CN115227869B - Artificial cochlea electrode with nerve stem cell transplanting and signal transmission enhancing functions and preparation method thereof - Google Patents
Artificial cochlea electrode with nerve stem cell transplanting and signal transmission enhancing functions and preparation method thereof Download PDFInfo
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- CN115227869B CN115227869B CN202210976866.0A CN202210976866A CN115227869B CN 115227869 B CN115227869 B CN 115227869B CN 202210976866 A CN202210976866 A CN 202210976866A CN 115227869 B CN115227869 B CN 115227869B
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an artificial cochlea electrode with nerve stem cell transplanting and signal transmission enhancing functions and a preparation method thereof. The preparation process comprises the following steps: 1) Adding phytic acid and aniline into the GO dispersion liquid in sequence, and polymerizing to obtain GO-PAni conductive filler; 2) Sequentially adding GO-PAni and nerve growth factor into the GelMA/photoinitiator solution, and loading nerve stem cells to obtain a gel electrode pre-solution; 3) Fixing an electrode substrate in a mould, pouring the gel electrode pre-solution in the step 2), and preparing the artificial cochlea electrode by using an ultraviolet curing process. The gel layer on the electrode surface of the artificial cochlea has low modulus, and can not cause damage to the cochlea when being implanted into the cochlea; the conductive component is favorable for signal transmission, has the function of transplanting the neural stem cells, and combines the nerve growth factor to promote the differentiation of the neural stem cells and the survival of residual acoustic nerves. The electrode can improve the number of auditory nerves and reduce the electrode resistance, so that the auditory experience of the artificial cochlea after implantation can be improved.
Description
Technical Field
The invention belongs to the field of artificial cochlea and hydrogel preparation, and particularly relates to an artificial cochlea electrode with nerve stem cell transplanting and signal transmission enhancing functions and a preparation method thereof.
Background
Hearing loss is an important sensory deficit, and places a heavy burden on individuals, families, and society. Cochlear implants have become the standard treatment for severe sensorineural hearing loss. The artificial cochlea converts external acoustic signals into electric signals, and the electric signals directly stimulate spiral ganglion cells through microelectrodes implanted in the scala tympani to transmit auditory information into the center. The residual spiral ganglion cells are small in number, the distance between the nerve fibers and the electrode is large, the spiral ganglion cells are damaged in the process of electrode implantation, fibrosis and ossification in the scala tympani caused by inflammatory reaction after implantation and the like are caused, and the hearing experience after the artificial cochlea is implanted is influenced by the resistance increase caused by the formation of fibrous tissue on the surface of the electrode. Currently, extensive researches such as intracochlear injection of stem cells have been developed aiming at improving the number of functional nerve cells, reducing electrode resistance, reducing electrode implantation damage and the like. There is still a lack of effective means to directly increase nerve cell numbers and promote their growth, differentiation, and formation of nerve fibers and synapses in the cochlea. Based on the prior researches, a novel artificial cochlea composite electrode based on biological materials can solve the problems.
Methylpropyl acylated gelatin (GelMA) has been used in the medical field for skin tissue engineering, bone repair, nerve injury repair, treatment of cardiovascular diseases, and the like. Because of the high permeability to oxygen and nutrient substances, the high water content and soft mechanical properties, the natural nerve cell growth agent is similar to the in-vivo nerve cell growth environment, and is beneficial to the nerve cell growth. And has RGD sequence of arginine-glycine-aspartic acid, which is favorable for cell adhesion and stem cell differentiation.
The conductive polymer has been widely proved in the aspects of promoting the growth of neurons and the formation of synapses, and the conductive hydrogel taking the conductive polymer as a raw material is used as an electrode coating, so that the electrode resistance can be reduced, the required electrical stimulation intensity is reduced, the cochlear injury is reduced, and the service life of the artificial cochlea is prolonged.
The composite electrode based on the methylpropyl acylated gelatin/graphene oxide-polyaniline (GelMA/GO-PAni) conductive hydrogel artificial cochlea can realize nerve cell transplantation, promote survival of transplanted cells and residual neurons, reduce electrode resistance, and prevent the flexible hydrogel from increasing intra-cochlear injury after implantation.
Disclosure of Invention
The invention aims to provide a GelMA/GO-PAni conductive hydrogel-based artificial cochlea electrode with nerve stem cell transplanting and signal transmission enhancing functions and a preparation method thereof, aiming at the problems that the number of functional auditory neurons is small after artificial cochlea implantation, and the electrode resistance is increased to influence hearing experience after implantation.
The technical scheme adopted by the invention is as follows:
the artificial cochlea electrode with the nerve stem cell transplanting and signal transmission enhancing functions is prepared by taking a platinum iridium electrode as a substrate, taking conductive hydrogel loaded with nerve stem cells and nerve growth factors as an electrode coating and adopting nano-composite, cell three-dimensional loading and ultraviolet curing processes; the conductive hydrogel is GelMA/GO-PAni hydrogel. The electrode utilizes GelMA to realize the transplantation of the neural stem cells, is favorable for the survival of the neural stem cells and reduces the intra-cochlear diffusion of the stem cells, and the combined nerve growth factors promote the differentiation of the neural stem cells and the survival of residual acoustic nerves, and utilizes the conductive GO-PAni nanofiller to reduce the electrode resistance and promote the growth and differentiation of the neural cells.
The preparation process comprises the following steps:
1) Preparing GO dispersion liquid with certain concentration under the condition of high-speed stirring, then sequentially dripping phytic acid and aniline monomers, and dispersing by using a triple dispersion process of intense stirring, vortex and ultrasonic for each raw material; then dropwise adding an initiator solution under the condition of intense stirring, performing free radical polymerization for 4-8 hours at normal temperature, dialyzing, drying to obtain GO-PAni conductive filler, and dispersing the GO-PAni conductive filler in deionized water to obtain dispersion liquid;
2) Under the condition of avoiding light, in a water bath at 60 ℃, gelMA is dissolved in a photoinitiator/PBS solution, after the solution is cooled to room temperature, a certain amount of the GO-PAni conductive filler prepared in the step 1) is added, and the GelMA/GO-PAni conductive filler is uniformly mixed under the double dispersion process of vortex and ultrasonic to obtain a GelMA/GO-PAni pre-solution;
3) Adding nerve growth factor into the GelMA/GO-PAni solution prepared in the step 2) under the light-shielding condition, fully and uniformly mixing under the vortex and ultrasonic double dispersion process, and centrifugally defoaming;
4) Adding the GelMA/GO-PAni pre-solution containing the nerve growth factor prepared in the step 3) into the neural stem cell sediment under the light-shielding condition, and fully and uniformly mixing to obtain a cell suspension;
5) Fixing the electrode substrate of the artificial cochlea in a mould, and rapidly transferring the cell suspension prepared in the step 4) into the mould under the light-proof condition, wherein the wavelength light source of 350-410nm is 20-40mW/cm 2 Irradiating for 15-40s, and demolding to obtain the artificial cochlea electrode.
In the technical scheme, further, the concentrations of GO, phytic acid, aniline and an initiator in the step 1) in the reaction solution are respectively 0.005-0.02wt%, 4-8wt%, 0.5-2wt%, 0.1-0.5wt%, and the balance of deionized water; the initiator is one or more of sodium persulfate, ammonium persulfate and potassium persulfate; the concentration of the obtained GO-PAni conductive filler dispersion liquid is 0.1-0.5wt%. GO is used as a substrate, and aniline is polymerized on the surface of the GO in situ, so that the problem of poor dispersibility of polyaniline is solved.
Further, the process of intense stirring, vortex and ultrasonic triple dispersion comprises the following steps: the stirring speed is 2000-3000 r/min, and the stirring time is 5-10min; vortex for 5-10min; ultrasonic power is 60-100W, and ultrasonic time is 5-10min; the double dispersion process comprises the following steps: vortex for 5-10min; the ultrasonic power is 60-100W, and the ultrasonic time is 5-10min. The dispersion process, especially the triple dispersion process, is adopted because GO is extremely easy to aggregate in an acid solution, and the dispersion effect is poor when a single dispersion process is adopted.
Further, the concentration of GelMA after dissolution in the step 2) is 5-10wt%, and the substitution degree of GelMA is 30-90%; the photoinitiator is Irgacure or Lithium phenyl (2, 4, 6-trimethylphenyl) phosphinate (LAP), the concentration is 0.1-0.5wt%, and the volume of the added GO-PAni dispersion liquid is 1-5% of the volume of the GelMA solution.
Further, the nerve growth factor added in the step 3) is one or more of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), and the concentration is 200-500ng/ml.
Further, the neural stem cell number in step 4) is 0.1-10×10 7 mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The preparation process of the neural stem cell sediment comprises the following steps: taking out cultured neural stem cells in a cell incubator, removing the culture medium, rinsing 1 time with 0.5-1ml phosphate buffer solution (DPBS) without calcium and magnesium ions, adding 0.35-0.65ml Accutase,37 ℃ for digestion for 1-3 minutes, stopping digestion with 3-5 times of DPBS, centrifuging at 1000-1500rpm for 3-5 minutes, and discarding the supernatant to obtain cell sediment.
Furthermore, in the step 5), the die is of a groove structure with an opening at the upper part, two side plates of the groove can be separated and detached from each other, the die is required to be disassembled before the electrode is placed, medical vaseline is uniformly coated on the inner side surface of the die, the coating process of the medical vaseline is one-way coating, the coating rate is 5-10mm/s, and each surface of the die is coated for 3 times; after demolding the samples were rinsed 3 times with PBS to elute the residual petrolatum from the surface.
Further, the inner dimension of the groove is attached to the electrode, the length is 18-22mm, the width is 0.4-1.0mm, and the height is 0.6-1.5mm.
Compared with the prior art, the invention has the following advantages:
1) The novel composite artificial cochlea electrode based on the composite conductive hydrogel is prepared by adopting a combined process of nano-composite, cell three-dimensional loading and ultraviolet curing. Compared with a clinically used platinum iridium electrode, the composite electrode organically combines the conductive hydrogel electrode with a platinum iridium electrode substrate, the platinum iridium electrode can efficiently and stably transmit signals, and the conductive hydrogel can further realize the transplantation of nerve stem cells and promote the growth and differentiation of the nerve stem cells in vivo on the basis of transmitting signals.
2) The artificial cochlea electrode takes GelMA/GO-PAni conductive hydrogel embedded with nerve stem cells and nerve growth factors as an electrode coating. The GelMA is used as a matrix for carrying cells and factors, so that the intracochlear transplantation of the neural stem cells can be realized, the diffusion of the stem cells is reduced, the provided three-dimensional culture environment can simulate the growth environment in the neural cells, the material exchange of the cells and the microenvironment where the cells are positioned is facilitated, the growth and differentiation of the neural cells are facilitated, and the embedded nerve growth factors can locally promote the differentiation of the neural stem cells and protect residual auditory nerve cells. The conductive polymer GO-PAni is used as the nano filler, so that the electrode resistance can be reduced, the required electric stimulation intensity is reduced, cochlear injury and fibrosis and ossification around the electrode caused by electric stimulation are reduced, and meanwhile, the service life of the artificial cochlea can be prolonged; the high dispersibility of the polymer can lead the polymer to be uniformly distributed in the hydrogel matrix, the stable conduction of the electrical signals of the artificial cochlea electrode in the hydrogel is realized; the conductive polymer may also promote nerve cell growth and synapse formation. Above, gelMA provides good nerve cell growth environment, local nerve growth factor, conductive polymer nanofiller, and electrical stimulation provided by electrode itself to promote growth and differentiation of transplanted nerve stem cells together, and protect residual acoustic nerve cells, or can solve the problem of poor hearing effect after artificial cochlea implantation due to lack of residual functional acoustic nerve cells in clinic at present, and can also be used for treating sensorineural deafness caused by acoustic neuropathy.
3) The GelMA/GO-PAni conductive hydrogel carrying stem cells is used as an artificial cochlea electrode composite material, has the advantages of excellent biocompatibility, cell carrying property, conductivity, flexibility, effective adhesion with a platinum iridium electrode and the like, and is synergistic, so that the composite conductive hydrogel is more suitable for the artificial cochlea electrode and is used for improving hearing experience after artificial cochlea implantation.
Drawings
FIG. 1 is a schematic cross-sectional view of a novel cochlear implant conductive hydrogel composite electrode prepared by the invention;
FIG. 2 is a schematic diagram of a mold used for casting the conductive hydrogel composite electrode, (a) a top view, (b) a side view;
Detailed Description
The invention will be further illustrated with reference to specific examples.
The artificial cochlea electrode with the neural stem cell transplanting and signal transmission enhancing functions based on GelMA/GO-PAni conductive hydrogel is prepared by a nano-composite, cell three-dimensional loading and ultraviolet curing process and comprises the following steps: 1) Under the action of a multiple dispersion process, sequentially adding phytic acid and aniline into Graphene Oxide (GO) dispersion liquid, and obtaining graphene oxide-polyaniline (GO-PAni) nano conductive functional filler through in-situ free radical polymerization; 2) Sequentially adding GO-PAni and nerve growth factor into the GelMA/photoinitiator solution under the condition of light shielding, and then using the solution to load nerve stem cells in a three-dimensional way to obtain a gel electrode pre-solution; 3) Fixing an artificial cochlea electrode substrate in a special die, pouring the gel electrode pre-solution in the step 2), and using an ultraviolet curing process to realize in-situ gelation of the solution to obtain the novel composite artificial cochlea electrode based on the composite conductive hydrogel. The gel layer on the electrode surface of the artificial cochlea has low modulus, and can not cause damage to the cochlea when being implanted into the cochlea; the conductive component is favorable for signal transmission, has the function of transplanting the neural stem cells, and combines the nerve growth factor to promote the differentiation of the neural stem cells and the survival of residual acoustic nerves. The artificial cochlea electrode can be used for improving the problem of small number of residual functional auditory nerves, reducing electrode resistance and improving auditory experience after artificial cochlea implantation.
Example 1:
1) Firstly, adding 1mg of GO into 10ml of deionized water, vigorously stirring for 10min at 2000 rpm, swirling for 10min, performing 100W ultrasonic dispersion for 10min to obtain GO solution, slowly dropwise adding 0.6g of phytic acid under 2000 rpm high-speed stirring, adding 0.1g of aniline, fully swirling after intense stirring consistent with the parameters, performing ultrasonic triple dispersion to obtain dispersion, and then adding 0.03g of ammonium persulfate under 2000 rpm high-speed stirring. Free radical polymerization is carried out for 4 hours under the condition of continuous and intense stirring, dialysis is carried out by a 8000 molecular weight dialysis bag, centrifugation and freeze-drying are carried out, GO-PAni is obtained, and 10ml of deionized water is added again for dispersion, thus obtaining GO-PAni dispersion.
2) 2ml of LAP (GelMA 5% by mass) was added to 0.1g of GelMA under dark conditions, and the mixture was dissolved in a water bath at 60℃for 15 minutes. After cooling to room temperature, 66ul of the GO-PAni dispersion liquid and 0.4ug of BDNF are added, and after 10min of vortex and 10min of 100W ultrasonic dispersion, the dispersion is uniform, and the GelMA/GO-PAni solution is obtained, and the bubbles are removed by centrifugation.
3) Taking good 4 th generation, and the number of cells in a 6-well plate is about 1×10 7 After washing 1 time with 1ml of DPBS, 0.5ml of Ackutase was added, and the mixture was digested in a 37℃cell incubator for 1 minute, and 1.5ml of DPBS was stopped, centrifuged at 1000rmp for 5 minutes, and the supernatant was discarded to obtain a cell pellet. And (3) re-suspending the cells with the GelMA/GO-PAni solution under the dark condition to obtain a cell suspension.
4) Disassembling a special die, uniformly and thinly coating medical Vaseline on the inner side surface of a groove of the die, unidirectional coating, wherein the coating speed is 8mm/s, each surface of the die is coated for 3 times, after the die is tightly combined, rapidly dripping a cell suspension into the die fixed with the artificial cochlea platinum iridium electrode under the light-shielding condition, and using 405nm and 30mW/cm 2 And irradiating for 15s by using an ultraviolet light source, and after demolding, flushing for 3 times by using PBS (phosphate buffer solution) to obtain the electrode coating of the artificial cochlea.
The compression strength of the electrode coating is 84.2kPa, the conductivity is 0.89S/m, the 14-day degradation rate is 63%, and after in-vitro nerve cell differentiation culture medium is partitioned for 1 week, immunofluorescence detection proves that the nerve cell differentiation is good.
Example 2
1) Firstly, adding 1mg of GO into 10ml of deionized water, vigorously stirring for 10min at 2000 rpm, swirling for 10min, performing 100W ultrasonic dispersion for 10min to obtain GO solution, slowly dropwise adding 0.6g of phytic acid under 2000 rpm high-speed stirring, adding 0.1g of aniline, fully swirling after intense stirring consistent with the parameters, performing ultrasonic triple dispersion to obtain dispersion, and then adding 0.03g of ammonium persulfate under 2000 rpm high-speed stirring. Free radical polymerization is carried out for 4 hours under the condition of continuous and intense stirring, dialysis is carried out by a 8000 molecular weight dialysis bag, centrifugation and freeze-drying are carried out, GO-PAni is obtained, and 10ml of deionized water is added again for dispersion, thus obtaining GO-PAni dispersion.
2) 2ml of LAP (GelMA mass percent 10%) was added to 0.2g of GelMA under dark conditions and dissolved in a water bath at 60℃for 15 min. After cooling to room temperature, 66ul of the GO-PAni dispersion liquid and 0.4ug of BDNF are added, and after 10min of vortex and 10min of 100W ultrasonic dispersion, the dispersion is uniform, and the GelMA/GO-PAni solution is obtained, and the bubbles are removed by centrifugation.
3) Taking good 4 th generation, and the number of cells in a 6-well plate is about 1×10 7 After washing 1 time with 1ml of DPBS, 0.5ml of Ackutase was added, and the mixture was digested in a 37℃cell incubator for 1 minute, and 1.5ml of DPBS was stopped, centrifuged at 1000rmp for 5 minutes, and the supernatant was discarded to obtain a cell pellet. And (3) re-suspending the cells with the GelMA/GO-PAni solution under the dark condition to obtain a cell suspension.
4) Disassembling a special die, uniformly and thinly coating medical Vaseline on the inner side surface of a groove of the die, unidirectional coating, wherein the coating speed is 8mm/s, each surface of the die is coated for 3 times, after the die is tightly combined, rapidly dripping a cell suspension into the die containing the artificial cochlea platinum iridium electrode under the light-shielding condition, and using 405nm and 30mW/cm 2 And irradiating for 15s by using an ultraviolet light source, and after demolding, flushing for 3 times by using PBS (phosphate buffer solution) to obtain the electrode coating of the artificial cochlea.
The hydrogel compressive strength increased, 92.3kPa, after increasing the gelma content compared to example 1; conductivity drops to 0.64S/m; after 14 days of degradation rate of 60%, in vitro nerve cell differentiation medium is divided into 1 week, immunofluorescence detection proves that nerve cell differentiation is good.
Example 3
1) Firstly, adding 1mg of GO into 10ml of deionized water, vigorously stirring for 10min at 2000 rpm, swirling for 10min, performing 100W ultrasonic dispersion for 10min to obtain GO solution, slowly dropwise adding 0.9g of phytic acid under 2000 rpm high-speed stirring, adding 0.2g of aniline, fully swirling after intense stirring consistent with the parameters, performing ultrasonic triple dispersion to obtain dispersion, and then adding 0.03g of ammonium persulfate under 2000 rpm high-speed stirring. Free radical polymerization is carried out for 4 hours under the condition of continuous and intense stirring, dialysis is carried out by a 8000 molecular weight dialysis bag, centrifugation and freeze-drying are carried out, GO-PAni is obtained, and 10ml of deionized water is added again for dispersion, thus obtaining GO-PAni dispersion.
2) 2ml of LAP (GelMA 5% by mass) was added to 0.1g of GelMA under dark conditions, and the mixture was dissolved in a water bath at 60℃for 15 minutes. After cooling to room temperature, 66ul of the GO-PAni dispersion liquid and 0.4ug of BDNF are added, and after 10min of vortex and 10min of 100W ultrasonic dispersion, the dispersion is uniform, and the GelMA/GO-PAni solution is obtained, and the bubbles are removed by centrifugation.
3) Taking good 4 th generation, and the number of cells in a 6-well plate is about 1×10 7 After washing 1 time with 1ml of DPBS, 0.5ml of Ackutase was added, and the mixture was digested in a 37℃cell incubator for 1 minute, and 1.5ml of DPBS was stopped, centrifuged at 1000rmp for 5 minutes, and the supernatant was discarded to obtain a cell pellet. And (3) re-suspending the cells with the GelMA/GO-PAni solution under the dark condition to obtain a cell suspension.
4) Disassembling a special die, uniformly and thinly coating medical Vaseline on the inner side surface of a groove of the die, unidirectional coating, wherein the coating speed is 8mm/s, each surface of the die is coated for 3 times, after the die is tightly combined, rapidly dripping a cell suspension into the die containing the artificial cochlea platinum iridium electrode under the light-shielding condition, and using 405nm and 30mW/cm 2 And irradiating for 15s by using an ultraviolet light source, and after demolding, flushing for 3 times by using PBS (phosphate buffer solution) to obtain the electrode coating of the artificial cochlea.
The compressive strength of the electrode coating increased after the aniline usage was increased, compared to example 1, to 87.4kPa; conductivity increase of 11.21S/m; after 14 days of degradation rate is 59%, in vitro nerve cell differentiation medium is divided into 1 week, immunofluorescence detection proves that nerve cell differentiation is good.
Example 4
1) Firstly, adding 1mg of GO into 10ml of deionized water, vigorously stirring for 10min at 2000 rpm, swirling for 10min, performing 100W ultrasonic dispersion for 10min to obtain GO solution, slowly dropwise adding 0.6g of phytic acid under 2000 rpm high-speed stirring, adding 0.1g of aniline, fully swirling after intense stirring consistent with the parameters, performing ultrasonic triple dispersion to obtain dispersion, and then adding 0.03g of ammonium persulfate under 2000 rpm high-speed stirring. Free radical polymerization is carried out for 4 hours under the condition of continuous and intense stirring, dialysis is carried out by a 8000 molecular weight dialysis bag, centrifugation and freeze-drying are carried out, GO-PAni is obtained, and 10ml of deionized water is added again for dispersion, thus obtaining GO-PAni dispersion.
2) 2ml of LAP (GelMA 5% by mass) was added to 0.1g of GelMA under dark conditions, and the mixture was dissolved in a water bath at 60℃for 15 minutes. After cooling to room temperature, 66ul of the GO-PAni dispersion liquid and 0.4ug of BDNF are added, and after 10min of vortex and 10min of 100W ultrasonic dispersion, the dispersion is uniform, and the GelMA/GO-PAni solution is obtained, and the bubbles are removed by centrifugation.
3) Taking good 4 th generation, and the number of cells in a 6-well plate is about 1×10 7 After washing 1 time with 1ml of DPBS, 0.5ml of Ackutase was added, and the mixture was digested in a 37℃cell incubator for 1 minute, and 1.5ml of DPBS was stopped, centrifuged at 1000rmp for 5 minutes, and the supernatant was discarded to obtain a cell pellet. And (3) re-suspending the cells with the GelMA/GO-PAni solution under the dark condition to obtain a cell suspension.
4) Disassembling a special die, uniformly and thinly coating medical Vaseline on the inner side surface of a groove of the die, unidirectional coating, wherein the coating speed is 8mm/s, each surface of the die is coated for 3 times, after the die is tightly combined, rapidly dripping a cell suspension into the die containing the artificial cochlea platinum iridium electrode under the light-shielding condition, and using 405nm and 30mW/cm 2 And irradiating for 40s by using an ultraviolet light source, and after demolding, flushing for 3 times by using PBS (phosphate buffer solution) to obtain the electrode coating of the artificial cochlea.
Compared with the example 1, the irradiation time of the light source in the ultraviolet curing process is increased, and the compression strength of the electrode coating is increased to be 101.3kPa; conductivity is 0.91S/m; the degradation rate is reduced to 56% after 14 days, and after 1 week in the in vitro nerve cell differentiation medium, immunofluorescence detection proves that the nerve cell differentiation is good.
Example 5
1) Firstly, adding 1mg of GO into 10ml of deionized water, vigorously stirring for 10min at 2000 rpm, swirling for 10min, performing 100W ultrasonic dispersion for 10min to obtain GO solution, slowly dropwise adding 0.6g of phytic acid under 2000 rpm high-speed stirring, adding 0.1g of aniline, fully swirling after intense stirring consistent with the parameters, performing ultrasonic triple dispersion to obtain dispersion, and then adding 0.03g of ammonium persulfate under 2000 rpm high-speed stirring. Under the condition of continuous and intense stirring, free radical polymerization is carried out for 4 hours, dialysis is carried out by a 8000 molecular weight dialysis bag, centrifugation and freeze-drying are carried out, GO-PAni is obtained, and 10ml of deionized water is added again for dispersion, thus obtaining GelMA/GO-PAni dispersion.
2) 2ml of LAP (GelMA 5% by mass) was added to 0.1g of GelMA under dark conditions, and the mixture was dissolved in a water bath at 60℃for 15 minutes. After cooling to room temperature, 80ul of the GO-PAni dispersion liquid and 0.4ug of BDNF are added, and after 10min of vortex and 10min of 100W ultrasonic dispersion, the dispersion is uniform, and the GelMA/GO-PAni solution is obtained, and the bubbles are removed by centrifugation.
3) Taking good 4 th generation, and the number of cells in a 6-well plate is about 1×10 7 After washing 1 time with 1ml of DPBS, 0.5ml of Ackutase was added, and the mixture was digested in a 37℃cell incubator for 1 minute, and 1.5ml of DPBS was stopped, centrifuged at 1000rmp for 5 minutes, and the supernatant was discarded to obtain a cell pellet. And (3) re-suspending the cells with the GelMA/GO-PAni solution under the dark condition to obtain a cell suspension.
4) Disassembling a special die, uniformly and thinly coating medical Vaseline on the inner side surface of a groove of the die, unidirectional coating, wherein the coating speed is 8mm/s, each surface of the die is coated for 3 times, after the die is tightly combined, rapidly dripping a cell suspension into the die containing the artificial cochlea platinum iridium electrode under the light-shielding condition, and using 405nm and 30mW/cm 2 And irradiating for 15s by using an ultraviolet light source, and after demolding, flushing for 3 times by using PBS (phosphate buffer solution) to obtain the electrode coating of the artificial cochlea.
The electrode coating had an increased compressive strength of 87.6kPa compared to example 1 with an increased amount of go-PAni dispersion; the conductivity is increased to 0.92S/m, the degradation rate is 60% after 14 days, and after the in vitro nerve cell differentiation culture medium is divided for 1 week, immunofluorescence detection proves that the nerve cell differentiation is good.
Claims (8)
1. The artificial cochlea electrode with the nerve stem cell transplanting and signal transmission enhancing functions is characterized in that: the artificial cochlea electrode takes a platinum iridium electrode as a substrate, takes conductive hydrogel loaded with nerve stem cells and nerve growth factors as an electrode coating, and is prepared through nano-composite, cell three-dimensional loading and ultraviolet curing processes; the conductive hydrogel is GelMA/GO-PAni hydrogel, the electrode utilizes GelMA to realize neural stem cell transplantation, thereby being beneficial to survival of the neural stem cells and reducing intra-cochlear diffusion of the stem cells, and the combined nerve growth factors promote differentiation of the neural stem cells and survival of residual auditory nerves, and the conductive GO-PAni nanofiller is utilized to reduce electrode resistance and promote growth and differentiation of the neural cells;
the preparation process comprises the following steps:
1) Preparing GO dispersion liquid with certain concentration under the condition of high-speed stirring, then sequentially dripping phytic acid and aniline monomers, and dispersing by using a triple dispersion process of intense stirring, vortex and ultrasonic for each raw material; then dropwise adding an initiator solution under the condition of intense stirring, performing free radical polymerization for 4-8 hours at normal temperature, dialyzing, drying to obtain GO-PAni conductive filler, and dispersing the GO-PAni conductive filler in deionized water to obtain dispersion liquid;
2) Under the condition of avoiding light, in a water bath at 60 ℃, gelMA is dissolved in a photoinitiator/PBS solution, after the solution is cooled to room temperature, a certain amount of GO-PAni conductive filler dispersion liquid prepared in the step 1) is added, and the mixture is uniformly mixed under the double dispersion process of vortex and ultrasonic to obtain GelMA/GO-PAni pre-solution;
3) Adding nerve growth factor into the GelMA/GO-PAni solution prepared in the step 2) under the light-shielding condition, fully and uniformly mixing under the vortex and ultrasonic double dispersion process, and centrifugally defoaming;
4) Adding the GelMA/GO-PAni pre-solution containing the nerve growth factor prepared in the step 3) into the neural stem cell sediment under the light-shielding condition, and fully and uniformly mixing to obtain a cell suspension;
5) Fixing the electrode substrate of the artificial cochlea in a mould, and rapidly transferring the cell suspension prepared in the step 4) into the mould under the light-proof condition, wherein the wavelength light source of 350-410nm is 20-40mW/cm 2 Irradiating for 15-40s, and demolding to obtain the artificial cochlea electrode.
2. The cochlear implant electrode with neural stem cell transplantation and signaling enhancement function according to claim 1, wherein: the concentration of GO, phytic acid, aniline and initiator in the step 1) in the reaction solution is respectively 0.005-0.02wt%, 4-8wt%, 0.5-2wt%, 0.1-0.5wt% and the balance deionized water; the initiator is one or more of sodium persulfate, ammonium persulfate and potassium persulfate; the concentration of the dispersion liquid of the GO-PAni conductive filler is 0.1-0.5-wt%.
3. The cochlear implant electrode with neural stem cell transplantation and signaling enhancement function according to claim 1, wherein: the process for triple dispersion by intense stirring, vortex and ultrasonic comprises the following steps: the stirring speed is 2000-3000 r/min, and the stirring time is 5-10min; vortex for 5-10min; ultrasonic power is 60-100W, and ultrasonic time is 5-10min; the double dispersion process comprises the following steps: vortex for 5-10min; the ultrasonic power is 60-100W, and the ultrasonic time is 5-10min.
4. The cochlear implant electrode with neural stem cell transplantation and signaling enhancement function according to claim 1, wherein: the concentration of GelMA in the step 2) is 5-10wt%, and the substitution degree of GelMA is 30-90%; the photoinitiator is phenyl (2, 4,6-trimethyl benzoyl) lithium phosphate (LAP) with the concentration of 0.1-0.5wt%, and the volume of the added GO-PAni dispersion liquid is 1-5% of the volume of the GelMA solution.
5. The cochlear implant electrode with neural stem cell transplantation and signaling enhancement function according to claim 1, wherein: the nerve growth factor added in the step 3) is one or more of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), and the concentration is 200-500ng/ml.
6. The cochlear implant electrode with neural stem cell transplantation and signaling enhancement function according to claim 1, wherein: step 4) the neural stem cell number is 0.1-10×10 7 mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The preparation process of the neural stem cell sediment comprises the following steps: taking out cultured neural stem cells in a cell incubator, removing the culture medium, rinsing 1 time with 0.5-1ml phosphate buffer solution (DPBS) without calcium and magnesium ions, adding 0.35-0.65ml Ackutase cell digestive juice, digesting for 1-3 minutes at 37 ℃, stopping digestion by 3-5 times volume of DPBS, centrifuging for 3-5 minutes at 1000-1500rpm, and discarding the supernatant to obtain cell sediment.
7. The cochlear implant electrode with neural stem cell transplantation and signaling enhancement function according to claim 1, wherein: in the step 5), the die is of a groove structure with an opening at the upper part, two side plates of the groove can be separated and detached from each other, the die is required to be disassembled before the electrode is placed, medical vaseline is uniformly coated on the inner side surface of the die, the coating process of the medical vaseline is one-way coating, the coating rate is 5-10mm/s, and each surface of the die is coated for 3 times; after demolding the samples were rinsed 3 times with PBS to elute the residual petrolatum from the surface.
8. The cochlear implant electrode with neural stem cell transplantation and signaling enhancement function of claim 7, wherein: the inner dimension of the groove is attached to the electrode, the length is 18-22mm, the width is 0.4-1.0mm, and the height is 0.6-1.5mm.
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