ABSTRACT Sensorineural hearing loss, resulting from damage to the hair cells of the inner ear, profoundly impairs quality of life. A widely used treatment involves cochlear implants (CIs), which electrically stimulate the remaining spiral ganglion neurons (SGNs) to partially restore auditory function. Despite considerable technological progress, improvements in implant performance are necessary. Local and controlled drug delivery represents a promising strategy to optimize the implant's biological environment and to support the preservation of SGNs. This study introduces a patented implant‐associated local drug delivery system, designed as a nanocomposite coating for electrode contacts. The composite consists of nanoporous silica nanoparticles embedded within nanoporous platinum synthesized via electrochemical deposition combined with templating techniques, resulting in a homogeneous and mechanically robust coating. The porous structure enhances electrochemical performance by increasing the specific surface area, while simultaneously improving drug loading capacity and enabling dexamethasone release. Cytocompatibility tests using fibroblasts and SGNs indicate good cytocompatibility, supporting its suitability for CI applications. Furthermore, reduced TNF‐α levels in stressed dendritic cells following exposure to released dexamethasone suggest a potential anti‐inflammatory effect and provide an initial proof‐of‐concept for the biological functionality. Ultimately, these features suggest that the proposed system may contribute to improved hearing outcomes in CI recipients.

A major challenge in cochlear implant (CI) therapy is postoperative inflammation, which can compromise long-term electrode function. Conventional interleukin-6 (IL-6) detection is challenging due to factors such as its inherent instability. In this study, we present a cost-effective detection strategy using epitope-specific molecularly imprinted polymer nanoparticles (nanoMIPs). These nanoMIPs enable sensitive detection of both IL-6 and its epitope, representing a scalable and economical alternative for inflammation monitoring. NanoMIPs were embedded in a biodegradable chitosan matrix and sprayed onto electrodes. To detect the biomarker, electrochemical impedance spectroscopy was used, a functionality which is already embedded in CI circuits. The sensors enabled reliable detection of IL-6 down to 29 pg/mL, which represents the lowest concentration investigated in this study, with improved sensitivity to the small epitope, as the larger protein causes higher impedance changes due to steric hindrance. Additionally, the sensors enabled concentration-dependent IL-6 detection in human perilymph samples down to 2 pg/mL, with higher signal responses for IL-6 relative to other perilymph components. Comparison with an immunoassay supported analytical accuracy and diagnostic relevance. The sensors were subsequently tested weekly for functionality over four weeks under physiological conditions, with nanoMIP:chitosan ratios of 1:6 and 1:8 being optimal for long-term monitoring. Moreover, the nanoMIPs have a diameter of 56 nm (a parameter that can influence physiological excretion) and cytotoxicity tests demonstrated their biocompatibility. This work presents an epitope-based nanoMIP sensor platform that overcomes economic and biochemical limitations of protein-based biosensors, enabling advanced, real-time self-monitoring CI systems for in-vivo inflammation tracking to reduce implant failure.

External ear canal (EEC) stenosis, often associated with cholesteatoma, carries a high risk of postoperative restenosis despite surgical intervention. While individualized implants offer promise in preventing restenosis, the high morphological variability of EECs and the lack of standardized definitions hinder systematic implant design. This study aimed to characterize individual EEC morphology and to develop a validated automated segmentation system for efficient implant preparation. Reference datasets were first generated by manual segmentation using 3D SlicerTM software version 5.2.2. Based on these, we developed a customized plugin capable of automatically identifying the maximal implantable region within the EEC and measuring its key dimensions. The accuracy of the plugin was assessed by comparing it with manual segmentation results in terms of shape, volume, length, and width. Validation was further performed using three temporal bone implantation experiments with 3D-Bioplotter©-fabricated EEC implants. The automated system demonstrated strong consistency with manual methods and significantly improved segmentation efficiency. The plugin-generated models enabled successful implant fabrication and placement in all validation tests. These results confirm the system’s clinical feasibility and support its use for individualized and systematic EEC implant design. The developed tool holds potential to improve surgical planning and reduce postoperative restenosis in EEC stenosis treatment.

A multifunctional copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) and diethyl-2-(methacrylamido)diethylphosphonate as a phosphonate-containing monomer was developed with the aim of applying a self-organizing hydrogel coating to titanium surfaces with the potential to be used for the functionalization of implants. For this purpose, a copolymer was prepared that can bind to the titanium via a phosphonate group. A photocrosslinkable group (phenyl azide) and hydrophilic groups were also incorporated to form a hydrogel. Coatings of these polymers produced by spin coating and subsequent photocrosslinking were characterized by laser ellipsometry, which yielded controlled dry layer thicknesses in the range of 40-140 nm. Furthermore, the swelling capacity was investigated by in situ ellipsometry in different aqueous media, resulting in a swelling up to three times the original dry film thickness, depending on their crosslinker content. We also validated long-term stability under physiological conditions and biocompatibility. Although the hydrogel layers and the silica nanoparticles have negative surface charges, different types of silica nanoparticles are strongly adsorbed on the surfaces. This shows the potential of the hydrogels for bonding with other hydrogels or soft tissue (nanogluing). Preliminary tests have shown that a PDMA hydrogel could in fact adhere to the surface-bound hydrogels by way of nanoparticles.Trial Registration: NA.

Purpose:After cochlear implantation, molecular processes at the electrode-nerve interface significantly influence the variability in clinical outcomes. The present study investigates molecular processes in a guinea pig model of cochlear implant (CI) using positron emission tomography/computed tomography (PET/CT) and correlates the imaging findings with histological analyses. Methods:Animals were examined with PET in the 3 weeks and 9-12 months post-implantation using the inflammation marker [18F]FDG and, at the later time points, [68Ga]FAPI-46 as a marker for fibrosis. Tracer accumulation in the cochlea was determined from PET imaging based on the co-registered CT. Nine animals (seven with unilateral CI) were included. Uptake in non-implanted cochleae served as reference. Tissue growth around the implant was evaluated histologically. Results:Post-implantation, [18F]FDG uptake was significantly increased when pooling early and late in investigation time points, while after 1 year, [68Ga]FAPI-46 uptake was increased inside the cochlear. Cochlear volumes measured by CT did not show significant differences between compared groups. Tissue growth around the implant was observed in all animals, with a trend toward increased growth associated with insertion depth. However, no clear correlation was observed between the extent of tissue growth and the uptake intensities of FDG and FAPI. Discussion:The data indicate that increased accumulation of PET biomarkers in the cochlea after implantation can be detected in guinea pigs using a dedicated PET/CT. Given the high resolution of current clinical PET/CT devices, this method is expected to be suitable for use in patients, particularly for assessing the effect of anti-inflammatory or anti-fibrotic therapies.

Introduction: Neurotrophic factors are widely known for their protective effect on spiral ganglion neurons (SGN) and the protection of these neurons is of great importance to optimize Cochlear Implants, which directly stimulate SGN in deaf patients. Previous studies have identified Cometin - also known as Meteroin-like - to be neuroprotective and beneficial for metabolic disorders. The aim of our study was to investigate the effects of different concentrations of recombinant human Cometin (hCometin) on SGN in regard to neuroprotection and neurite outgrowth and to evaluate its neurite guidance potential using a neurite outgrowth chamber. Methods: Human Cometin was initially tested in two separate dosing experiments: 5, 10, and 15 µg/ml (medium dose group) and 10, 25, and 50 µg/ml (high dose group). The hCometin was added to dissociated neonatal murine SGN. The number, morphology, and neurite length of SGN treated with hCometin were compared to untreated (negative control, NC) and brain-derived neurotrophic factor treated (50 ng/ml) (positive control, PC) cells. Subsequently, to investigate a potential effect on neurite guidance, 10 µg/ml hCometin was delivered via osmotic pumps to neonatal murine spiral ganglion explants (SGE) cultured in a neurite outgrowth chamber to experimentally mimic the scala tympani and the Rosenthal’s canal. The amount of pump-released hCometin was measured by Enzyme-linked Immunosorbent Assay and neurite growth was quantified and compared to a Cometin-free NC. Results: All medium dose group concentrations of hCometin resulted in significant neuronal protection, whereas high dose group concentrations (25 and 50 µg/ml) were neurotoxic. The medium doses significantly increased the number of monopolar neurons compared to NC, and 10 and 15 µg/ml hCometin increased the number of neurons with a physiological bipolar morphology to an even greater extent than BDNF. For neurite length, 5 and 10 µg/ml hCometin had the greatest effect, which was comparable with the BDNF-PC. The osmotic-pump based delivery of 10 µg/ml hCometin to SGE had no or an adverse effect on the number, extent, or orientation of outgrowing neurites in the culture set up used. Conclusion: A concentration of 10 µg/ml hCometin significantly protects dissociated SGN from degeneration and significantly increases the outgrowth of neurites, which is favourable in view of induced neurite outgrowth towards cochlear electrode arrays for future optimisation of the nerve-electrode-interface. The study failed to detect a guided neurite outgrowth by pump-based drug release, which may be due to the experimental set up, which could be improved in future studies.

The anatomical and structural complexity and variability of the hearing organ pose significant challenges for efficient drug delivery to treat inner ear disorders. To address this issue, we developed a local drug delivery implant (LDDI) fabricated with 3D printing technology. The implant is personalized based on the individual patient's anatomy of the round window niche from where drugs can diffuse into the inner ear. A key challenge is to find materials that are not only printable and flexible but also both biocompatible and biodegradable. While alginate hydrogels are known for biocompatibility and biodegradability, low viscosity and suboptimal mechanical properties limit their suitability for 3D printing applications. In this study, alginate hydrogels were refined by incorporating polycaprolactone microparticles (PCL-MPs), developing a micro-composite hydrogel with enhanced printability and mechanical strength. Rheological assessments and compression tests demonstrated the hydrogels' suitability for extrusion-based 3D printing. Swelling, degradation, and shape stability studies confirmed the durability under physiological conditions. Atomic force microscopy and scanning electron microscopy revealed a uniform microstructure, supporting in vitro drug release profiles quantified via high-performance liquid chromatography. Cytotoxicity studies showed biocompatibility, the reinforced hydrogel containing dexamethasone induced anti-inflammatory effects, and the composite material was used to 3D-print a round window niche implant. This work highlights the potential of PCL-MP-reinforced alginate hydrogels in fabricating drug-loaded, personalized, and biodegradable LDDIs, offering a promising strategy for targeted inner ear therapy. The findings expand the applications of 3D bioprinting in precision medicine and demonstrate the feasibility of this approach for clinical translation.

An atraumatic implantation of the Cochlear Implant (CI) to prevent, inter alia, loss of spiral ganglion neurons (SGN) and residual hearing, might be achieved by a flexible, smooth, and hydrophilic coating of the electrode. Such a coating needs to be long-term stable to avoid swelling or shrinking, which cause micro-movements of the electrode. In parallel, it has to be harmless to hair cells (HC) and SGN. Due to its inert and biocompatible character, barium cross-linked ultra-high viscosity alginate is a promising candidate for this propose and was tested for stability and biocompatibility under simulated inner ear conditions. CI dummies were coated with alginate by using a cone-shaped semipermeable membrane. Alginate sol was injected in these membrane cones, dummies inserted, and all covered with barium cross-linking solution for ionic gelation. Coated dummies were incubated in artificial perilymph and observed for one year with weekly medium change and photo documentation for stability assessment. Additionally, beads of alginate hydrogel were co-cultivated with murine inner ear tissue for one week to test biocompatibility with SGN and HC. Alginate hydrogel coating stayed attached over the one-year observation period. Small variations in the diameter of coated dummies were detectable, likely due to rotation of the floating dummies, but no clear shrinking or swelling was seen. Neither number of SGN nor number of HC was significantly reduced by co-culture with alginate beads. In simulated inner ear conditions, alginate hydrogel was stable as coating for one year without greater changes of shape and had no neuro- or ototoxic effect. These promising results support testing of alginate as lubricant-coating in vivo as a further step towards translation to clinical use for CI improvement.

Implantable drug delivery systems (IDDS) hold great promise for sustained therapeutic administration, particularly for deep tissues like the inner ear. However, the obstruction of delivery systems induced by foreign body reactions (FBRs) remains a significant challenge to long-term implantation. Here, we developed a bio-inspired zwitterionic nanocoating (PDA-PSB) for IDDS. Experimental results showed that the PDA-PSB coating significantly improved the hydrophilicity, reduced protein and cell adhesion, and effectively suppressed inflammatory responses. To evaluate the long-term performance, we implanted PDA-PSB-coated microcatheters subcutaneously and in the tympanic bullae of rats for six months. Dynamic observations revealed that, in the uncoated group, fibrotic tissues resulting from the FBRs gradually infiltrated the lumen of the microcatheter, ultimately causing complete occlusion. In contrast, the PDA-PSB-coated microcatheters significantly reduced the fibrosis and prevented obstruction. Pressure measurements further demonstrated that the PDA-PSB-coated microcatheters maintained low drug delivery pressure after long-term implantation, ensuring sustained patency and continuous drug delivery. Mechanistic studies revealed that the PDA-PSB coating inhibited early macrophage M1 polarization and prevented macrophage transition into myofibroblasts (MMT), thereby reducing collagen deposition. This study provides a novel solution for improving the performance of IDDS and highlights its considerable potential for long-term application.

More than 5% of the global population suffers from disabling hearing loss, primarily sensorineural hearing loss (SNHL). SNHL is often caused by factors such as vascular disorders, viral infections, ototoxic drugs, systemic inflammation, age-related labyrinthine membrane degeneration, and noise-induced hearing loss (NIHL). NIHL, in particular, leads to changes in blood-labyrinth-barrier (BLB) physiology, increased permeability, and various health issues, including cardiovascular disease, hypertension, diabetes, neurological disorders, and adverse reproductive outcomes. Recent advances in neuromodulation and vector-based approaches offer hope for overcoming biological barriers such as the BLB in the development of innovative treatments. Computational methods, including molecular docking, molecular dynamics simulations, QSAR/QSPR analysis with machine/deep learning algorithms, and network pharmacology, hold potential for identifying drug candidates and optimizing their interactions with BLB transporters, such as the glutamate transporter. This paper provides an overview of NIHL, focusing on its pathophysiology; its impact on membrane transporters, ion channels, and BLB structures; and associated symptoms, comorbidities, and emerging therapeutic approaches. Recent advancements in neuromodulation and vector-based strategies show great promise in overcoming biological barriers such as BLB, facilitating the development of innovative treatment options. The primary aim of this review is to examine NIHL in detail and explore its underlying mechanisms, physiological effects, and cutting-edge therapeutic strategies for its effective management and prevention.

Background There exists an unfulfilled requirement for effective cochlear pharmacotherapy. Controlled local drug delivery could lead to effective bioavailability. The round window niche (RWN), a cavity in the middle ear, is connected to the cochlea via a membrane through which drug can diffuse. We are developing individualized drug-eluting RWN implants (RNIs). To test their effectiveness in guinea pigs, a commonly used model in cochlear pharmacology studies, it is first necessary to develop guinea pig RNIs (GP-RNI).Methods Since guinea pigs do not have a RWN such as it is present in humans and to reduce the variables in in vivo studies, a one-size-fits-all GP-RNI model was designed using 12 data sets of Dunkin-Hartley guinea pigs. The model was 3D-printed using silicone. The accuracy and precision of printing, distribution of the sample ingredient dexamethasone (DEX), biocompatibility, bio-efficacy, implantability and drug release were tested in vitro. The GP-RNI efficacy was validated in cochlear implant-traumatized guinea pigs in vivo.Results The 3D-printed GP-RNI was precise, accurate and fitted in all tested guinea pig RWNs. DEX was homogeneously included in the silicone. The GP-RNI containing 1% DEX was biocompatible, bio-effective and showed a two-phase and sustained DEX release in vitro, while it reduced fibrous tissue growth around the cochlear implant in vivo.Conclusions We developed a GP-RNI that can be used for precise inner ear drug delivery in guinea pigs, providing a reliable platform for testing the RNI’s safety and efficacy, with potential implications for future clinical translation.

Background The human inner ear (IE) contains complex sensitive cellular structures. These structures inside the cochlea are vulnerable to e.g. toxic substances, aging, diseases and inflammatory processes. Sensorineural hearing loss can be induced by a wide range of molecular and cellular pathologic changes. These pathologic changes and disease specific biomarkers can be identified in low volume perilymph (PL) samples by performing ultrasensitive analytic methods like proteomics [1], metabolomics and multiplex protein array (MPA) [2].

Sensorineural hearing loss is one of the most prevalent sensory deficits. Spiral ganglion neurons (SGNs) exhibit very limited regeneration capacity and their degeneration leads to profound hearing loss. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) have been demonstrated to repair tissue damage in various degenerative diseases. However, the effects of MSC-sEV on SGN degeneration remain unclear. In this study, we investigated the efficacy of MSC-sEV for protection against ouabain-induced SGN degeneration. MSC-sEV were derived from rat bone marrow and their components related to neuron growth were determined by proteomic analysis. In primary culture SGNs, MSC-sEV significantly promoted neurite growth and growth cone development. The RNA-Seq analysis of SGNs showed that enriched pathways include neuron development and axon regeneration, consistent with proteomics. In ouabain induced SGN degeneration rat model, MSC-sEV administration via intratympanic injection significantly enhanced SGN survival and mitigated hearing loss. Furthermore, after ouabain treatment, SGNs displayed evident signs of apoptosis, including nuclei condensation and fragmentation, with numerous cells exhibiting TUNEL-positive. However, administration of MSC-sEV effectively decreased the number of TUNEL-positive cells and reduced caspase-3 activation. In conclusion, our findings demonstrate the potential of MSC-sEV in preventing SGN degeneration and promoting neural growth, suggesting intratympanic injection of MSC-sEV is a specific and efficient strategy for neural hearing loss.

There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides osteoinduction, there is growing evidence of also anti-inflammatory, angiogenetic and neuroprotective effects of biodegradable magnesium-based biomaterials. Their use for the treatment of cartilage lesions in contrast is not well-evaluated yet.Mg-cylinders were analysed in an in vitro and in vivo OA model. In vitro, SCP-1 stem cell line was analysed under inflammatory conditions and Mg-impact. In vivo, small Mg- and WE43 alloy-cylinders (1mm × 0,5mm) were implanted into the subchondral bone of the knee joint of 24 NZW rabbits after establishment of OA. As control, another 12 rabbits received only drill-holes. µCT-scan were performed and assessed for changes in bone volume and density. After euthanasia, cartilage was evaluated macroscopically and histologically after Safranin-O-staining. Furthermore, staining with CD271 directed antibody was performed to assess neuro-reactivity.In vitro, an increased gene expression of extracellular matrix proteins as collagen II or aggrecan even under inflammatory conditions was observed under Mg-impact. In vivo, µCT evaluation revealed twice-elevated values for bone volume in femoral condyles with Mg-cylinders compared to controls while density remained unchanged. Cartilage showed no significant differences between the groups. Mg- and WE-samples showed significantly lower levels of CD271+ cells in the cartilage and bone of the operated joints than in non-operated joints, which was not the case in the Drilling-group. Furthermore, bone in operated knees of Drilling-group showed a strong trend to an increase in CD271+ cells compared to both Cylinder-groups. Counting of CD271+ vessels revealed that this difference was attributable to a higher amount of these vessels.The in vitro results indicate a potential cartilage regenerative activity of the degradable Mg-based material. While so far there was no positive effect on the cartilage itself in vivo, implantation of Mg-cylinders seemed to reduce pain-mediating vessels.Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 404534760). We thank Björn Wiese for production of the cylinders.

Introduction A lubricant coating of the cochlear implant (CI) electrode might be a useful tool to improve implantation procedure. The implantation can cause trauma of the inner ear tissue leading to e.g. loss of spiral ganglion neurons (SGN) or residual hearing. A coating with hydrogel could cover the electrode with a hydrophilic, smooth, and flexible layer that softens implantation and reduces trauma. Ultra-high viscosity (UHV) alginate and its barium chloride (BaCl2)-crosslinking are generally described as long-term stable and biocompatible. In previous studies, we showed the overall feasibility of an alginate-coating of the CI but detailed studies for toxicity of BaCl2 on inner ear tissue are lacking. Therefore, safety of BaCl2 and BaCl2-cross-linked alginate (Ba-Alg) was tested in vitro.

With its main features of cartilage degeneration, subchondral bone sclerosis and osteophyte formation, osteoarthritis represents a multifactorial disease with no effective treatment options. As biomechanical shift in the trabecular network may be a driver for further cartilage degeneration, bone enhancement could possibly delay OA progression. Magnesium is known to be osteoconductive and already showed positive effects in OA models. We aimed to use magnesium cylinders to enhance subchondral bone quality, condition of cartilage and pain sensation compared to sole drilling in vivo. After eight weeks of implantation in rabbits, significant increase in subchondral bone volume and trabecular thickness with constant bone mineral density was found indicating favored biomechanics. As representative for pain, a higher number of CD271+ vessels were present in control samples without magnesium. However, this result could not be confirmed by sensitive, objective lameness evaluation using a pressure sensing mat and no positive effect could be shown on either cartilage degeneration evaluated by OARSI score nor the presence of regenerative cells in CD271-stained samples. The presented results show a relevant impact of implanted magnesium on key structures in OA pain with missing clinical relevance regarding pain. Further studies with shifted focus should examine additional structures as joint capsule or osteophytes.

Hintergrund Reduktion postoperativer Stenosen des äußeren Gehörgangs (EGC) ist nach Canaloplastiken entscheidend. Ziel: Machbarkeit dreidimensionaler, medikamentenfreisetzender Implantate (DEX, Cipro) als postoperative Stents.

Background: Preserving residual hearing after cochlear implant (CI) surgery remains a crucial challenge. The application of dexamethasone (DEX) has been proven to positively affect residual hearing. To deliver DEX in a localized and controlled way, a round window niche implant (RNI), allowing drug diffusion via the round window membrane into the cochlea, may be used. To prove this concept, an RNI for guinea pigs as a CI-trauma model was manufactured by molding and tested for its drug release in vitro and biological effects in vivo. Methods: The RNIs were molded using silicone containing 10% DEX. Release was analyzed over time using high-performance liquid chromatography (HPLC). Fourteen adult guinea pigs were randomly assigned to two groups (CI or CI + RNI group). All animals received a unilateral CI electrode insertion trauma followed by CI insertion. The CI + RNI group was additionally implanted with an RNI containing 10% DEX. Animals were followed up for 4 weeks. Acoustically evoked auditory brainstem response and impedance measurement, micro-computed tomography (µCT) imaging, and histology were performed for evaluation. Results: DEX was released for more than 250 days in vitro, with an initial burst followed by a slower release over time. Comparing the hearing threshold shift (from day 0 to day 28) of the CI and CI + RNI groups, significant differences were observed at 32 and 40 kHz. The impedance shift at basal contacts was lower in the CI + RNI group than in the CI group. Moreover, the fibrosis in the lower basal turn was reduced in the CI + RNI group in contrast to the CI group. Conclusions: The RNI containing 10% DEX has anti-inflammatory potential concerning fibrosis inhibition and has beneficial effects on hearing preservation at high frequencies.

For treating idiopathic sudden sensorineural hearing loss, prednisolone is commonly used. However, systemic or middle ear injections often lead to insufficient drug delivery to the inner ear, causing ineffective treatment and systemic side effects. An implant inserted into the middle ear and delivering the drug directly to the inner ear offers a promising solution, providing controlled, long-term drug release with potentially better efficacy and fewer side effects. Individualized implants made of prednisolone-containing silicone can optimize inner ear treatment by fitting the patient's middle ear anatomy. To gauge the properties of prednisolone-21- hydrogen succinate containing silicone, samples with different geometries and drug concentrations have been 3D-printed. The shore hardness of samples with three different drug concentrations was assessed. Three different shapes with four different drug concentrations were incubated in artificial perilymph for up to 56 days to evaluate the release rates. The resulting eluates were analyzed via Ultra high precision liquid chromatography coupled with a time-of-flight micro-mass spectrometer. Samples were softer when a higher drug concentration was used. A high burst release of prednisolone after one hour was measured. Afterward, the release rates decreased and reached a relatively constant rate after ten days and stayed there for at least another 46 days. The release rates were multiple times higher when the samples had a higher surface-to-volume ratio. The softer the sample, the higher the release rate, unproportional to the concentration increase.

Cisplatin is an election chemotherapeutic agent used for many cancer treatments. Its cytotoxicity against neoplastic cells is mirrored by that taking place in healthy cells and tissues, resulting in serious adverse events. A very frequent one is ototoxicity, causing hearing loss which may permanently affect quality of life after successful oncologic treatments. Exacerbated oxidative stress is a main cytotoxic mechanism of cisplatin, including ototoxicity. Previous reports have shown antioxidant protection against cisplatin ototoxicity, but there is a lack of comparative studies on the otoprotectant activity and mechanism of antioxidant formulations. Here, we show evidence that a cocktail of vitamins A, C, and E along with Mg++ (ACEMg), previously shown to protect against noise-induced hearing loss, reverses auditory threshold shifts, promotes outer hair cell survival, and attenuates oxidative stress in the cochlea after cisplatin treatment, thus protecting against extreme cisplatin ototoxicity in rats. The addition of 500 mg N-acetylcysteine (NAC), which, administered individually, also shows significant attenuation of cisplatin ototoxicity, to the ACEMg formulation results in functional degradation of ACEMg otoprotection. Mg++ administered alone, as MgSO4, also prevents cisplatin ototoxicity, but in combination with 500 mg NAC, otoprotection is also greatly degraded. Increasing the dose of NAC to 1000 mg also results in dramatic loss of otoprotection activity compared with 500 mg NAC. These findings support that single antioxidants or antioxidant combinations, particularly ACEMg in this experimental series, have significant otoprotection efficacy against cisplatin ototoxicity. However, an excess of combined antioxidants and/or elevated doses, above a yet-to-be-defined “antioxidation threshold”, results in unrecoverable redox imbalance with loss of otoprotectant activity.