Electroacoustic Responsive Cochlea-on-a-Chip.

Organ-on-chips can highly simulate the complex physiological functions of organs, exhibiting broad application prospects in developmental research, disease simulation, as well as new drug research and development. However, there is still less concern about effectively constructing cochlea-on-chips. Here, a novel cochlear organoids-integrated conductive hydrogel biohybrid system with cochlear implant electroacoustic stimulation (EAS) for cochlea-on-a-chip construction and high-throughput drug screening, is presented. Benefiting from the superior biocompatibility and electrical property of conductive hydrogel, together with cochlear implant EAS, the inner ear progenitor cells can proliferate and spontaneously shape into spheres, finally forming cochlear organoids with good cell viability and structurally mature hair cells. By incorporating these progenitor cells-encapsulated hydrogels into a microfluidic-based cochlea-on-a-chip with culture chambers and a concentration gradient generator, a dynamic and high-throughput evaluation of inner ear disease-related drugs is demonstrated. These results indicate that the proposed cochlea-on-a-chip platform has great application potential in organoid cultivation and deafness drug evaluation.

Thermal Aging Degradation of High-Viscosity Asphalt Based on Rheological Methods.

With the acceleration of the construction of sponge cities in China, porous asphalt pavement (PA) is has been widely used. High-viscosity asphalt (HVA) is the core material in building PA because it has good rheology properties, which can provide good raveling and rutting resistance. However, due to the open-graded structure of PA, HVA was more susceptible to rapid aging, which significantly affects the durability of PA. To investigate the thermal aging effect on the rheological properties of self-modified HVA (SHVA), five types of asphalts were aged using a rolling thin film oven (RTFO) and pressure aging vessel (PAV). Then, rheological tests were adopted, such as temperature sweep test (TS), repeated creep and recovery test (RCR), and bending beam rheometer test (BBR). The results indicate that during the aging process, the oxidation-induced hardening effect of neat asphalt and the degradation-induced softening effect of the modifier changes the rheology properties of HVA significantly. As the aging progresses, the contribution of the modifiers of HVA to anti-aging performance is greatly reduced. At high temperatures, HVA demonstrates better anti-aging performance than conventional styrene-butadiene-styrene (SBS)-modified asphalt (Guo Chuang, GC). The change of the high-temperature rheological indices of the two HVA types (SHVA and TAFPACK-super HVA (TPS)) showed a smaller activation energy index (EAI), a more considerable viscous component of binder creep stiffness (Gv), and more minor accumulated stain (racc), indicating a more significant anti-short-term and long-term aging performance, which is beneficial to the high-temperature performance of asphalts. However, the changes in low-temperature rheological properties do not align with those in high-temperature rheological properties after long-term aging. The BBR test results reveal that TPS exhibits worse low-temperature performance than GC and SHVA. During the thermal aging process, the contribution rate of the modifiers in SHVA against RTFO and PAV aging is higher than that of the modifiers in TPS, which contributes to the superior anti-aging property. Overall, SHVA demonstrates the best anti-aging performance among the five asphalts tested.

Increased mitophagy protects cochlear hair cells from aminoglycoside-induced damage.

Aminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It is well known that damaged mitochondria are degraded by mitophagy, an important mitochondrial quality control system that maintains mitochondrial homeostasis and ensures cell survival. However, it is unclear whether dysregulation of mitophagy contributes to aminoglycoside-induced hair cell injury. In the current study, we found that PINK1-PRKN-mediated mitophagy was impaired in neomycin-treated hair cells. Our data suggested that mitochondrial recruitment of PRKN and phagophore recognition of damaged mitochondria during mitophagy were blocked following neomycin treatment. In addition, the degradation of damaged mitochondria by lysosomes was significantly decreased as indicated by the mitophagic flux reporter mt-mKeima. Moreover, we demonstrated that neomycin disrupted mitophagy through transcriptional inhibition of Pink1 expression, the key initiator of mitophagy. Moreover, we found that neomycin impaired mitophagy by inducing ATF3 expression. Importantly, treatment with a mitophagy activator could rescue neomycin-treated hair cells by increasing mitophagy, indicating that genetic modulation or drug intervention in mitophagy may have therapeutic potential for aminoglycoside-induced hearing loss.Abbreviations: AAV: adeno-associated virus; ABR: auditory brainstem response; ATF3: activating transcription factor 3; ATOH1/MATH1: atonal bHLH transcription factor 1; BafA1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; COX4I1/COXIV: cytochrome c oxidase subunit 4I1; CTBP2/RIBEYE: C-terminal binding protein 2; DFP: deferiprone; EGFP: enhanced green fluorescent protein; FOXO3: forkhead box O3; GRIA2/GLUR2: glutamate receptor, ionotropic, AMPA2 (alpha 2); HC: hair cell; HSPD1/HSP60: heat shock protein 1 (chaperonin); IHC: inner hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MYO7A: myosin VIIA; OPTN: optineurin; OMM: outer mitochondrial membrane; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RT-qPCR: real-time quantitative polymerase chain reaction; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; USP30: ubiquitin specific peptidase 30; XBP1: X-box binding protein 1.

3D Ti3C2Tx MXene-Matrigel with Electroacoustic Stimulation to Promote the Growth of Spiral Ganglion Neurons.

Cochlear implantation has become the most effective treatment method for patients with profound and total hearing loss. However, its therapeutic efficacy is dependent on the number and normal physiological function of cochlear implant-targeted spiral ganglion neurons (SGNs). Electrical stimulation can be used as an effective cue to regulate the morphology and function of excitatory cells. Therefore, it is important to develop an efficient cochlear implant electroacoustic stimulation (EAS) system to study the behavior of SGNs. In this work, we present an electrical stimulation system constructed by combining a cochlear implant and a conductive Ti3C2Tx MXene-matrigel hydrogel. SGNs were cultured in the Ti3C2Tx MXene-matrigel hydrogel and exposed to electrical stimulation transduced by the cochlear implant. It was demonstrated that low-frequency stimulation promoted the growth cone development and neurite outgrowth of SGNs as well as signal transmission between cells. This work may have potential value for the clinical application of the Ti3C2Tx MXene hydrogel to optimize the postoperative listening effect of cochlear implantation and benefit people with sensorineural hearing loss.

2D Ti3C2TxMXene couples electrical stimulation to promote proliferation and neural differentiation of neural stem cells.

Preclinical studies involving stem cells require efficient physiochemical regulations on the fate of such cells. Because of their unique planar structure, metallic conductivity, and flexible surface functionalization, MXenes show potential for modulating stem cell fate. Here, the Ti3C2TxMXenenanosheets are dispersed on tissue culture polystyrene (TCPS). When primary mouse neural stem cells (NSCs) are cultured on laminin-coated Ti3C2TxMXene film, they form stable adhesion, retain their proliferative ability, and show extensive spreading of terminal extensions. With respect to their functional activity, NSCs cultured on Ti3C2TxMXene films form more active and synchronous network activity than those cultured on TCPS substrates. Moreover, Ti3C2TxMXene film significantly promotes the neural differentiation and the neurons have longer neurites and greater numbers of branch points and branch tips. NSC-derived neurons grown on the Ti3C2Tx MXene film preserved normal synapse development. Finally, electrical stimulation coupled with Ti3C2TxMXene film significantly enhances the proliferation of NSCs. These results indicate that Ti3C2TxMXene is an efficient interface for the proliferation and neural differentiation of NSC and the maturation of NSC-derived neurons, which expands the potential uses of the MXene family of materials and provides new strategies for stem cell studies. STATEMENT OF SIGNIFICANCE: The 2DTi3C2TxMXenenanosheets were applied to be an interface for regulating neural stem cells (NSCs). NSCs cultured on Ti3C2TxMXene film possessed higher proliferative ability with higher and more synchronous electrical activities. Moreover, Ti3C2TxMXene film significantly promoted the neural differentiation ratio of NSCs, and the neurons derived from NSCs cultured on Ti3C2TxMXene film had longer neurites and greater numbers of branch points and branch tips.When electrical stimulation was applied to NSCs via the Ti3C2TxMXene film, it significantly enhanced the proliferation of NSCs. This work expands the potential uses of the MXene family of materials and provides new strategies for stem cell studies.

Cochlear implant-based electric-acoustic stimulation modulates neural stem cell-derived neural regeneration.

Cochlear implantation is considered to be the best therapeutic method for profound sensorineural hearing loss, but insufficient numbers of functional spiral ganglion neurons hinder the clinical effects of cochlear implantation. Stem cell transplantation has the potential to provide novel strategies for spiral ganglion neuron regeneration after injury. However, some obstacles still need to be overcome, such as low survival and uncontrolled differentiation. Several novel technologies show promise for modulating neural stem cell behaviors to address these issues. Here, a device capable of electrical stimulation was designed by combining a cochlear implant with a graphene substrate. Neural stem cells (NSCs) were cultured on the graphene substrate and subjected to electrical stimulation transduced from sound waves detected by the cochlear implant. Cell behaviors were studied, and this device showed good biocompatibility for NSCs. More importantly, electric-acoustic stimulation with higher frequencies and amplitudes induced NSC death and apoptosis, and electric-acoustic stimulation could promote NSCs to proliferate and differentiate into neurons only when low-frequency stimulation was supplied. The present study provides experimental evidence for understanding the regulatory role of electric-acoustic stimulation on NSCs and highlights the potentials of the above-mentioned device in stem cell therapy for hearing loss treatment.

Neurite Extension and Orientation of Spiral Ganglion Neurons Can Be Directed by Superparamagnetic Iron Oxide Nanoparticles in a Magnetic Field.

INTRODUCTION: Neuroregeneration is a major challenge in neuroscience for treating degenerative diseases and for repairing injured nerves. Numerous studies have shown the importance of physical stimulation for neuronal growth and development, and here we report an approach for the physical guidance of neuron orientation and neurite growth using superparamagnetic iron oxide (SPIO) nanoparticles and magnetic fields (MFs). METHODS: SPIO nanoparticles were synthesized by classic chemical co-precipitation methods and then characterized by transmission electron microscope, dynamic light scattering, and vibrating sample magnetometer. The cytotoxicity of the prepared SPIO nanoparticles and MF was determined using CCK-8 assay and LIVE/DEAD assay. The immunofluorescence images were captured by a laser scanning confocal microscopy. Cell migration was evaluated using the wound healing assay. RESULTS: The prepared SPIO nanoparticles showed a narrow size distribution, low cytotoxicity, and superparamagnetism. SPIO nanoparticles coated with poly-L-lysine could be internalized by spiral ganglion neurons (SGNs) and showed no cytotoxicity at concentrations less than 300 µg/mL. The neurite extension of SGNs was promoted after internalizing SPIO nanoparticles with or without an external MF, and this might be due to the promotion of growth cone development. It was also confirmed that SPIO can regulate cell migration and can direct neurite outgrowth in SGNs preferentially along the direction imposed by an external MF. CONCLUSION: Our results provide a fundamental understanding of the regulation of cell behaviors under physical cues and suggest alternative treatments for sensorineural hearing loss caused by the degeneration of SGNs.

Fiber-Reinforcing Effect in the Mechanical and Road Performance of Cement-Emulsified Asphalt Mixtures.

Cement-emulsified asphalt mixture (CEAM), a kind of cold mix asphalt mixture, has the advantages of energy conservation and emission reduction as well as easy construction. However, the performance of CEAM is not as good as hot mixed asphalt mixtures. Hence, in this study, two different fibers were adopted as the reinforcing phase to improve the comprehensive properties of CEAM. The results indicated that the addition proportion and curing time were crucial to fiber-reinforced cement-emulsified asphalt mixture (FRCEAM). The compressive strengths, water stability, and raveling resistances of FRCEAM preparations with polyester or brucite fibers (FRCEAM-PF and -BF, respectively) were enhanced significantly. FRCEAM-PF had the maximum flexural tensile strength and strain, which meant that its low-temperature performance was the best compared to FRCEAM-PF and CEAM. However, the contribution of PF to CEAM high-temperature stability was greater than that of BF. Fiber addition to CEAM not only enhanced the cycles of fatigue loading but also reduced sensitivity to changes in stress level. Furthermore, FRCEAM-BF durability was slightly better than that of FRCEAM-PF. SEM analysis indicated that fibers provided bridging and meshing effects. Although PF and BF showed different enhancement effects, both mixtures met the requirements for hot mixed asphalt mixtures.

Biomimetic 3D bacterial cellulose-graphene foam hybrid scaffold regulates neural stem cell proliferation and differentiation.

Neural stem cell (NSC)-based therapy is a promising candidate for treating neurodegenerative diseases and the preclinical researches call an urgent need for regulating the growth and differentiation of such cells. The recognition that three-dimensional culture has the potential to be a biologically significant system has stimulated an extraordinary impetus for scientific researches in tissue engineering and regenerative medicine. Here, A novel scaffold for culturing NSCs, three-dimensional bacterial cellulose-graphene foam (3D-BC/G), which was prepared via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam has been reported. 3D-BC/G not only supports NSC growth and adhesion, but also maintains NSC stemness and enhances their proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G induces NSCs to selectively differentiate into neurons, forming a neural network in a short amount of time. The scaffold has good biocompatibility with primary cortical neurons enhancing the neuronal network activities. To explore the underlying mechanisms, RNA-Seq analysis to identify genes and signaling pathways was performed and it suggests that 3D-BC/G offers a more promising three-dimensional conductive substrate for NSC research and neural tissue engineering, and the repertoire of gene expression serves as a basis for further studies to better understand NSC biology.

Superparamagnetic Iron Oxide Nanoparticles and Static Magnetic Field Regulate Neural Stem Cell Proliferation.

Neural stem cells (NSCs) transplantation is a promising approach for the treatment of various neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIOs) are reported to modulate stem cell behaviors and are used for medical imaging. However, the detailed effects of SPIOs under the presence of static magnetic field (SMF) on NSCs are not well elucidated. In this study, it was found that SPIOs could enter the cells within 24 h, while they were mainly distributed in the lysosomes. SPIO exhibited good adhesion and excellent biocompatibility at concentrations below 500 µg/ml. In addition, SPIOs were able to promote NSC proliferation in the absence of SMF. In contrast, the high intensity of SMF (145 ± 10 mT) inhibited the expansion ability of NSCs. Our results demonstrate that SPIOs with SMF could promote NSC proliferation, which could have profound significance for tissue engineering and regenerative medicine for SPIO applications.

Transcriptomic profiling of neural stem cell differentiation on graphene substrates.

Graphene exhibits excellent mechanical strength, electrical conductivity and good biocompatibility, which make it a suitable candidate as a neural interfacing material in regenerative medicine and tissue engineering. Graphene is reported to promote both of neural stem cells (NSCs) proliferation and differentiation. However, the transcriptomes of 2D graphene-regulated NSC differentiation have not yet been investigated. To identify candidate genes, on which graphene may affect, we used next-generation RNA sequencing to analyze the transcriptome of NSCs differentiated for 21 days on a graphene substrate. These NSCs displayed highly enriched and differentially expressed genes compared with traditional cell culture in vitro. Of these, we identified motor protein genes that might regulate NSC differentiation, including cytoplasmic dynein and axonemal dynein genes, Ccdc108, Dnah5, and Dnah11. Furthermore, we analyzed the cell signaling pathway genes that might regulate NSC differentiation, and we constructed a protein-protein interaction network for the genes that are differentially expressed in NSCs on graphene compared to commercial tissue culture polystyrene substrates. We have identified genes potentially regulating the differentiation and migration of NSCs on graphene substrates, and our findings provide mechanistic evidence for the biological activities of graphene, especially in view of graphene-stem cell interactions.

Deletion of Limk1 and Limk2 in mice does not alter cochlear development or auditory function.

Inherited hearing loss is associated with gene mutations that result in sensory hair cell (HC) malfunction. HC structure is defined by the cytoskeleton, which is mainly composed of actin filaments and actin-binding partners. LIM motif-containing protein kinases (LIMKs) are the primary regulators of actin dynamics and consist of two members: LIMK1 and LIMK2. Actin arrangement is directly involved in the regulation of cytoskeletal structure and the maturation of synapses in the central nervous system, and LIMKs are involved in structural plasticity by controlling the activation of the actin depolymerization protein cofilin in the olfactory system and in the hippocampus. However, the expression pattern and the role of LIMKs in mouse cochlear development and synapse function also need to be further studied. We show here that the Limk genes are expressed in the mouse cochlea. We examined the morphology and the afferent synapse densities of HCs and measured the auditory function in Limk1 and Limk2 double knockout (DKO) mice. We found that the loss of Limk1 and Limk2 did not appear to affect the overall development of the cochlea, including the number of HCs and the structure of hair bundles. There were no significant differences in auditory thresholds between DKO mice and wild-type littermates. However, the expression of p-cofilin in the DKO mice was significantly decreased. Additionally, no significant differences were found in the number or distribution of ribbon synapses between the DKO and wild-type mice. In summary, our data suggest that the Limk genes play a different role in the development of the cochlea compared to their role in the central nervous system.

Two-Photon Image Tracking of Neural Stem Cells via Iridium Complexes Encapsulated in Polymeric Nanospheres.

Iridium(III) complexes have been shown to be promising probes in two-photon imaging to real-time track the transplanted cells in stem-cell-based therapy. Here, we report on polymeric nanocapsules loaded with red phosphorescence dye of bis(2-methyldibenzo[f,h]quinoxaline) (acetylacetonate) iridium(III) (Ir(MDQ)2acac) with excellent stability created by the double emulsion method. The Ir(MDQ)2acac nanocapsules present high biocompatibility and an efficient fluorescent labeling rate when incubated with cultured mouse neural stem cells (NSCs). More importantly, the Ir(MDQ)2acac nanocapsules had both one- and two-photon imaging properties with stable phosphorescence lasting for 72 h. Furthermore, data from in vivo tracking in nude mice demonstrated that the photoluminescence from Ir(MDQ)2acac nanocapsules in NSCs could be stably monitored for up to 21 days. Our data shed light on the potential clinical application of iridium complexes encapsulated in polymeric nanospheres for two-photon imaging in real-time tracking of the transplanted stem cells.

Development and Application of Cochlear Implant-Based Electric-Acoustic Stimulation of Spiral Ganglion Neurons.

Cochlear implants are currently the most effective treatment for profound sensorineural hearing loss. However, their therapeutic effect is limited by the survival and proper physiological function of spiral ganglion neurons (SGNs), which are targeted by the cochlear implant. It is therefore critical to explore the mechanism behind the effect of electric-acoustic stimulation (EAS) on the targeted SGNs. In this work, a biocompatible cochlear implant/graphene EAS system was created by combining a cochlear implant to provide the electrically transformed sound stimulation with graphene as the conductive neural interface. SGNs were cultured on the graphene and exposed to EAS from the cochlear implant. Neurite extension of SGNs was accelerated with long-term stimulation, which might contribute to the development of growth cones. Our system allows us to study the effects of cochlear implants on SGNs in a low-cost and time-saving way, and this might provide profound insights into the use of cochlear implants and thus be of benefit to the populations suffering from sensorineural hearing loss.