Construction Of Organ Of Corti Organoids To Study The Protective Mechanism Of Auditory Cell Injury

Around 200 million people currently suffer from varying degrees of hearing loss in China.The main cause of hearing loss is the non-spontaneous regeneration of the hair cells or the spiral ganglion neurons in the mammalian inner ear.Previous studies have shown that the organ of Corti and the attached basilar membrane in the cochlea of the mammalian inner ear are extremely fine three-dimensional(3D)structure composed of collagen,laminin and proteoglycan.The physiological anatomy of the mammalian cortical apparatus is so deep in the head and its loss cannot be accomplished by surgical repair or tissue engineering techniques such as transplanting a replacement.Therefore,if we can construct organ of Corti organoids in vitro which are very similar in structure and performance to those in vivo,and then study the injury and protective effect of auditory cells,it will open up a new idea for the clinical treatment of deafness in the future.In this study,gelatin(Gel),sodium alginate(SA)and polyvinyl alcohol(PVA)were used to prepare composite bioinks(PVA/Gel/SA).Combined with 3D bioprinting technology,the biomimetic 3D scaffolds were constructed,and organ of Corti of neonatal mice separated in situ were implanted into the 3D scaffolds for culture,and then organ of Corti organoids were constructed in vitro to simulate the microenvironment in vivo.The organ of Corti organoids were used to study the protection and intervention of auditory hair cells and spiral ganglion cells.Finally,a mouse animal model was constructed to study the protective effect of berberine(BBR)in the process of auditory cell injury and its possible mechanism,which could provide experimental basis for realizing the regeneration of auditory cells and exploring new ideas for the treatment of hearing loss.The main research contents include the following aspects:(1)Preparation and characterization of PVA/Gel/SA bionic 3D scaffolds.By adjusting the ratio of PVA in Gel/SA bio-ink,the best PVA content was selected.Scanning electron microscope(SEM)results showed that the aperture of the bionic 3D scaffold is 150 μm to 200μm,which is suitable for cell growth;PVA/Gel/SA bionic 3D scaffold has good biocompatibility and no cytotoxicity;The mechanical properties showed that the mechanical properties of PVA/Gel/SA biomimetic 3D scaffold were better when the content of PVA was 1-2%.The results of degradation experiment showed that the degradation of scaffold was slowest when the content of PVA was 1%.In view of the above experimental results,the 1%PVA/Gel/SA bionic 3D scaffold with good comprehensive performance was finally selected for follow-up experiments.(2)The protective effect of hair cell injury was studied based on the constructed organ of Corti organoids.Cochlear organ of Corti with the associated basement membrane tissues of neonatal Institute of Cancer Research(ICR)mice were cultured on PVA/Gel/SA biomimetic3 D scaffold to construct organ of Corti organoids simulating the microenvironment of organ of Corti in vivo.After 5 days of continuous culture with organ of Corti organoids,the growth behavior of hair cells was observed by immunofluorescence staining and SEM.The results showed that the 3D structure of organ of Corti organoid was similar to that of cochlear organ of Corti of normal mice,and the number and morphology of hair cells cultured on the organ of Corti organoid were not significantly different from that of the control group.Then,the injury effects of neomycin on hair cells and the protection of BBR on cell damage were studied based on the organ of Corti organoids.Preliminary experimental results showed that the application of BBR could reduce neomycin-induced hair cell damage.(3)Protective effect of BBR on neomycin-induced auditory cell injury.Firstly,neomycin was used to establish hair cell damage model in vitro,and then 16 8-day-old ICR mice were divided into 4 groups,and each group was injected intraperitoneally with neomycin(200mg/kg),BBR(50 mg/kg)and neomycin(200 mg/kg),BBR(50 mg/kg)and the normal saline for 7 days.Immunofluorescence staining,q PCR,DCFH-DA staining and TUNEL staining showed that BBR could reduce neomycin induced hair cell damage and apoptosis by activating PI3K/AKT signaling pathway,and reduce ROS levels in hair cells.In vivo experiments also showed that BBR could reduce ototoxicity induced by neomycin.To sum up,the damage,intervention and protection of auditory cells were studied by constructing organ of Corti organoids similar to the microenvironment of the organ of Corti in vivo.The organ of Corti organoids can well simulate the structure of the organ of Corti in the cochlea of mammals,and it is expected to be a multifunctional model to study the damage,protection and regeneration of auditory hair cells.BBR,as an antioxidant and antiinflammatory drug,can significantly reduce neomycin-induced auditory cell damage,and BBR is expected to be an effective drug for hearing loss in the future.