A reduced graphene oxide nerve conduit and a device for promoting peripheral nerve regeneration

Abstract

The present invention discloses a reduced graphene oxide nerve conduit and a device for promoting peripheral nerve regeneration, belonging to the technical field of tissue engineering. The raw materials for preparing the reduced graphene oxide nerve conduit of the present invention include paramagnetic materials and gels. The present invention constructs a nerve conduit that mimics the hierarchical structure of peripheral nerves, greatly improving the bionic degree and biocompatibility of the nerve conduit. The present invention uses 3D printing technology. On the premise of simulating the inherent biological components of the neurilemma, rGO is added to the composite material to enhance electrophysiological properties, and different formulations of composite materials are constructed to promote the recovery of electrophysiological disorders caused by nerve injuries. The present invention utilizes the fact that a magnetic field can be used as a non-invasive and controllable biological stimulus to guide nerve growth, and its combination with rGO can produce a synergistic effect to enhance the biological effect of rGO. The possibility of postoperative complications is reduced, and the repair effect after nerve injury repair can be increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering, and particularly relates to a reduced graphene oxide nerve conduit and a device for promoting peripheral nerve regeneration. Background Art

[0002] Trauma and surgery can cause peripheral nerve injury. Without timely and reasonable treatment, patients will suffer from long-term disabilities, affecting their physical and mental health. Long-distance nerve injuries are often accompanied by a large amount of loss of nerve tissue and cannot be anastomosed end to end, requiring surgical transplantation repair to restore function. Autologous nerve transplantation has become the gold standard for surgical repair of peripheral nerve defects. The transplanted nerve itself cannot perform functions, but provides the best scaffold for axon elongation. However, autologous nerve transplantation has limitations such as donor site complications, limited donors, and mismatched diameters, resulting in an unsatisfactory overall repair effect.

[0003] Therefore, a variety of artificial nerve guidance conduits made of natural or synthetic materials and similar to the natural nerve structure and components began to appear. However, the simple physical connection of nerve conduits cannot achieve good nerve regeneration, and the regeneration effect can be improved after introducing biological stimulation. Physical stimulation can provide "bioactivity" as biological stimulation at a certain intensity. Especially in the field of nerve regeneration, people widely recognize the important role of electrical stimulation in the nerve recovery process. Electrical stimulation is a part of electromagnetic force, and the other part is magnetic force, and the two interact with each other. The flux of electromagnetic force can change with displacement, which is called an electromagnetic field. A pulsed electromagnetic field is an electromagnetic field with a specific amplitude and waveform. Various electromagnetic fields have been reported to regulate cell proliferation, DNA replication, wound healing, cytokine expression, and cell differentiation. The main reason why pulsed electromagnetic fields can cause biological changes is that they are easy to penetrate cells, and their biological basis mainly depends on protein synthesis, ion channel regulation, and growth factor secretion.

[0004] Due to the important role of electrical stimulation, many conductive materials have been used in nerve tissue engineering to obtain electrophysiological properties. Graphene is a two-dimensional crystal composed of sp2 hybridized carbon atoms arranged in hexagonal rings. It and its derivatives have become a hot topic in the field of biomaterials due to their unique physical, chemical, mechanical, electrical, thermal, and biological properties. However, the hydrophobicity and cytotoxicity of graphene limit its application in tissue engineering. Graphene oxide is an important derivative of graphene and has excellent dispersibility in certain polar solvents due to the carboxyl groups on its surface. However, the oxidation of graphene depletes its aromaticity, resulting in a weakening of the electrical properties of the material. Converting graphene oxide to reduced graphene oxide by chemical or heat treatment can increase the electrical properties of graphene oxide by restoring the sp2-carbon bonds. In recent years, the functions of graphene have received attention in the fields of cell and tissue engineering. In addition, some researchers have noticed the synergistic effect of pulsed magnetic field stimulation combined with graphene-based materials in osteogenesis and neural differentiation. However, there are still few reports on the combined application of rGO (reduced graphene oxide) and PEMF (pulsed electromagnetic fields) in neurogenesis, and few people have applied their combination to the repair of peripheral nerve defects. Summary of the Invention

[0005] The object of the present invention is to provide a conductive and paramagnetic reduced graphene oxide nerve conduit composite system that matches pulsed electromagnetic fields and a preparation method thereof. This conduit composite system has the function of promoting angiogenesis, can better guide nerve growth, and promote the regeneration and repair of nerves, thereby overcoming the defects of the existing technology.

[0006] The first aspect of the present invention discloses a reduced graphene oxide nerve conduit, and the raw materials for preparation include a paramagnetic material and a gel.

[0007] In some embodiments of the present invention, the reduced graphene oxide nerve conduit is fabricated by three-dimensional bioprinting.

[0008] In some embodiments of the present invention, the paramagnetic material is reduced graphene oxide.

[0009] In some embodiments of the present invention, the gel in the raw materials for preparation is at least one of silk fibroin, gelatin, and sodium alginate.

[0010] In some embodiments of the present invention, the reduced graphene oxide nerve conduit is provided with at least one axial independent channel, and the independent channel penetrates both ends of the reduced graphene oxide nerve conduit.

[0011] In some embodiments of the present invention, the cross-section of the reduced graphene oxide nerve conduit is circular, and the diameter is 4 - 8 mm;

[0012] The cross-section of the independent channel is square with a side length of 400 - 500 μm.

[0013] In some embodiments of the present invention, the weight ratio of the reduced graphene oxide, silk fibroin, gelatin, and sodium alginate is 0.01 - 0.05:0.5 - 3:0.2 - 1.5:0.05 - 0.5.

[0014] The second aspect of the present invention is to disclose a method for preparing the reduced graphene oxide nerve conduit described in the first aspect, including the following steps:

[0015] S1. Disinfect silk fibroin, gelatin, sodium alginate, and reduced graphene oxide.

[0016] S2. Add the gelatin and sodium alginate obtained in step S1 to a PBS solution for mixing and heat treatment.

[0017] S3. Add the silk fibroin obtained in step S1 to a PBS solution for mixing.

[0018] S4. Mix the gelatin - sodium alginate solution obtained in step S2, the silk fibroin solution obtained in step S3, and the reduced graphene oxide obtained in step S1, and perform heat treatment to obtain a mixed solution.

[0019] S5. Transfer the mixed solution obtained in step S4 to a syringe and let it return to room temperature to form a gel.

[0020] S6. Transfer the gel obtained in step S5 to a 3D bioprinter, connect the printing needle, and perform 3D bioprinting using the nerve conduit model designed by a computer to obtain the reduced graphene oxide nerve conduit.

[0021] In some embodiments of the present invention, in step S2, the mass ratio of the gelatin to the sodium alginate is 1 - 5:1.

[0022] In step S2, in the gelatin - sodium alginate mixed solution, the concentration of the gelatin is 0.1 - 0.5 g / ml.

[0023] In step S3, the concentration of the silk fibroin solution is 0.1 - 0.5 g / ml.

[0024] In step S4, the concentration of the silk fibroin in the mixed solution is 0.1 - 0.5 g / ml.

[0025] In step S4, the concentration of the reduced graphene oxide in the mixed solution is 0.001 - 0.005 g / ml.

[0026] In step S6, the temperature of the composite gel in the syringe is 27 - 29°C, the ambient temperature in the three-dimensional bioprinter is 14 - 18°C, the inner diameter of the printing needle is 200 - 400 μm, and during the three-microorganism printing process, the extrusion rate of the syringe is 0.1 - 0.3 ml / min, and the moving speed of the needle is 2 - 5 mm / s.

[0027] The third aspect of the present invention lies in disclosing a device for promoting peripheral nerve regeneration, including the reduced graphene oxide nerve conduit and the magnetic field generating device described in the first aspect.

[0028] In some embodiments of the present invention, the magnetic field generating device includes:

[0029] At least one coil, the center distance between the coils being equal to the radius of the coil;

[0030] A function signal generator for adjusting the intensity and frequency of the pulsed magnetic electric field;

[0031] A gaussmeter for measuring the intensity of the pulsed electromagnetic field.

[0032] In some embodiments of the present invention, the diameter of the coil is 200 - 300 mm, and the height is 30 - 70 mm;

[0033] The output frequency of the function signal generator is 40 - 60 Hz;

[0034] The pulsed magnetic field intensity between the two coils is 1 - 5 mT.

[0035] Beneficial effects:

[0036] (1) The present invention constructs a nerve conduit simulating the hierarchical structure of the peripheral nerve, greatly improving the bionic degree and biocompatibility of the nerve conduit.

[0037] (2) The present invention uses 3D printing technology. On the premise of simulating the inherent biological components of the neurilemma, rGO is added to the composite material to enhance the electrophysiological performance, and different formulations of composite materials are constructed to promote the recovery of electrophysiological disorders caused by nerve injury.

[0038] (3) The present invention utilizes that the magnetic field can be used as a non-invasive and controllable biological stimulus to guide nerve growth, and its combination with rGO can produce a synergistic effect to enhance the biological effect of rGO. The possibility of postoperative complications is reduced, and the repair effect after nerve injury repair can be increased. Description of the Drawings

[0039] Figure 1 It is a scanning electron microscope image of the reduced graphene oxide composite nerve conduit according to an embodiment of the present invention;

[0040] Figure 2Schematic diagram of the structure of a reduced graphene oxide composite nerve conduit according to an embodiment of the present invention; a is a top view, and b is a three-dimensional view;

[0041] Figure 3 Schematic diagram of a magnetic field generating device;

[0042] Figure 4 Sciatic nerve indices at different times after sciatic nerve repair in rats according to an embodiment of the present invention. Among them, the average value of the magnetic field + rGO conduit group at 8 weeks was -46.107, which was higher than the average value of the autograft group (-55.462) and that of the rGO conduit (-72.519), and the functional recovery effect was the best;

[0043] Figure 5 Compound muscle action potential of the gastrocnemius muscle at 8 weeks after sciatic nerve repair in rats according to an embodiment of the present invention. Among them, the average value of the magnetic field + rGO conduit group was 1.66 mV, and the recovery effect was closer to that of the autograft (5.47 mV), far exceeding that of the rGO conduit group (0.44 mV), showing a more excellent electrophysiological recovery effect;

[0044] Figure 6 The reduced graphene oxide composite material according to an embodiment of the present invention exhibits paramagnetism;

[0045] Figure 7 Cytotoxicity test of the original graphene oxide composite material according to an embodiment of the present invention. Among them, after treatment with the extract for 7 days, the absorbance (cell proliferation) had no significant difference from that of the blank control group. Specific embodiments

[0046] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0047] Unless otherwise specified, the examples and comparative examples are parallel tests with the same components, component contents, preparation steps, and preparation parameters. The PBS solution is 0.1 M and pH 7.2. The gelatin is purchased from Merck (V900863). The average molecular weight of the silk fibroin is 100 KDa. The reduced graphene oxide is purchased from Xianfeng Nano (100022, XF032) and has paramagnetism.

[0048] A reduced graphene oxide nerve conduit

[0049] It includes a cylindrical nerve conduit body with multiple axially arranged independent channels inside. The independent channels are arranged at intervals of each other and penetrate through both ends of the nerve conduit body. As Figure 2 shown. The cross-section of the reduced graphene oxide nerve conduit is circular with a diameter of 4 - 8 mm; the cross-section of the independent channel is square with a side length of 400 - 500 μm.

[0050] A device for promoting peripheral nerve regeneration

[0051] It includes a reduced graphene oxide nerve conduit and a magnetic field generating device (as Figure 3 shown). The magnetic field generating device includes two coils, a function signal generator and a gaussmeter. The center distance between the coils is equal to the radius of the coils. The function signal generator is used to adjust the intensity and frequency of the pulsed magnetic field. The gaussmeter can be used to measure the intensity of the pulsed electromagnetic field. The diameter of the coil is 250 mm and the height is 50 mm. The output frequency of the function signal generator is 50 Hz. The pulsed magnetic field intensity between the two coils is 2 mT.

[0052] Example 1

[0053] Preparation of the reduced graphene oxide nerve conduit:

[0054] (1) Irradiate silk fibroin, gelatin, sodium alginate, and reduced graphene oxide with cobalt-60 for 30 minutes;

[0055] (2) Weigh 0.8 g of sterile gelatin and 0.2 g of sodium alginate in a laminar flow hood. Connect the front ends of two 10-ml syringes through an adapter, transfer the gelatin-sodium alginate mixture to one of the syringes, add 5 ml of PBS solution, exhaust the air, mix well, and place it in an incubator at 37°C for 2 hours;

[0056] (3) Weigh 1.25 g of silk fibroin in a laminar flow hood, transfer it to a 15-ml centrifuge tube, add 5 ml of PBS solution, vortex and mix well, and place it in an incubator at 37°C for 2 hours; Weigh 0.02 g of reduced graphene oxide in a laminar flow hood, add it to the obtained silk fibroin solution and then add them together to the syringe containing the gelatin-sodium alginate solution, mix well, and place it in an incubator at 37°C for 2 hours; and transfer the obtained mixed solution to a 1-ml syringe and return it to room temperature;

[0057] (4) Transfer the 1-ml syringe to a three-dimensional bioprinter and perform three-dimensional bioprinting using a computer-designed nerve conduit model. Use a Musashi printing needle with an inner diameter of 300 μm, adjust the temperature of the composite gel in the syringe to 28.3 °C, the environmental temperature in the three-dimensional bioprinter to 16 °C, the syringe extrusion rate to 0.15 ml / min, and the needle movement speed to 3.5 mm / s. Design the nerve conduit as a cylinder with a diameter of 4 mm and a height of 10 mm, and an internal filling density of 15%. Divide the designed model into layers with a layer thickness of 0.3 mm. After printing, the reduced graphene oxide nerve conduit is obtained.

[0058] Magnetic detection:

[0059] To detect the magnetic properties of the reduced graphene oxide nerve conduit material, use an MPMS-XL5 superconducting quantum magnetometer system (SQUID, Quantum Design, USA) for detection. After freeze-drying the reduced graphene oxide nerve conduit, grind it into powder. As shown in the scanning electron microscope Figure 1 Put the powder in the sample chamber, with a magnetic field range of -2 mT to 2 mT and a resolution of 0.01 mT, and plot the hysteresis loop. As shown in Figure 6 Shown.

[0060] Cytotoxicity detection:

[0061] Use a cell counting kit (CCK8, Japan, LK815) to detect the cell proliferation on the 1st, 3rd, 5th, and 7th days after treatment with the extract of the reduced graphene oxide nerve conduit. The initial sample cell count is 20,000 for all, and the control group has no extract. Mix the CCK8 solution with the culture medium at a ratio of 1:9 to obtain the working solution. Incubate the samples at each time point with 550 μl of the working solution for 1 hour. Transfer 110 μl of the supernatant to a 96-well plate and measure the absorbance value of the plate at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (BioTek ELX800, VT, USA). As shown in Figure 7 Shown.

[0062] Example 2

[0063] Use of the reduced graphene oxide nerve conduit

[0064] (1) Animal model construction:

[0065] Six SD rats (male, SPF grade, weighing 200 - 250 g) were selected for in vivo experiments. They were anesthetized by intraperitoneal injection of sodium pentobarbital at 30 mg / kg. The right sciatic nerve was exposed through an incision in the gluteal muscle. A 15-mm long nerve segment was isolated, transected, and a 10-mm nerve defect area was created. The 10-mm nerve conduit prepared in Example 1 of reduced graphene oxide was placed in the nerve defect area and sutured with 9-0 nylon suture. Then the muscle and skin were sutured. The rats were stimulated in a 2-mT, 50-Hz pulsed magnetic field for 20 minutes daily. Gait evaluation was performed at 2 weeks, 4 weeks, and 8 weeks after surgery for each group. All animals were cultured in an environment with the same stable temperature and circadian cycle. All animals were immediately injected with 10 5 units of penicillin intraperitoneally after surgery. All rats were sacrificed at 8 weeks after surgery.

[0066] (2) Analysis of regenerated nerve function:

[0067] Analysis of the walking track was performed. Briefly, black ink was applied to the bilateral hindlimbs of the rats, and the footprints of the rats walking on white paper were collected for walking track analysis. The toe spread (TS), paw length (PL), and intermediate toe spread (IT) of the experimental side (E) and the normal side (N) were measured, and the sciatic nerve function index (SFI) was calculated as follows:

[0068] SFI = 109.5(ETS - NTS) / NTS - 38.3(EPL - NPL) / NPL + 13.3(EIT - NIT) / NIT - 8.8

[0069] Autologous transplantation and transplantation without pulsed magnetic field were used as controls, and the results are as Figure 4 shown.

[0070] (3) Electrophysiological analysis:

[0071] Electrophysiological analysis was performed on SD rats at 8 weeks after surgery. The right sciatic nerve was exposed again under anesthesia. Bipolar electrodes were fixed at the proximal end of the regenerated nerve to transmit single electrical signals. A receiving electrode was implanted in the belly of the gastrocnemius muscle to record the evoked electromyogram. The latency and the distance between the stimulating electrode and the receiving electrode were recorded, and the nerve conduction velocity and compound muscle action potential were measured. The experiment was repeated 5 times. Autologous transplantation and transplantation without pulsed magnetic field were used as controls, and the results are as Figure 5 shown.

[0072] Example 3

[0073] The difference from Example 1 was that in step (3) of the preparation of the reduced graphene oxide composite nerve conduit, different amounts of reduced graphene oxide were added. It was used according to the method of Example 2, and analysis of regenerated nerve function and electrophysiological analysis were performed.

[0074] Table 1 Influence of the amount of reduced graphene oxide used

[0075]

[0076] Example 4

[0077] The difference from Example 1 lies in that in (2) of the preparation of the reduced graphene oxide composite nerve conduit, different weights of silk fibroin are weighed in a clean bench, transferred to a 15 ml centrifuge tube, and 5 ml of PBS solution is added.

[0078] Table 2 Influence of the dosage of silk fibroin

[0079] Dosage of silk fibroin <![CDATA[Viscosity Pa·s (25°C, 100 s -1 )]]> 1.5g 1.15 1.25 g (Example 1) 0.79 0.5g 0.36

[0080] Example 5

[0081] The difference from Example 2 lies in the different intensities of pulsed magnetic field stimulation.

[0082] Table 3 Influence of pulsed magnetic field stimulation

[0083] Strength mT Frequency Hz Time min Cell viability 1 50 20 98.4％ 2 (Example 2) 50 (Example 2) 20 (Example 2) 122.7％ 4 50 20 105.7％ 8 50 20 83.9％

[0084] The specific preferred embodiments and examples of the present invention are described in detail above. However, the present invention is not limited to the above embodiments and examples. Various changes can be made without departing from the concept of the present invention within the scope of knowledge possessed by those skilled in the art.

Claims

1. A reduced graphene oxide nerve conduit, characterized in that, the raw materials for preparation include a paramagnetic material and a gel; the raw materials for preparing the gel include silk fibroin, gelatin and sodium alginate; the paramagnetic material is reduced graphene oxide; the weight ratio of the paramagnetic material, silk fibroin, gelatin and sodium alginate is 0.01 - 0.05: 0.5 - 3: 0.2 - 1.5: 0.05 - 0.

5.

2. The reduced graphene oxide nerve conduit according to claim 1 or, characterized in that, the cross-section of the reduced graphene oxide nerve conduit is circular, with a diameter of 4 - 8 mm.

3. A method for preparing the reduced graphene oxide nerve conduit according to claim 1 or 2, characterized in that, it includes the following steps: S1. Disinfect silk fibroin, gelatin, sodium alginate and reduced graphene oxide; S2. Add the gelatin and sodium alginate obtained in step S1 to a PBS solution for mixing and heat treatment; S3. Add the silk fibroin obtained in step S1 to a PBS solution for mixing; S4. Mix the gelatin-sodium alginate solution obtained in step S2, the silk fibroin solution obtained in step S3 and the reduced graphene oxide obtained in step S1, and perform heat treatment to obtain a mixed solution; S5. Transfer the mixed solution obtained in step S4 to a syringe and return it to room temperature to form a gel; S6. Transfer the gel obtained in step S5 to a three-dimensional bio-printer, connect the printing needle, and perform three-dimensional bio-printing using the nerve conduit model designed by a computer to obtain the reduced graphene oxide nerve conduit.

4. The method for preparing the reduced graphene oxide nerve conduit according to claim 3, characterized in that, in step S2, in the gelatin-sodium alginate mixed solution, the gelatin concentration is 0.1 - 0.5 g / ml; in step S3, the concentration of the silk fibroin solution is 0.1 - 0.5 g / ml; in step S4, the concentration of silk fibroin in the mixed solution is 0.1 - 0.5 g / ml; in step S4, the concentration of reduced graphene oxide in the mixed solution is 0.001 - 0.005 g / ml; in step S6, the temperature of the composite gel in the syringe is 27 - 29 °C, the environmental temperature in the three-dimensional bio-printer is 14 - 18 °C, the inner diameter of the printing needle is 200 - 400 μm, and during the three-dimensional bio-printing process, the extrusion rate of the syringe is 0.1 - 0.3 ml / min, and the moving speed of the needle is 2 - 5 mm / s.

5. A device for promoting peripheral nerve regeneration, comprising the reduced graphene oxide nerve conduit according to claim 1 or 2 and a magnetic field generating device.

6. The device for promoting peripheral nerve regeneration according to claim 5, characterized in that, the magnetic field generating device includes: at least one coil, the center distance between the coils being equal to the radius of the coil; a function signal generator for adjusting the intensity and frequency of the pulsed magnetic electric field; a gaussmeter for measuring the intensity of the pulsed electromagnetic field.

7. The device for promoting peripheral nerve regeneration according to claim 5 or 6, characterized in that, the diameter of the coil is 200 - 300 mm, and the height is 30 - 70 mm; The output frequency of the function signal generator is 40 - 60 Hz; The pulsed magnetic field intensity between the coils is 1 - 8 mT.

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