| Sensorineural hearing loss（SNHL）is caused by noise,ototoxic drugs,inflammation,aging and genetic mutations that result in the loss or lesion of sensory hair cells（HCs）or connected spiral ganglion neurons（SGNs）.The current clinical treatment for SNHL is a cochlear implant（CI）.However,the effectiveness of the CI depends on the degree of preservation of the SGNs,and the CI is ineffective in patients with severe neuronal damage or loss.SGNs are highly differentiated terminal cells that play an important role in auditory conduction,and damage to the neurons has been identified as a major cause of SNHL.Therefore,repairing or protecting damaged SGNs is an effective way to treat SNHL at present,and is an important research topic in the field of auditory regeneration.In recent years,neural tissue engineering has evolved rapidly and important advances have been made in the study of nerve cell injury and regeneration.However,the structure and function of the mammalian inner ear,including the human inner ear,is exceptionally complex,and neural tissue engineering rarely involves the regeneration of SGNs.In the inner ear,SGNs grow and extend from the modiolus to organ of Corti and establish synaptic connections with HCs.Therefore,the use of neural tissue engineering techniques to prepare bionic scaffolds that can mimic the microenvironment of neuronal and nerve fiber tissue in the Organ of Corti to guide neuronal cell growth is a key step to achieve SGNs regeneration.Electrospinning（ES）technology for the preparation of nanofiber scaffolds has been widely used in neural tissue engineering,especially the study of highly oriented nanofiber scaffolds for the regeneration of sciatic and spinal nerves is a current hot topic in neural tissue engineering application.The main component of the organ of Corti’s basement membrane is collagen IV.Gelatin is mainly derived from the denaturation of collagen in animals,which is highly biocompatible,biodegradable,non-immunogenic and has the same components and biological properties as collagen,and is widely used in tissue engineering.Previous studies have shown that poly（L-lactic acid）（PLLA）has a beneficial effect on the protection and repair of a variety of nerves,especially the oriented arrangement of PLLA fibers can guide the growth and extension of nerve fibers.In recent years,conductive polymers have been widely used in neural tissue engineering due to their good biocompatibility and ability to transmit electrical signals to contacting tissues.Among them,polyaniline（PANI）has shown good potential for neural repair in tissue engineering applications and can be used as a material for building bioactive scaffolds to support neural stem cell differentiation and neurite growth.The oriented composite nanofibrous scaffold composed of gelatin,PLLA and PANI can accurately mimic the organ of Corti’s basement membrane in terms of composition and structure,providing a suitable microenvironment for the growth and adhesion of SGNs.The main research objectives of this study are to establish the SGNs growth microenvironment in vitro,which can simulate the organ of Corti’s basement membrane in vivo.Electrospinning technology was selected to prepare oriented nanofiber composite scaffolds combined with inducing factors to comprehensively.Then to study the growth and differentiation of SGNs through the established model.The project will initially investigate the potential mechanism of induced SGNs regeneration,may be beneficial to find effective measures to protect and repair the damaged SGNs,and provide a new idea for mammalian auditory reconstruction.First,preparing composite oriented nanofiber scaffolds.The PLLA/Gelatin oriented composite nanofiber scaffolds doped with different ratios of PANI were prepared by electrospinning technique,and characterized by morphology,mechanical properties,hydrophilic properties,elemental composition and cytocompatibility analyses.The results showed that the scaffolds doped with 2%w/v PANI had the best characterization results and would to be used as the ideal scaffolds for subsequent studies.Second,evaluating the effects of oriented nanofibers on the growth of SGNs.We isolated SGNs from the modiolus cochlea of mice at 3 days of age and performed primary culture,and identified SGNs by immunofluorescence staining after 7 days of culture.The primary extracted spiral ganglion precursor cells were cultured in tissue culture plates,PLLA/Gelatin and 2%PANI/PLLA/Gelatin,respectively.The cell differentiation rate and neurites growth were analyzed by immunofluorescence staining after 1,4 and 7 days of culture.In addition,the fit of neurites to the oriented fiber axis was observed by SEM.The results showed that the SGNs grew along the fiber orientation and the angle between the neurites and the fiber axis was less than 15°.Compared with the control group,the length of neurites and the cell differentiation rate increased significantly with the increase of culture time,indicating that the 2%PANI/PLLA/Gelatin fiber scaffolds played a significant role in promoting the growth and differentiation of SGNs,which was conducive to cell adhesion and nerve fiber extension.Third,investigating the mechanism of the regeneration of SGNs.SGNs in injury group were given 50μmol/L neomycin for 48 h,and the protective intervention group was pretreated with induction cultures containing 50 ng/m L and 500 ng/m L NGF for 2 h for SGNs,followed by co-treatment with neomycin for 48 h.For the regenerative repair group,cultures containing 50 ng/m L and 500 ng/m L NGF were induced for 72 h after neomycin damage to SGNs,respectively.The damage and repair status of the cells were observed by immunofluorescence staining,and the expression of SGN-associated marker genes Tuj 1,NF200 and autophagy regulator BECN 1,PINK 1 were analyzed by quantitative PCR.The results showed that SGNs survival rate was significantly reduced after neomycin treatment,cell numbers decreased with the increasing culture time,and neurite growth was slow or stagnant.In the protective intervention group,the degree of cell injury was significantly reduced and the neurite grew normally.The length of neurite was significantly restored during SGNs regeneration induced with NGF.The results of gene analysis showed that the expression level of neuronal marker genes Tuj 1 and neurofilament protein NF 200 were significantly reduced by SGNs damage,while the expression of NF 200 was increased after NGF induction.Meanwhile,both neuronal marker genes were highly expressed under the protection of NGF.At the level of autophagy,cell damage resulted in the down regulation of two autophagy regulators,BECN 1 and PINK 1,which inhibited the autophagic process.However,activation or enhancement of cellular autophagy and improvement of SGNs damage under NGF protection and induced regeneration may be associated with timely removal of intracellular error proteins,reduction of apoptotic factor aggregation,and improvement of mitochondrial damage.The experimental results showed that NGF could well protect SGNs from neomycin induced damage,and may improve SGNs degenerative lesions and maintain neuroinvironmental homeostasis by activating or enhancing autophagy.In summary,the PANI/PLLA/Gelatin oriented nanofibrous scaffolds fabricated by electrospinning technique were suitable for mimic the microenvironment of the organ of Corti’s basement membrane.We used the simulation model to study the growth,repair and regeneration of SGNs in vitro.Our results showed that the oriented nanofiber scaffolds mimicking the organ of Corti’s basement membrane,which could promote the adhesion,growth and differentiation of SGNs,as well as play a good role in promoting and guiding the extension of neurites.In the meantime,we found that the oriented nanofiber scaffolds combined with NGF application could effectively inhibit the damage of neomycin,and further promoted to repair the damaged SGNs.Our studies may provide a successful model for the treatment of auditory SGNs damage,which is expected to be a feasible way for clinical therapeutics of SNHL in future. |
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