The importance of mechanical and biological cues of tympanic membrane grafts to ensure optimal regeneration.

Chronic suppurative otitis media (CSOM) is often associated with permanent tympanic membrane (TM) perforation and conductive hearing loss. The current clinical gold standard, using autografts and allografts, suffers from several drawbacks. Artificial replacement materials can help to overcome these drawbacks. Therefore, scaffolds fabricated through digital light processing (DLP) were herein created to support TM regeneration. Various UV-curable printing inks, including gelatin methacryloyl (GelMA), gelatin-norbornene-norbornene (GelNBNB) (crosslinked with thiolated gelatin (GelSH)) and alkene-functionalized poly-ε-caprolactone (E-PCL) (crosslinked with pentaerythritol tetrakis(3-mercaptopropionate) (PETA4SH)) were optimized regarding photo-initiator (PI) and photo-absorber (PA) concentrations through viscosity characterization, photo-rheology and the establishment of working curves for DLP. Our material platform enabled the development of constructs with a range of mechanical properties (plateau storage modulus varying between 15 and 119 kPa). Excellent network connectivity for the GelNBNB and E-PCL constructs was demonstrated (gel fractions >95 %) whereas a post-crosslinking step was required for the GelMA constructs. All samples showed excellent biocompatibility (viability >93 % and metabolic activity >88 %). Finally, in vivo and ex vivo assessments, including histology, vibration and deformation responses measured through laser doppler vibrometry and digital image correlation respectively, were performed to investigate the effects of the scaffolds on the anatomical and physiological regeneration of acute TM perforations in rabbits. The data showed that the most efficient healing with the best functional quality was obtained when both mechanical (obtained with the PCL-based resin) and biological (obtained with the gelatin-based resins) material properties were taken into account.

Rational design of a genomically humanized mouse model for dominantly inherited hearing loss, DFNA9.

DFNA9 is a dominantly inherited form of adult-onset progressive hearing impairment caused by mutations in the COCH gene. COCH encodes cochlin, a crucial extracellular matrix protein. We established a genomically humanized mouse model for the Dutch/Belgian c.151C>T founder mutation in COCH. Considering upcoming sequence-specific genetic therapies, we exchanged the genomic murine Coch exons 3-6 for the corresponding human sequence. Introducing human-specific genetic information into mouse exons can be risky. To mitigate unforeseen consequences on cochlin function resulting from the introduction of the human COCH protein-coding sequence, we converted all human-specific amino acids to mouse equivalents. We furthermore optimized the recognition of the human COCH exons by the murine splicing machinery during pre-mRNA splicing. Subsequent observations in mouse embryonic stem cells revealed correct splicing of the hybrid Coch transcript. The inner ear of the established humanized Coch mice displays correctly-spliced wild-type and mutant humanized Coch alleles. For a comprehensive study of auditory function, mice were crossbred with C57BL/6 Cdh23753A>G mice to remove the Cdh23ahl allele from the genetic background of the mice. At 9 months, all humanized Coch genotypes showed hearing thresholds comparable to wild-type C57BL/6 Cdh23753A>G mice. This indicates that both the introduction of human wildtype COCH, and correction of Cdh23ahl in the humanized Coch lines was successful. Overall, our approach proved beneficial in eliminating potential adverse events of genomic humanization of mouse genes, and provides us with a model in which sequence-specific therapies directed against the human mutant COCH alle can be investigated. With the hearing and balance defects anticipated to occur late in the second year of life, a long-term follow-up study is ongoing to fully characterize the humanized Coch mouse model.

Cochlin Deficiency Protects Aged Mice from Noise-Induced Hearing Loss.

Several studies have shown that type IV fibrocytes, located in the spiral ligament, degenerate first after noise exposure. Interestingly, this is the region where Coch expression is most abundant. As it is suggested that cochlin plays a role in our innate immune system, our goal is to investigate hearing thresholds and inner ear inflammation after noise exposure in Coch knockout (Coch-/-) mice compared to Coch wildtype (Coch+/+) mice. Animals were randomly allocated to a noise exposure group and a control group. Vestibular and auditory testing was performed at 48 h and one week after noise exposure. Whole mount staining and cryosectioning of the cochlea was performed in order to investigate hair cells, spiral ganglion neurons, inner ear inflammation, Coch expression and fibrocyte degeneration. Hearing assessment revealed that Coch+/+ mice had significantly larger threshold shifts than Coch-/- mice after noise exposure. We were unable to identify any differences in hair cells, neurons, fibrocytes and influx of macrophages in the inner ear between both groups. Interestingly, Coch expression was significantly lower in the group exposed to noise. Our results indicate that the absence of Coch has a protective influence on hearing thresholds after noise exposure, but this is not related to reduced inner ear inflammation in the knockout.

Transduction Efficiency and Immunogenicity of Viral Vectors for Cochlear Gene Therapy: A Systematic Review of Preclinical Animal Studies.

Background: Hearing impairment is the most frequent sensory deficit, affecting 466 million people worldwide and has been listed by the World Health Organization (WHO) as one of the priority diseases for research into therapeutic interventions to address public health needs. Inner ear gene therapy is a promising approach to restore sensorineural hearing loss, for which several gene therapy applications have been studied and reported in preclinical animal studies. Objective: To perform a systematic review on preclinical studies reporting cochlear gene therapy, with a specific focus on transduction efficiency. Methods: An initial PubMed search was performed on April 1st 2021 using the PRISMA methodology. Preclinical in vivo studies reporting primary data regarding transduction efficiency of gene therapy targeting the inner ear were included in this report. Results: Thirty-six studies were included in this review. Transduction of various cell types in the inner ear can be achieved, according to the viral vector used. However, there is significant variability in the applied vector delivery systems, including promoter, viral vector titer, etc. Conclusion: Although gene therapy presents a promising approach to treat sensorineural hearing loss in preclinical studies, the heterogeneity of methodologies impedes the identification of the most promising tools for future use in inner ear therapies.

Attitudes of Potential Participants Towards Potential Gene Therapy Trials in Autosomal Dominant Progressive Sensorineural Hearing Loss.

BACKGROUND: Advances in gene therapeutic approaches to treat sensorineural hearing loss (SNHL) confront us with future challenges of translating these animal studies into clinical trials. Little is known on patient attitudes towards future innovative therapies. OBJECTIVE: We aimed to better understand the willingness of patients with progressive SNHL and vestibular function loss of autosomal dominant (AD) inheritance to participate in potential gene therapy trials to prevent, stabilize, or slow down hearing loss. METHODS: A survey was performed in carriers of the P51S and G88E pathogenic variant in the COCH gene (DFNA9). Various hypothetical scenarios were presented while using a Likert scale. RESULTS: Fifty three participants were included, incl. 49 symptomatic patients, one presymptomatic patient, and three participants at risk. Their attitude towards potential trials studying innovative therapies was overall affirmative, even if the treatment would only slow down the decline of hearing and vestibular function, rather than cure the disease. Among the different potential scenarios, the less invasive and less frequent treatments increased the likelihood to enroll. Daily oral medication and annual intravenous infusion were awarded the highest scores. The more invasive, more frequent, and more at-risk treatments were still likely to be accepted but decreased the willingness to participate. The presence of a placebo arm was met with the lowest scores of willingness to participate. CONCLUSIONS: Overall, most symptomatic DFNA9 patients would likely consider participation in future innovative inner ear therapy trials, even if it would only slow down the decline of hearing and vestibular function.

On the pathophysiology of DFNA9: Effect of pathogenic variants in the COCH gene on inner ear functioning in human and transgenic mice.

DeaFNess Autosomal Dominant 9 (DFNA9) is a dominant hereditary non-syndromic form of progressive sensorineural hearing loss often associated with vestibular dysfunction. DFNA9 is caused by pathogenic variants in the COCH gene. This gene encodes for cochlin, a protein that is abundantly expressed in the spiral ligament and spiral limbus of the inner ear but the function of cochlin is still not fully understood. There are 22 known pathogenic variants located in different domains of the COCH gene that can cause DFNA9, all expressing slightly different phenotypes. It is believed that COCH mutations affect the intracellular trafficking of cochlin which could explain the characteristic pathology seen in temporal bones of DFNA9 patients. This pathology involves a widespread accumulation of acellular eosinophilic deposits throughout the labyrinth. To gain a better understanding of the pathology underlying DFNA9, different mouse models were developed. The objective of this review is to describe the different pathogenic variants in the COCH gene and their effect on intracellular trafficking, associated phenotypes and histopathological findings in both patients and mouse models.

Effect of Oral Allylnitrile Administration on Cochlear Functioning in Mice Following Comparison of Different Anesthetics for Hearing Assessment.

Background: Allylnitrile is a compound found in cruciferous vegetables and has the same lethality and toxic effects as the other nitriles. In 2013, a viable allylnitrile ototoxicity mouse model was established. The toxicity of allylnitrile was limited through inhibition of CYP2E1 with trans-1,2-dichloroethylene (TDCE). The allylnitrile intoxication model has been extensively tested in the 129S1 mouse strain for vestibular function, which showed significant HC loss in the vestibular organ accompanied by severe behavioral abnormalities. However, the effect of allylnitrile on auditory function remains to be evaluated. Commonly used anesthetics to conduct hearing measurements are isoflurane and ketamine/xylazine anesthesia but the effect of these anesthetics on hearing assessment is still unknown. In this study we will evaluate the otovestibular effects of oral allylnitrile administration in mice. In addition, we will compare the influence of isoflurane and ketamine/xylazine anesthesia on hearing thresholds. Methods and Materials: Fourteen Coch+/- CBACa mice were randomly allocated into an allylnitrile (n = 8) and a control group (n = 6). Baseline measurements were done with isoflurane and 1 week later under ketamine/xylazine anesthesia. After baseline audiovestibular measurements, mice were co-administered with a single dose of allylnitrile and, to reduce systemic toxicity, three intraperitoneal injections of TDCE were given. Hearing loss was evaluated by recordings of auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE). Specific behavioral test batteries for vestibular function were used to assess alterations in vestibular function. Results: Hearing thresholds were significantly elevated when using isoflurane anesthesia compared to ketamine/xylazine anesthesia for all frequencies of the ABR and the mid-to-high frequencies in DPOAE. Allylnitrile-treated mice lacked detectable ABR thresholds at each frequency tested, while DPOAE thresholds were significantly elevated in the low-frequency region of the cochlea and completely lacking in the mid-to high frequency region. Vestibular function was not affected by allylnitrile administration. Conclusion: Isoflurane anesthesia has a negative confounding effect on the measurement of hearing thresholds in mice. A single oral dose of allylnitrile induced hearing loss but did not significantly alter vestibular function in mice. This is the first study to show that administration of allylnitrile can cause a complete loss of hearing function in mice.

On the Role of Fibrocytes and the Extracellular Matrix in the Physiology and Pathophysiology of the Spiral Ligament.

The spiral ligament in the cochlea has been suggested to play a significant role in the pathophysiology of different etiologies of strial hearing loss. Spiral ligament fibrocytes (SLFs), the main cell type in the lateral wall, are crucial in maintaining the endocochlear potential and regulating blood flow. SLF dysfunction can therefore cause cochlear dysfunction and thus hearing impairment. Recent studies have highlighted the role of SLFs in the immune response of the cochlea. In contrast to sensory cells in the inner ear, SLFs (more specifically type III fibrocytes) have also demonstrated the ability to regenerate after different types of trauma such as drug toxicity and noise. SLFs are responsible for producing proteins, such as collagen and cochlin, that create an adequate extracellular matrix to thrive in. Any dysfunction of SLFs or structural changes to the extracellular matrix can significantly impact hearing function. However, SLFs may prove useful in restoring hearing by their potential to regenerate cells in the spiral ligament.