the mean variation of the average of the auditory brainstem response thresholds between the three groups in the in vivo ototoxicity study, thus confirming its safety profile regarding auditory function. MI503 does not demonstrate any deleterious effects on murine hair cells when assessed by immunostaining. CONCLUSION: These findings demonstrate an encouraging safety profile for the use of menin-MLL inhibitor for the conservative treatment of cholesteatoma.

Radiological and audiological prediction for hearing outcome in cholesteatoma recidivism surgery.

PURPOSE: In cholesteatoma recidivism, achieving satisfactory hearing outcome after revision surgery remains challenging. The presence of concomitant recidivism pathology or related anatomical abnormalities can impact revision reconstruction of the sound transmission system. The current study aimed to identify prognostic factors affecting hearing outcomes after surgery for cholesteatoma recidivism. METHODS: This retrospective cohort study included consecutive patients whose ears required initial surgery for recidivism between January 2016 and December 2021. Patients followed up for < 6 months and those not indicated for ossiculoplasty were excluded. The impact of preoperative otoscopic findings, computed tomography (CT) features, and hearing levels on the prediction of satisfactory hearing (postoperative air-bone gap [ABG] ≤ 20 dB) was evaluated using univariate and multivariate logistic regression analyses. RESULTS: Overall, 102 patients were included, with a mean follow-up of 24.8 months. Multivariate logistic regression analysis revealed the following independent predictive factors for satisfactory hearing: presence of aeration in the tympanic cavity (odds ratio [OR] [95% confidence interval {CI}]: 13.287 [1.113-158.604], p = 0.0409), absence of soft-tissue density occupying the oval window (OR [95% CI]: 13.445 [3.178-56.887], p = 0.0040), and ≤ 22.5 dB preoperative ABG in four-frequency average (OR [95% CI]: 9.339 [2.026-43.050], p = 0.0042). CONCLUSIONS: For cholesteatoma recidivism, reliable preoperative prediction based on CT and ABG would facilitate decision-making regarding the probability of efficient revision ossiculoplasty or appropriate preoperative counseling, including early hearing rehabilitation using hearing aids or implementation of simultaneous implantable hearing equipment during surgery for recidivism.

Combined analysis of finite element model and audiometry provides insights into the pathogenesis of conductive hearing loss.

The middle ear transmits sound to the inner ear via vibrations in the eardrum and ossicles, and damage to the middle ear results in conductive hearing loss. Although conductive hearing loss can be corrected by surgery, the currently available clinical investigations cannot always diagnose the ossicular chain pathology underlying the conductive hearing loss, and even intraoperative findings can be equivocal. Acoustic analysis using finite element models (FEMs) can simulate the sound pressure change at an arbitrary site for each frequency. FEMs are used in acoustic engineering to simulate the frequency-dependent sound pressure distribution at discrete cells in a simulated model and analyze the effects of specific parameters on the audiogram. However, few reports have compared the numerical results obtained using FEMs with data from clinical cases. We used FEMs to simulate audiograms of the air-bone gap (ABG) for various ossicular chain defects and compared these with preoperative audiograms obtained from 44 patients with a normal tympanic membrane who had otosclerosis, middle ear malformations or traumatic ossicular disruption. The simulated audiograms for otosclerosis and attic fixation of the malleus-incus complex both exhibited an up-slope but could be distinguished from each other based on the ABG at 1000 Hz. The simulated audiogram for incudostapedial joint discontinuity exhibited a peak at around 750 Hz and a down-slope above 1000 Hz. In general, the simulated audiograms for otosclerosis, attic fixation and incudostapedial joint discontinuity were consistent with those obtained from clinical cases. Additional simulations indicated that changes in ossicular mass had relatively small effects on ABG. Furthermore, analyses of combination pathologies suggested that the effects of one defect on ABG were added to those of the other defect. These FEM-based findings provide insights into the pathogenesis of conductive hearing loss due to otosclerosis, middle ear malformations and traumatic injury.

Otic Organoids Containing Spiral Ganglion Neuron-like Cells Derived from Human-induced Pluripotent Stem Cells as a Model of Drug-induced Neuropathy.

The spiral ganglion of the cochlea is essential for hearing and contains primary bipolar neurons that relay action potentials generated by mechanosensory hair cells. Injury to spiral ganglion neurons (SGNs) causes permanent hearing loss because these cells have limited regenerative capacity. Establishment of human cell-derived inner ear tissue in vitro could facilitate the development of treatments for hearing loss. Here, we report a stepwise protocol for differentiating human-induced pluripotent stem cells (hiPSCs) into otic organoids that contain SGN-like cells and demonstrate that otic organoids have potential for use as an experimental model of drug-induced neuropathy. Otic progenitor cells (OPCs) were created by 2D culture of hiPSCs for 9 days. Otic spheroids were formed after 2D culture of OPCs for 2 days in a hypoxic environment. Otic organoids were generated by 3D culture of otic spheroids under hypoxic conditions for 5 days and normoxic conditions for a further 30 days or more. The protein expression profile, morphological characteristics, and electrophysiological properties of SGN-like cells in otic organoids were similar to those of primary SGNs. Live-cell imaging of AAV-syn-EGFP-labeled neurons demonstrated temporal changes in cell morphology and revealed the toxic effects of ouabain (which causes SGN-specific damage in animal experiments) and cisplatin (a chemotherapeutic drug with ototoxic adverse effects). Furthermore, a cyclin-dependent kinase-2 inhibitor suppressed the toxic actions of cisplatin on SGN-like cells in otic organoids. The otic organoid described here is a candidate novel drug screening system and could be used to identify drugs for the prevention of cisplatin-induced neuropathy.

Anatomical and Surgical Evaluation of the Common Marmoset as an Animal Model in Hearing Research.

Recent studies have indicated that direct administration of viral vectors or small compounds to the inner ear may aid in the treatment of Sensorineural hearing loss (SNHL). However, due to species differences between humans and rodents, translating experimental results into clinical applications remains challenging. The common marmoset (Callithrix jacchus), a New World monkey, is considered a pre-clinical animal model. In the present study, we describe morphometric data acquired from the temporal bone of the common marmoset in order to define the routes of topical drug administration to the inner ear. Dissection and diffusion tensor tractography (DTT) were performed on the fixed cadaverous heads of 13 common marmosets. To investigate potential routes for drug administration to the inner ear, we explored the anatomy of the round window, oval window (OW), semicircular canal, and endolymphatic sac (ES). Among these, the approach via the round window with posterior tympanotomy appeared feasible for delivering drugs to the inner ear without manipulating the tympanic membrane, minimizing the chances of conductive hearing loss. The courses of four critical nerves [including the facial nerve (FN)] were visualized using three-dimensional (3D) DTT, which may help to avoid nerve damage during surgery. Finally, to investigate the feasibility of actual drug administration, we measured the volume of the round window niche (RWN), which was approximately 0.9 µL. The present findings may help to establish experimental standards for evaluating new therapies in this primate model.