Multi-layer integrated cell-loaded nerve repair conduit with conductive, anti-inflammatory and neural differentiation promoting functions and preparation method of multi-layer integrated cell-loaded nerve repair conduit with conductive, anti-inflammatory and neural differentiation promoting functions

Abstract

The invention discloses a multi-layer integrated cell-loaded nerve repair conduit with conductive, anti-inflammatory and neural differentiation promoting functions and a preparation method thereof. The preparation method comprises the following steps: 1) preparing PLGA fibers doped with graphene, and weaving the PLGA fibers to obtain a layer of cylindrical fiber net; 2) preparing an HAMA mixed solution mixed with microspheres loaded with nerve growth promoting factors and manganese dioxide nanoparticles, and an HAMA solution loaded with neural stem cells; 3) fixing the cylindrical fiber net in a cylindrical groove mold, pouring an HAMA mixed solution, and freeze-drying after UV curing to obtain a two-layer porous composite scaffold; and (4) dipping in a neural stem cell-loaded HAMA solution, quickly curing to form three cell-loaded gel layers, and culturing in vitro for 4-8W to obtain the multi-layer integrated cell-loaded neural repair conduit. The nerve repair catheter has the functions of conducting electricity, resisting inflammation and promoting neural differentiation, and a brand new strategy is provided for nerve and spinal cord injury repair.

Description

Technical Field

[0001] The present invention relates to a preparation technology of a nerve repair material, and in particular to a multi-layered cell-loaded nerve repair conduit with the functions of conducting electricity, resisting inflammation and promoting nerve differentiation, and a preparation method thereof. Background Art

[0002] Peripheral nerve injury (PNI) is a very common disease in clinical practice, mostly caused by traffic accidents, falls, earthquakes, firearm injuries, and iatrogenic injuries. Statistics show that about 2.8% of trauma patients will have peripheral nerve involvement, and nearly 40% of patients will have symptoms of organic nerve damage, mainly manifested as partial or complete loss of motor and sensory functions, chronic pain, and target muscle atrophy, which brings huge physical and psychological burdens to patients.

[0003] With the development of tissue engineering technology, nerve repair materials have gradually developed. Specifically, they should have the following characteristics: good biocompatibility and biodegradability; anti-inflammatory properties; bionic structure to achieve morphological bionic purposes; release of growth factors related to nerve regeneration; and convenience for clinical operation. At the same time, studies have shown that electrical stimulation promotes nerve regeneration. However, the current related nerve repair materials usually only have some of the above properties, which makes them unable to fully match the actual needs of nerve repair during use, resulting in insignificant repair effects.

[0004] In view of the above problems, the present invention designs a multi-layered cell-loaded nerve repair conduit with the functions of conducting electricity, anti-inflammation and promoting nerve differentiation and its preparation method. The above nerve conduit is provided with a conductive fiber layer, a freeze-dried composite scaffold layer and a cell-loaded hydrogel layer from the inside to the outside, and at the same time, anti-inflammation and nerve differentiation-promoting drugs are organically loaded therein, so that the material has multiple functions at the same time, providing a new strategy for the repair of nerve and spinal cord injuries. Summary of the invention

[0005] The purpose of the present invention is to provide a multilayer cell-loaded nerve repair conduit with the functions of conducting electricity, anti-inflammation and promoting nerve differentiation, and a preparation method thereof, in order to solve the problem that the traditional nerve conduit has a poor repair effect. The nerve repair conduit has the functions of conducting electricity, anti-inflammation and promoting nerve differentiation, and provides a new strategy for the repair of nerve and spinal cord injuries.

[0006] The present invention is implemented by the following technical solution, and the specific steps include:

[0007] A method for preparing a multilayer cell-loaded nerve repair conduit having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation, comprises the following steps:

[0008] 1) firstly preparing PLGA fibers doped with graphene by a melt spinning process, and weaving the fibers to obtain a cylindrical fiber mesh;

[0009] 2) Prepare methacryloylated hyaluronic acid (HAMA) microspheres and manganese dioxide nanoparticles loaded with nerve growth promoting drugs using microfluidic technology and hydrothermal method, respectively;

[0010] 3) preparing a HAMA mixed solution containing HAMA microspheres loaded with a drug that promotes nerve growth and manganese dioxide nanoparticles, and a HAMA solution loaded with neural stem cells;

[0011] 4) fixing the cylindrical fiber mesh in a cylindrical groove mold as the first conductive fiber base layer, pouring the HAMA mixed solution, vibrating and UV curing, demoulding, and freeze-drying to obtain the second porous composite scaffold layer;

[0012] 5) Immerse it in a neural stem cell-carrying HAMA solution, quickly UV-curing it to form a third cell-carrying gel layer, and after culturing it in vitro for 4-8 weeks, a multi-layered collective cell-carrying nerve repair conduit is obtained, which is implanted into the nerve defect area for use.

[0013] Furthermore, in step 1), in the process of preparing the PLGA fiber doped with graphene, the PLGA is PLGA50:50 or PLGA75:25; the size of the graphene is 2-3 microns, the concentration is 0.1-0.5wt%, and the fiber diameter is about 30-100 microns;

[0014] The cylindrical fiber web has a length of about 1-5 cm and a diameter of 1-3 mm.

[0015] Furthermore, in step 2), during the preparation of the HAMA microspheres loaded with the nerve growth promoting drug, the microfluidic fluid channel is assembled, and the channel diameter is between 30-100 μm; then a HAMA solution with a concentration of 2-10 wt% is placed in a microfluidic injection pump, and the HAMA solution is injected into the fluid channel at a flow rate of 0.1-1.0 mL / h using a microfluidic system, and a mixed solution of Span80 and paraffin oil is dripped in, UV curing, the temperature is 20-25° C., and the pH value is 6.5-7.5; after the end, acetone, ethanol and deionized water are used in sequence. The HAMA microspheres are washed with water and then immersed in a nerve growth promoting drug solution to obtain HAMA microspheres loaded with nerve growth promoting drugs, with a concentration of 0.1-1.0wt%; the nerve growth promoting drugs are one or more of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial cell-derived neurotrophic factor (GDNF), ganglioside, vitamin B1, citicoline, and oxiracetam, with a concentration of 100ng / ml-0.1g / ml.

[0016] Furthermore, in step 2), during the preparation of the manganese dioxide nanoparticles, a mixed solution of sodium dodecylbenzene sulfonate and manganese chloride is prepared, and a potassium permanganate solution is added dropwise to the mixed solution under stirring conditions. After the reaction is completed, the manganese dioxide nanoparticles are washed by high-speed centrifugation to obtain the manganese dioxide nanoparticles;

[0017] The concentration of the sodium dodecyl sulfate is 1-2 g / L, the concentration of the manganese chloride is 100-200 g / L, the concentration of the potassium permanganate is 15-80 g / L, the reaction temperature is 80-100° C., the stirring rate is 800-1000 r / min, and the reaction time is 3-8 h; the particle size of the manganese dioxide nanoparticles is 20-30 nm, and the concentration is 0.05-0.25 wt %.

[0018] Furthermore, in step 3), the prepared HAMA mixed solution contains a photoinitiator, the substitution degree of the HAMA is 30-60%, the concentration is 7-20wt%, the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, the concentration is 0.25-0.5wt%, and the solvent of the HAMA mixed solution is PBS;

[0019] The HAMA solution loaded with neural stem cells contains a photoinitiator, the generation of neural stem cells is 2-3 generations, and the concentration is 0.1-10×10 7 mL -1 ; The concentration of HAMA is 5-10wt%, the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, with a concentration of 0.25-0.4wt%, and the solvent is PBS.

[0020] Further, in step 4), the cylindrical groove mold is made of PMMA or glass material, including a base and two cylinders, the base has a diameter of 1.5-3.5 mm, and two concentric circular grooves are arranged on the base, each groove has a width of 100-200 microns, a depth of 1-2 mm, and a spacing of 0.5-1.5 mm between the two grooves. The thickness of each cylinder is consistent with the width of the groove, and the two cylinders are respectively embedded in the two grooves, with a height of 1.1-5.2 cm;

[0021] During the UV curing process, the UV wavelength is 405nm, the power is 20-30W, and the time is 5-12min;

[0022] During the freeze-drying process, the freezing is carried out at 0-20° C. and the freeze-drying time is 5-10 hours.

[0023] Furthermore, in step 4), during the UV curing process, the UV wavelength is 405 nm, the power is 20-30 W, and the time is 10-20 s; and the in vitro culture uses a neural differentiation-promoting culture medium.

[0024] A multi-layered cell-loaded nerve repair conduit with the functions of conducting electricity, resisting inflammation and promoting nerve differentiation is obtained by using any of the above-mentioned preparation methods.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1) The present invention uses graphene, PLGA, HAMA, nerve growth promoting drugs, manganese dioxide and the like as raw materials, and combines melt spinning, fiber weaving, hydrothermal method, in-situ casting, nanocomposite, freeze drying and other combined processes to produce a multilayered cell-loaded nerve repair catheter material that has the functions of being conductive, anti-inflammatory and promoting nerve differentiation. The above raw material selection and process combination are original to the present invention.

[0027] 2) The nerve repair catheter in the present invention has a three-layer composite structure: a cylindrical fiber mesh layer, a second porous composite scaffold layer and a third cell-carrying gel layer. The first cylindrical fiber mesh layer is the innermost layer, providing mechanical support and conductive properties for the catheter. The fiber is laid in it to ensure that the outer scaffold and gel layer will not break due to surgical operations, traction and other effects during use; the second porous composite scaffold layer is the main bioactive substance storage layer, which is loaded with microspheres and manganese dioxide nanoparticles that promote nerve growth factors. The above two substances will gradually release growth factors and manganese ions to promote nerve regeneration and fight against neuroinflammation, respectively, and the porous surface after freeze-drying is conducive to the adhesion of the subsequent third gel layer, increasing the mechanical bite between the two; the third cell-carrying gel layer is loaded with neural stem cells, and the porous structure is also provided to facilitate the exchange of nutrients and oxygen for stem cells, reduce cell mortality, and promote their growth. The above three-layer composite structure cooperates with each other to provide different aspects of protection for nerve regeneration, and the structure is obviously innovative.

[0028] 3) In order to solve the problem of long nerve repair cycle, the present invention sets up the release of drugs in the microspheres that promote nerve differentiation and growth of stem cells through the nerve differentiation culture medium and the load of nerve growth factor in the early stage; in the later stage, after the nerve conduit is implanted in the body, the internal conductive cylindrical fiber mesh is connected with the nerve stump to transmit the electrophysiological signals in the human body, and further promote the repair and regeneration of nerves through bioelectric stimulation. It is the synergistic effect of the above two effects that makes this nerve repair conduit meet the cycle requirements of nerve repair for more than 3 months.

[0029] 4) In the present invention, a casting method is used to wrap the cylindrical fiber mesh in the HAMA matrix to form a sandwich structure. The above structure is similar to the natural nerve structure, with tissue on the outside and a neural network on the inside. After the nerve conduit is subsequently connected to the broken end of the damaged nerve, this structure can achieve good connection and conduction of the conductive network, which is beneficial to the subsequent transmission of electrophysiological signals; after the HAMA is further freeze-dried to form a porous scaffold, the neural stem cell-carrying HAMA solution is adsorbed into the porous structure in combination with the impregnation method. After the neural stem cell-carrying HAMA solution forms a gel, it forms a mechanical interlock through the porous structure and firmly adheres to the outermost layer. At the same time, the gel layer formed by in-situ UV curing can cleverly introduce a cell source for the nerve conduit to make up for the problem of scarce nerve cell sources in the nerve defect area. The above-mentioned combined processes work together to produce a new type of nerve conduit material that meets the complex requirements of nerve repair, which is a new application of the old process.

[0030] 5) The nerve repair conduit in the present invention has the functions of being degradable, high-strength, anti-inflammatory, conductive, and promoting nerve differentiation, which can meet the various needs of nerve injury repair. First of all, the raw materials selected by the present invention are all degradable and highly biocompatible raw materials, so there will be no toxicity during use, and no secondary surgery is required; at the same time, the internal cylindrical fiber mesh structure is used as the bottom layer to ensure the strength and toughness of the nerve conduit, so that it will not be easily broken and damaged during use; in view of the problem that nerve damage is prone to inflammation, the present invention introduces manganese dioxide nanoparticles with anti-inflammatory function, which continuously release manganese ions through its degradation to inhibit inflammation and provide a suitable microenvironment for nerve injury repair; at the same time, in the process of promoting the regeneration and repair of damaged nerves by the nerve conduit, the nerve growth factor can promote the differentiation of stem cells into nerves, and at the same time, the connection between nerves and nerve conduits is achieved through the graphene in the PLGA fiber mesh in the later stage, so as to achieve the purpose of smooth conduction of bioelectric signals. It is precisely because of the synergy between the above functions that the complex needs of nerve injury regeneration and repair can be met, and the above performance is carefully designed by the applicant according to the actual symptoms of the disease, which has obvious innovation.

[0031] 6) The size and length of the nerve repair catheter in the present invention can be prepared by selecting a suitable mold according to the condition of the patient with nerve damage. It is convenient, operable and universal, and can meet the various needs of different patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention is a schematic diagram of the preparation process of a multilayer cell-loaded nerve repair catheter material having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation. DETAILED DESCRIPTION

[0033] The present invention is further described below with reference to specific examples and accompanying drawings.

[0034] Comparative Example 1:

[0035] 1) Preparation of HAMA microspheres mixed with a loaded nerve growth promoting drug using microfluidic technology: First, assemble a microfluidic fluid channel with a channel diameter of 50 μm, then put a HAMA solution with a concentration of 7.5 wt% (substitution degree 60%) into a microfluidic injection pump, inject the HAMA solution into the fluid channel at a flow rate of 0.4 mL / h using a microfluidic system, drop a mixed solution of Span80 and paraffin oil, UV cure at a temperature of 25° C. and a pH value of 7.2, and then wash with acetone, ethanol and deionized water in turn. After washing, the obtained HAMA solution is AMA microspheres are then immersed in a 100ng / ml NGF solution for immersion adsorption to obtain HAMA microspheres mixed with a loaded nerve growth promoting drug; at the same time, a hydrothermal method is used to prepare manganese dioxide nanoparticles: first, a mixed solution of 1g / L sodium dodecylbenzene sulfonate and 125g / L manganese chloride is prepared, and a potassium permanganate solution is added dropwise to the above solution under a high-speed stirring condition of 1000r / min, and the potassium permanganate concentration in the final solution is 25g / L. After reacting at 80°C for 7h, high-speed centrifugation and washing are performed to obtain manganese dioxide nanoparticles with a particle size of about 20-30nm;

[0036] 2) Under the conditions of light protection and heating at 60°C, two HAMA solutions with a concentration of 7.5wt% (degree of substitution 60%) were prepared using PBS as a solvent and 0.25wt% LAP as a photoinitiator; HAMA microspheres with a concentration of 0.4wt% loaded with a nerve growth promoting drug and 0.1wt% manganese dioxide nanoparticles were mixed with a HAMA solution with a concentration of 7.5wt%; neural stem cells with a substitution number of 2 were selected and 5×10 7 mL -1 The concentration of HAMA solution was added to another HAMA solution having a concentration of 7.5 wt % and preheated to 37° C., and dispersed evenly by blowing to obtain a HAMA solution loaded with neural stem cells;

[0037] 3) A HAMA mixed solution containing HAMA microspheres loaded with nerve growth promoting drugs and manganese dioxide nanoparticles was poured into a cylindrical groove mold (composed of a base and two cylinders of different diameters, the base having a diameter of 2 mm, two concentric circular grooves being arranged on the base, the width of the groove being 200 microns, the depth being 1 mm, the interval between the two grooves being 0.5 mm, the thickness of the cylinder being consistent with the groove width, and the height being 2.6 cm), after oscillation and UV curing (UV wavelength being 405 nm, power being 20 W, and time being 5 min), demolding and freeze drying (freezing at -20°C for 5 h) was performed to obtain a porous composite scaffold layer;

[0038] 4) Immerse it in a neural stem cell-carrying HAMA solution, quickly UV cure (UV wavelength of 405nm, power of 20W, time of 20s) to form a cell-carrying gel layer, and culture it in vitro for 8W using a neural differentiation-promoting culture medium to obtain a cell-carrying neural repair conduit, which is implanted into the nerve defect area for use.

[0039] The extract of the above materials was prepared and co-cultured with neural stem cells for 7 days before CCK-8 detection. The results showed that the cell survival rate was about 92.5%; the neural stem cells on the nerve conduit had a good differentiation effect after 8 weeks of differentiation culture. When they were implanted into a nerve injury model, the conduit ruptured during the suturing process. After one month of feeding, the nerve regeneration rate was acceptable; but the nerve regeneration and repair effect after 3 months was average, and no neuroinflammation occurred.

[0040] Embodiment 1:

[0041] 1) Using PLGA 50:50 and graphene (0.25 wt%) with a size of about 2-3 microns as raw materials, a melt spinning process was used to prepare poly (D, L-lactide-co-glycolide) fibers doped with graphene, i.e., PLGA fibers, with a fiber diameter of about 80 microns, and the fibers were woven to obtain a cylindrical fiber mesh with a length of about 1.5 cm and a diameter of about 2 mm;

[0042] 2) Preparation of HAMA microspheres mixed with loaded nerve growth promoting drugs using microfluidic technology: First, assemble the microfluidic fluid channel, the channel diameter is between 50 μm, then put the HAMA solution with a concentration of 7.5wt% (substitution degree 60%) into the injection pump, inject the HAMA solution into the fluid channel at a flow rate of 0.4mL / h using the microfluidic system, drip Span80 and paraffin oil mixed solution, UV curing, temperature 25°C, pH 7.2, and then use acetone, ethanol and deionized water to wash in turn, and obtain to HAMA microspheres, and then immersed them in a NGF solution with a concentration of 100ng / ml, and then immersed and adsorbed to obtain HAMA microspheres mixed with a loaded nerve growth promoting drug; at the same time, a mixed solution of 1g / L sodium dodecylbenzene sulfonate and 125g / L manganese chloride was prepared by a hydrothermal method, and a potassium permanganate solution was added dropwise to the above solution under a high-speed stirring condition of 1000r / min, and the potassium permanganate concentration in the final solution was 25g / L. After reacting at 80°C for 7h, high-speed centrifugation and washing were performed to obtain manganese dioxide nanoparticles with a particle size of about 20-30nm;

[0043] 3) Two HAMA solutions with a concentration of 7.5 wt% (degree of substitution 60%) were prepared under light-proof and 60°C heating conditions using PBS as a solvent and 0.25 wt% LAP as a photoinitiator; HAMA microspheres with a concentration of 0.4 wt% loaded with a nerve growth promoting drug and 0.1 wt% manganese dioxide nanoparticles were mixed with a HAMA solution with a concentration of 7.5 wt%; neural stem cells with a substitution number of 2 were selected and 5×10 7 mL -1 The concentration of HAMA solution was added to another HAMA solution having a concentration of 7.5 wt % and preheated to 37° C., and dispersed evenly by blowing to obtain a HAMA solution loaded with neural stem cells;

[0044] 4) The cylindrical fiber mesh is fixed in a cylindrical groove mold (composed of a base and two cylinders of different diameters, the base has a diameter of 2 mm, two concentric circular grooves are arranged on the base, the width of the groove is 200 microns, the depth is 1 mm, the interval between the two grooves is 0.5 mm, the thickness of the cylinder is consistent with the width of the groove, and the height is 2.6 cm) as the first conductive fiber base layer, and a HAMA mixed solution containing HAMA microspheres loaded with nerve growth-promoting drugs and manganese dioxide nanoparticles is poured, and after oscillation and UV curing (UV wavelength of 405 nm, power of 20 W, time of 5 min), demolding and freeze drying (freezing at -20°C, freeze drying time of 5 h) is obtained to obtain a second porous composite scaffold layer;

[0045] 5) Immerse it in a neural stem cell-carrying HAMA solution, and quickly UV cure it (UV wavelength is 405nm, power is 20W, time is 20s) to form a third cell-carrying gel layer. After culturing in vitro for 8 weeks using a neural differentiation-promoting culture medium, a multilayered collective cell-carrying neural repair conduit is obtained, which is implanted into the nerve defect area for use.

[0046] Compared with comparative example 1, this embodiment adds a conductive cylindrical fiber mesh. The conductivity of the conductive fiber is about 5.69s / cm; the extract of the above material is prepared, and CCK-8 test is performed after co-culture with neural stem cells for 7 days. The results show that the cell survival rate is about 90.1%; the neural stem cells on the nerve conduit have good differentiation effect after 8 weeks of differentiation culture. They are implanted into the nerve injury model, the conduit is intact during the suturing process, and the nerve injury is basically repaired after 3 months of feeding, and no neuroinflammation occurs.

[0047] Embodiment 2:

[0048] 1) Using PLGA 50:50 and graphene (0.25 wt%) with a size of about 2-3 microns as raw materials, a melt spinning process was used to prepare PLGA fibers doped with graphene, the fiber diameter was about 80 microns, and the fiber was woven to obtain a cylindrical fiber mesh with a length of about 1.5 cm and a diameter of about 2 mm;

[0049] 2) Preparation of HAMA microspheres mixed with loaded nerve growth promoting drugs using microfluidic technology: First, assemble the microfluidic fluid channel, the channel diameter is between 50 μm, then put a HAMA solution with a concentration of 7.5wt% (substitution degree 60%) into the injection pump, inject the HAMA solution into the fluid channel at a flow rate of 0.4mL / h using the microfluidic system, drip into the mixed solution of Span80 and paraffin oil, UV cure, temperature 25°C, pH 7.2, and then use acetone, ethanol and deionized water to wash in turn, and obtain HAMA after complete washing The microspheres were then immersed in a 100ng / ml NGF solution to obtain HAMA microspheres mixed with a drug that promotes nerve growth. At the same time, a hydrothermal method was used to prepare manganese dioxide nanoparticles: first, a mixed solution of 1g / L sodium dodecylbenzene sulfonate and 125g / L manganese chloride was prepared, and a potassium permanganate solution was added dropwise to the above solution under a high-speed stirring condition of 1000r / min. The potassium permanganate concentration in the final solution was 25g / L. After reacting at 80°C for 7h, high-speed centrifugation and washing were performed to obtain manganese dioxide nanoparticles with a particle size of about 20-30nm.

[0050] 3) A HAMA solution with a concentration of 7.5 wt% (degree of substitution 60%) was prepared by using PBS as a solvent and 0.25 wt% LAP as a photoinitiator under light-proof and 60° C.; HAMA microspheres with a concentration of 0.4 wt% loaded with a nerve growth promoting drug and 0.1 wt% manganese dioxide nanoparticles were uniformly mixed with the HAMA solution with a concentration of 7.5 wt%;

[0051] 4) The cylindrical fiber mesh is fixed in a cylindrical groove mold (consisting of a base and two cylinders of different diameters, the base has a diameter of 2 mm, and two concentric circular grooves are arranged on the base. The width of the groove is 200 microns, the depth is 1 mm, the interval between the two grooves is 0.5 mm, the thickness of the cylinder is consistent with the groove width, and the height is 2.6 cm) as the first layer of conductive fiber base layer, and a HAMA mixed solution containing HAMA microspheres and manganese dioxide nanoparticles loaded with nerve growth-promoting drugs is poured. After oscillation and UV curing (UV wavelength is 405 nm, power is 20 W, and time is 5 min), the second layer of porous composite scaffold layer is obtained by demolding and freeze-drying (freezing at -20°C for 5 h), which is then implanted in the nerve defect area for use.

[0052] Compared with Example 1, this embodiment reduces the design of the outermost cell-carrying gel layer. The conductivity of the conductive fiber is about 5.48s / cm; the extract of the material is prepared, and CCK-8 is tested after co-culture with neural stem cells for 7 days. The results show that the cell survival rate is about 91.4%; it is implanted into a nerve injury model, the catheter is intact during the suturing process, and some cells migrate to the catheter after 1 month of feeding; the nerve regeneration and repair effect after 3 months is average, and no neuroinflammation occurs.

[0053] Embodiment 3:

[0054] 1) Using PLGA 50:50 and graphene (0.25 wt%) with a size of about 2-3 microns as raw materials, a melt spinning process was used to prepare PLGA fibers doped with graphene, the fiber diameter was about 80 microns, and the fiber was woven to obtain a cylindrical fiber mesh with a length of about 1.5 cm and a diameter of about 2 mm;

[0055] 2) Using microfluidic technology to prepare HAMA microspheres mixed with drugs that promote nerve growth: first, assemble the fluid channel of the microfluidic system, the channel diameter is between 50 μm, then put a HAMA solution with a concentration of 7.5wt% (substitution degree 60%) into the injection pump, use the microfluidic system to inject the HAMA solution into the fluid channel at a flow rate of 0.4mL / h, drop Span80 and paraffin oil mixed solution, UV curing, temperature 25°C, pH 7.2, after completion, use acetone, ethanol and deionized water to wash in turn, after washing, obtain HAMA microspheres, then immerse them in a NGF solution with a concentration of 100ng / ml, and immerse and adsorb to obtain HAMA microspheres mixed with drugs that promote nerve growth;

[0056] 3) Two HAMA solutions with a concentration of 7.5 wt% (degree of substitution 60%) were prepared under light-proof and 60°C heating conditions using PBS as a solvent and 0.25 wt% LAP as a photoinitiator; HAMA microspheres loaded with a nerve growth promoting drug with a concentration of 0.4 wt% and a HAMA mixed solution with a concentration of 7.5 wt% were mixed evenly; neural stem cells with a substitution number of 2 were selected and 5×10 7 mL -1 The concentration of HAMA solution was added to another HAMA solution having a concentration of 7.5 wt % and preheated to 37° C., and dispersed evenly by blowing to obtain a HAMA solution loaded with neural stem cells;

[0057] 4) The cylindrical fiber mesh is fixed in a cylindrical groove mold (composed of a base and two cylinders of different diameters, the base has a diameter of 2 mm, two concentric circular grooves are arranged on the base, the width of the groove is 200 microns, the depth is 1 mm, the interval between the two grooves is 0.5 mm, the thickness of the cylinder is consistent with the width of the groove, and the height is 2.6 cm) as the first conductive fiber base layer, and a HAMA mixed solution containing HAMA microspheres loaded with nerve growth-promoting drugs and manganese dioxide nanoparticles is poured, and after oscillation and UV curing (UV wavelength of 405 nm, power of 20 W, time of 5 min), demolding and freeze drying (freezing at -20°C, freeze drying time of 5 h) is obtained to obtain a second porous composite scaffold layer;

[0058] 5) Immerse it in a neural stem cell-carrying HAMA solution, and quickly UV cure it (UV wavelength is 405nm, power is 20W, time is 20s) to form a third cell-carrying gel layer. After culturing in vitro for 8 weeks using a neural differentiation-promoting culture medium, a multilayered collective cell-carrying neural repair conduit is obtained, which is implanted into the nerve defect area for use.

[0059] Compared with Example 1, this example reduces the addition of manganese dioxide nanoparticles. The conductivity of the conductive fiber is about 5.77s / cm; the extract of the material is prepared, and CCK-8 test is performed after co-culture with neural stem cells for 7 days. The results show that the cell survival rate is about 93.3%; the neural stem cells on the nerve conduit have good differentiation effect after 8 weeks of differentiation culture. They are implanted into a nerve injury model. The conduit is intact during the suturing process. Inflammation occurs in the nerve injury area after 1 month of feeding, and the nerve repair effect is poor after 3 months.

Claims

1. A method for preparing a multilayer cell-loaded nerve repair conduit having the functions of conducting electricity, anti-inflammation and promoting nerve differentiation, characterized in that: The following steps are involved: 1) firstly preparing PLGA fibers doped with graphene by a melt spinning process, and weaving the fibers to obtain a cylindrical fiber mesh; 2) Prepare methacryloylated hyaluronic acid (HAMA) microspheres and manganese dioxide nanoparticles loaded with nerve growth promoting drugs using microfluidic technology and hydrothermal method, respectively; 3) preparing a HAMA mixed solution containing HAMA microspheres loaded with a drug that promotes nerve growth and manganese dioxide nanoparticles, and a HAMA solution loaded with neural stem cells; 4) Fixing the cylindrical fiber mesh in a cylindrical groove mold as the first conductive fiber base layer, pouring the HAMA mixed solution, demolding after oscillation and UV curing, and freeze-drying to obtain the second porous composite scaffold layer; 5) Immersing it in the neural stem cell-carrying HAMA solution, quickly UV curing to form the third cell-carrying gel layer, and culturing it in vitro for 4-8 weeks to obtain a multi-layered collective cell-carrying nerve repair conduit.

2. The method for preparing the multi-layer cell-loaded nerve repair conduit having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation according to claim 1, characterized in that: In step 1), during the preparation of the PLGA fiber doped with graphene, the PLGA is PLGA50:50 or PLGA75:25; the graphene has a size of 2-3 microns, a concentration of 0.1-0.5wt%, and a fiber diameter of about 30-100 microns; The cylindrical fiber web has a length of about 1-5 cm and a diameter of 1-3 mm.

3. The method for preparing the multi-layer cell-loaded nerve repair conduit having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation according to claim 1, characterized in that: In step 2), during the preparation of the HAMA microspheres loaded with the nerve growth promoting drug, the microfluidic fluid channel is assembled, and the channel diameter is between 30-100 μm; then a HAMA solution with a concentration of 2-10 wt% is placed in a microfluidic injection pump, and the HAMA solution is injected into the fluid channel at a flow rate of 0.1-1.0 mL / h using a microfluidic system, and a mixed solution of Span80 and paraffin oil is dripped into it, UV curing is performed, the temperature is 20-25° C., and the pH value is 6.5-7.5; after the end, acetone, ethanol and deionized water are used in turn. The HAMA microspheres are washed and then immersed in a nerve growth promoting drug solution to obtain HAMA microspheres loaded with nerve growth promoting drugs, with a concentration of 0.1-1.0wt%; the nerve growth promoting drugs are one or more of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial cell-derived neurotrophic factor (GDNF), ganglioside, vitamin B1, citicoline, and oxiracetam, with a concentration of 100ng / ml-0.1g / ml.

4. The method for preparing the multi-layer cell-loaded nerve repair conduit having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation according to claim 1, characterized in that: In step 2), during the preparation of the manganese dioxide nanoparticles, a mixed solution of sodium dodecylbenzene sulfonate and manganese chloride is prepared, and a potassium permanganate solution is added dropwise to the mixed solution under stirring conditions. After the reaction is completed, high-speed centrifugation and washing are performed to obtain the manganese dioxide nanoparticles; The concentration of the sodium dodecyl sulfate is 1-2 g / L, the concentration of the manganese chloride is 100-200 g / L, the concentration of the potassium permanganate is 15-80 g / L, the reaction temperature is 80-100° C., the stirring rate is 800-1000 r / min, and the reaction time is 3-8 h; the particle size of the manganese dioxide nanoparticles is 20-30 nm, and the concentration is 0.05-0.25 wt %.

5. The method for preparing the multi-layer cell-loaded nerve repair conduit having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation according to claim 1, characterized in that: In step 3), the prepared HAMA mixed solution contains a photoinitiator, the substitution degree of the HAMA is 30-60%, the concentration is 7-20wt%, the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, the concentration is 0.25-0.5wt%, and the solvent of the HAMA mixed solution is PBS; The HAMA solution loaded with neural stem cells contains a photoinitiator, the generation of neural stem cells is 2-3 generations, and the concentration is 0.1-10×10 7 mL -1 ; The concentration of HAMA is 5-10wt%, the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, with a concentration of 0.25-0.4wt%, and the solvent is PBS.

6. The method for preparing the multi-layer cell-loaded nerve repair conduit having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation according to claim 1, characterized in that: In step 4), the cylindrical groove mold is made of PMMA or glass material, including a base and two cylinders, the base has a diameter of 1.5-3.5 mm, and two concentric circular grooves are arranged on the base, each groove has a width of 100-200 microns, a depth of 1-2 mm, and a spacing of 0.5-1.5 mm between the two grooves. The thickness of each cylinder is consistent with the width of the groove, and the two cylinders are respectively embedded in the two grooves, with a height of 1.1-5.2 cm; During the UV curing process, the UV wavelength is 405nm, the power is 20-30W, and the time is 5-12min; During the freeze-drying process, the freezing is carried out at 0-20° C. and the freeze-drying time is 5-10 hours.

7. The method for preparing the multi-layer cell-loaded nerve repair conduit having the functions of conducting electricity, resisting inflammation and promoting nerve differentiation according to claim 1, characterized in that: In step 4), during the UV curing process, the UV wavelength is 405 nm, the power is 20-30 W, and the time is 10-20 s; the in vitro culture uses a neural differentiation-promoting culture medium.

8. A multi-layered cell-loaded nerve repair catheter with the functions of conducting electricity, resisting inflammation and promoting nerve differentiation, characterized in that: The method is adopted as described in any one of claims 1 to 7.

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