Ti₃C₂Tx-MXene Potentiates The Regeneration Of Functional Hair Cells In Lgr5-Positive Cochlea-Organoids And Its Mechanism

Background:Hearing is the sense that is perceived by sensory structures within the inner ear.The sensory epithelium is composed of mechanosensory hair cells and non-sensory supporting cells.Hair cells detect mechanical motions induced by physical motion or sound waves and produce graded responses in neurotransmitter release.Those signals are sent to the central nervous system（CNS）and form the sense of hearing.Once human cochlear hair cells are lost,they cannot regenerate in vivo.According to the hearing report of World Health Organization（WHO）,sensorineural hearing loss is currently one of the most frequent health problems,especially in the aged,and it is mainly caused by the degradation of cochlear hair cells.Cochlear stem cells have been discovered in the sensory epithelium of the postnatal mice,especially Lgr5+stem cells,which have abilities to proliferate and differentiate into hair cells.Cochlea-organoids（Cochlea-Orgs）can be formed through 3D in vitro expansion of Lgr5+progenitor cells,whereas the differentiation efficiency of hair cell in Cochlea-Orgs is not enough.In our previous work,we found that Ti₃C₂T_x-MXene film enhanced NSCs development,resulting in neuron differentiation and maturation.Other studies have demonstrated MXenes promote osteogenic differentiation of various stem cells.Therefore,the aim of this study was to investigate the effect of Ti₃C₂T_x-MXene on the development and maturation of Cochlea-Orgs,especially on hair cell regeneration.Methods:Herein,we constructed a 3D hydrogel culture system by incorporating an appropriate amount of Ti₃C₂T_x-MXene into Matrigel.Firstly,the properties of Ti₃C₂T_x-MXene were characterized by using XRD,XPS,Raman,SEM and TEM.The mechanical strength,electrical conductivity and hydrophilicity of MXene-Matrigel were detected to determine whether MXene enhanced properties of Matrigel.The toxicity of MXene to Cochlea-Orgs was detected in a dose-response manner.Then,EdU staining and CCK-8 assay were conducted to investigate whether MXene-Matrigel promoted the proliferation of Cochlea-Orgs.Immunofluorescence and q-PCR analysis were conducted to investigate whether MXene-Matrigel can promote the differentiation of Cochlea-Orgs that derived from stem cells of Atoh1-GFP mice.The differentially expressed genes of Cochlea-Orgs in the MXene group were detected by m RNA-seq,and the relationship between related signaling pathways and hair cell regeneration was analyzed.Using the whole-cell patch-clamp,the electrophysiological characteristics of regenerated hair cells in Cochlea-Orgs was recorded to evaluate the function and maturity of regenerated hair cells.Furthermore,agonists or inhibitors of related signaling pathways were used to elucidate the molecular mechanism of MXene potentiated differentiation by immunoblotting and immunofluorescence.Finally,a co-culture system between Cochlea-Orgs and modiolus were established to investigate whether MXene could facilitate synaptic reconstruction between regenerated hair cells and SNGs.Results:Through material characterization,it confirmed that synthetic MXene had typical surface morphology,elemental composition and crystal surface characteristics,indicating that MXene was successfully prepared.The physical and chemical properties of MXene-Matrigel were detected,and it was found that MXene improved various properties of Matrigel,including electrical conductivity and mechanical properties.To clarify the toxicity of MXene to Cochlea-Orgs,CCK-8 assay was performed and showed that MXene≤300μg/m L had good biocompatibility to Cochlea-Orgs.After 7-10 days of proliferation,EdU staining showed MXene-Matrigel did not affect the proportion of EdU+proliferating cells in Cochlea-Orgs or the formation rate of Cochlea-Orgs.q-PCR analysis illustrated that MXene-Matrigel did not significantly change the relative expression levels of some stem cell markers in Cochlea-Orgs,confirming MXene did not affect the proliferation of Cochlea-Orgs.After differentiation,the proportion of Atoh1+Cochlea-Orgs was significantly increased in the MXene-Matrigel group,suggesting that MXene promote the differentiation of organoid hair cells.The immunofluorescence results of Cochlea-Orgs showed that the proportion of Atoh1+newly formed hair cells and Myo7a+hair cells in Cochlea-Orgs of the MXene-Matrigel group were significantly raised,confirming that MXene enhanced the differentiation abilities of Cochlea-Orgs.In addition,morphology of regenerated hair cells in the MXene-Matrigel group seemed more mature with Myo7a and Phalloidin stanning when compared to the Matrigel group,particularly the hair bundles presented better organized structures.Taking together,it suggested that MXene also promote the maturation of regenerated hair cells.By m RNA-seq and q-PCR analysis,it was found that the hair cell differentiation and hair bundle related genes were significantly up-regulated in the MXene-Matrigel group,confirming that MXene could not only potentiate the regeneration of hair cells,but also accelerate the maturation of hair cells.The electrophysiological characteristics of regenerated hair cells were recorded by patch clamp,and it was found that the regenerated hair cells in the MXene-Matrigel group showed better electrophysiological properties when compared to the Matrigel group.Some characteristics of regenerated hair cells of the MXene-Matrigel group were even better than the native P2 mouse inner hair cells,proving the maturity of regenerated hair cells were at least comparable to native P2 mouse inner hair cells.Taking together,it was verified that MXene could promote the maturation of functional regenerated hair cells.GO analysis was conducted to find out the most significantly up-regulated GO items,and it was found that mTOR and other signaling pathways may be related to hair cell regeneration.The results of immunoblot and immunofluorescent demonstrated the protein levels of several important proteins in the mTOR signaling were significantly higher in the MXene-Matrigel group compared with the Matrigel group,indicating that MXene activated mTOR signaling of Cochlea-Orgs.To determine whether mTOR activity are crucial for hair cell formation,mTOR activity was potentiated or attenuated by agonist MHY1485 and antagonist Rapamycin,respectively.By immunoblotting and immunofluorescence,it was verified that mTOR signaling activating potentiated hair cell regeneration and mTOR signaling inhibition restrained hair cell regeneration.Taking together,it confirmed that mTOR signaling pathway is involved in the regulation of hair cell regeneration.The co-culture system of Cochlear-Orgs and cochlear modiolus was also successfully established in this work.The co-localization of synaptic markers PSD95 and CTBP2 was observed by immunofluorescence,confirming that MXene-Matrigel promoted spiral ganglion neurons（SGNs）innervation of regenerated hair cells,and facilitate the formation of synaptic-like contacts.Conclusion:The purpose of this thesis was to promote the regeneration of cochlear hair cells.Herein,we described a new method of promoting the regeneration of cochlear functional hair cells through incorporating of MXene,which potentiated the regeneration and maturity of cochlear organoid functional hair cells.Furthermore,this work revealed a new mechanism of promoting hair cell regeneration,elucidated the role of mTOR signaling pathway in the regulation of hair cell differentiation.In addition,we established a co-culture system of Cochlea-Orgs and modiolus,in which MXene-Matrigel facilitated innervation establishment and synapse formation between regenerated hair cells and SGNs in our co-culture system.This approach overcomes some limitations of the Matrigel-dependent culture system and greatly accelerates the application of nanomaterials in organoid development and research on therapies for hearing loss.