Preparation method of anisotropic nerve scaffold loaded with magnetic responsive nerve cells

Abstract

The invention discloses a preparation method of an anisotropic nerve scaffold loaded with magnetic responsive nerve cells. The preparation method comprises the following steps: preparing a PDMS (Polydimethylsiloxane) mold with a regular topological geometric structure by adopting a micro-molding method; pouring a biological material into the surface of the PDMS mold to form an anisotropic biological material scaffold; the magnetic nanoparticles are subjected to surface modification and internalized in nerve cells through a co-culture method, and the magnetic responsive nerve cells are obtained; and culturing the magnetic responsive nerve cells on the anisotropic nerve scaffold to obtain the anisotropic nerve scaffold loaded with the magnetic responsive nerve cells. According to the method, micro-molding and magnetic response cell therapy are combined, the stent is easy to prepare, the loaded nerve cells have good magnetic responsiveness, after the stent is transplanted into a human body, rapid elongation and migration of the nerve cells and rapid communication of injured nerves can be achieved under the combined action of an external magnetic field and an anisotropic topological structure, and the stent has a good application prospect. And repair and functional reconstruction of long-distance peripheral nerve injury are promoted.

Description

Technical Field

[0001] The invention relates to a method for preparing a medical biomaterial, in particular to a method for preparing an implantable biomaterial. Background Art

[0002] The nervous system is an important part of the human body, regulating and controlling the functional activities of other systems, making the organism a complete and unified whole. Nerve damage will cause functional disorders and chronic pain in the body, and in severe cases, it will even affect self-care ability and even life. For longer-distance defects, common nerve grafts include autologous nerves, allogeneic nerves, xenogeneic nerves, artificial nerve grafts, etc. Among them, autologous nerve transplantation is known as the gold standard and has a high transplantation success rate, but it is clinically limited by problems such as mismatch between the size of the donor and the recipient repair site, insufficient sources, and permanent loss of donor site function; allogeneic or xenogeneic nerve transplantation expands the source, but has the risk of immune rejection and disease transmission; tissue engineering artificial nerve grafts have therefore been widely studied, but their repair effect is still not as good as autologous nerve grafts, especially for the repair of long-distance peripheral nerve injuries. The efficacy is poor, mainly because the graft lacks sufficient bionic factors. Therefore, it is urgent to develop a functional artificial nerve graft with excellent long-distance peripheral nerve injury repair. Summary of the invention

[0003] Purpose of the invention: In view of the above-mentioned prior art, a method for preparing an anisotropic neural scaffold loaded with magnetically responsive neural cells is proposed to solve the problems of lack of bionic signals and difficulty in long-distance rapid regeneration of damaged nerves in the prior art.

[0004] Technical solution: A method for preparing an anisotropic neural scaffold loaded with magnetically responsive neural cells, comprising: Step 1: Fully mix the dimethylsiloxane monomer and the cross-linking agent, and pour them into a plane, curved surface or cylindrical mold with a regular anisotropic topological structure on the surface, vacuum degas and cross-link to obtain a plane, curved surface or cylindrical PDMS mold with a micro / nano anisotropic topological structure on the surface; Step 2: The natural or synthetic biomaterial solution is uniformly infused or coated on the surface of the PDMS mold, and after air drying or cross-linking and curing, a planar, curved or tubular biomaterial scaffold with a micro / nano anisotropic topological structure is prepared; Step 3: functionalizing the magnetic nanoparticles by a surface modification method to obtain magnetic nanoparticles with high biocompatibility; Step 4: internalizing the surface-modified magnetic nanoparticles into the nerve cells through a co-culture method to obtain magnetically responsive nerve cells; Step 5: Cultivate the magnetically responsive neural cells on the biomaterial scaffold to obtain an anisotropic neural scaffold loaded with the magnetically responsive neural cells.

[0005] Preferably, the topological structure is grooves, ridges, recesses or protrusions; wherein the high part of the groove-ridge structure is a ridge, the low part is a groove, the vertical distance between the groove and the ridge is 0.1-10 μm, the width of the groove and the ridge is 0.1-50 μm, and the connection between the groove and the ridge is smooth or vertical.

[0006] Preferably, the biomaterial scaffold has a thickness of 0.5-3 mm. If the biomaterial scaffold is tubular, the inner diameter is 0.5-10 mm and the length is 10-100 mm.

[0007] Preferably, in step 3, the magnetic nanoparticles used are one or a mixture of Fe2O3, MFe2O4 (M=Co, Mn, Ni), Fe3O4, NdFeB, and the particle size of the modified magnetic nanoparticles is 10-100 nm.

[0008] Preferably, in step 4, the neural cells may be one or more of Schwann cells, neurons, neural stem cells, astrocytes, microglia, olfactory ensheathing cells, etc.; the concentration of the magnetic nanoparticles during co-culture is 10-500 μg / mL, and the co-culture time is 4-48 h.

[0009] Preferably, in step 5, the co-culture time is 0.5-2 days, and the density of the magnetically responsive neural cells is 500-5000 / μm 2 .

[0010] Beneficial effects: The main body of the anisotropic nerve stent of the present invention is preferably natural macromolecular materials such as ovalbumin, silk fibroin, chitosan, etc., which have excellent biocompatibility and degradability and good mechanical strength. Compared with traditional nerve grafts, the stent of the present invention is easy to customize according to the actual defect environment, and the anisotropic topological structure can promote the directional elongation and migration of broken nerve cells in patients. The innovative addition of magnetically responsive nerve cells as seed cells can further provide the biochemical clues required for regeneration and may replace the function of damaged nerves to a certain extent. It can also remotely and non-invasively manipulate the magnetically responsive cells in the catheter through the external magnetic field, avoiding the potential damage of secondary surgery. Specifically: (1) The present invention uses micromolding technology to prepare a neural scaffold, which is easy to operate, can adapt to the complex environment of the injury site, and can achieve customization of size and shape.

[0011] (2) The surface of the neural scaffold prepared by the present invention has a regular micrometer and / or nanometer-scale anisotropic topological structure, which can effectively guide the directional migration and elongation of nerve cells, facilitate the regulation of cell behavior and physiological functions at the cellular and molecular levels, and accelerate damage repair.

[0012] (3) The neural scaffold prepared by the present invention is loaded with neural cells, which can provide biochemical clues for the patient's nerve regeneration on the one hand, and on the other hand may replace part of the function of the damaged nerves and promote the early recovery of nerve function.

[0013] (4) The nerve cells loaded on the neural scaffold prepared by the present invention have internalized magnetic nanoparticles. The magnetic nanoparticles can function as a separate magnet on the one hand, and can also realize remote non-destructive manipulation of the nerve cells through an external magnetic field after implantation. That is, the magnetically responsive nerve cells loaded on the scaffold are used as seed cells. On the one hand, the cells themselves play a unique biological role in nerve regeneration, and on the other hand, the external magnetic field can accelerate the migration and elongation of magnetically labeled cells non-invasively at a long distance, thereby promoting the rapid connection and functional recovery of long-distance damaged nerves.

[0014] (5) The anisotropic neural scaffold loaded with magnetically responsive neural cells constructed by the present invention can promote nerves and accelerate long-distance peripheral nerve regeneration through the synergistic action of multiple factors by simulating the topological microenvironment of nerves, loading seed cells and applying an external magnetic field to provide magnetic stimulation. During treatment, the scaffold of the present invention is implanted into the nerve defect site, and an external magnetic field is applied to achieve the anisotropic topological structure and magnetically responsive neural cells to synergistically accelerate the repair of long-distance peripheral nerve damage. Among them, the applied external magnetic field can be one or more of a static magnetic field, an alternating magnetic field, and a pulsed magnetic field generated by a permanent magnet, an electromagnet, etc., and the magnetic field strength is 10-1000 mT. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the process of preparing a PDMS mold using micromolding technology in step 1 of the present invention; Figure 2 This is a schematic diagram of the process of preparing planar or tubular biomaterial scaffolds in step 2 of the present invention; Figure 3 It is a schematic diagram of the process of step 3 to step 5 of the present invention; Figure 4 This is a photo of a columnar PDMS mold according to an embodiment of the present invention; the mold has a regular anisotropic topological structure and is elastic, which is convenient for reuse and large-scale production; Figure 5 This is a photo of an anisotropic biomaterial tubular scaffold according to an embodiment of the present invention; the scaffold surface well replicates the topological structure of the PDMS mold, and the scaffold thickness is uniform; Figure 6This is an uptake quantitative experiment; A is the Prussian blue staining image of APTES and PDA modified MNPs at different time points, B is the Prussian blue absorbance standard curve of MNPs, and C is the uptake of APTES modified MNPs at different times; n=5, ns p>0.05, indicating no significant difference; Figure 7 This is a photo of magnetically responsive neural cells loaded on an anisotropic topological scaffold according to an embodiment of the present invention; the scaffold has excellent biocompatibility, and the neural cells grow well and are evenly distributed thereon, and present an elongated shape; Figure 8 Figure 3 is the morphological analysis of SCs under different magnetic and topological conditions; A is the immunofluorescence staining image of SCs in the Control, 6% PEG, MAG and 6% PEG+MAG groups (green fluorescence represents actin), B is the statistics of cell orientation angles, and C is the statistics of cell aspect ratios; ****p<0.0001 indicates that each group is significantly different from the Control group.

[0016] Fig. 9 It is the topological characterization of the catheter morphology and the analysis of the growth behavior and mechanical properties of cells in the catheter; A is the catheter cross section, A' is the microscopic image of the catheter cross section, A'' is the optical microscope morphology of the inner wall of the catheter, B is the macroscopic image of catheters of different thicknesses, C is the distance-load curve of the catheter suture strength test, D is the maximum load of the catheter, E is the observation of cell culture in the catheter (green fluorescence represents actin), and F is the 3D reconstruction of the fluorescence intensity of cells in the catheter; ***p<0.001, *p<0.01 represents a significant difference between the groups; Fig.10 Evaluation of muscle and nerve function recovery in rats after surgery; A is the statistical chart of gastrocnemius wet weight ratio 8 weeks after surgery, B is the H&E staining image of gastrocnemius on the surgical side of rats 8 weeks after surgery, C is the statistics of muscle fiber diameter, DE is the statistics of plantar heat pain reaction latency on the surgical side of rats 2 weeks and 8 weeks after surgery, FG is the statistics of sciatic nerve function index on the surgical side of rats 2 weeks and 8 weeks after surgery, ****p<0.0001, ***p<0.001, **p<0.01 represent significant differences between the groups, ns represents no significant differences between the groups. DETAILED DESCRIPTION

[0017] The present invention will be further explained below in conjunction with the accompanying drawings. Example

[0018] An anisotropic neural scaffold loaded with magnetically responsive neural cells, the specific preparation method comprising the following steps: (1) After fully mixing dimethylsiloxane monomer and cross-linking agent in a volume ratio of 10:1, the mixture was poured onto a polymer sleeve mold with an anisotropic groove-ridge structure with a depth of 10 μm and a width of 50 μm. The mixture was vacuum degassed for 1 h and cross-linked at 90 °C for 3 h. After peeling, a columnar PDMS mold with an anisotropic groove-ridge structure was obtained.

[0019] (2) Weigh 20 g of ovalbumin and dissolve it in 100 mL of anhydrous formic acid. After stirring and dissolving evenly, apply the material solution on the surface of the PDMS mold and air-dry to obtain an anisotropic ovalbumin tubular scaffold with the above-mentioned anisotropic groove-ridge structure on the surface; the thickness of the scaffold is 3 mm. If the biomaterial scaffold is tubular, the inner diameter is 10 mm and the length is 100 mm.

[0020] (3) Add 0.2423 g of Tris (tris(hydroxymethyl)aminomethane) powder to 200 mL of triple-distilled water. After it is evenly dissolved, add an appropriate amount of 0.01 M NaOH solution to adjust the pH value of the solution to 8.5. Then add 400 mg of dopamine powder to obtain a 2 mg / mL dopamine solution.

[0021] (4) Immediately add 2 g of Fe3O4 particles with a particle size of 20 nm into 200 mL of dopamine solution and react on a shaker at room temperature for one day. Then, the dopamine-modified magnetic nanoparticles are obtained by magnet attraction and repeated washing with double distilled water.

[0022] (5) The magnetic nanoparticles were mixed into the culture medium of Schwann cells at a concentration of 500 μg / mL. After culturing for 4 h, the cells were thoroughly washed with phosphate buffer to remove the unabsorbed magnetic nanoparticles. The Schwann cells were digested with trypsin to remove them from the culture dish. The cell suspension was then centrifuged at 500 rpm for 3 min, and the solution was removed by aspiration to obtain magnetically responsive Schwann cells.

[0023] (6) Magnetically responsive Schwann cells were placed at 5000 / μm 2 The density of cells was inoculated on the fully sterilized anisotropic ovalbumin scaffold, and after culturing for 12 h and complete adhesion, the anisotropic ovalbumin scaffold loaded with magnetically responsive Schwann cells was obtained.

[0024] Applying a 10 mT static magnetic field on both sides of the prepared scaffold at intervals or for a long time can achieve non-contact and non-destructive manipulation of magnetically responsive Schwann cells. The scaffold was implanted into the long-distance sciatic nerve injury (10 mm) of rats, and the sciatic nerve regenerated significantly after 50 days, with good functional recovery. Example

[0025] An anisotropic neural scaffold loaded with magnetically responsive neural cells, the specific preparation method comprising the following steps: (1) After fully mixing dimethylsiloxane monomer and cross-linking agent in a volume ratio of 10:1, pour them onto a polymer flat mold with an anisotropic groove-ridge structure with a depth of 0.1 μm and a width of 0.1 μm. Degassing was performed in vacuum for 1 h, cross-linking was performed at 90 °C for 3 h, and the PDMS mold with an anisotropic groove-ridge structure on the surface was obtained after peeling.

[0026] (2) Weigh 20 g of chitosan and dissolve it in 100 mL of 2% (w / v) acetic acid solution. After stirring and dissolving evenly, the material solution is poured onto the surface of the PDMS mold. After air drying, an anisotropic chitosan planar scaffold with the above-mentioned anisotropic groove-ridge structure on the surface is obtained. The thickness of the scaffold is 0.5 mm.

[0027] (3) Add 1 mL of acetic acid solution to 100 mL of triple distilled water, and then dissolve 2.5 g of chitosan in the above solution to obtain a 2.5% chitosan solution.

[0028] (4) Immediately add 2 g of Fe2O3 particles with a particle size of 40 nm into 200 mL of chitosan solution and react on a shaker at room temperature for one day. Then, the chitosan-modified magnetic nanoparticles are obtained by magnet attraction and repeated washing with double distilled water.

[0029] (5) The magnetic nanoparticles were mixed into the culture medium of the neuronal cells at a concentration of 100 μg / mL. After culturing for 6 h, the cells were thoroughly washed with phosphate buffer to remove the unabsorbed magnetic nanoparticles. The neuronal cells were digested with trypsin to separate them from the culture dish. The cell suspension was then centrifuged at 500 rpm for 3 min, and the solution was removed by aspiration to obtain magnetically responsive neuronal cells.

[0030] (6) Magnetically responsive neurons were cultured at 500 / μm 2 The density of the cells was inoculated on the fully sterilized anisotropic chitosan scaffold, and after culturing for 24 hours, the anisotropic chitosan scaffold loaded with magnetic responsive neuronal cells was obtained after complete attachment to the wall.

[0031] Applying a 500 mT alternating magnetic field on both sides of the prepared scaffold at intervals or for a long time can achieve non-contact and non-destructive manipulation of magnetically responsive neuronal cells. The scaffold was implanted into the long-distance sciatic nerve injury (10 mm) site of rats, and the sciatic nerve regenerated significantly after 2 months, and the function recovered well. Example

[0032] An anisotropic neural scaffold loaded with magnetically responsive neural cells, the specific preparation method comprising the following steps: (1) After fully mixing dimethylsiloxane monomer and cross-linking agent in a volume ratio of 10:1, pour them onto a polymer flat mold with an anisotropic groove-ridge structure with a depth of 5 μm and a width of 25 μm. Degassing was performed in vacuum for 1 h, cross-linking was performed at 60 °C for 12 h, and peeling was performed to obtain a flat PDMS mold with an anisotropic groove-ridge structure on the surface.

[0033] (2) Weigh 20 g of collagen and dissolve it in 100 mL of 2% (w / v) acetic acid solution. After stirring and dissolving evenly, pour the material solution onto the surface of the PDMS mold. After air drying, an anisotropic collagen planar scaffold with the above-mentioned anisotropic groove-ridge structure on the surface is obtained. The thickness of the scaffold is 1.5 mm.

[0034] (3) Add 2 mL of silane coupling agent solution to 100 mL of anhydrous ethanol to obtain 2% silane coupling agent.

[0035] (4) Immediately add 2 g of Fe2O3 and NdFeB mixed particles with a particle size of 10 nm (mass ratio 1:1) into 200 mL of silane coupling agent solution and react on a shaker at room temperature for one day. Then, the mixture is attracted by a magnet and repeatedly washed with deionized water to obtain silane coupling agent-modified magnetic nanoparticles.

[0036] (5) Magnetic nanoparticles were mixed into the culture medium of neural stem cells at a concentration of 10 μg / mL. After culturing for 48 h, the cells were thoroughly washed with phosphate buffer to remove the unabsorbed magnetic nanoparticles. The neuronal cells were digested with trypsin to separate them from the culture dish. The cell suspension was then centrifuged at 500 rpm for 3 min, and the solution was removed by aspiration to obtain magnetically responsive neural stem cells.

[0037] (6) Magnetic responsive neural stem cells were cultured at 2500 / μm 2 The density of the cells was inoculated on a fully sterilized anisotropic collagen scaffold, and after culturing for 36 h until they were completely attached to the wall, anisotropic collagen scaffold loaded with magnetic responsive neural stem cells was obtained.

[0038] Applying 1000 mT pulsed magnetic field on both sides of the prepared scaffold at intervals or for a long time can achieve non-contact and non-destructive manipulation of magnetically responsive neuronal cells. The scaffold was implanted into the long-distance sciatic nerve injury (10 mm) of rats, and the sciatic nerve regenerated significantly after 70 days, and the function was well restored. Example

[0039] An anisotropic neural scaffold loaded with magnetically responsive neural cells, the specific preparation method comprising the following steps: (1) After fully mixing dimethylsiloxane monomer and cross-linking agent in a volume ratio of 10:1, pour them onto a polymer arc-shaped mold with an anisotropic groove-ridge structure with a depth of 10 μm and a width of 30 μm. Vacuum degassing for 1 h, cross-linking at 60 °C for 12 h, and peeling off to obtain an arc-shaped PDMS mold with an anisotropic groove-ridge structure on the surface.

[0040] (2) Weigh 20 g of silk fibroin and dissolve it in 100 mL of ultrapure water. After stirring and dissolving evenly, apply the material solution on the surface of the PDMS mold and air-dry to obtain an anisotropic silk fibroin curved surface scaffold with the above-mentioned anisotropic groove-ridge structure on the surface. The thickness of the scaffold is 2 mm.

[0041] (3) Add 2 mL of silane coupling agent to 100 mL of anhydrous ethanol to obtain a 2% silane coupling agent solution.

[0042] (4) Immediately add 2 g of Fe2O3 and Fe3O4 mixed particles with a particle size of 10 nm (mass ratio 1:1) into 200 mL of silane coupling agent solution and react on a shaker at room temperature for one day. Then, attract the mixture with a magnet and wash it repeatedly with deionized water to obtain silane coupling agent-modified magnetic nanoparticles.

[0043] (5) The magnetic nanoparticles were mixed into the culture medium of astrocytes at a concentration of 300 μg / mL. After culturing for 48 h, the cells were thoroughly washed with phosphate buffer to remove the uningested magnetic nanoparticles. The astrocytes were digested with trypsin to remove them from the culture dish. The cell suspension was then centrifuged at 500 rpm for 3 min, and the solution was removed by aspiration to obtain magnetically responsive astrocytes.

[0044] (6) Magnetically responsive astrocytes were cultured at 1000 / μm 2 The density of the cells was inoculated on a fully sterilized anisotropic collagen scaffold, and after culturing for 48 h until they were completely attached to the wall, anisotropic silk fibroin scaffold loaded with magnetically responsive neural stem cells was obtained.

[0045] Applying a 300 mT static magnetic field on both sides of the prepared scaffold at intervals or for a long time can achieve non-contact and non-destructive manipulation of magnetically responsive neuronal cells. The scaffold was implanted into the long-distance sciatic nerve injury (10 mm) of rats, and the sciatic nerve regenerated significantly after 45 days, with good functional recovery. Example

[0046] An anisotropic neural scaffold loaded with magnetically responsive neural cells, the specific preparation method comprising the following steps: (1) After fully mixing dimethylsiloxane monomer and cross-linking agent in a volume ratio of 10:1, the mixture was poured onto a polymer flat mold with a protrusion structure with a depth of 1 μm and a diameter of 5 μm on the surface. The protrusions were arranged in an oriented manner with a spacing of 10 μm in the horizontal direction and 20 μm in the vertical direction. The mixture was vacuum degassed for 1 h and cross-linked at 90 °C for 3 h. After peeling, a flat PDMS mold with an anisotropic concave hole structure on the surface was obtained.

[0047] (2) Weigh 20 g of gelatin and dissolve it in 100 mL of 60°C ultrapure water. After stirring and dissolving evenly, apply the material solution on the surface of the PDMS mold. After air drying, an anisotropic gelatin planar scaffold with the above-mentioned anisotropic protrusion structure on the surface is obtained. The thickness of the scaffold is 1.5 mm.

[0048] (3) Add 1 mL of acetic acid solution to 100 mL of triple distilled water, and then dissolve 2.5 g of chitosan in the above solution to obtain a 2.5% chitosan solution.

[0049] (4) Immediately add 2 g of mixed particles of MnFe2O4 and NiFe2O4 with a particle size of 100 nm (mass ratio 1:1) into 200 mL of chitosan solution and react on a shaker at room temperature for one day. Then, the mixture was attracted by a magnet and repeatedly washed with double distilled water to obtain chitosan-modified magnetic nanoparticles.

[0050] (5) Magnetic nanoparticles were mixed into the culture medium of microglia at a concentration of 100 μg / mL. After culturing for 6 h, the cells were thoroughly washed with phosphate buffer to remove the uningested magnetic nanoparticles. The microglia were digested with trypsin to remove them from the culture dish. The cell suspension was then centrifuged at 500 rpm for 3 min, and the solution was removed by aspiration to obtain magnetically responsive microglia.

[0051] (6) Magnetically responsive microglia were cultured at 500 / μm 2 The density of the cells was inoculated on a fully sterilized anisotropic gelatin scaffold, and after culturing for 12 h until they were completely adhered to the wall, anisotropic gelatin scaffolds loaded with magnetically responsive microglia were obtained.

[0052] Applying a 500 mT static magnetic field on both sides of the prepared scaffold at intervals or for a long time can achieve non-contact and non-destructive manipulation of magnetically responsive microglia. The scaffold was implanted into the long-distance sciatic nerve injury (10 mm) of rats, and the sciatic nerve regenerated significantly after 90 days, with good functional recovery. Example

[0053] An anisotropic neural scaffold loaded with magnetically responsive neural cells, the specific preparation method comprising the following steps: (1) After fully mixing dimethylsiloxane monomer and cross-linking agent in a volume ratio of 10:1, the mixture was poured onto a polymer flat mold with an anisotropic concave pore structure with a depth of 2 μm and a diameter of 10 μm. The concave pores were arranged in an oriented manner with an interval of 30 μm in the horizontal direction and 100 μm in the vertical direction. The mixture was vacuum degassed for 1 h and cross-linked at 90 °C for 3 h. After peeling, a flat PDMS mold with an anisotropic convex structure on the surface was obtained.

[0054] (2) Weigh 20 g of ovalbumin and dissolve it in 100 mL of anhydrous formic acid. After stirring and dissolving evenly, pour the material solution onto the surface of the PDMS mold. After air drying, an anisotropic ovalbumin planar scaffold with the above-mentioned anisotropic concave pore structure on the surface is obtained. The thickness of the scaffold is 2 mm.

[0055] (3) Add 0.2423 g of Tris (tris(hydroxymethyl)aminomethane) powder to 200 mL of triple-distilled water. After it is evenly dissolved, add an appropriate amount of 0.01 M NaOH solution to adjust the pH value of the solution to 8.5. Then add 400 mg of dopamine powder to obtain a 2 mg / mL dopamine solution.

[0056] (4) Immediately add 2 g of CoFe2O4 particles with a particle size of 20 nm into 200 mL of dopamine solution and react on a shaker at room temperature for one day. Then, the dopamine-modified magnetic nanoparticles are obtained by magnet attraction and repeated washing with double distilled water.

[0057] (5) Magnetic nanoparticles were mixed into the culture medium of OECs at a concentration of 100 μg / mL. After culturing for 4 h, the cells were thoroughly washed with phosphate buffer to remove the uningested magnetic nanoparticles. The OECs were digested with trypsin to remove them from the culture dish. The cell suspension was then centrifuged at 500 rpm for 3 min, and the solution was removed by aspiration to obtain magnetically responsive OECs.

[0058] (6) Magnetically responsive olfactory ensheathing cells were cultured at 5000 / μm 2 The density of the cells was inoculated on the fully sterilized anisotropic ovalbumin scaffold, and after culturing for 12 h and complete adhesion, the anisotropic ovalbumin scaffold loaded with magnetically responsive olfactory ensheathing cells was obtained.

[0059] Applying a 100 mT static magnetic field on both sides of the prepared scaffold at intervals or for a long time can achieve non-contact and non-destructive manipulation of magnetically responsive olfactory ensheathing cells. The scaffold was implanted into the long-distance sciatic nerve injury (10 mm) of rats, and the sciatic nerve regenerated significantly after 80 days, with good functional recovery.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an anisotropic neural scaffold loaded with magnetically responsive neural cells, characterized in that: include: Step 1: Prepare a PDMS mold with a regular anisotropic topological structure on the surface by a micromolding method; Step 2: Infusing or coating the surface of the PDMS mold with natural or synthetic biomaterials, and forming a planar, curved or tubular biomaterial scaffold with an anisotropic topological structure after air drying or cross-linking curing; Step 3: functionalizing the magnetic nanoparticles by a surface modification method to obtain magnetic nanoparticles with high biocompatibility; Step 4: internalizing the surface-modified magnetic nanoparticles into nerve cells by a co-culture method to obtain magnetically responsive nerve cells; Step 5: The magnetically responsive neural cells are loaded onto the biomaterial scaffold by a co-culture method to obtain an anisotropic neural scaffold loaded with magnetically responsive neural cells.

2. The preparation method according to claim 1, characterized in that: The topological structure is grooves and ridges, concave holes or protrusions; wherein the high part of the groove and ridge structure is a ridge, the low part is a groove, the vertical distance between the groove and the ridge is 0.1-10 μm, the width of the groove and the ridge is 0.1-50 μm, and the connection between the groove and the ridge is smooth or vertical.

3. The preparation method according to claim 1 or 2, characterized in that: The thickness of the biomaterial scaffold is 0.5-3 mm. If the biomaterial scaffold is tubular, the inner diameter is 0.5-10 mm and the length is 10-100 mm.

4. The preparation method according to claim 1, characterized in that: In step 3, the magnetic nanoparticles used are one or a mixture of Fe2O3, MFe2O4 (M = Co, Mn, Ni), Fe3O4, NdFeB, and the particle size of the modified magnetic nanoparticles is 10-100 nm.

5. The preparation method according to claim 1, characterized in that: In step 4, the neural cells are one or more of Schwann cells, neurons, neural stem cells, astrocytes, microglia, and olfactory ensheathing cells; the concentration of the magnetic nanoparticles during co-culture is 10-500 μg / mL, and the co-culture time is 4-48 h.

6. The preparation method according to claim 1, characterized in that: In step 5, the co-culture time is 0.5-2 days, and the density of the magnetically responsive neural cells is 500-5000 / μm 2 .

7. An anisotropic neural scaffold prepared according to any one of claims 1-6.

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