A multilayered cell-loaded nerve repair conduit with conductive, anti-inflammatory and neural differentiation-promoting functions and its preparation method

Abstract

The present invention discloses 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. The preparation method is as follows: 1) preparing PLGA fibers doped with graphene, weaving them to obtain a layer of cylindrical fiber mesh; 2) preparing a HAMA mixed solution mixed with microspheres loaded with nerve growth factor and manganese dioxide nanoparticles, and a HAMA solution loaded with neural stem cells; 3) fixing the cylindrical fiber mesh in a cylindrical groove mold, pouring the HAMA mixed solution, and freeze-drying after UV curing to obtain a two-layer porous composite scaffold; 4) immersing it in the HAMA solution loaded with neural stem cells, rapidly curing to form a three-layer cell-loaded gel layer, and culturing in vitro for 4-8 weeks to obtain a multi-layered cell-loaded nerve repair conduit. The nerve repair conduit has the functions of conducting electricity, resisting inflammation and promoting nerve differentiation, and provides a new strategy for the repair of nerve and spinal cord injuries.

Description

Technical Field

[0001] The present invention relates to a preparation technology of nerve repair materials, 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 clinical condition, often caused by traffic accidents, falls, earthquakes, firearm injuries, and iatrogenic injuries. Statistics show that approximately 2.8% of trauma patients will have peripheral nerve involvement, and nearly 40% of these patients will experience symptoms of organic neurological damage, mainly manifested as partial or complete loss of motor and sensory function, chronic pain, and target muscle atrophy, which imposes a significant physical and psychological burden on patients.

[0003] With the advancement of tissue engineering technology, neural repair materials have gradually been developed. Specifically, they should possess the following characteristics: good biocompatibility and biodegradability; anti-inflammatory properties; biomimetic structures to achieve morphological biomimetic effects; release of growth factors related to neural regeneration; and ease of clinical operation. Studies have also shown that electrical stimulation can promote neural regeneration. However, current neural repair materials typically only possess some of these properties, resulting in them not fully meeting the actual needs of neural repair during use, resulting in insignificant repair effects.

[0004] To address these challenges, the present invention has designed a multi-layered, integrated, cell-loaded nerve repair conduit with electrical conductivity, anti-inflammatory properties, and neurodifferentiation-promoting properties, as well as a method for its preparation. This conduit comprises a conductive fiber layer, a freeze-dried composite scaffold layer, and a cell-loaded hydrogel layer, arranged from the inside out. Furthermore, it is organically loaded with anti-inflammatory and neurodifferentiation-promoting drugs, resulting in a multifunctional material offering a novel strategy for repairing nerve and spinal cord injuries. Summary of the Invention

[0005] The present invention aims to address the ineffectiveness of conventional nerve conduits for repair by providing a multilayered, cell-loaded nerve repair conduit with electrical conductivity, anti-inflammatory properties, and the ability to promote neural differentiation, as well as its preparation method. This conduit offers a novel strategy for repairing nerve and spinal cord injuries, combining electrical conductivity, anti-inflammatory properties, and the ability to promote neural differentiation.

[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 with conductive, anti-inflammatory and neurodifferentiation-promoting functions comprises the following steps:

[0008] 1) First, PLGA fibers doped with graphene are prepared by a melt spinning process, and the fibers are woven into 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 nerve growth-promoting drug 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, shaking and UV curing, then demolding, and freeze-drying to obtain the second porous composite scaffold layer;

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

[0013] Furthermore, in step 1), during the preparation of the graphene-doped PLGA fibers, the PLGA is PLGA 50:50 or PLGA 75:25; the graphene has a size of 2-3 microns, a concentration of 0.1-0.5 wt%, and a fiber diameter of 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, a 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 the fluid channel, and UV curing is performed at a temperature of 20-25° C. and a pH value of 6.5-7.5; after the completion, acetone, ethanol and deionized water are used in sequence. The HAMA microspheres are washed with water to obtain HAMA microspheres, which are then immersed in a nerve growth-promoting drug solution to obtain HAMA microspheres loaded with nerve growth-promoting drugs at a concentration of 0.1-1.0 wt%; 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 line-derived neurotrophic factor (GDNF), ganglioside, vitamin B1, citicoline, and oxiracetam, with a concentration of 100 ng / ml-0.1 g / ml.

[0016] Furthermore, in step 2), during the preparation of the manganese dioxide nanoparticles, a mixed solution of sodium dodecylbenzenesulfonate and manganese chloride is prepared, and a potassium permanganate solution is added dropwise to the mixed solution under stirring. After the reaction is completed, the mixture is centrifuged and washed at high speed 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, wherein the degree of substitution of the HAMA is 30-60% and the concentration is 7-20 wt %. The photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, and the concentration is 0.25-0.5 wt %. The solvent of the HAMA mixed solution is PBS.

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

[0020] Furthermore, in step 4), the cylindrical groove mold is made of PMMA or glass and comprises a base and two cylinders. The base has a diameter of 1.5-3.5 mm and is provided with two concentric circular grooves. Each groove has a width of 100-200 microns and a depth of 1-2 mm. The interval between the two grooves is 0.5-1.5 mm. The thickness of each cylinder is consistent with the width of the groove. 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 h.

[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 utilizes graphene, PLGA, HAMA, a nerve growth-promoting drug, and manganese dioxide as raw materials, and combines melt spinning, fiber weaving, hydrothermal treatment, in-situ casting, nanocomposite, and freeze-drying processes to produce a multilayered, cell-laden nerve repair conduit material that exhibits electrical conductivity, anti-inflammatory properties, and the ability to promote neural differentiation. This raw material selection and process combination are unique 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-loaded gel layer. The first cylindrical fiber mesh layer serves as the innermost layer, providing mechanical support and electrical conductivity for the catheter. By laying a foundation of fibers therein, it can ensure that the outer scaffold and gel layer will not break during use due to surgical manipulation, traction, etc. The second porous composite scaffold layer serves as the main bioactive substance storage layer, internally 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 combat neuroinflammation, respectively. 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-loaded 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 significantly innovative.

[0028] 3) This invention addresses the long nerve repair cycle by initially releasing drugs from neural differentiation-promoting culture media and microspheres loaded with nerve growth factors to promote stem cell differentiation and growth. Later, after the nerve conduit is implanted in the body, the conductive cylindrical fiber mesh inside connects to the nerve stump, transmitting electrophysiological signals within the body and further promoting nerve repair and regeneration through bioelectric stimulation. It is the synergistic effect of these two actions that enables this nerve repair conduit to meet the requirements of a nerve repair cycle of more than three months.

[0029] 4) The present invention uses a casting method to wrap the cylindrical fiber mesh within 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 nerve injury, 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 immersion 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 into 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 an 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 damage 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 need for secondary surgery; at the same time, the internal cylindrical fiber mesh structure serves as the bottom layer to ensure the strength and toughness of the nerve conduit, so that it will not be easily broken or damaged during use; in response to 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 damage repair; at the same time, in the process of promoting the regeneration and repair of damaged nerves through the nerve conduit, the nerve growth factor can promote the differentiation of stem cells into nerves, and at the same time, the connection between the nerve and the nerve conduit 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 damage regeneration and repair are met, and the above performance is carefully designed by the applicant based on the actual symptoms of the disease, which is obviously innovative.

[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 This is a schematic diagram of the preparation process of the multi-layered cell-laden nerve repair catheter material of the present invention that has 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 loaded with a drug promoting nerve growth using microfluidic technology: First, a microfluidic fluid channel was assembled, with a channel diameter of 50 μm. Then, a HAMA solution with a concentration of 7.5 wt% (degree of substitution 60%) was placed in a microfluidic syringe pump. The HAMA solution was injected into the fluid channel at a flow rate of 0.4 mL / h using the microfluidic system. A mixed solution of Span 80 and paraffin oil was added dropwise. UV curing was performed at a temperature of 25°C and a pH of 7.2. After completion, the microspheres were washed with acetone, ethanol, and deionized water in sequence. After thorough washing, the obtained HAMA solution was removed. The AMA microspheres were then immersed in a 100 ng / ml NGF solution for adsorption to obtain HAMA microspheres loaded with a drug that promotes nerve growth. Simultaneously, a hydrothermal method was used to prepare manganese dioxide nanoparticles: a mixed solution of 1 g / L sodium dodecylbenzenesulfonate and 125 g / L manganese chloride was prepared. Potassium permanganate solution was added dropwise to the solution under high-speed stirring at 1000 rpm, resulting in a potassium permanganate concentration of 25 g / L. The solution was reacted at 80°C for 7 hours, then centrifuged and washed at high speed to obtain manganese dioxide nanoparticles with a particle size of approximately 20-30 nm.

[0036] 2) In the dark and heated at 60°C, two 7.5 wt% HAMA solutions (degree of substitution 60%) were prepared using PBS as a solvent and 0.25 wt% LAP as a photoinitiator. 0.4 wt% HAMA microspheres loaded with a nerve growth promoting drug and 0.1 wt% manganese dioxide nanoparticles were mixed with one 7.5 wt% HAMA solution. At the same time, neural stem cells of passage 2 were selected and 5×10 7 mL -1 The concentration of the HAMA solution was added to another HAMA solution with a concentration of 7.5 wt % and preheated to 37° C., and the HAMA solution loaded with neural stem cells was uniformly dispersed by pipetting;

[0037] 3) A HAMA mixed solution containing HAMA microspheres loaded with a nerve growth-promoting drug and manganese dioxide nanoparticles was poured into a cylindrical groove mold (composed of a base and two cylinders of different diameters; the base diameter was 2 mm, with two concentric circular grooves on the base. The grooves were 200 μm wide and 1 mm deep, separated by 0.5 mm. The cylinders were 2.6 cm thick and the height matched the groove width). After shaking and UV curing (UV wavelength of 405 nm, power of 20 W, time of 5 minutes), the solution was demolded and freeze-dried (frozen at -20°C for 5 hours) to obtain a porous composite scaffold layer.

[0038] 4) Immerse it in a neural stem cell-loaded HAMA solution and rapidly UV cure it (UV wavelength of 405 nm, power of 20 W, time of 20 s) to form a cell-loaded gel layer. After culturing in vitro for 8 weeks using a neural differentiation-promoting medium, a cell-loaded neural repair conduit is obtained, which is then 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 culture, the nerve regeneration rate was acceptable; but the nerve regeneration and repair effect after 3 months was average, and no neuroinflammation occurred.

[0040] Example 1:

[0041] 1) melt-spinning graphene-doped poly(D,L-lactide-co-glycolide) fibers (PLGA fibers) of approximately 80 μm in diameter using a 50:50 ratio of PLGA and 0.25 wt% graphene (2-3 μm) as raw materials. The fibers were then braided into a cylindrical fiber web approximately 1.5 cm in length and 2 mm in diameter.

[0042] 2) Preparation of HAMA microspheres loaded with a drug that promotes nerve growth using microfluidic technology: First, a microfluidic fluid channel was assembled with a channel diameter of 50 μm. Then, a HAMA solution with a concentration of 7.5 wt% (degree of substitution 60%) was placed in a syringe pump. The HAMA solution was injected into the fluid channel at a flow rate of 0.4 mL / h using the microfluidic system. A mixed solution of Span80 and paraffin oil was added dropwise. UV curing was performed at a temperature of 25°C and a pH of 7.2. After completion, the microspheres were cleaned with acetone, ethanol, and deionized water. The HAMA microspheres were then immersed in a 100 ng / ml NGF solution for adsorption to obtain HAMA microspheres loaded with a drug that promotes nerve growth. Simultaneously, a mixed solution of 1 g / L sodium dodecylbenzenesulfonate and 125 g / L manganese chloride was prepared using a hydrothermal method. A potassium permanganate solution was added dropwise to the solution under high-speed stirring at 1000 rpm, resulting in a potassium permanganate concentration of 25 g / L. After reacting at 80°C for 7 hours, the solution was washed by high-speed centrifugation to obtain manganese dioxide nanoparticles with a particle size of approximately 20-30 nm.

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

[0044] 4) The cylindrical fiber mesh was fixed in a cylindrical groove mold (composed of a base and two cylinders of different diameters; the base had a diameter of 2 mm and two concentric circular grooves on the base; the grooves were 200 μm wide and 1 mm deep, separated by 0.5 mm; the cylinders were 2.6 cm tall and the thickness of the grooves matched the groove width) as the first conductive fiber base layer. A HAMA mixed solution containing HAMA microspheres and manganese dioxide nanoparticles loaded with a nerve growth-promoting drug was poured into the mold. After shaking and UV curing (UV wavelength of 405 nm, power of 20 W, time of 5 min), the mold was demolded and freeze-dried (frozen at -20°C for 5 h) to obtain the second porous composite scaffold layer.

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

[0046] Compared to Comparative Example 1, this example added a conductive cylindrical fiber mesh. The conductivity of the conductive fiber was approximately 5.69 s / cm. An extract of the material was prepared and co-cultured with neural stem cells for 7 days before CCK-8 analysis. The results showed a cell survival rate of approximately 90.1%. The neural stem cells cultured on the nerve conduit showed good differentiation after 8 weeks. When implanted into a nerve injury model, the conduit remained intact during suturing. After 3 months of culture, the nerve damage was essentially repaired, with no neuroinflammation.

[0047] Example 2:

[0048] 1) melt-spinning PLGA fibers doped with graphene (about 80 microns in diameter) at a ratio of 50:50 and 0.25 wt% graphene (about 2-3 microns) as raw materials to produce cylindrical fiber webs about 1.5 cm in length and 2 mm in diameter.

[0049] 2) Preparation of HAMA microspheres loaded with a drug promoting nerve growth using microfluidic technology: First, a microfluidic fluid channel was assembled with a channel diameter of 50 μm. Next, a 7.5 wt% HAMA (60% substitution) solution was placed in a syringe pump. The HAMA solution was injected into the fluid channel at a flow rate of 0.4 mL / h using the microfluidic system. A mixed solution of Span 80 and paraffin oil was added dropwise. UV curing was performed at a temperature of 25°C and a pH of 7.2. After completion, the microspheres were cleaned with acetone, ethanol, and deionized water in sequence. After thorough cleaning, HAMA was obtained. The microspheres were then immersed in a 100 ng / ml NGF solution to obtain HAMA microspheres loaded with a nerve growth-promoting drug. Simultaneously, a hydrothermal method was used to prepare manganese dioxide nanoparticles: a mixed solution of 1 g / L sodium dodecylbenzenesulfonate and 125 g / L manganese chloride was prepared. Potassium permanganate solution was added dropwise to the solution under high-speed stirring at 1000 rpm, resulting in a potassium permanganate concentration of 25 g / L in the solution. The solution was reacted at 80°C for 7 hours and then centrifuged and washed at high speed to obtain manganese dioxide nanoparticles with a particle size of approximately 20-30 nm.

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

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

[0052] Compared to Example 1, this example eliminated the outermost cell-carrying gel layer. The conductive fiber exhibited an electrical conductivity of approximately 5.48 s / cm. An extract of the material was prepared and co-cultured with neural stem cells for seven days before CCK-8 analysis, demonstrating a cell survival rate of approximately 91.4%. The extract was implanted into a nerve injury model, maintaining intact catheters during suturing. After one month of culture, some cells migrated into the catheters. Three months later, the nerve regeneration and repair effect was moderate, with no neuroinflammation observed.

[0053] Example 3:

[0054] 1) melt-spinning PLGA fibers doped with graphene (about 80 microns in diameter) at a ratio of 50:50 and 0.25 wt% graphene (about 2-3 microns) as raw materials to produce cylindrical fiber webs about 1.5 cm in length and 2 mm in diameter.

[0055] 2) Preparation of HAMA microspheres loaded with a drug that promotes nerve growth using microfluidic technology: First, a microfluidic fluid channel was assembled with a channel diameter of 50 μm. Next, a 7.5 wt% HAMA (60% substitution) solution was placed in a syringe pump. The HAMA solution was injected into the fluid channel at a flow rate of 0.4 mL / h using the microfluidic system. A mixed solution of Span 80 and paraffin oil was added dropwise. UV curing was performed at a temperature of 25°C and a pH of 7.2. After completion, the microspheres were washed with acetone, ethanol, and deionized water, respectively. After thorough washing, the microspheres were immersed in a 100 ng / ml NGF solution for immersion adsorption to obtain HAMA microspheres loaded with the drug that promotes nerve growth.

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

[0057] 4) The cylindrical fiber mesh was fixed in a cylindrical groove mold (composed of a base and two cylinders of different diameters; the base had a diameter of 2 mm and two concentric circular grooves on the base; the grooves were 200 μm wide and 1 mm deep, separated by 0.5 mm; the cylinders were 2.6 cm tall and the thickness of the grooves matched the groove width) as the first conductive fiber base layer. A HAMA mixed solution containing HAMA microspheres and manganese dioxide nanoparticles loaded with a nerve growth-promoting drug was poured into the mold. After shaking and UV curing (UV wavelength of 405 nm, power of 20 W, time of 5 min), the mold was demolded and freeze-dried (frozen at -20°C for 5 h) to obtain the second porous composite scaffold layer.

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

[0059] Compared to Example 1, this example reduces the addition of manganese dioxide nanoparticles. The conductivity of the conductive fiber was approximately 5.77 s / cm. An extract of the material was prepared and co-cultured with neural stem cells for 7 days, followed by CCK-8 analysis. The results showed a cell survival rate of approximately 93.3%. The neural stem cells cultured on the nerve conduit showed good differentiation after 8 weeks of culture. When implanted into a nerve injury model, the conduit remained intact during suturing. However, inflammation occurred in the nerve injury area after one month of culture, and poor nerve repair was observed after three months.

Claims

1. A method for preparing a multi-layered cell-loaded nerve repair conduit with electrical conductivity, anti-inflammatory and neural differentiation-promoting functions, characterized in that: The following steps are involved: 1) First, PLGA fibers doped with graphene are prepared by a melt spinning process, and the fibers are woven into 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 nerve growth-promoting drug 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, shaking and UV curing, then demolding, and freeze-drying to obtain the second porous composite scaffold layer; 5) Immersing the conduit in a neural stem cell-loaded HAMA solution, rapidly UV-curing it to form a third cell-loaded gel layer, and culturing it in vitro for 4-8 weeks to obtain a multi-layered, cell-loaded neural repair conduit; In step 2), during the preparation of the HAMA microspheres loaded with the nerve growth promoting drug, a 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 syringe 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 dropped into the fluid channel, and UV curing is performed at a temperature of 20-25°C and a pH value of 6.5-7.5; after completion, acetone, ethanol, and deionized water are used in sequence. 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 at a concentration of 0.1-1.0 wt%; 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 line-derived neurotrophic factor (GDNF), ganglioside, vitamin B1, citicoline, and oxiracetam at a concentration of 100 ng / ml-0.1 g / ml; In step 2), during the preparation of the manganese dioxide nanoparticles, a mixed solution of sodium dodecylbenzenesulfonate and manganese chloride is prepared, and a potassium permanganate solution is added dropwise to the mixed solution under stirring. After the reaction is completed, the mixture is centrifuged and washed at high speed 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 %.

2. The method for preparing a multi-layered cell-loaded nerve repair conduit with conductive, anti-inflammatory and neurodifferentiation-promoting functions according to claim 1, characterized in that: In step 1), during the preparation of the graphene-doped PLGA fibers, the PLGA is PLGA 50:50 or PLGA 75:25; the graphene has a size of 2-3 microns, a concentration of 0.1-0.5 wt%, 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 a multi-layered cell-loaded nerve repair conduit with conductive, anti-inflammatory and neurodifferentiation-promoting functions according to claim 1, characterized in that: In step 3), the prepared HAMA mixed solution contains a photoinitiator, wherein the degree of substitution of the HAMA is 30-60% and the concentration is 7-20 wt %. The photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, and the concentration is 0.25-0.5 wt %. The solvent of the HAMA mixed solution is PBS; The HAMA solution loaded with neural stem cells contains a photoinitiator, the generation number of the neural stem cells is 2-3 generations, and the concentration is 0.1-10×10 7 mL -1 HAMA concentration is 5-10wt%, the photoinitiator is LAP, Irgacure, photoinitiator 2959 one or more, the concentration is 0.25-0.4wt%, the solvent is PBS.

4. The method for preparing a multi-layered cell-loaded nerve repair conduit with conductive, anti-inflammatory and neurodifferentiation-promoting functions according to claim 1, characterized in that: In step 4), the cylindrical groove mold is made of PMMA or glass and consists of a base and two cylinders. The base has a diameter of 1.5-3.5 mm and is provided with two concentric circular grooves. Each groove has a width of 100-200 microns and a depth of 1-2 mm. The interval between the two grooves is 0.5-1.5 mm. The thickness of each cylinder is consistent with the groove width. 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 h.

5. The method for preparing a multi-layered cell-loaded nerve repair conduit with conductive, anti-inflammatory and neurodifferentiation-promoting functions 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 medium.

6. A multi-layered cell-loaded nerve repair conduit with electrical conductivity, anti-inflammatory and neurodifferentiation-promoting functions, characterized in that: The method is described in any one of claims 1 to 5.

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