A multi-channel cell-laden nerve repair catheter with a double-patterned structure and its preparation method

Abstract

The present invention discloses a multi-channel cell-loaded nerve repair catheter with a double-patterned structure and a preparation method thereof. The preparation method comprises: ① preparing conductive fibers by wet spinning and in-situ surface polymerization to form conductive fiber bundles; ② designing a casting mold; ③ coating a release agent on the inside of the mold, and fixing the prepared fiber bundle in the mold, adding a UV-curable polymer solution to the mold, and curing it into a gel; ④ after the gel is aged, demoulding; after removing the surface release agent, using an immersion method to introduce a protein / polypeptide material that can promote cell adhesion on the surface of the nerve catheter, drying and sterilizing to obtain a multi-channel nerve repair catheter material with a double-patterned structure; combining the co-culture technology of neural stem cells, after the cells adhere and migrate in a directional manner in the tube, combining the pulsed direct current stimulation technology to achieve neural differentiation, and it can be used to replace autologous nerve transplant materials. The catheter material can be used in the fields of nerve and spinal cord repair.

Description

Technical Field

[0001] The present invention relates to the field of nerve repair materials, and in particular to a multi-channel cell-laden nerve repair conduit with a double-patterned structure and a preparation method thereof. Background Art

[0002] Peripheral nerve injury (PNI) is a common clinical disease caused by traffic accidents, iatrogenic injuries, falls, and other causes. According to statistics, approximately 5 million patients worldwide will suffer from peripheral nerve injury each year. Currently, for defects smaller than a few millimeters, direct suture surgery is generally used, while for defects with larger gaps, autologous transplantation has become the gold standard for clinical treatment. However, issues such as donor sources and the risk of morbidity at the donor site have limited the further development of autologous transplantation.

[0003] In recent years, with the rapid advancement of tissue engineering, artificial nerve guidance conduits (NGCs) have been developed as an alternative to autologous nerve grafts. The development of NGCs has primarily focused on three aspects: raw materials, structure, and function. Regarding raw materials, the first generation of NGCs was made from inert, non-degradable biomaterials such as silicone. However, the need for secondary surgical removal led to their gradual replacement by bioresorbable materials such as polylactide and type I collagen. Regarding structure, early NGCs primarily had a single-channel structure, but comparative studies have shown that multi-channel structures can promote reinnervation. Regarding function, traditional nerve guidance conduits lack various active functions and serve merely as a filling medium to connect damaged nerves. Functionalities such as promoting cell adhesion, cell proliferation, and cell differentiation into neurons are lacking, and have gradually gained momentum in recent years. Despite the rapid advancement of nerve repair conduit materials, nerve repair is an extremely complex process, leading to a growing demand for comprehensive and all-round development of nerve guidance conduits.

[0004] Based on the above problems, the present invention introduces a dual patterned structure and combines technologies or structures such as fiber reinforcement, cell loading, cell adhesion promotion, and electrical stimulation to provide a highly integrated and efficient nerve repair strategy through the synergy of technology, structure and function, targeting the actual physiological repair process of nerves. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the existing technology and combine the advantages of various materials and processes to provide a multi-channel cell-laden nerve repair catheter with a double-patterned structure and a preparation method thereof.

[0006] The present invention is achieved by adopting the following technical solutions:

[0007] A method for preparing a multi-channel cell-laden nerve repair conduit with a double-patterned structure comprises the following steps:

[0008] 1) preparing a chitosan / sodium alginate mixed spinning solution, preparing fiber filaments by a continuous multi-step wet spinning method, immersing the fiber filaments in an aniline / phytic acid solution for in-situ surface polymerization to prepare conductive fibers, and forming conductive fiber bundles by a twisting process;

[0009] 2) Designing a casting mold capable of imparting a dual-patterned and multi-channel structure to a nerve repair conduit that promotes directional migration of neural stem cells; the mold can provide the nerve repair conduit with multiple channels, with the surface of the nerve repair conduit and the surfaces of each channel being patterned;

[0010] 3) applying a release agent to the interior of the mold, arranging the fiber bundles prepared in step 1) longitudinally and fixing them in the mold, adding a UV-curable polymer solution from an injection port on the mold, curing the solution to form a gel having a double network structure, and then curing the solution with UV light;

[0011] 4) After the gel solidifies, it is demolded using a freeze-thaw cycle; after removing the surface release agent through a rapid rinse-dialysis process, a protein / peptide material that promotes cell adhesion is introduced into the surface of the nerve conduit by an impregnation method. After low-temperature airflow drying and irradiation sterilization, the gel conduit is obtained, thereby obtaining a multi-channel nerve repair conduit material with a dual-patterned structure;

[0012] 5) Combined with the co-culture experiment of neural stem cells, after the cells adhere and migrate in a directional manner in the tube, they are combined with pulsed direct current stimulation technology to achieve neural differentiation, which can be used to replace autologous neural transplant materials.

[0013] Furthermore, in step 1), chitosan is dissolved in an acetic acid aqueous solution containing urea, glycerol and sodium ethoxide to prepare a chitosan solution, and then sodium alginate powder is slowly added and stirred to completely dissolve to obtain the desired chitosan / sodium alginate mixed spinning solution, wherein the concentration of chitosan is 2-5wt%, the molecular weight of chitosan is 1200000-2000000, the degree of deacetylation is 70-80%, and the concentration of the acetic acid aqueous solution is 2-4wt%; the viscosity of sodium alginate (10g / L, 20°C) is 0.02-0.1Pa·s, and the concentration is 0.1-0.5wt%; the amount of urea added is 2-3wt%, the amount of glycerol added is 2-5wt%, and the amount of sodium ethoxide added is 0.5-1wt%; the stirring rate during the preparation of the above spinning solution is 400-600rpm / min, and the stirring time is 2-4 hours.

[0014] Furthermore, in step 1), the spinneret parameters during the wet spinning process are: spinning flow rate 40-80 mL / h, spinning temperature 30-45°C. The fiber-forming stage mainly passes through 7 solutions: a single coagulation bath, a single cleaning solution, a double cleaning solution, a double coagulation bath, a triple cleaning solution, and a quadruple cleaning solution. The single coagulation bath is 3-5wt% NaOH or KOH, 0.2-0.5wt% Na2SO4 dissolved in ethanol / water solution (1:1 v:v); the double coagulation bath is 5-10wt% calcium chloride aqueous solution; the cleaning solutions are all deionized water; the coagulation bath temperature is 20-50°C, and the cleaning solution temperature is 40-50°C.

[0015] Furthermore, in step 1), the conductive fiber preparation process is as follows: immersing the prepared fiber filaments in an aniline / phytic acid solution containing 0.01-0.03 g / ml of aniline, 0.06-0.12 g / ml of phytic acid, and 0.002-0.01 g / ml of an initiator; wherein the initiator is one of potassium persulfate, sodium persulfate, and ammonium persulfate, and then in situ self-polymerizing under shaking conditions at room temperature for 2-4 hours;

[0016] Furthermore, in step 2), the mold is a hollow cylindrical structure composed of two halves, and has a removable bottom cover and top cover. The inner wall of the hollow column is provided with a plurality of axially parallel micron-scale grooves; the bottom cover of the mold is provided with a plurality of upright posts, each of which has a plurality of axially parallel micron-scale grooves on its outer surface; the upper and bottom covers are provided with corresponding small holes for the conductive fiber bundle to pass through, and the upper cover is provided with a liquid injection port. The mold is preferably made of a transparent material.

[0017] Furthermore, the mold is preferably made of PMMA, cylindrical in shape, with a longitudinal groove structure on the inner surface, a groove width of 100-200 μm, a height of 80-150 μm, and a spacing of 100-200 μm between the grooves; the inside of the mold is arranged with small cylindrical structures with a diameter of 400-500 μm, and the columns are evenly arranged with a spacing of 800-1000 μm. The surface also has the same longitudinal groove structure as the inner surface of the mold; a liquid injection port is provided on the edge of the upper cover of the mold; at the same time, the upper cover of the mold has small holes with a diameter of 200-600 μm and a spacing of 800-1000 μm, and the bottom cover of the mold is provided with small holes of the same number and position as the upper cover, and the conductive fiber bundle passes through the holes on the upper and bottom covers of the mold, and the ends are exposed for fixed arrangement and subsequent application; a sealing gasket can be provided under the bottom cover of the mold to prevent leakage during solution casting;

[0018] Furthermore, in step 3), the release agent may be one of glycerol and ethylene glycol; the polymer in the UV-curable polymer solution may be two of GelMA, Alg-MA, and Sil-MA, with a total concentration of 10-20 wt% and a usage ratio of 1:3-3:1; the photoinitiator may be one or more of LAP, I2959, and Irgacure, with a concentration of 0.25-2.5 wt%; the UV curing wavelength is 405 nm, the power is 5-20 W, and the curing time is 3-5 min;

[0019] Furthermore, in step 4), the freeze-thaw cycle process is as follows: place the mold in a -80°C freezer, freeze for 5-10 minutes, then remove and thaw naturally for 10-15 minutes; repeat the freeze-thaw steps 3-5 times. Use PBS solution pre-cooled to 4°C as the rinsing and dialyzing fluid. Rinse the gel 3-5 times, then place it in PBS solution, changing the solution every 30-60 minutes, and repeat 5-8 times. The adhesive protein can be one or more of Fibronectin, Laminin, ColⅢ, etc., with a concentration of 0.1-0.5wt%. The immersion time is 1-2 hours, and after the immersion is completed, rinse with PBS.

[0020] Furthermore, in step 5), the neural stem cells of P2-P4 and the gel conduit were placed in a low-adhesion plate and co-cultured for 2-3 days, with the cell density in the culture medium being 1-2*10 5 / ml, and then connected to the two ends of the conductive fiber bundle of the gel catheter with a wire, and pulsed direct current stimulation was applied to it, with a voltage of 1-3V, a pulse direction of positive and negative pulses, a pulse width of 100-300ms, and a pulse frequency of 3-10Hz; the stimulation lasted for 30-60min / day, and a total of 7-14 days.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1) The present invention uses UV-curable polymers, conductive polymers, adhesion-promoting proteins / peptides, etc. as raw materials, and a two-stage patterned mold as a tool. At the same time, it combines casting method, template method, in-situ UV curing process, dipping method, cell loading, electrical stimulation and other combined processes to prepare a multi-channel cell-loaded nerve repair catheter material with a dual-patterned structure. This raw material selection and process combination to prepare the nerve repair catheter material is the first of its kind in the present invention.

[0023] 2) The present invention innovatively designs and prepares a two-stage patterned transparent casting mold for the preparation of the nerve repair catheter. First, the inner wall of the mold is provided with axially parallel grooves at the micron level, which serves as a mold for patterning the outer surface of the nerve catheter (single patterning); secondly, the interior of the mold is provided with columns arranged regularly according to the drawing, and the outer surface of the columns is also provided with axially parallel grooves at the micron level. The columns not only provide the possibility for the preparation of the nerve catheter channel, but also introduce a patterned structure (double patterning) into the nerve catheter channel. At the same time, when the mold is transparent and the side of the cover plate is provided with an injection port, the reason why the injection port is provided on the side of the cover plate is that the polymer solution can flow down along the mold wall when injected into the mold, reducing the generation of bubbles and defects (at the same time, the transparency of the mold can observe whether there are bubbles inside), ensuring the integrity of the material structure, and the transparent mold can ensure the feasibility of in-situ UV curing of the polymer solution in the mold.

[0024] 3) The present invention uses the above mold to prepare a multi-channel cell-loaded nerve repair conduit material with a double-patterned structure. The material has a multiple composite structure: wherein the nerve conduit is provided with a regularly arranged channel structure, which can provide space for the growth of cells in the conduit and also provides the possibility for the exchange of cell substances (oxygen, nutrients, waste, etc.). Moreover, the inner surface of the channel and the outer surface of the conduit are simultaneously provided with a longitudinally parallel micron-scale groove structure, which has the function of inducing longitudinal migration of cells and can promote the migration and growth of cells on the inner and outer surfaces of the nerve conduit. At the same time, a conductive fiber bundle structure is provided inside. On the one hand, the fiber bundle structure can provide a reinforcement effect on the nerve conduit matrix material. Through the mechanical engagement between the rough structure on the surface of the fiber bundle and the polymer matrix, a good physical bond between the fiber bundle and the matrix is ​​achieved, and then the matrix is ​​reinforced by the pulling effect of the fiber bundle; and the conductive effect of the fiber bundle can serve as a "transmission chain" for the later electrical stimulation effect. After the electrical stimulation is applied at both ends of the nerve conduit, it can act on the cells through the conductive fiber bundle to achieve the induction of cell neural differentiation. The above-mentioned structural designs are all obtained through organic and synergistic combinations based on the actual needs of nerve repair, realizing the innovative application and functional amplification of traditional structures.

[0025] 4) The multi-channel cell-laden nerve repair conduit with a double-patterned structure prepared in the present invention has the functions of promoting cell adhesion on the surface, inducing cell growth into the nerve conduit channel, promoting cell differentiation into neurons, and being biodegradable. First, the purpose of inducing neural stem cells to adhere to the nerve conduit is achieved by having proteins / peptides on the surface that promote cell adhesion; secondly, the adhered cells can be induced to grow longitudinally on the surface or in the pores of the nerve conduit by the induction of the patterned structure; then, the conductive polymer fiber bundle and electrical stimulation are used to induce the differentiation of neural stem cells into neurons; finally, the cell-laden and fully differentiated nerve conduit material is connected to the broken and damaged nerves, and it is expected that the synergy of the above functions can achieve efficient repair of nerve damage.

[0026] 5) The present invention innovatively completes the in vitro construction of "human nerves" by introducing stem cells into nerve conduits, inducing stem cell migration, and using electrical stimulation to induce stem cells to differentiate into neurons in vitro. This provides an efficient "substitute" for autologous transplantation to repair nerve damage and addresses actual clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Conductive fiber SEM;

[0028] Figure 2 3D schematic diagram of an example structure of a mold for preparing a multi-channel cell-laden nerve repair conduit with a double patterning structure;

[0029] Figure 3 3D schematic diagram of a multi-channel cell-laden nerve repair conduit with a double-patterned structure;

[0030] Figure 4 Schematic diagram of the operation of a multi-channel cell-laden nerve repair conduit with a dual-patterned structure combined with electrical stimulation technology to promote neural differentiation;

[0031] Figure 5 PCR data of neural stem cell differentiation induced by multi-channel cell-laden nerve repair conduits with a double-patterned structure combined with electrical stimulation (day 7, Group C represents Comparative Example 1, Group E represents Example 1). DETAILED DESCRIPTION

[0032] The technical solutions and effects of the present invention are further described below with reference to the accompanying drawings and specific examples.

[0033] The preparation process of the multi-channel cell-loaded nerve repair conduit with a double patterned structure of the present invention is as follows: Figure 4As shown, the mold is a hollow cylindrical structure composed of two halves, with a removable bottom cover and top cover. The inner wall of the hollow column is provided with several axially parallel micron-scale grooves. The bottom cover is provided with several uprights, each of which has several axially parallel micron-scale grooves on its outer surface. The top and bottom covers are provided with corresponding small holes for the conductive fiber bundle to pass through, and the top cover is provided with a liquid injection port. The mold is preferably made of a transparent material. Figure 2 This is a schematic diagram of a structural portion of a mold used in one embodiment of the present invention. In other embodiments, other mold structures that meet the above requirements may also be used.

[0034] Comparative Example 1

[0035] 1) Design a casting mold that can endow a neural repair conduit with a dual-patterned, multi-channel structure that promotes the directional migration of neural stem cells. The mold is a hollow cylinder made of PMMA (composed of two halves) with a longitudinal groove structure on the inner surface. The grooves are 200 μm wide and 150 μm high, with a spacing of 200 μm between the grooves. The mold is internally arranged with small cylindrical structures with a diameter of 500 μm, evenly spaced 1000 μm apart, and the surface also has the same longitudinal groove structure as the inner surface of the mold. The mold has a removable bottom cover and top cover, and the edge of the top cover is provided with a liquid injection port.

[0036] 2) Coat the inside of the mold with 0.1 ml of glycerol. Add a mixed solution of GelMA and Sil-MA (w:w = 1:1) containing 0.25% LAP through the mold's injection port to a total concentration of 10 wt %. Then, cure with 405 nm 10 W UV for 5 min to form a gel with a double network structure.

[0037] 3) After the gel solidifies, the mold is placed in a -80°C freezer and quick-frozen for 10 minutes, then removed and naturally thawed for 10 minutes; the freeze-thaw step is repeated three times, followed by demolding; the gel is then rinsed three times with a PBS solution precooled to 4°C, and then placed in a PBS solution, changing the solution every 30 minutes, and repeated eight times to remove excess release agent; the gel is then immersed in a 0.1 wt% Fibronectin solution for 2 hours, a protein material that promotes cell adhesion is introduced onto the surface, and the gel is dried in a low-temperature airflow and sterilized by irradiation to obtain a multi-channel nerve repair conduit material with a dual-patterned structure;

[0038] 4) Use P3 neural stem cells with a cell density of 2*10 5 The cells were co-cultured with nerve conduit gel in low-adhesion well plates for 3 days. After the cells adhered and migrated in the tube, they were differentiated for 14 days.

[0039] The conductivity of the composite nerve conduit hydrogel obtained in this comparative example was 0.09 S / m, and the degradation rate in vitro after 30 days was 72%. After 14 days of co-culture with neural stem cells, Confocal imaging of live and dead cell fluorescence staining showed that the proportion of live cells was 94%, and some cells could grow into the nerve conduit. Due to the lack of the introduction of conductive fibers and pulsed direct current stimulation, the neural stem cells had poor neuronal differentiation (the PCR data are shown in Figure 2). Figure 5 Confocal imaging of NF200 immunofluorescence staining showed a relative fluorescence area of ​​9%.

[0040] Comparative Example 2

[0041] 1) 5 wt% chitosan with a molecular weight of 1,200,000 and a degree of deacetylation of 80% is dissolved in a 3% acetic acid solution containing 3 wt% urea, 3 wt% glycerol, and 1 wt% sodium ethoxide to prepare a chitosan solution. Sodium alginate powder is then slowly added and stirred until completely dissolved. The viscosity of the sodium alginate (10 g / L, 20°C) is 0.1 Pa·s, and the concentration is 0.5 wt%. This yields the desired chitosan / sodium alginate mixed spinning solution. Conductive fibers are then prepared using a continuous multi-step wet spinning method and in-situ surface polymerization. The continuous multi-step wet spinning method parameters are as follows: a spinning flow rate of 50 mL / h and a spinning temperature of 40°C. The fiber-forming stage primarily involves seven solutions: a single coagulation bath, a single rinse, a double rinse, a double coagulation bath, a triple rinse, and a quadruple rinse. The first coagulation bath is 5wt% NaOH and 0.5wt% Na2SO4 dissolved in ethanol / water solution (1:1 v:v); the second coagulation bath is 5wt% calcium chloride aqueous solution; the cleaning solution is deionized water; the coagulation bath temperature is 40°C, and the cleaning solution temperature is 50°C; the prepared fiber filaments are immersed in an aniline / phytic acid solution, containing 0.02g / ml aniline, 0.1g / ml phytic acid, and 0.01g / ml initiator; the initiator is potassium persulfate, and then in situ self-polymerization is carried out under shaking conditions at room temperature for 4h; after cleaning and drying, the conductive fiber bundles are formed by twisting process;

[0042] 2) Design a casting mold that can endow the nerve repair conduit with a dual-patterned and multi-channel structure that promotes the directional migration of neural stem cells. The mold is a hollow cylinder made of PMMA (composed of two halves) with a longitudinal groove structure on the inner surface. The grooves are 200 μm wide and 150 μm high, and the spacing between the grooves is 200 μm. The mold has a removable upper cover and bottom cover, and the edge of the upper cover is provided with a liquid injection port. The upper and lower covers of the mold have small holes with a diameter of 500 μm and a spacing of 1000 μm. The conductive fiber bundles are inserted through the holes in the mold, arranged axially with both ends exposed.

[0043] 3) Coat the inside of the mold with 0.1 ml of glycerol. Add a mixed solution of GelMA and Sil-MA (w:w = 1:1) containing 0.25% LAP through the injection port on the mold to a total concentration of 10 wt %. Then, cure with 405 nm 10 W UV for 5 min to form a gel with a double network structure.

[0044] 4) After the gel solidifies, the mold is placed in a -80°C freezer and quick-frozen for 10 minutes, then removed and thawed naturally for 10 minutes; the freeze-thaw step is repeated three times, followed by demolding; the gel is then rinsed three times with a PBS solution pre-cooled to 4°C, and then placed in a PBS solution, changing the solution every 30 minutes, repeated eight times to remove excess release agent; the gel is then immersed in a 0.1 wt% Fibronectin solution for 2 hours, a protein material that promotes cell adhesion is introduced onto the surface, and the gel is dried in a low-temperature airflow and sterilized by irradiation to obtain a multi-channel nerve repair conduit material with a dual-patterned structure;

[0045] 5) Use P3 neural stem cells with a cell density of 2*10 5 / ml, and co-cultured with nerve conduit gel in a low-adhesion well plate for 3 days. After the cells adhered and migrated in the tube, they were combined with pulsed direct current stimulation technology to achieve neural differentiation, where the voltage was 3V, the pulse direction was positive and negative, the pulse width was 200ms, and the pulse frequency was 10Hz; the stimulation lasted for 60min / day, and the stimulation lasted for 14 days in total.

[0046] Compared with Comparative Example 1, conductive fibers were introduced and the multi-channel structure was eliminated. The conductivity of the hydrogel obtained in this comparative example was 0.72 S / m, the 30-day in vitro degradation rate was 65%, and after co-culture with neural stem cells for 14 days, Confocal imaging of live and dead cell fluorescence staining showed that the proportion of live cells was 92%. Due to the lack of a multi-channel microcatheter structure, the cells all adhered to the surface, and the neural differentiation of neural stem cells was poor. Confocal imaging of NF200 immunofluorescence staining showed that the relative fluorescence area was 17%.

[0047] Example 1

[0048] 1) 5 wt% chitosan with a molecular weight of 1,200,000 and a degree of deacetylation of 80% is dissolved in a 3% acetic acid solution containing 3 wt% urea, 3 wt% glycerol, and 1 wt% sodium ethoxide to prepare a chitosan solution. Sodium alginate powder is then slowly added and stirred until completely dissolved. The viscosity of the sodium alginate (10 g / L, 20°C) is 0.1 Pa·s, and the concentration is 0.5 wt%. This yields the desired chitosan / sodium alginate mixed spinning solution. Conductive fibers are then prepared using a continuous multi-step wet spinning method and in-situ surface polymerization. The continuous multi-step wet spinning method parameters are as follows: a spinning flow rate of 50 mL / h and a spinning temperature of 40°C. The fiber-forming stage primarily involves seven solutions: a single coagulation bath, a single rinse, a double rinse, a double coagulation bath, a triple rinse, and a quadruple rinse. The first coagulation bath is 5wt% NaOH and 0.5wt% Na2SO4 dissolved in ethanol / water solution (1:1 v:v); the second coagulation bath is 5wt% calcium chloride aqueous solution; the cleaning liquid is deionized water; the coagulation bath temperature is 40℃, and the cleaning liquid temperature is 50℃; the prepared fiber filaments are immersed in aniline / phytic acid solution, aniline 0.02g / ml, phytic acid 0.1g / ml, initiator 0.01g / ml; the initiator is potassium persulfate, and then in situ self-polymerization is carried out under room temperature shaking for 4h; the conductive fiber obtained is as follows Figure 1 As shown, after cleaning and drying, a conductive fiber bundle is formed through a twisting process;

[0049] 2) Designing a casting mold capable of endowing the neural repair conduit with a dual-patterned and multi-channel structure that promotes the directional migration of neural stem cells. The mold is a hollow cylinder made of PMMA (composed of two halves) with an inner surface having a longitudinal groove structure with a width of 200 μm, a height of 150 μm, and a spacing of 200 μm between the grooves. The interior of the mold is arranged with small cylindrical structures with a diameter of 500 μm, evenly spaced with a spacing of 1000 μm, and the surface also has the same longitudinal groove structure as the inner surface of the mold. The mold has a removable bottom cover and a top cover, and the edge of the top cover is provided with a liquid injection port. The top and bottom covers of the mold have corresponding small holes with a diameter of 500 μm and a spacing of 1000 μm. The small holes in the mold are used to fix the conductive fiber bundles through the holes and arrange them axially, with both ends protruding from the mold.

[0050] 3) Coat the inside of the mold with 0.1 ml of glycerol. Add a mixed solution of GelMA and Sil-MA (w:w = 1:1) containing 0.25% LAP through the injection port on the mold to a total concentration of 10 wt %. Then, cure with 405 nm 10 W UV for 5 min to form a gel with a double network structure.

[0051] 4) After the gel solidifies, the mold is placed in a -80°C freezer and quick-frozen for 10 minutes, then removed and thawed naturally for 10 minutes; the freeze-thaw step is repeated three times, followed by demolding; the gel is then rinsed three times with a PBS solution pre-cooled to 4°C, and then placed in a PBS solution, changing the solution every 30 minutes, repeated eight times to remove excess release agent; the gel is then immersed in a 0.1 wt% Fibronectin solution for 2 hours, a protein material that promotes cell adhesion is introduced onto the surface, and the gel is dried in a low-temperature airflow and sterilized by irradiation to obtain a multi-channel nerve repair conduit material with a dual-patterned structure;

[0052] 5) Use P3 neural stem cells with a cell density of 2*10 5 / ml, and co-cultured with nerve conduit gel in a low-adhesion well plate for 3 days. After the cells adhered and migrated in the tube, they were combined with pulsed direct current stimulation technology to achieve neural differentiation, where the voltage was 3V, the pulse direction was positive and negative, the pulse width was 200ms, and the pulse frequency was 10Hz; the stimulation lasted for 60min / day, and the stimulation lasted for 14 days in total.

[0053] The conductivity of the composite nerve conduit hydrogel obtained in this example was 0.78 S / m, and the degradation rate in vitro was 68% after 30 days. After co-culture with neural stem cells for 14 days, Confocal imaging of live and dead cell fluorescence staining showed that the proportion of live cells was 90%, and a large number of cells migrated into the nerve conduit and arranged in a directional manner. The neural stem cells differentiated well into neurons (the PCR data are shown in FIG). Figure 5 Confocal imaging of NF200 immunofluorescence staining showed a relative fluorescence area of ​​49%.

[0054] Example 2

[0055] 1) 5 wt% chitosan with a molecular weight of 1,200,000 and a degree of deacetylation of 80% is dissolved in a 3% acetic acid solution containing 3 wt% urea, 3 wt% glycerol, and 1 wt% sodium ethoxide to prepare a chitosan solution. Sodium alginate powder is then slowly added and stirred until completely dissolved. The viscosity of the sodium alginate (10 g / L, 20°C) is 0.1 Pa·s, and the concentration is 0.5 wt%. This yields the desired chitosan / sodium alginate mixed spinning solution. Conductive fibers are then prepared using a continuous multi-step wet spinning method and in-situ surface polymerization. The continuous multi-step wet spinning method parameters are as follows: a spinning flow rate of 50 mL / h and a spinning temperature of 40°C. The fiber-forming stage primarily involves seven solutions: a single coagulation bath, a single rinse, a double rinse, a double coagulation bath, a triple rinse, and a quadruple rinse. The first coagulation bath is 5wt% NaOH and 0.5wt% Na2SO4 dissolved in ethanol / water solution (1:1 v:v); the second coagulation bath is 5wt% calcium chloride aqueous solution; the cleaning solution is deionized water; the coagulation bath temperature is 40°C, and the cleaning solution temperature is 50°C; the prepared fiber filaments are immersed in an aniline / phytic acid solution, containing 0.03g / ml aniline, 0.1g / ml phytic acid, and 0.01g / ml initiator; the initiator is potassium persulfate, and then in situ self-polymerization is carried out under shaking conditions at room temperature for 4 hours; after cleaning and drying, the conductive fiber bundles are formed by twisting process;

[0056] 2) Designing a casting mold that can impart a dual-patterned, multi-channel structure to the neural repair conduit, which can promote the directional migration of neural stem cells. The mold is a hollow cylinder made of PMMA (composed of two halves) with an inner surface having a longitudinal groove structure with a width of 200 μm, a height of 150 μm, and a spacing of 200 μm between the grooves. The interior of the mold is arranged with small cylindrical structures with a diameter of 500 μm, evenly spaced with a spacing of 1000 μm, and the surface also has the same longitudinal groove structure as the inner surface of the mold. The mold has a removable bottom cover and a top cover, and the edge of the top cover is provided with a liquid injection port. The top and bottom covers of the mold have corresponding small holes with a diameter of 500 μm and a spacing of 800 μm. The small holes in the mold are used to pass the conductive fiber bundle, and are arranged axially with both ends exposed.

[0057] 3) Coat the inside of the mold with 0.1 ml of glycerol. Add a mixed solution of GelMA and Sil-MA (w:w = 1:1) containing 0.25% LAP through the injection port on the mold to a total concentration of 10 wt %. Then, cure with 405 nm 10 W UV for 5 min to form a gel with a double network structure.

[0058] 4) After the gel solidifies, the mold is placed in a -80°C freezer and quick-frozen for 10 minutes, then removed and thawed naturally for 10 minutes; the freeze-thaw step is repeated three times, followed by demolding; the gel is then rinsed three times with a PBS solution pre-cooled to 4°C, and then placed in a PBS solution, changing the solution every 30 minutes, repeated eight times to remove excess release agent; the gel is then immersed in a 0.1 wt% Fibronectin solution for 2 hours, a protein material that promotes cell adhesion is introduced onto the surface, and the gel is dried in a low-temperature airflow and sterilized by irradiation to obtain a multi-channel nerve repair conduit material with a dual-patterned structure;

[0059] 5) Use P3 neural stem cells with a cell density of 2*10 5 / ml, and co-cultured with nerve conduit gel in a low-adhesion well plate for 3 days. After the cells adhered and migrated in the tube, they were combined with pulsed direct current stimulation technology to achieve neural differentiation, where the voltage was 3V, the pulse direction was positive and negative, the pulse width was 200ms, and the pulse frequency was 10Hz; the stimulation lasted for 60min / day, and the stimulation lasted for 14 days in total.

[0060] Compared with Example 1, the aniline concentration was increased to increase the thickness of the conductive coating and the number of conductive fibers. The conductivity of the composite nerve conduit hydrogel obtained in this example was 0.93 S / m, and the in vitro degradation rate was 64% after 30 days. After co-culture with neural stem cells for 14 days, Confocal imaging of live-dead cell fluorescence staining showed that the proportion of live cells was 86%, a large number of cells migrated into the nerve conduit and arranged in a direction, the neural stem cells differentiated well into neurons, and Confocal imaging of NF200 immunofluorescence staining showed a relative fluorescence area of ​​61%.

[0061] Example 3

[0062] 1) 5 wt% chitosan with a molecular weight of 1,200,000 and a degree of deacetylation of 80% is dissolved in a 3% acetic acid solution containing 3 wt% urea, 3 wt% glycerol, and 1 wt% sodium ethoxide to prepare a chitosan solution. Sodium alginate powder is then slowly added and stirred until completely dissolved. The viscosity of the sodium alginate (10 g / L, 20°C) is 0.1 Pa·s, and the concentration is 0.5 wt%. This yields the desired chitosan / sodium alginate mixed spinning solution. Conductive fibers are then prepared using a continuous multi-step wet spinning method and in-situ surface polymerization. The continuous multi-step wet spinning method parameters are as follows: a spinning flow rate of 50 mL / h and a spinning temperature of 40°C. The fiber-forming stage primarily involves seven solutions: a single coagulation bath, a single rinse, a double rinse, a double coagulation bath, a triple rinse, and a quadruple rinse. The first coagulation bath is 5wt% NaOH and 0.5wt% Na2SO4 dissolved in ethanol / water solution (1:1 v:v); the second coagulation bath is 5-wt% calcium chloride aqueous solution; the cleaning solution is deionized water; the coagulation bath temperature is 40°C, and the cleaning solution temperature is 50°C; the prepared fiber filaments are immersed in an aniline / phytic acid solution, containing 0.02g / ml aniline, 0.1g / ml phytic acid, and 0.01g / ml initiator; the initiator is potassium persulfate, and then in situ self-polymerization is carried out under shaking conditions at room temperature for 4h; after washing and drying, the conductive fiber bundles are formed by twisting process;

[0063] 2) Designing a casting mold that can endow the neural repair conduit with a dual-patterned and multi-channel structure that can promote the directional migration of neural stem cells. The mold is a hollow cylinder made of PMMA (composed of two halves) with a longitudinal groove structure on the inner surface. The grooves are 100 μm wide, 100 μm high, and spaced 100 μm apart. The interior of the mold is arranged with small cylindrical structures with a diameter of 500 μm, evenly spaced with 1000 μm spacing, and the surface also has the same longitudinal groove structure as the inner surface of the mold. The mold has a removable upper cover and bottom cover, and the edge of the upper cover is provided with a liquid injection port. The upper and bottom covers of the mold have corresponding small holes with a diameter of 500 μm and a spacing of 1000 μm. The small holes in the mold are used to pass the conductive fiber bundle, and are arranged axially with both ends exposed.

[0064] 3) Coat the inside of the mold with 0.1 ml of glycerol. Add a mixed solution of GelMA and Sil-MA (w:w = 1:1) containing 0.25% LAP through the injection port on the mold to a total concentration of 10 wt %. Then, cure with 405 nm 10 W UV for 5 min to form a gel with a double network structure.

[0065] 4) After the gel solidifies, the mold is placed in a -80°C freezer and quick-frozen for 10 minutes, then removed and thawed naturally for 10 minutes; the freeze-thaw step is repeated three times, followed by demolding; the gel is then rinsed three times with a PBS solution pre-cooled to 4°C, and then placed in a PBS solution, changing the solution every 30 minutes, repeated eight times to remove excess release agent; the gel is then immersed in a 0.1 wt% Fibronectin solution for 2 hours, a protein material that promotes cell adhesion is introduced onto the surface, and the gel is dried in a low-temperature airflow and sterilized by irradiation to obtain a multi-channel nerve repair conduit material with a dual-patterned structure;

[0066] 5) Use P3 neural stem cells with a cell density of 2*10 5 / ml, and co-cultured with nerve conduit gel in a low-adhesion well plate for 3 days. After the cells adhered and migrated in the tube, they were combined with pulsed direct current stimulation technology to achieve neural differentiation, where the voltage was 3V, the pulse direction was positive and negative, the pulse width was 200ms, and the pulse frequency was 10Hz; the stimulation lasted for 60min / day, and the stimulation lasted for 14 days in total.

[0067] Compared with Example 1, the surface morphology of the nerve conduit micropattern was changed to make the pattern denser. The conductivity of the composite nerve conduit hydrogel obtained in this example was 0.76 S / m, and the in vitro degradation rate was 67% after 30 days of co-culture with neural stem cells. Confocal imaging of live and dead cell fluorescence staining showed that the proportion of live cells was 92%, and a large number of cells migrated into the nerve conduit and arranged in a direction. The cell arrangement was more orderly than in Example 1, and the neural stem cells had good neuronal differentiation. Confocal imaging of NF200 immunofluorescence staining showed a relative fluorescence area of ​​57%.

[0068] The foregoing description is merely a partial list of preferred embodiments of the present invention, intended only to facilitate understanding of the present invention and not to limit the present invention. It should be noted that variations and improvements are possible without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A method for preparing a multi-channel cell-laden nerve repair catheter with a double-patterned structure, characterized in that: The steps include: 1) A chitosan / sodium alginate mixed spinning solution was prepared, and fibers were prepared by a continuous multi-step wet spinning method. The fibers were then immersed in an aniline / phytic acid solution for in-situ surface polymerization to prepare conductive fibers, and the conductive fiber bundles were formed by a twisting process. 2) Design a casting mold capable of imparting dual patterning and a multi-channel structure to the nerve repair conduit, i.e., enabling the conduit to have multiple channels, with both the conduit surface and the surfaces of each channel patterned. The mold is a hollow cylindrical structure composed of two halves, with a removable bottom cover and top cover. The inner wall of the hollow column is provided with a plurality of axially parallel micron-scale grooves. The bottom cover is provided with a plurality of axially parallel columns, each of which has a plurality of axially parallel micron-scale grooves on its outer surface. The top cover and bottom surface are provided with corresponding small holes for the conductive fiber bundle to pass through, and the top cover is provided with a liquid injection port. 3) Coating a mold release agent on the inside of the mold, arranging the conductive fiber bundles prepared in step 1) longitudinally and fixing them in the mold, adding a UV-curable polymer solution from the injection port on the mold, curing the solution to form a gel having a double network structure, and then curing the solution with UV light; 4) After the gel solidifies, it is demolded using a freeze-thaw cycle. After removing the surface release agent through a rapid rinse-dialysis process, a protein / peptide material that promotes cell adhesion is introduced onto the sample surface by an impregnation method. After low-temperature airflow drying and irradiation sterilization, the gel catheter is obtained, thereby obtaining a multi-channel nerve repair catheter material with a dual-patterned structure. 5) Combined with the co-culture experiment of neural stem cells, after the cells adhered and migrated in the tube, pulsed direct current stimulation technology was used to achieve neural differentiation, and a multi-channel cell-loaded neural repair conduit with a double-patterned structure was obtained; specifically, P2-P4 neural stem cells and gel conduits were placed in low-adhesion well plates and co-cultured for 2-3 days, with a cell density of (1-2)*10 in the culture medium. 5 / ml, and then connected to the two ends of the conductive fiber bundle of the gel catheter with a wire, and pulsed direct current stimulation was applied to it, with a voltage of 1-3V, a pulse direction of positive and negative pulses, a pulse width of 100-300ms, and a pulse frequency of 3-10Hz; the stimulation lasted for 30-60min / day, and a total of 7-14 days.

2. The preparation method according to claim 1, characterized in that In step 1), chitosan is dissolved in an acetic acid aqueous solution containing urea, glycerol, and sodium ethoxide to prepare a chitosan solution, and then sodium alginate powder is slowly added and stirred to completely dissolve to obtain the chitosan / sodium alginate mixed spinning solution, wherein the chitosan concentration is 2-5wt%, the chitosan molecular weight is 1200000-2000000, the deacetylation degree is 70-80%, and the concentration of the acetic acid aqueous solution is 2-4wt%; The viscosity of sodium alginate at 10 g / L and 20° C. is 0.02-0.1 Pa·s, and the concentration is 0.1-0.5 wt %. The amount of urea added is 2-3 wt %, the amount of glycerol added is 2-5 wt %, and the amount of sodium ethoxide added is 0.5-1 wt %. The stirring rate during the preparation of the above spinning solution is 400-600 rpm / min, and the stirring time is 2-4 hours.

3. The preparation method according to claim 1, characterized in that In step 1), the spinneret parameters during the wet spinning process are: spinning flow rate 40-80 mL / h, spinning temperature 30-45°C; the fiber forming stage mainly passes through 7 solutions: including a coagulation bath, a cleaning solution, a second cleaning solution, a second coagulation bath, a triple cleaning solution, and a quadruple cleaning solution, the first coagulation bath is 3-5wt% NaOH or KOH, 0.2-0.5wt% Na2SO4 dissolved in ethanol / water solution, and the volume ratio of ethanol to water is 1:1; the second coagulation bath is 5-10wt% calcium chloride aqueous solution; the cleaning solution is deionized water; the coagulation bath temperature is 20-50°C, and the cleaning solution temperature is 40-50°C.

4. The preparation method according to claim 1, characterized in that In step 1), the conductive fiber preparation process is as follows: the fiber filaments are immersed in an aniline / phytic acid solution containing 0.01-0.03 g / ml of aniline, 0.06-0.12 g / ml of phytic acid, and 0.002-0.01 g / ml of an initiator; wherein the initiator is one of potassium persulfate, sodium persulfate, and ammonium persulfate, and then in situ self-polymerization is carried out under shaking conditions at room temperature for 2-4 hours.

5. The preparation method according to claim 1, wherein: The mold is made of PMMA and is cylindrical in shape. The groove width is 100-200 μm, the height is 80-150 μm, and the spacing between grooves is 100-200 μm. The diameter of the columns is 400-500 μm, and the columns are evenly arranged with a spacing of 800-1000 μm.

6. The preparation method according to claim 1, wherein: In step 3), the release agent is one of glycerol and ethylene glycol; the polymers in the UV-curable polymer solution are two of GelMA, Alg-MA, and Sil-MA, with a total concentration of 10-20wt% and a mass ratio of the two polymers of 1:3-3:1; the photoinitiator in the solution is one or more of LAP, I2959, and Irgacure, and the concentration of the photoinitiator is 0.25-2.5wt%; The UV curing wavelength is 405nm, the power is 5-20W, and the curing time is 3-5min.

7. The preparation method according to claim 1, wherein: In step 4), the freeze-thaw cycle process is as follows: place the mold in a -80°C freezer, quick-freeze for 5-10 minutes, then take it out and thaw naturally for 10-15 minutes; repeat the above freeze-thaw steps 3-5 times; use PBS solution pre-cooled to 4°C as the flushing and dialysate, rinse the gel 3-5 times, place it in PBS solution, change the solution every 30-60 minutes, and repeat 5-8 times; the adhesion protein is one or more of Fibronectin, Laminin, and Col Ⅲ, with a concentration of 0.1-0.5wt%, the immersion time is 1-2 hours, and it is rinsed with PBS after the immersion is completed.

8. A multi-channel cell-laden nerve repair catheter with a double-patterned structure, characterized in that: The method is prepared by the method according to any one of claims 1 to 7.

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