Bioactive artificial cochlea electrode based on stem cells and preparation method thereof

Abstract

The invention relates to the technical field of auditory sense and neuroscience, and particularly discloses a stem cell-based bioactive artificial cochlea electrode and a preparation method thereof, and the stem cell-based bioactive artificial cochlea electrode comprises an artificial cochlea electrode completely or partially formed by stem cell combined I-type collagen conductive hydrogel; a conductive material is wrapped in the I-type collagen conductive hydrogel; stem cells are wrapped in the type I collagen conductive hydrogel. The hydrogel is implanted into the cochlea through the round window, and the stem cells in the hydrogel effectively regenerate functional auditory neurons under the electroacoustic stimulation of the artificial cochlea, so that the auditory function is recovered. The bioactive artificial cochlea electrode based on the conductive material takes the conductive material and the I-type collagen as raw materials, is easy to obtain, low in cost, safe and non-toxic, and combines factors for promoting nerve regeneration by a three-dimensional nanotechnology, directional induction and electroacoustic stimulation.

Description

Technical Field

[0001] The present invention relates to the fields of hearing and neuroscience, and in particular to a stem cell-based bioactive cochlear implant electrode and a preparation method thereof. Background Art

[0002] Hearing impairment is a common ear condition and has become a global health issue with significant sociopolitical and economic implications. With the aging population, the abuse of ototoxic drugs, noise, and environmental pollution, the number of people suffering from deafness and hearing loss is increasing year by year. Sensorineural hearing loss is a common type of hearing loss, and patients often experience difficulty spontaneously regenerating spiral ganglion neurons. Cochlear implantation is an effective treatment for sensorineural hearing loss. However, the number and function of remaining spiral ganglion neurons are key factors in determining the feasibility of cochlear implantation and the effectiveness of postoperative recovery. Preserving and restoring the function and number of spiral ganglion neurons remains a key challenge. Furthermore, in the presence of background noise, the extensive electric field generated by the considerable distance between the cochlear implant electrode and the spiral ganglion neurons can adversely affect speech perception. The gap between the electrode and inner ear neurons hinders optimal electrical stimulation of the cochlear implant. The electrode contact point is surrounded by lymphatic vessels within the tympanic membrane, preventing sufficient bridging with the SGNs. When physical contact is established, a lower current can reach the threshold for stimulating adjacent auditory neurons.

[0003] Biohybrid implants, with their excellent biocompatibility, enhanced attraction to hearing-related cells, and ability to reduce chronic inflammation, make them very suitable for addressing this challenge. Studies have shown that neurons implanted with living electrodes can remain at the implant site, with axons extending and terminating at different locations, thereby establishing a dynamic and stable interaction between the device and the tissue, ultimately improving the effectiveness of the cochlear implant. Stem cell transplantation plays an important role in the treatment of degenerative neuropathy. Many studies have shown that exogenous cells can survive in the inner ear environment and migrate to functionally relevant areas. However, inner ear cell regeneration through cell transplantation faces considerable challenges in terms of survival, migration, and differentiation efficiency. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the present invention proposes a bioactive cochlear implant electrode based on stem cells and a preparation method thereof.

[0005] The purpose of the present invention can be achieved by adopting the following technical solutions:

[0006] A bioactive cochlear implant electrode, comprising a cochlear implant electrode composed entirely or partially of stem cells combined with type I collagen conductive hydrogel;

[0007] The type I collagen conductive hydrogel is wrapped with a conductive material;

[0008] Stem cells are encapsulated in the type I collagen conductive hydrogel.

[0009] Optionally, the cochlear implant electrode can generate electrical signals to promote stem cells to differentiate into neurons.

[0010] Optionally, the stem cells are neural stem cells, mesenchymal stem cells, iPSCs, ESCs, including cell spheres and organoids differentiated therefrom.

[0011] Optionally, the conductive material is one or more of PEDOT:PSS, MXenes, and graphene.

[0012] A method for preparing a bioactive cochlear implant electrode comprises the following steps:

[0013] Step 1: resuspending the conductive material in PBS and diluting it to obtain a diluted conductive material solution; then adding it to a neutralizing reagent to obtain a conductive material neutralizing reagent solution;

[0014] Step 2: The obtained conductive material neutralization reagent solution is mixed with a type I collagen stock solution on ice to obtain a type I collagen conductive hydrogel system. The in vitro cultured stem cells are then added to the type I collagen conductive hydrogel system. After dilution, the conductive material is cross-linked with the type I collagen stock solution to obtain a stem cell-loaded type I collagen conductive hydrogel.

[0015] Step 3: Wrap the type I collagen conductive hydrogel loaded with stem cells on the surface of the cochlear implant electrode to finally obtain a bioactive cochlear implant electrode.

[0016] Optionally, in step 1, the volume ratio of the conductive material solution to the neutralizing agent is 1:9.

[0017] Optionally, in step 2, the ratio of the conductive material neutralizing reagent solution to the type I collagen stock solution is 1:9; and the final concentration of the conductive material is 100 μg / mL.

[0018] Optionally, the cross-linking conditions in step 2 are: temperature 37° C., time 20 to 30 minutes.

[0019] Optionally, the neutralizing agent is sodium bicarbonate.

[0020] The application of bioactive cochlear implant electrodes in the manufacture of products for treating hearing loss.

[0021] Beneficial effects of the present invention:

[0022] (1) The present invention uses a composite conductive hydrogel with excellent biocompatibility and conductivity as a raw material to construct a bioactive cochlear implant electrode. The three-dimensional environment provided by this conductive hydrogel is conducive to the growth, adhesion, and differentiation of stem cells. Combined with the cochlear implant electrode, it promotes the generation of new neurons.

[0023] (2) The present invention uses a conductive hydrogel with a three-dimensional porous structure to prepare a bioactive electrode without using complex and expensive raw materials and technologies, which greatly reduces time and economic costs.

[0024] (3) The bioactive electrode designed by the present invention, which combines a cochlear implant with stem cell transplantation, has a simple operation method. The entire system has good flexibility, adhesion, conductivity, and biocompatibility, and has good repeatability. The present invention has great application potential in the treatment of sensorineural hearing loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below with reference to the accompanying drawings.

[0026] Figure 1 Schematic diagram of the process for fabricating bioactive cochlear implant electrodes according to some examples of the present application;

[0027] Figure 2 Transmission electron microscope images of conductive PEDOT:PSS used in some examples of this application;

[0028] Figure 3 Scanning electron microscope images of conductive PEDOT:PSS collagen hydrogels in some examples of this application;

[0029] Figure 4 Scanning electron microscope images of neural stem cells adhering and growing in conductive PEDOT:PSS collagen hydrogels in some examples of this application;

[0030] Figure 5 The biocompatibility of conductive PEDOT:PSS collagen hydrogels was evaluated in some examples of this application. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. The following embodiments only belong to some cases involved in the present invention, not all implementation cases. Based on the implementation methods and implementation cases designed in the present invention, all non-creative work embodiments of other persons are within the scope of protection of the present invention.

[0032] Example 1

[0033] like Figure 1FIG2 is a schematic diagram of a stem cell-based bioactive cochlear implant electrode and a preparation method thereof. The bioactive cochlear implant electrode is realized by the following steps:

[0034] The conductive material was resuspended in PBS and diluted to an initial concentration of 10 mg / mL to obtain a diluted conductive material solution. The transmission electron microscope image of the conductive material example PEDOT:PSS is shown in FIG. Figure 2 As shown. The conductive material can also be selected from one or more of PEDOT:PSS, MXenes, and graphene. In this embodiment, PEDOT:PSS is selected. PBS (phosphate buffered saline) is a buffer system widely used in biochemical research. Its core function is to maintain solution pH stability and promote conductivity.

[0035] The diluted conductive material solution was mixed with a neutralizing agent at a volume ratio of 1:9 to obtain a conductive material neutralizing agent solution, wherein the neutralizing agent was 2.2 g / L sodium bicarbonate. The conductive material neutralizing agent solution was then mixed with a type I collagen stock solution at a volume ratio of 1:9 on ice to obtain a final concentration of the conductive material of 100 μg / mL, thereby obtaining a type I collagen conductive hydrogel system. The scanning electron microscope image is shown in FIG. Figure 3 As shown. The neural stem cells cultured in vitro were added to the above-mentioned type I collagen conductive hydrogel system. At a set temperature, the diluted conductive material was cross-linked with the type I collagen stock solution. The cross-linking conditions were: temperature 37°C, time 20 to 30 minutes, and the type I collagen conductive hydrogel loaded with neural stem cells was obtained. Figure 4 The scanning electron microscope images shown show that neural stem cells adhere to and grow in the conductive PEDOT:PSS collagen hydrogel.

[0036] The surface of the cochlear implant electrode is wrapped with type I collagen conductive hydrogel loaded with neural stem cells, and the cochlear implant is implanted to finally obtain a bioactive cochlear implant electrode, that is, the cochlear implant electrode has bioactivity, and the overall cross-section is as follows Figure 1 As shown. The neural stem cells in the type I collagen conductive hydrogel of the present invention can differentiate into neurons under electrical stimulation, that is, the neural stem cells in the hydrogel effectively regenerate functional auditory neurons under the electrical and acoustic stimulation of the cochlear implant, thereby establishing connections between neurons, such as Figure 1 As shown in .

[0037] Example 2

[0038] Validation object / sample: mouse

[0039] Pre-treatment methods for test samples: in vitro culture of neural stem cells, preoperative auditory brainstem response (ABR) of mice, mouse nerve injury model, post-model auditory brainstem response of mice, and preoperative auditory brainstem response of mice.

[0040] Experimental steps and results: Neural stem cells were extracted from hippocampal tissue of E18-19 mice in pre-cooled PBS. After culturing for 3 days, they were passaged and digested into single cells for inoculation after another 3 days. The inoculation density was 10 6 pieces / cm 2 . Through the above steps, a bioactive cochlear implant electrode was made. Anesthesia was performed with 1% sodium pentobarbital at a dose of 10 mg / kg. The ABR recording electrode, reference electrode and ground electrode were placed on the top of the skull, behind the test ear and behind the contralateral ear of the guinea pig respectively. The TDT system was used to collect ABR signals at preoperative sound frequencies of 4, 8, 12, 16, 24 and 32 kHz. The skin behind the mouse's ear was incised, the muscle tissue was peeled off, and the round window was exposed. 8 μL of 1 mM ouabain was injected into the round window niche to create a nerve injury model. ABR signals were collected again 3 days later to obtain nerve injury mice. The nerve injury mice were randomly divided into 3 groups, the round window was exposed again, and then sham surgery (no implant, only the round window was exposed), simple electrode implantation, and bioactive electrode implantation were performed respectively. The muscle tissue and skin of the mice were sutured with 8-0 sutures. ABR signals were collected again 14 days after surgery. The results showed that the hearing of mice in the sham operation group and the simple electrode implantation group did not recover, while the hearing of mice in the bioactive electrode implantation group recovered.

[0041] Example 3

[0042] Figure 5 The results showed that mouse neural stem cells were loaded into collagen hydrogel and conductive hydrogel respectively by the above method, and the cells were detected by dead-alive staining ( Figure 5 A) and CCK8 assay ( Figure 5 B) Evaluation of the effect of the conductive hydrogel system on the activity of neural stem cells. The experimental results showed that almost no dead cells were observed after live-death staining ( Figure 5 A), there was no significant difference in cell activity between the two groups ( Figure 5 B, the vertical axis is cell activity, and the horizontal axis is the control group and the experimental group). Therefore, the conductive hydrogel system exemplified above has good biocompatibility with mouse neural stem cells.

[0043] Unless otherwise specified, the reagents and instruments used in the present invention can be obtained from the market.

[0044] It is understood that in this embodiment, the above technical effects can be achieved by wrapping the conductive hydrogel loaded with neural stem cells on the surface of the cochlear implant electrode. Therefore, adding other materials or cells to the entire system will not affect the realization of the above effects.

[0045] Throughout this specification, references to "one embodiment," "example," "specific example," and other reference terms indicate that the specific materials, structures, features, or characteristics described in that example or embodiment encompass at least one example or embodiment of the present invention. The specific materials, structures, features, or characteristics exemplarily described in these terms may be combined in any suitable manner in one or more examples or embodiments.

[0046] The above content explains the basic principles, main features and advantages of the present invention. Researchers or technicians in related fields should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are merely based on the ideas and principles of the present invention. Based on the original basis of the present invention, various changes and improvements will be made to the present invention in the future, and these changes and improvements are all within the scope of the invention claimed.

Claims

1. A bioactive cochlear implant electrode, characterized in that: Cochlear implant electrodes comprising in whole or in part stem cells combined with type I collagen conductive hydrogel; The type I collagen conductive hydrogel is wrapped with a conductive material; Stem cells are encapsulated in the type I collagen conductive hydrogel.

2. The bioactive cochlear implant electrode according to claim 1, characterized in that: The cochlear implant electrode can generate electrical signals to promote the differentiation of stem cells into neurons.

3. The bioactive cochlear implant electrode according to claim 1, characterized in that: The stem cells are neural stem cells, mesenchymal stem cells, iPSCs, ESCs, including cell spheres and organoids differentiated therefrom.

4. The bioactive cochlear implant electrode according to claim 1, wherein: The conductive material is one or more of PEDOT:PSS, MXenes, and graphene.

5. The method for preparing the bioactive cochlear implant electrode according to claims 1 to 3, characterized in that: The following steps are involved: Step 1: resuspending the conductive material in PBS and diluting it to obtain a diluted conductive material solution; then adding it to a neutralizing reagent to obtain a conductive material neutralizing reagent solution; Step 2: The obtained conductive material neutralization reagent solution is mixed with a type I collagen stock solution on ice to obtain a type I collagen conductive hydrogel system. The in vitro cultured stem cells are then added to the type I collagen conductive hydrogel system. After dilution, the conductive material is cross-linked with the type I collagen stock solution to obtain a stem cell-loaded type I collagen conductive hydrogel. Step 3: Wrap the type I collagen conductive hydrogel loaded with stem cells on the surface of the cochlear implant electrode to finally obtain a bioactive cochlear implant electrode.

6. The method for preparing a bioactive cochlear implant electrode according to claim 5, wherein: In the step 1, the volume ratio of the conductive material solution to the neutralizing agent is 1:

9.

7. The method for preparing a bioactive cochlear implant electrode according to claim 5, wherein: In the step 2, the ratio of the conductive material neutralizing reagent solution to the type I collagen stock solution is 1:9; the final concentration of the conductive material is 100 μg / mL.

8. The method for preparing a bioactive cochlear implant electrode according to claim 5, wherein: The cross-linking conditions in step 2 are: temperature 37° C., time 20 to 30 minutes.

9. The method for preparing a bioactive cochlear implant electrode according to claim 5, wherein: The neutralizing agent is sodium bicarbonate.

10. Use of the bioactive cochlear implant electrode according to any one of claims 1 to 9 in the manufacture of a product for treating hearing loss.

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