A nerve regeneration scaffold and its preparation method and application

Abstract

The present invention discloses a nerve regeneration scaffold and its preparation method and application, belonging to the technical field of biomedical materials. The nerve regeneration scaffold provided by the present invention comprises a stacked first gel layer and a second gel layer; the raw materials for preparing the first gel layer comprise protein-coated germanium nanoparticles and a first hydrogel prepolymer, the raw materials for preparing the second gel layer comprise reduced graphene oxide and a second hydrogel prepolymer, and the first hydrogel prepolymer and the second hydrogel prepolymer are the same or different. The present invention adopts protein-coated germanium nanoparticles and reduced graphene oxide as active ingredients of the first gel layer and the second gel layer, respectively, which can effectively exert the photoacoustic effect of germanium nanoparticles and the mechanical properties and electrochemical activity of reduced graphene oxide, and jointly promote the repair and regeneration of nerves. The nerve regeneration scaffold provided by the present invention has a wide range of applications in the preparation of nerve repair materials, especially spinal nerve repair materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a nerve regeneration scaffold and a preparation method and application thereof. Background Art

[0002] Nerve regeneration scaffold is an important material used to repair damaged nerves. An ideal nerve regeneration scaffold should have the following characteristics: ① Good biocompatibility or biosafety: the scaffold or its degradation products are non-toxic to the body and have a low host immune response; ② Appropriate structure and good permeability: support the transport of oxygen and nutrients, and achieve and maintain cell-to-cell interactions; ③ A biodegradation rate that matches the growth rate of tissue cells in the repair area and can be completely absorbed or safely excreted from the body; ④ Appropriate mechanical strength and plasticity: The scaffold plays a mechanical support role and must have good mechanical properties.

[0003] In the study of nerve injury, spinal cord injury is a serious post-traumatic complication of the nervous system, which can easily cause nerve dysfunction below the injured segment, with high disability rate and difficulty in functional recovery. At present, the clinical treatment after spinal cord injury mainly involves surgery to relieve compression, stabilize the spinal structure and hormone therapy, etc. There is still a lack of effective treatment plans to promote nerve regeneration and restore function. How to effectively repair the injured spinal cord has become one of the most challenging scientific issues in clinical research. Summary of the invention

[0004] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide a nerve regeneration scaffold having good nerve repair and regeneration effects.

[0005] A second object of the present invention is to provide a method for preparing the above-mentioned nerve regeneration scaffold.

[0006] The third object of the present invention is to provide an application of the above-mentioned nerve regeneration scaffold in the preparation of nerve repair materials.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a nerve regeneration scaffold, comprising a stacked first gel layer and a second gel layer; the raw materials for preparing the first gel layer include protein-coated germanium nanoparticles and a first hydrogel prepolymer, and the raw materials for preparing the second gel layer include reduced graphene oxide and a second hydrogel prepolymer, and the first hydrogel prepolymer and the second hydrogel prepolymer are the same or different.

[0009] In the present invention, germanium nanoparticles are used as efficient photoacoustic agents, and the photoacoustic stimulation they generate is a non-invasive and precise neural stimulation to neurons, which can convert light into sound waves, promote axon elongation, and further promote nerve regeneration and functional recovery; the use of protein to coat the germanium nanoparticles can effectively reduce the cytotoxicity of the nanoparticles, improve the biocompatibility of the material, and significantly enhance its photothermal stability. At the same time, it has a high molar extinction coefficient (i.e., higher light absorption capacity) in a wide spectral range. Therefore, compared with uncoated germanium nanoparticles, protein-coated germanium nanoparticles have a stronger photoacoustic effect. In addition, the reduced graphene oxide (RGO) of the present invention is obtained by reducing graphene oxide (GO). Compared with graphene and GO, RGO has better biocompatibility, biological activity and dispersibility, and on the basis of retaining the good bioaffinity of GO, it has more excellent electrical, thermal and mechanical properties, and its oxidation and destructive effects on biofilms, etc. are significantly improved compared to GO. Therefore, compared with graphene and GO, RGO has obvious superiority in the present invention, which is more conducive to transmitting the photoacoustic stimulation generated by germanium nanoparticles, and exerting photoacoustic efficacy with greater efficiency. The present invention can effectively promote the regeneration of damaged nerves by coordinating germanium nanoparticles and reduced graphene oxide.

[0010] Preferably, the protein is albumin; further preferably, the albumin comprises bovine serum albumin (BSA), human serum albumin (HSA) or a combination thereof.

[0011] BSA is a widely used, easily available, simple to prepare, and reasonably priced protein. Germanium nanoparticles coated with BSA can not only promote nerve regeneration to a large extent, but also have a low cost, which is an important advantage in achieving clinical transformation and industrialization. Other albumins such as HSA can also achieve good coating effects.

[0012] Preferably, the first hydrogel prepolymer and the second hydrogel prepolymer each independently include at least one of a gelatin gel prepolymer, a polysaccharide gel prepolymer or a polypeptide gel prepolymer; further preferably, the first hydrogel prepolymer and the second hydrogel prepolymer each independently include at least one of methacryloyl gelatin, chitosan, hyaluronic acid or conductive polypyrrole; even more preferably, the first hydrogel prepolymer and the second hydrogel prepolymer each independently are selected from methacryloyl gelatin (Gel MA).

[0013] Using hydrogel as a carrier for germanium nanoparticles and reduced graphene oxide can achieve good material dispersion without affecting its photoacoustic effect. The preparation process is simple and the application advantages are obvious.

[0014] Preferably, the mass ratio of the first gel layer to the second gel layer is 1:(0.1-5); more preferably 1:(0.2-4); further preferably 1:(0.3-3); non-limiting examples include 1:0.5, 1:1, 1:2 or 1:2.5.

[0015] Preferably, in the raw materials for preparing the first gel layer, the mass ratio of the protein-coated germanium nanoparticles to the first hydrogel prepolymer is 1:(50-150); more preferably 1:(80-120); non-limiting examples include 1:90, 1:100 or 1:120.

[0016] Preferably, in the raw materials for preparing the second gel layer, the mass ratio of the reduced graphene oxide to the second hydrogel prepolymer is 1:(50-150); more preferably 1:(80-120); non-limiting examples include 1:90, 1:100 or 1:120.

[0017] The second aspect of the present invention provides a method for preparing a nerve regeneration scaffold as described in the first aspect of the present invention, comprising the following steps: subjecting a first mixed solution containing the protein-coated germanium nanoparticles and the first hydrogel prepolymer to a first curing to obtain the first gel layer; subjecting a second mixed solution containing the reduced graphene oxide and the second hydrogel prepolymer to a second curing to obtain the second gel layer; and stacking the first gel layer and the second gel layer to obtain the nerve regeneration scaffold.

[0018] Preferably, in the first mixed solution, the concentration of the protein-coated germanium nanoparticles is 0.1-5 mg / mL; more preferably 0.2-4 mg / mL; further preferably 0.3-3 mg / mL; non-limiting examples include 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL or 2.5 mg / mL.

[0019] Preferably, in the second mixed solution, the concentration of the reduced graphene oxide is 0.1-3 mg / mL; more preferably 0.2-2.5 mg / mL; more preferably 0.3-2 mg / mL; non-limiting examples include 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL or 1.8 mg / mL.

[0020] Preferably, the first curing and the second curing are each independently selected from photocuring.

[0021] Preferably, the first mixed solution further includes a first photoinitiator; in some specific embodiments of the present invention, the first photoinitiator is selected from phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP).

[0022] Preferably, the first mixed solution further includes a first solvent; in some specific embodiments of the present invention, the first solvent is selected from phosphate buffered saline (PBS).

[0023] Preferably, the second mixed liquid further includes a second photoinitiator; in some specific embodiments of the present invention, the second photoinitiator is selected from phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP).

[0024] Preferably, the second mixed solution further comprises a second solvent; in some specific embodiments of the present invention, the first solvent is selected from phosphate buffered saline (PBS).

[0025] The third aspect of the present invention provides a use of the nerve regeneration scaffold as described in the first aspect of the present invention in the preparation of nerve repair materials.

[0026] Preferably, the first gel layer in the nerve regeneration scaffold serves as the surface layer of the nerve repair material, and the second gel layer in the nerve regeneration scaffold serves as the deep layer of the nerve repair material.

[0027] The second gel layer containing RGO as the deep layer of the nerve repair material has a suitable elastic modulus and does not affect photoacoustic transmission. It can resist the stress stimulation generated by germanium nanoparticles, provide a significant microenvironment that promotes nerve regeneration, and is beneficial to regulating the behavior of Schwann cells, the extension of axons and the phenotype of macrophages, thereby accelerating the process of nerve regeneration.

[0028] Preferably, the nerve repair material is a spinal nerve repair material.

[0029] The beneficial effects of the present invention are as follows: the present invention adopts protein-coated germanium nanoparticles and reduced graphene oxide as active ingredients of the first gel layer and the second gel layer, respectively, which can effectively exert the photoacoustic effect of the germanium nanoparticles and the mechanical properties and electrochemical activity of the reduced graphene oxide, and jointly promote the repair and regeneration of nerves. The nerve regeneration scaffold provided by the present invention has a wide range of applications in the preparation of nerve repair materials, especially spinal nerve repair materials.

[0030] Specifically, compared with the prior art, the present invention has the following advantages:

[0031] 1. Germanium nanoparticles are a highly efficient photoacoustic agent that can provide repeatable photoacoustic stimulation, absorb infrared light and convert it into heat energy, then expand and contract and radiate sound waves outward. This kind of sound wave stimulation is conducive to the elongation of neuronal axons. It is worth noting that germanium nanoparticles also have antioxidant properties, which can inhibit oxidative stress in nerve injury, especially spinal cord injury, and can effectively reduce the level of intracellular reactive oxygen species in nerve cells, reduce the damage of reactive oxygen species to cells, and ultimately promote nerve regeneration. Moreover, germanium nanoparticles have good conductivity, biocompatibility, biodegradability and antibacterial activity, and are a promising tissue engineering material. The use of germanium nanoparticles can be used as an alternative to electrical stimulation and optogenetics, and does not require percutaneous wire connection or implantation of neural conductive devices. It is not only easy to operate, but also reduces the risk of infection. In addition, as a photoacoustic agent, germanium nanoparticles can use sound waves as a carrier for imaging, which can provide functional information such as new blood vessels and tissue congestion in spinal cord injury, which is conducive to evaluating the therapeutic effect of nerve regeneration scaffolds and has the function of monitoring nerve recovery.

[0032] 2. Since the toxicity of nanomaterials is the key factor limiting their clinical application, the present invention uses proteins (such as BSA) to coat germanium nanoparticles, which can effectively reduce the cytotoxicity of germanium nanoparticles and improve biocompatibility. The photothermal stability of germanium nanoparticles is significantly enhanced, and they have a high molar extinction coefficient (i.e., higher light absorption capacity) in a wide spectral range, a stronger photoacoustic effect, and better nerve repair and regeneration effects.

[0033] 3. Reduced graphene oxide (RGO) is obtained by reducing graphene oxide (GO). Its dispersibility, solubility and conductivity are better than those of graphene oxide, and it has good biomechanical properties, biocompatibility, antibacterial activity and angiogenesis-promoting ability. Compared with graphene, RGO has better biosafety and bioactivity. Due to the presence of a large number of impurities in the synthesis process of graphene, its biosafety and bioactivity are poor, and its biological application range is narrow; compared with GO, since RGO is obtained by reducing GO, a large number of oxygen vacancies and defects will be formed on the surface during the reduction process, so RGO has higher bioactivity, and its oxidation and destructive effects on biofilms are also improved, and it has better electrical, thermal and mechanical properties. In addition, RGO has a mechanical strength similar to that of the surrounding damaged tissue, strong tissue compliance, can promote the migration and adhesion of macrophages in the blood, make them aggregate at the site of nerve damage, and accelerate the process of nerve regeneration.

[0034] 4. The first hydrogel and the second hydrogel are used as carriers. The porous structure on their surfaces is conducive to the uniform distribution of germanium nanoparticles and RGO, which is conducive to the absorption of near-infrared light by germanium nanoparticles and increases the photoacoustic conversion efficiency; and is conducive to RGO to fully exert its electrical and chemical activity and more efficiently exert the neural stimulation brought by photoacoustic stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a flow chart for preparing the nerve regeneration scaffold in Example 1.

[0036] Figure 2 This is a comparison diagram of the states of Ge NPs@Gel MA in Example 1 before and after photocuring.

[0037] Figure 3 Schematic diagram of the process of using nerve regeneration scaffolds for spinal cord nerve repair.

[0038] Figure 4 Schematic diagram of histological fluorescence staining of the lesion area after spinal cord injury surgery using the nerve regeneration scaffolds of Example 2 and Comparative Example 3.

[0039] Figure 5 This is a behavioral evaluation diagram of the effects of the nerve regeneration scaffolds of Example 2 and Comparative Example 3 on motor function after SCI.

[0040] Figure 6 The graph shows the neuroelectrophysiological evaluation of the nerve regeneration scaffolds of Example 2 and Comparative Example 3 on motor function after SCI. DETAILED DESCRIPTION

[0041] The content of the present invention is further described in detail below through specific examples. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the principles set forth in the present invention all belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific data exemplified below. The raw materials, reagents or devices used in the following examples and comparative examples, unless otherwise specified, can all be obtained from conventional commercial sources, or can be obtained by existing known methods.

[0042] In the present invention, nanoparticles refer to particles with a particle size ranging from 1 to 100 nm.

[0043] In some embodiments of the present invention, the particle size of the germanium nanoparticles ranges from 1 to 100 nm. In the following embodiments and comparative examples, the particle size of the germanium nanoparticles (Ge NPs) is 50 nm.

[0044] Example 1

[0045] A nerve regeneration scaffold, the preparation process diagram is as follows Figure 1 As shown, the specific preparation process includes the following:

[0046] Step 1: Preparation of BSA-coated Ge NPs: A crystalline Ge sheet (about 0.6 g) was placed in a zirconia grinding jar containing about 80 g of zirconia beads and 25 mL of isopropanol (IPA). The crystalline Ge sheet was first ground for 3 h using 10 mm zirconia beads to crush it into a coarse powder, and then ground for 20 h using 3 mm zirconia beads to further grind it into Ge NPs. The resulting Ge NPs were etched in a mixture of 10 wt% nitric acid and 3 wt% HF, then washed by ultrasonic treatment and dispersed in isopropanol. Centrifugation at 3000 rcf for 1 min precipitated Ge NPs with relatively large size, while the remaining Ge NPs in the supernatant were collected for the next step of treatment, treated with 8 wt% HF to remove surface oxides. The HF-treated Ge NPs were centrifugally precipitated at 10000 rcf, and then dispersed in deoxygenated pure undecylenic acid (UDA), and a small amount of chloroplatinic acid was added as a catalyst. UDA was reacted as an olefin at 130°C for 20 h to undergo a hydrosilylation reaction to obtain UDA-passivated Ge NPs, so that the surface of the Ge NPs was modified with carboxylic acid, and then centrifuged, hexane was added as an antisolvent, washed several times in tetrahydrofuran, and dispersed in dimethyl sulfoxide by ultrasonic treatment to obtain a solution to be treated. In order to conjugate with BSA, 100 mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC hydrochloride) and 50 mM N-hydroxysulfosuccinimide (Sulfo-NHS) were first added to the solution to be treated, reacted at DMSO and 60°C for 48 h, activated the carboxylic acid in the UDA ligand, and then added to 0.1 M phosphate buffer (PBS) with a BSA concentration of 20 mg / mL and a pH of 7.4, and ultrasonically treated. Finally, the excess BSA was washed to remove the excess BSA, and a stock solution of BSA-coated Ge NPs with a concentration of 10 mg / mL was prepared and stored at 4°C.

[0047] Step 2: Preparation of reduced graphene oxide (RGO): Ascorbic acid was added to monolayer graphene oxide (mass ratio of ascorbic acid: graphene oxide was 10:1), and the mixture was reacted in a 60°C water bath for 3 h. After the dispersion turned black and the particles precipitated, the RGO dispersion was stirred for 48 h and then ultrasonically treated for 2 h to prepare a reduced graphene oxide stock solution with a concentration of 10 mg / mL, which was stored at 4°C.

[0048] Step 3: Preparation of Gel MA: Take 20mL PBS and add it to a brown bottle containing initiator LAP (containing 0.05g LAP); heat in a 40-50℃ water bath to dissolve for 15min, shaking several times during the period. The prepared 0.25% (w / v) initiator standard solution is stored at 4℃ away from light. Take 100mg of Gel MA and put it into a centrifuge tube; take 1mL of the initiator standard solution and add it to the above centrifuge tube, shake to make Gel MA fully infiltrated; heat in a 60-70℃ water bath away from light to dissolve for 20-30min, shaking several times during the period; the prepared 10% (w / v) Gel MA solution is immediately sterilized with a 0.22μm sterile syringe filter and stored at -20℃.

[0049] Step 4: Construction of nerve regeneration scaffold: Dilute the BSA-coated Ge NPs stock solution and the reduced graphene oxide stock solution with PBS to 1 mg / mL Ge NPs and 1 mg / mL RGO, respectively. Dissolve 10% (w / v) Gel MA in a water bath at 60-70°C in the dark for 20-30 min, oscillate several times during the process, add BSA-coated GeNPs suspension and reduced graphene oxide solution at a volume ratio of 1:1 to prepare 0.5 mg / mL Ge NPs@Gel MA and 0.5 mg / mL RGO@Gel MA, respectively, and photocure for 10 s. After solidification, cut into appropriate sizes, and finally overlap Ge NPs@Gel MA and RGO@Gel MA to prepare a nerve regeneration scaffold. Figure 2 This is a comparison diagram of the states of Ge NPs@Gel MA before and after photocuring in Example 1. It can be seen that good curing effect can be achieved through photocuring.

[0050] Example 2

[0051] This example relates to a nerve regeneration scaffold, which is different from Example 1 in that:

[0052] In step 4, the concentration of Ge NPs@Gel MA is 1 mg / mL. Other raw materials and steps are the same as those in Example 1.

[0053] Example 3

[0054] This example relates to a nerve regeneration scaffold, which is different from Example 1 in that:

[0055] In step 4, the concentration of Ge NPs@Gel MA is 1.5 mg / mL. Other raw materials and steps are the same as those in Example 1.

[0056] Example 4

[0057] This example relates to a nerve regeneration scaffold, which is different from Example 1 in that:

[0058] In step 4, the concentration of RGO@Gel MA is 1 mg / mL. Other raw materials and steps are the same as those in Example 1.

[0059] Example 5

[0060] This example relates to a nerve regeneration scaffold, which is different from Example 1 in that:

[0061] In step 4, the concentration of Ge NPs@Gel MA is 1 mg / mL, and the concentration of RGO@Gel MA is 1 mg / mL. Other raw materials and steps are the same as those in Example 1.

[0062] Example 6

[0063] This example relates to a nerve regeneration scaffold, which is different from Example 1 in that:

[0064] In step 4, the concentration of Ge NPs@Gel MA is 1.5 mg / mL, and the concentration of RGO@Gel MA is 1 mg / mL. Other raw materials and steps are the same as those in Example 1.

[0065] Comparative Example 1

[0066] This example involves a photoacoustic germanium nanoparticle nerve regeneration scaffold, including the following preparation method:

[0067] Step 1: Preparation of BSA-coated Ge NPs: Same as step 1 of Example 1.

[0068] Step 2: Preparation of Gel MA: the same as step 3 of Example 1.

[0069] Step 3: Construction of nerve regeneration scaffold: dilute the BSA-coated Ge NPs stock solution with PBS to 1 mg / mL Ge NPs, dissolve 10% (w / v) Gel MA in a water bath at 60-70°C in the dark for 20-30 min, oscillate several times during the process, add BSA-coated Ge NPs suspension at a volume ratio of 1:1 to prepare 0.5 mg / mL Ge NPs@Gel MA, photocure for 10 s, and cut into appropriate sizes after solidification to obtain the nerve regeneration scaffold.

[0070] Comparative Example 2

[0071] This example relates to a reduced graphene oxide nerve regeneration scaffold, including the following preparation method:

[0072] Step 1: Preparation of reduced graphene oxide (RGO): the same as step 2 of Example 1.

[0073] Step 2: Preparation of Gel MA: the same as step 3 of Example 1.

[0074] Step 3: Construction of nerve regeneration scaffold: dilute the reduced graphene oxide stock solution with PBS to 1 mg / mL RGO, dissolve 10% (w / v) Gel MA in a water bath at 60-70°C in the dark for 20-30 min, oscillate several times during the process, add reduced graphene oxide solution at a volume ratio of 1:1 to prepare 0.5 mg / mL RGO@Gel MA, photocuring for 10 s, and cutting into appropriate sizes after solidification to obtain the nerve regeneration scaffold.

[0075] Comparative Example 3

[0076] This example relates to a nerve regeneration scaffold, including the following preparation method:

[0077] Step 1: Preparation of Gel MA: Same as step 3 of Example 1.

[0078] Step 2: Construction of nerve regeneration scaffold: Dissolve 10% (w / v) Gel MA in a water bath at 60-70°C in the dark for 20-30 min, oscillate several times, and light cure for 10 s. After solidification, cut into appropriate sizes to obtain the nerve regeneration scaffold.

[0079] The raw material compositions of Examples 1 to 6 and Comparative Examples 1 to 3 are summarized in Table 1.

[0080] Table 1 Raw material composition of Examples 1 to 6 and Comparative Examples 1 to 3

[0081]

[0082]

[0083] Application Examples

[0084] The nerve regeneration scaffolds of Examples 1 to 6 and Comparative Examples 1 to 3 were used as nerve repair materials, and the specific process was as follows:

[0085] Establishment of the spinal cord hemi-transection model: Male SD rats weighing 200-250g were selected, and the SD rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50mg·kg-1). A 3 cm long longitudinal incision was made on the back of the rat with C5 as the center, and attention was paid to protecting the subcutaneous fat pad (containing rich blood supply). After careful separation of the paravertebral muscles, the C5 vertebral lamina was completely exposed in the spinal canal. The right vertebral lamina was incised to expose the spinal cord, and the right spinal cord was cut into a 3 mm inverted triangle spinal cord defect area by a medical blade. After irrigation with physiological saline, a 2 mm×3 mm nerve regeneration scaffold prepared in Examples 1 to 6 and Comparative Examples 1 to 3 was placed in the injured area. The schematic diagram of the process of using the nerve regeneration scaffold for spinal cord nerve repair is shown in the figure. Figure 3 The experimental group used the nerve regeneration scaffold of Example 2, and the control group used the nerve regeneration scaffold of Comparative Example 3. 12 weeks after surgery, the SD rats were killed, and histological staining was performed, and the sagittal sections of the SCI injury were analyzed by tissue immunofluorescence method. Figure 4 This is a schematic diagram of histological fluorescence staining of the lesion area after spinal cord injury surgery using the nerve regeneration scaffolds of Example 2 and Comparative Example 3. It can be seen that, compared with Comparative Example 3, after treatment with the nerve regeneration scaffold of Example 2, the fluorescence level of tyrosine hydroxylase (TH) at the spinal cord injury site is significantly enhanced, indicating that axons extend through the lesion area and extend to the caudal spinal cord. The spinal cord regeneration ability of Example 2 is stronger than that of the control group.

[0086] Figure 5 The behavioral evaluation diagram of the effect of the nerve regeneration scaffold of Example 2 and Comparative Example 3 on the motor function after SCI, wherein (A) is a cylinder climbing experiment to evaluate the use rate of the affected forelimb of rats, and (B) is a combing experiment to evaluate the combing function of rats. It can be seen that the forelimb function scores of rats after SCI surgery gradually increased from 2 weeks to 12 weeks, but were still significantly lower than the preoperative level. In the second week after surgery, there was no significant difference in the forelimb motor function scores of rats in Example 2 and Comparative Example 3. At 12 weeks after surgery, the forelimb motor function scores of the experimental group were significantly higher than those of the control group.

[0087] Figure 6 The figure is a neuroelectrophysiological evaluation diagram of the motor function after SCI by the nerve regeneration scaffold of Example 2 and Comparative Example 3, wherein (A) is the motor evoked potential (MEP) waveform, (B) is the MEP amplitude, and (C) is the MEP latency. It can be seen that the motor evoked potential induced by cranial electrical stimulation, i.e., MEP, is detected by electromyographic signals to evaluate whether the regenerated axons can partially restore the signal transmission of cortical neurons. Compared with Comparative Example 3, after the nerve regeneration scaffold of Example 2 was used to treat SCI postoperative rats, the amplitude of the MEP induced by the motor cortical neurons after electrical stimulation was significantly higher than that of the control group, and the latency was significantly lower than that of the control group, indicating that the motor function was enhanced.

[0088] The nerve regeneration scaffold in the embodiment of the present invention comprises photoacoustic germanium nanoparticles, which are efficient photoacoustic agents, can provide repeatable photoacoustic stimulation, absorb infrared light and convert it into heat energy, then expand and contract due to heat and radiate sound waves outward, and the stimulation of such sound waves is beneficial to the axon regeneration of neurons; the nerve regeneration scaffold in the embodiment of the present invention has antioxidant properties, can inhibit oxidative stress in nerve tissue damage, can effectively reduce the intracellular reactive oxygen level of nerve cells, reduce the damage of reactive oxygen to cells, and ultimately is beneficial to nerve regeneration; the nerve regeneration scaffold in the embodiment of the present invention uses a gel layer containing reduced graphene oxide as a deep layer, has good mechanical properties and elastic modulus, can simulate normal extracellular matrix, promote the adhesion and extension of regenerated neurons, and ultimately promote regeneration after spinal cord injury; the nerve regeneration scaffold in the embodiment of the present invention is based on Gel MA, has excellent physical and chemical properties, biocompatibility, permeability, mechanical strength and suitable biodegradability, and the preparation method is simple and easy, has broad application prospects in the field of tissue engineering, and is easy to achieve industrial production and clinical transformation.

[0089] In summary, the present invention uses protein-coated germanium nanoparticles and reduced graphene oxide as the active ingredients of the first gel layer and the second gel layer, respectively, which can effectively exert the photoacoustic effect of germanium nanoparticles and the mechanical properties and electrochemical activity of reduced graphene oxide, and jointly promote nerve repair and regeneration. The nerve regeneration scaffold provided by the present invention has a wide range of applications in the preparation of nerve repair materials, especially spinal nerve repair materials.

Claims

1. A nerve regeneration scaffold, characterized in that: It comprises a first gel layer and a second gel layer stacked together; the raw materials for preparing the first gel layer comprise protein-coated germanium nanoparticles and a first hydrogel prepolymer, the raw materials for preparing the second gel layer comprise reduced graphene oxide and a second hydrogel prepolymer, the first hydrogel prepolymer and the second hydrogel prepolymer are the same or different; the protein is albumin.

2. The nerve regeneration scaffold according to claim 1, characterized in that: The albumin comprises bovine serum albumin, human serum albumin or a combination thereof; And / or, the first hydrogel prepolymer and the second hydrogel prepolymer each independently include at least one of a gelatin-based gel prepolymer, a polysaccharide-based gel prepolymer or a polypeptide-based gel prepolymer.

3. The nerve regeneration scaffold according to claim 2, characterized in that: The first hydrogel prepolymer and the second hydrogel prepolymer each independently include at least one of methacrylated gelatin, chitosan, hyaluronic acid, or conductive polypyrrole.

4. The nerve regeneration scaffold according to claim 3, characterized in that: The first hydrogel prepolymer and the second hydrogel prepolymer are each independently methacrylated gelatin.

5. The nerve regeneration scaffold according to claim 1, characterized in that: The mass ratio of the first gel layer to the second gel layer is 1:(0.1-5); and / or, in the raw materials for preparing the first gel layer, the mass ratio of the protein-coated germanium nanoparticles to the first hydrogel prepolymer is 1:(50-150); And / or, in the raw materials for preparing the second gel layer, the mass ratio of the reduced graphene oxide to the second hydrogel prepolymer is 1:(50-150).

6. A method for preparing a nerve regeneration scaffold according to any one of claims 1 to 5, characterized in that: The following steps are involved: After first curing a first mixed solution containing the protein-coated germanium nanoparticles and the first hydrogel prepolymer, the first gel layer is obtained; The second mixed solution containing the reduced graphene oxide and the second hydrogel prepolymer is subjected to a second curing to obtain the second gel layer; the first gel layer and the second gel layer are stacked to obtain the nerve regeneration scaffold.

7. The preparation method according to claim 6, characterized in that: In the first mixed solution, the concentration of the protein-coated germanium nanoparticles is 0.1 to 5 mg / mL; And / or, in the second mixed solution, the concentration of the reduced graphene oxide is 0.1 to 3 mg / mL.

8. The preparation method according to claim 6, characterized in that: The first curing and the second curing are each independently selected from photocuring.

9. Use of the nerve regeneration scaffold according to any one of claims 1 to 5 in the preparation of nerve repair materials.

10. The use according to claim 9, characterized in that: The first gel layer in the nerve regeneration scaffold serves as the surface layer of the nerve repair material, and the second gel layer in the nerve regeneration scaffold serves as the deep layer of the nerve repair material.

11. The use according to claim 9, characterized in that: The nerve repair material is a spinal nerve repair material.

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