Neural networked composite gel material for spinal cord injury repair and preparation method thereof

Abstract

The invention discloses a neural networked composite gel material for spinal cord injury repair and a preparation method of the neural networked composite gel material. Comprising the following steps: 1) preparing a shell layer mixed microsphere solution, a core layer matrix solution and a core layer network solution; 2) taking the core layer matrix solution as a matrix printing material, taking the core layer network solution as a printing material of a network structure distributed in a matrix, and performing 3D printing and in-situ UV curing according to a required drawing to obtain composite gel with a neural differentiation pre-network structure distributed in the matrix; taking the composite gel as a core layer structure, printing a shell layer mixed microsphere solution on the surface of the composite gel, and curing to form a shell layer structure; and (3) performing nerve formation culture on the material obtained in the step (2) at 37 DEG C to obtain the neural network composite gel material for spinal cord injury repair, and during culture, thermally sacrificing gelatin in a core layer structure to form a pore network. A neural network structure is arranged in the gel, neuroinflammation can be inhibited, and a brand new strategy is provided for nerve injury repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of spinal cord injury repair materials, and relates to a composite gel material and a preparation method thereof, and specifically relates to a neural network composite gel material for spinal cord injury repair and a preparation method thereof. Background Art

[0002] Spinal cord injury (SCI) is a serious neurological disease, which is mostly caused by traffic accidents, falls, sports injuries, violent incidents, etc. Statistics show that spinal cord injury has a high disability rate. Patients often experience symptoms such as loss of motor and sensory functions, autonomic dysfunction, chronic pain, and secondary complications, which have a serious impact on the patient's quality of life and mental health.

[0003] With the advancement of regenerative medicine and bioengineering technology, spinal cord injury repair materials have been developed. Excellent biocompatibility and degradability, good mechanical properties and bionic structure, the ability to effectively support the growth of nerve cells and the regeneration of complexes, the gradual release of neurotrophic factors, and convenient clinical application are all basic design requirements for spinal cord injury repair materials. However, due to the limitations of the physical space of the material, it is difficult to re-form a complete and continuous neural network structure similar to that in the body inside the material. In addition, studies have found that inflammatory reactions are very likely to occur at the site of spinal cord injury, and the inflammatory environment also greatly limits the differentiation of nerve cells supported by various biomaterials, resulting in further obstruction of the spinal cord injury repair and regeneration process.

[0004] In response to the above problems, the present invention designs a neural network composite gel material that can be used for spinal cord injury repair. Combining 3D printing and material sacrifice technology, the network structure that is conducive to neural differentiation is successfully set up in the hydrogel, and neural differentiation-promoting and anti-inflammatory drugs are used at the same time, so that the material has both neural differentiation-promoting and anti-inflammatory effects, providing a new strategy for nerve and spinal cord injury repair. Summary of the invention

[0005] The purpose of the present invention is to provide a neural network composite gel material for repairing spinal cord injury and a preparation method thereof in view of the deficiencies of the prior art.

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

[0007] A method for preparing a neural network composite gel material for repairing spinal cord injury comprises the following steps:

[0008] 1) using microfluidics technology to prepare methacrylylated hyaluronic acid HAMA gel microspheres loaded with anti-inflammatory drugs, and adding the methacrylylated hyaluronic acid HAMA gel microspheres to a high concentration HAMA solution to obtain a shell mixed microsphere solution;

[0009] A low-concentration HAMA solution loaded with nerve growth promoting factor is prepared to obtain a core layer matrix solution; the high concentration and low concentration are relative to each other;

[0010] A mixed solution of gelatin and HAMA is prepared using a neural differentiation promoting medium as a solvent, and neural stem cells are added thereto to obtain a core layer network solution;

[0011] 2) Using the core layer matrix solution as the matrix printing material, using the core layer network solution as the printing material for the network structure distributed in the matrix, 3D printing is performed according to the required drawings and in-situ UV curing is performed to obtain a composite gel having a pre-network structure for neural differentiation distributed in the matrix; then using the composite gel as the core layer structure, and printing the shell layer mixed microsphere solution on its surface and curing it to form a shell layer structure;

[0012] 3) The material obtained in 2) is subjected to neural culture at 37° C. to obtain a neural network composite gel material for repairing spinal cord injury. During the culture, the gelatin in the core layer structure is thermally sacrificed to form a pore network.

[0013] In the above technical solution, further, the high concentration HAMA solution in 1) is a PBS solution containing a photoinitiator and HAMA, the concentration of the HAMA is 10-20wt%, and the concentration of the photoinitiator is 0.1-0.25wt%. When preparing the solution, HAMA should be added and heated to dissolve in the dark, and the dissolution temperature can usually be 50-80°C, and then the photoinitiator is added and mixed.

[0014] Furthermore, the HAMA gel microspheres loaded with anti-inflammatory drugs are prepared by microfluidic technology and then combined with UV light curing. The anti-inflammatory drugs are added to the high-concentration HAMA solution as the water phase, and the Span80 / paraffin oil mixed solution is used as the oil phase. A microfluidic chip can be used, the material is PDMS, the channel width is 50-100μm, the water phase flow rate is controlled to be 1-5μL / min, the oil phase flow rate is 10-20μL / min, the temperature is 20-25°C, the wavelength of the UV light curing light source is 405nm, and the power is 20W / m 2 , exposure time is 60-600s.

[0015] Furthermore, the anti-inflammatory drugs loaded in the gel microspheres can generally be one or more of dexamethasone, indomethacin and aspirin, and their corresponding concentrations in the aqueous phase can be controlled to be 4-10 ng / ml, 200-500 ng / ml and 1000-2000 ng / ml, respectively;

[0016] Furthermore, the low-concentration HAMA solution in 1) is a PBS solution containing a photoinitiator and HAMA, the concentration of HAMA is 4-7wt%, and the concentration of the photoinitiator is 0.05-0.12wt%; the nerve growth factor is one or more of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial cell-derived neurotrophic factor (GDNF), ganglioside, and vitamin B1, and the concentration of the nerve growth factor is 100ng / ml-0.1g / ml.

[0017] Further, 1) the preparation method of the core layer network solution is as follows: the neural differentiation promoting medium is one or more of NBM, DMEM / F12 or advanced DMEM, gelatin and HAMA are added thereto, wherein the gelatin concentration is 10-18wt%, the HAMA concentration is 1-2.5wt%, after dissolving in the dark, a photoinitiator is added, and the photoinitiator concentration is 0.1-0.25wt%; after the above solution is completely dissolved, neural stem cells of the P2-P5 generations are added at 37-40°C, with a density of 1-3×10 7 / mL, and mix them evenly by blowing to obtain a core layer network solution.

[0018] Furthermore, the core layer structure is columnar, with the core layer matrix solution as the matrix, and the core layer network solution is distributed in the matrix according to the designed network structure, and is formed by in-situ UV light curing after 3D printing, wherein the core layer network solution accounts for 1 / 20-1 / 10 of the total volume of the core layer structure printing solution.

[0019] Furthermore, the shell structure has a thickness of 50-250 microns.

[0020] Furthermore, according to a specific example of the present invention, the structure in the design drawing of the 3D printing is a cylindrical structure with a core layer and a shell layer. The core layer structure is a columnar matrix, in which a number of wire columns are arranged axially, and the wire columns are interconnected to form a network structure, and the shell layer structure is wrapped around the columnar surface of the core layer structure; the core layer structure is composed of a core layer matrix solution and a core layer network solution that are printed simultaneously and then in-situ UV cured. Typically, the columnar diameter of the core layer structure can be 0.5-1.5 cm, and the length can be 0.5-2 cm. The volume of the core layer network solution accounts for 1 / 20-1 / 10 of the total volume of the core layer structure printing solution. The process parameters of 3D printing can usually be as follows: before printing, turn on the plasma sterilization mode to sterilize the printing chamber; during printing, the temperature in the chamber is 30-33°C, the humidity is 70%, the oxygen content is 20%, and the carbon dioxide content is 5%; using the extrusion printing mode, the nozzle can print in the XY axis, the movement rate is 5-15mm / s, the working distance is 50-80mm, the Z-axis platform up and down movement time is set based on the time required for the first layer of printing, and each layer is moved down 500-1000 microns after printing, the curing wavelength is 405nm, and the light intensity is 20W / m 2 , the curing time is 30s; then print another layer.

[0021] Furthermore, in the neuroblast culture, the basal culture medium used is one or more of NBM, DMEM / F12 or advancedDMEM, and is added with drugs that promote nerve growth, wherein the drugs that promote nerve growth are one or more of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), glial cell-derived neurotrophic factor (GDNF), ganglioside, and vitamin B1, with a concentration of 100ng / ml-0.1g / ml; the differentiation is cultured in a constant temperature incubator at 37°C and 5% CO2 for 14-21 days.

[0022] Furthermore, the photoinitiator described in the present invention can generally be one or more of LAP, Irgacure 1173, Irgacure 2959, Irgacure 819, and the like.

[0023] The neural network composite gel material obtained by any of the above methods can be used to prepare spinal cord injury repair drugs or devices.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1) The present invention uses HAMA, gelatin, neurotrophic factors, anti-inflammatory drugs, neural stem cells, etc. as raw materials, and combines microfluidics, blending, biological 3D printing, material sacrifice, in vitro three-dimensional culture and other processes to produce a unique neural network composite gel material that can be used for spinal cord injury repair.

[0026] 2) The neural network composite gel material for spinal cord injury repair in the present invention has a double-layer core-shell structure: the core layer is made of a mixed solution of low-concentration HAMA loaded with nerve growth factor and gelatin and HAMA loaded with neural stem cells through biological 3D printing and in-situ UV light curing process, and the shell layer is made of a high-concentration HAMA solution loaded with HAMA gel microspheres containing anti-inflammatory drugs, which is 3D printed outside the core layer. The composite gel material formed by the core-shell structure has the combined functions of the core layer promoting nerves and the shell layer anti-inflammation, which significantly optimizes the effect of traditional spinal cord injury repair methods. The core layer can promote the regeneration of nerve cells and the extension of axons, and provide necessary support and nutrition for the damaged spinal cord. At the same time, the shell layer contains anti-inflammatory ingredients, which can effectively inhibit the common inflammatory response after nerve injury and reduce secondary damage to the injured area.

[0027] 3) Aiming at the characteristics of the course of spinal cord injury, the present invention uses a high-concentration HAMA solution to construct a shell layer outside the core layer. First, as the outermost protective layer, it maintains the stability of the composite gel structure and prevents the gel from rupturing due to physical compression of spinal cord edema; secondly, the shell structure also slows down the release of nerve growth factor and gel degradation rate in the core layer through physical barrier effect, so that the degradation cycle matches the nerve injury repair process. The organic synergy of the above core-layer structure runs through the different needs in the complex course of spinal cord injury, thereby establishing a full-process anti-inflammatory-promoting regeneration treatment plan, which brings the possibility of higher-quality spinal cord injury repair.

[0028] 4) The composite gel material in the present invention has the functions of promoting nerve regeneration and repair and anti-inflammatory. Its function of promoting nerve regeneration and repair comes from multiple designs that promote the generation of neural networks: first, a HAMA / gelatin pre-network structure with heat-induced sacrificial ability is set in the core layer of the composite gel, in which the amount of gelatin is much greater than HAMA. During biological 3D printing, a small amount of HAMA allows the pre-network structure to be maintained. When printing is completed and neural culture is carried out, the gelatin is dissolved by heat to form an axially arranged and interconnected pore structure, providing a 3D space for the formation of a neural network, which is the first design; secondly, the neural differentiation medium used as the solvent of the HAMA / gelatin solution, when the gelatin is dissolved, the neural stem cells therein are immediately "bathed" in the environment of neural differentiation, which is the second design; moreover, the low-concentration HAMA matrix in the core layer is mixed with a neural growth factor, which plays the third function of promoting the efficient generation of neural networks; the composite gel is cultured for 14-21 days in vitro for neuralization, and after the neural network prototype is formed, it is implanted in a spinal cord injury model for use. Through the mutual coordination of the above-mentioned multiple designs, the neural network structure can be efficiently prepared in vitro, and at the same time, an excellent spinal cord injury repair effect is obtained. In addition, in order to address the secondary damage caused by inflammation and edema of spinal cord injuries in traditional spinal cord injuries, HAMA gel microspheres loaded with anti-inflammatory drugs are added to the shell layer of the gel material. Since the anti-inflammatory drugs are located in the microspheres, they need to diffuse out of the microspheres when released, and then further diffuse out of the shell layer of the composite gel to provide anti-inflammatory drugs around the implanted tissue, thereby inhibiting edema of the surrounding tissues and reducing secondary nerve damage, providing a good extracellular environment for the differentiation of core nerves. Under the organic synergy of anti-inflammatory and promoting nerve regeneration and repair functions, this composite hydrogel can achieve efficient repair of spinal cord injuries.

[0029] 5) The composite hydrogel in the present invention is achieved by using blending, biological 3D printing, and in-situ UV light curing to simultaneously load neural stem cells, neural differentiation culture medium and neural growth factor, and is provided with a composite hydrogel core layer of a pre-neural network, and after in vitro culture, the generation of a 3D spatial pore structure for neural network growth is achieved through thermal sacrificial technology, and finally an embedded neural network structure is obtained inside the gel. And the outer shell layer is also formed by 3D printing and in-situ UV curing, and at the same time, the drug-loaded gel microsphere material prepared by microfluidics and blending technology is loaded inside, so that the subsequent long-acting anti-inflammatory purpose can be achieved. The present invention achieves the successful preparation of the composite gel material for efficient repair of spinal cord injury through the organic synergy between the above-mentioned processes.

[0030] 6) The composite gel material in the present invention can be customized in gel form according to the size of the patient's spinal cord injury and actual needs. It has a high degree of universality and convenience. Implant gels of different sizes can be customized for different injury sites to meet the actual needs of the patient as much as possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the preparation process of the neural network composite gel material for spinal cord injury repair of the present invention;

[0032] Figure 2 The survival rate results of the material of the present invention and neural stem cells after 3D culture;

[0033] Figure 3 The repair effect of the material of the present invention on the mouse spinal cord injury model (A is the blank control (no surgery), B is Example 3, and C is Example 1);

[0034] Figure 4 The HE staining results of the material of the present invention on the repair of spinal cord injury (A is Example 3, B is Example 1). DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0036] Comparative Example 1

[0037] 1) In a light-proof condition and a 50°C water bath, a 10wt% high-concentration HAMA solution was prepared using a PBS solvent containing 0.25wt% LAP initiator. It was divided into two parts, and 10ng / ml dexamethasone was added to one part; then, at a constant temperature of 25°C, in a PDMS microfluidic chip with a channel width of 50μm, a Span 80 / paraffin oil mixed solution was used as the oil phase, and the obtained HAMA solution containing dexamethasone was used as the water phase. The oil phase flow rate was adjusted to 20μL / min and the water phase flow rate was 1μL / min. The wavelength was 405nm and the power was 20W / m 2 HAMA gel microspheres containing anti-inflammatory drugs were prepared under the conditions of a UV light curing light source and an exposure time of 300 seconds. Acetone, ethanol, and deionized water were used to quickly wash and remove the organic phase. Another portion of the high-concentration HAMA solution was mixed with the HAMA gel microspheres containing anti-inflammatory drugs to obtain a shell layer mixed microsphere solution for later use.

[0038] 2) In a dark environment and at 50°C in a water bath, a 4 wt% HAMA solution was prepared using a PBS solvent containing 0.25 wt% LAP initiator, to which 10 ng / ml NGF, 10 ng / ml BDNF, 10 ng / ml NT-3 and 10 ng / ml GDNF were added to obtain a HAMA solution containing nerve growth factor. 7 / mL) was added into the above solution and gently pipetted to mix well to obtain a core layer matrix solution.

[0039] 3) 3D printing is performed based on the design drawings. The printing solution is the core layer matrix solution. The printing diameter is 1 cm and the length is 1 cm. The printing process parameters are: before printing, the plasma sterilization mode is turned on to sterilize the printing chamber; during printing, the chamber temperature is 33°C, the humidity is 70%, the oxygen content is 20%, and the carbon dioxide content is 5%; the printing mode is extrusion, the XY axial movement rate is 5 mm / s, the working distance is 60 mm, and the Z-axis platform up and down movement time is set based on the time required for the first layer of printing. After each layer is printed, it moves down 500 microns. The curing wavelength is 405 nm and the light intensity is 20 W / m 2 , the curing time is 30s; the shell layer thickness is 250 microns, which is obtained by printing the shell layer mixed microsphere solution on the surface of the core layer structure using the same printing process.

[0040] 4) The above gel structure was placed in a constant temperature incubator at 37°C and 5% CO2, in NBM culture medium containing 100 ng / ml BDNF, 100 ng / ml NT-3, and 100 ng / ml GDNF, for 14 days to culture the neural tissue.

[0041] 5) After 7 days of culture, the cells in the gel were subjected to live-dead cell fluorescence staining, and the results showed that the cell survival rate was about 48%; after 21 days of culture, the cell-loaded material after neuroblast differentiation was implanted into a mouse spinal cord injury model, and the material was relatively completely integrated into the injured spinal cord structure without large-scale disintegration. Immunofluorescence staining was performed after frozen sections were used, and neurons were marked with NeuN and β-III Tubulin, and cell nuclei were marked with DAPI. The results showed that the neuroblast differentiation and repair effects were poor.

[0042] Example 1

[0043] 1) In a light-proof condition and a 50°C water bath, a 10wt% high-concentration HAMA solution was prepared using a PBS solvent containing 0.25wt% LAP initiator. It was divided into two parts, and 10ng / ml dexamethasone was added to one part; then, at a constant temperature of 25°C, in a PDMS microfluidic chip with a channel width of 50μm, a Span 80 / paraffin oil mixed solution was used as the oil phase, and the obtained HAMA solution containing dexamethasone was used as the water phase. The oil phase flow rate was adjusted to 20μL / min and the water phase flow rate was 1μL / min. The wavelength was 405nm and the power was 20W / m 2 HAMA gel microspheres containing anti-inflammatory drugs were prepared under the conditions of a UV light curing light source and an exposure time of 300 seconds. Acetone, ethanol, and deionized water were used to quickly wash and remove the organic phase. Another portion of the high-concentration HAMA solution was mixed with the HAMA gel microspheres containing anti-inflammatory drugs to obtain a shell layer mixed microsphere solution for later use.

[0044] 2) Under light-proof conditions and a 50°C water bath, a 4wt% HAMA solution was prepared using a PBS solvent containing 0.25wt% LAP initiator, to which 10ng / ml NGF, 10ng / ml BDNF, 10ng / ml NT-3 and 10ng / ml GDNF were added to obtain a HAMA solution containing nerve growth factor, i.e., a core layer matrix solution.

[0045] 3) In a dark environment and a 45°C water bath, a mixed solution of 1 wt% HAMA and 10 wt% gelatin was prepared using NBM medium containing 0.1 wt% LAP; after the solution was cooled to 37°C, mature neural stem cells of generation P3 (density 3×10 7 / mL) were added to the above solution, and gently pipetted to mix well to obtain a HAMA / gelatin mixed solution containing neural stem cells, that is, a core layer network solution.

[0046] 4) 3D printing was performed based on the design drawings, and the overall structure was a cylindrical structure with a core layer and a shell layer. The internal structure of the core layer was a low-concentration HAMA matrix wrapped in a gelatin / HAMA gel column loaded with neural stem cells that was arranged axially and interconnected. The printing solutions were the core layer matrix solution and the core layer network solution, respectively. The core layer network solution, i.e., the HAMA / gelatin mixed solution containing neural stem cells, accounted for 1 / 20 by volume, and the printing diameter was 1 cm and the length was 1 cm. The printing process parameters were: before printing, the plasma sterilization mode was turned on to sterilize the printing chamber; during printing, the chamber temperature was 33°C, the humidity was 70%, the oxygen content was 20%, and the carbon dioxide content was 5%; the printing mode was extrusion, the XY axial movement rate was 5 mm / s, the working distance was 60 mm, and the up and down movement time of the Z-axis platform was set based on the time required for the first layer to be printed. After each layer was printed, it moved down 500 microns, the curing wavelength was 405 nm, and the light intensity was 20 W / m 2 , the curing time is 30s; the shell layer thickness is 250 microns, which is obtained by printing the shell layer mixed microsphere solution on the surface of the core layer structure using the same printing process.

[0047] 5) The above gel structure was placed in a constant temperature incubator at 37°C and 5% CO2, in NBM culture medium containing 100 ng / ml BDNF, 100 ng / ml NT-3, and 100 ng / ml GDNF, for 14 days to culture the neural tissue.

[0048] 6) Compared with Comparative Example 1, this embodiment introduces a neural network pre-pore structure into the composite gel. The cells in the above gel were subjected to live and dead cell fluorescence staining after 7 days of culture, and the results showed that the cell survival rate was about 76%; after the cell-loaded material was implanted into the mouse spinal cord injury model after neural differentiation culture and cultured for 21 days, the material was relatively completely integrated into the damaged spinal cord structure, and no large-scale disintegration occurred. Immunofluorescence staining was performed after frozen sections were used, and neurons were labeled with NeuN and β-III Tubulin, and cell nuclei were labeled with DAPI. The results showed that the neuronal density increased by about 70% compared with Comparative Example 1, indicating that the shell layer retained the integrity of the core layer and avoided the waste of neural stem cells due to the disintegration of the gel structure. H&E staining showed that the tissue at the site of injury was mildly inflammatory infiltrated, with occasional inflammatory cells, mild edema, and slightly swollen local tissues, indicating that the gel microspheres of anti-inflammatory factors in the shell layer improved the inflammatory environment of the tissue at the site of injury through anti-inflammatory effects, provided a good neural stem cell differentiation environment, and greatly improved the survival rate of neurons.

[0049] Example 2

[0050] 1) In a light-proof condition and a 50°C water bath, a 10wt% high-concentration HAMA solution was prepared using a PBS solvent containing 0.25wt% LAP initiator. It was divided into two parts, and 10ng / ml dexamethasone was added to one part; then, at a constant temperature of 25°C, in a PDMS microfluidic chip with a channel width of 50μm, a Span 80 / paraffin oil mixed solution was used as the oil phase, and the obtained HAMA solution containing dexamethasone was used as the water phase. The oil phase flow rate was adjusted to 20μL / min and the water phase flow rate was 1μL / min. The wavelength was 405nm and the power was 20W / m 2 HAMA gel microspheres containing anti-inflammatory drugs were prepared under the conditions of a UV light curing light source and an exposure time of 300 seconds. Acetone, ethanol, and deionized water were used to quickly wash and remove the organic phase. Another portion of the high-concentration HAMA solution was mixed with the HAMA gel microspheres containing anti-inflammatory drugs to obtain a shell layer mixed microsphere solution for later use.

[0051] 2) Under light-proof conditions and a 50°C water bath, a 4 wt% HAMA solution was prepared using a PBS solvent containing 0.25 wt% LAP initiator, to which 10 ng / ml NGF, 10 ng / ml BDNF, 10 ng / ml NT-3 and 10 ng / ml GDNF were added to obtain a HAMA solution containing nerve growth factor to obtain a core layer matrix solution.

[0052] 3) In a dark environment and a 45°C water bath, a mixed solution of 1 wt% HAMA and 10 wt% gelatin was prepared using NBM medium containing 0.1 wt% LAP; after the solution was cooled to 37°C, mature neural stem cells of generation P3 (density 3×10 7 / mL) were added to the above solution, and gently pipetted to mix well to obtain a HAMA / gelatin mixed solution containing neural stem cells, that is, a core layer network solution.

[0053] 4) 3D printing was performed based on the design drawings, and the overall structure was a cylindrical structure with a core layer and a shell layer. The internal structure of the core layer was a low-concentration HAMA matrix wrapped in a gelatin / HAMA gel column loaded with neural stem cells that was arranged axially and interconnected. The printing solutions were the core layer matrix solution and the core layer network solution, respectively. The core layer network solution, i.e., the HAMA / gelatin mixed solution containing neural stem cells, accounted for 1 / 10 by volume, and the printing diameter was 1 cm and the length was 1 cm. The printing process parameters were: before printing, the plasma sterilization mode was turned on to sterilize the printing chamber; during printing, the chamber temperature was 33°C, the humidity was 70%, the oxygen content was 20%, and the carbon dioxide content was 5%; the printing mode was extrusion, the XY axial movement rate was 5 mm / s, the working distance was 60 mm, and the up and down movement time of the Z-axis platform was set based on the time required for the first layer to be printed. After each layer was printed, it moved down 500 microns, the curing wavelength was 405 nm, and the light intensity was 20 W / m 2 , the curing time is 30s; the shell layer thickness is 250 microns, which is obtained by printing the shell layer mixed microsphere solution on the surface of the core layer structure using the same printing process.

[0054] 5) The above gel structure was placed in a constant temperature incubator at 37°C and 5% CO2, in NBM culture medium containing 100 ng / ml BDNF, 100 ng / ml NT-3, and 100 ng / ml GDNF, for 14 days to culture the neural tissue.

[0055] 6) Relative to Example 1, the volume ratio of the HAMA / gelatin mixed solution loaded with neural stem cells to the core layer during printing was increased from 1 / 20 to 1 / 10. The cells in the above gel were subjected to live and dead cell fluorescence staining after 7 days of culture, and the results showed that the cell survival rate was about 81%; after being implanted in a mouse spinal cord injury model and cultured for 21 days, the material was relatively completely integrated into the damaged spinal cord structure, and no large-scale disintegration occurred. Immunofluorescence staining was performed after frozen sections were used, and neurons were labeled with NeuN and β-III Tubulin, and cell nuclei were labeled with DAPI. The results showed that the neuronal density increased by about 37% compared with that in real-time Example 1, and the fluorescence signals of nerve cell-related markers were more dense in the slice observation, indicating that compared with Example 1, the volume ratio of the HAMA / gelatin mixed solution loaded with neural stem cells increased, the content of gelatin in the core layer was increased, and the transport structure produced after the dissolution sacrifice was more conducive to the differentiation of neurons. H&E staining showed mild inflammatory infiltration in the injured tissue, occasional inflammatory cells, mild edema, and slight swelling of the local tissue, indicating that the gel microspheres of anti-inflammatory factors in the shell layer improved the inflammatory environment of the injured tissue through anti-inflammatory effects, provided a good environment for neural stem cell differentiation, and greatly improved the survival rate of neurons.

[0056] Example 3

[0057] 1) In a dark environment and at 50°C in a water bath, a 4 wt% HAMA solution was prepared using a PBS solvent containing 0.25 wt% LAP initiator, to which 10 ng / ml NGF, 10 ng / ml BDNF, 10 ng / ml NT-3 and 10 ng / ml GDNF were added to obtain a HAMA solution containing nerve growth factor, i.e., a core layer matrix solution.

[0058] 2) In a dark environment and a 45°C water bath, a mixed solution of 1 wt% HAMA and 10 wt% gelatin was prepared using NBM medium containing 0.1 wt% LAP; after the solution was cooled to 37°C, mature neural stem cells of generation P3 (density 3×10 7 / mL) were added to the above solution, and gently pipetted to mix well to obtain a HAMA / gelatin mixed solution containing neural stem cells, that is, a core layer network solution.

[0059] 3) 3D printing was performed based on the design drawings, and the overall structure was a cylindrical structure with a core layer and a shell layer. The internal structure of the core layer was a low-concentration HAMA matrix wrapped in a gelatin / HAMA gel column loaded with neural stem cells that was arranged axially and interconnected. The printing solutions were the core layer matrix solution and the core layer network solution, respectively. The core layer network solution, i.e., the HAMA / gelatin mixed solution containing neural stem cells, accounted for 1 / 20 by volume, and the printing diameter was 1 cm and the length was 1 cm. The printing process parameters were: before printing, the plasma sterilization mode was turned on to sterilize the printing chamber; during printing, the chamber temperature was 33°C, the humidity was 70%, the oxygen content was 20%, and the carbon dioxide content was 5%; the printing mode was extrusion, the XY axial movement rate was 5 mm / s, the working distance was 60 mm, and the up and down movement time of the Z-axis platform was set based on the time required for the first layer to be printed. After each layer was printed, it moved down 500 microns, the curing wavelength was 405 nm, and the light intensity was 20 W / m 2 , the curing time is 30s.

[0060] 4) The above gel structure was placed in a constant temperature incubator at 37°C and 5% CO2, in NBM culture medium containing 100 ng / ml BDNF, 100 ng / ml NT-3, and 100 ng / ml GDNF, for 14 days to culture the neural tissue.

[0061] 5) Compared with Example 1, the protective shell structure on the surface of the composite gel material was removed. The cells in the above gel were subjected to live and dead cell fluorescence staining after 7 days of culture, and the results showed that the cell survival rate was about 79%; after the cell-loaded material was implanted into the mouse spinal cord injury model after neural differentiation culture and cultured for 21 days, it was found that the gel structure was severely disintegrated and not well integrated into the damaged spinal cord structure, indicating that the structure could not maintain a good structure in the swollen spinal cord. Immunofluorescence staining was performed after frozen sections were used, and neurons were marked with NeuN and β-III Tubulin, and cell nuclei were marked with DAPI. The results showed that only a small number of neurons grew in the broken end of the spinal cord. H&E staining showed that the tissue in the injured area was severely infiltrated, a large number of inflammatory cells were aggregated and diffusely distributed, the local tissue was moderately edematous, the tissue swelling was obvious, and a large number of secondary neurons in the broken segment of the spinal cord were also observed. The secondary loss of a large number of neurons was also observed, indicating that the tissue inflammatory environment greatly reduced the survival rate of neural stem cells and further caused secondary damage to the tissue.

[0062] The above-described embodiments are only some of the preferred solutions of the present invention, but they are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for preparing a neural network composite gel material for repairing spinal cord injury, characterized in that: The following steps are involved: 1) using microfluidics technology to prepare methacrylylated hyaluronic acid HAMA gel microspheres loaded with anti-inflammatory drugs, and adding the methacrylylated hyaluronic acid HAMA gel microspheres to a high concentration HAMA solution to obtain a shell mixed microsphere solution; A low-concentration HAMA solution loaded with nerve growth promoting factor is prepared to obtain a core layer matrix solution; the high concentration and low concentration are relative to each other; A mixed solution of gelatin and HAMA is prepared using a neural differentiation promoting medium as a solvent, and neural stem cells are added thereto to obtain a core layer network solution; 2) Using the core layer matrix solution as the matrix printing material, using the core layer network solution as the printing material for the network structure distributed in the matrix, 3D printing is performed according to the required drawings and in-situ UV curing is performed to obtain a composite gel having a pre-network structure for neural differentiation distributed in the matrix; then using the composite gel as the core layer structure, and printing the shell layer mixed microsphere solution on its surface and curing it to form a shell layer structure; 3) The material obtained in 2) is subjected to neural culture at 37° C. to obtain a neural network composite gel material for repairing spinal cord injury. During the culture, the gelatin in the core layer structure is thermally sacrificed to form a pore network.

2. The method for preparing the neural network composite gel material for spinal cord injury repair according to claim 1, characterized in that: The high concentration HAMA solution in 1) is a PBS solution containing a photoinitiator and HAMA, the concentration of the HAMA is 10-20wt%, and the concentration of the photoinitiator is 0.1-0.25wt%.

3. The method for preparing the neural network composite gel material for spinal cord injury repair according to claim 1, characterized in that: The HAMA gel microspheres loaded with anti-inflammatory drugs are prepared by microfluidic technology and then combined with UV light curing. The anti-inflammatory drugs are added to the high-concentration HAMA solution as the water phase, and the Span 80 / paraffin oil mixed solution is used as the oil phase. The water phase flow rate is 1-5μL / min, the oil phase flow rate is 10-20μL / min, the temperature is 20-25°C, the wavelength of the UV light curing light source is 405nm, and the power is 20W / m 2 , exposure time is 60-600s.

4. The method for preparing the neural network composite gel material for spinal cord injury repair according to claim 1, characterized in that: 1) The low concentration HAMA solution is a PBS solution containing a photoinitiator and HAMA, the concentration of HAMA is 4-7wt%, and the concentration of the photoinitiator is 0.05-0.12wt%; the nerve growth factor is one or more of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial cell-derived neurotrophic factor (GDNF), ganglioside, and vitamin B1, and the concentration of the nerve growth factor is 100ng / ml-0.1g / ml.

5. The method for preparing the neural network composite gel material for spinal cord injury repair according to claim 1, characterized in that: 1) The preparation method of the core layer network solution is as follows: the neural differentiation promoting medium is one or more of NBM, DMEM / F12 or advanced DMEM, gelatin and HAMA are added thereto, wherein the gelatin concentration is 10-18wt%, and the HAMA concentration is 1-2.5wt%, after dissolving in the dark, a photoinitiator is added, and the photoinitiator concentration is 0.1-0.25wt%; after the above solution is completely dissolved, neural stem cells of the P2-P5 generations are added at 37-40°C, with a density of 1-3×10 7 / mL, and mix them evenly by blowing to obtain a core layer network solution.

6. The method for preparing the neural network composite gel material for spinal cord injury repair according to claim 1, characterized in that: The core layer structure is columnar, with a core layer matrix solution as the matrix, a core layer network solution distributed in the matrix according to the designed network structure, and in-situ UV light curing after 3D printing, wherein the core layer network solution accounts for 1 / 20-1 / 10 of the total volume of the core layer structure printing solution.

7. The method for preparing a neural network composite gel material for repairing spinal cord injury according to claim 1, characterized in that: The thickness of the shell structure is 50-250 microns.

8. The method for preparing the neural network composite gel material for spinal cord injury repair according to claim 1, characterized in that: In the neuroblast culture, the basic culture medium used is one or more of NBM, DMEM / F12 or advanced DMEM, and is added with drugs that promote nerve growth. The drugs that promote nerve growth are one or more of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), glial cell-derived neurotrophic factor (GDNF), ganglioside, and vitamin B1, with a concentration of 100ng / ml-0.1g / ml; the differentiation is cultured in a constant temperature incubator at 37°C and 5% CO2 for 14-21 days.

9. A neural network composite gel material obtained by the method according to any one of claims 1 to 8.

10. Use of the neural network composite gel material according to claim 9 in the preparation of spinal cord injury repair drugs or devices.

Images