Multifunctional composite material for repairing spinal cord injury as well as preparation method and application of multifunctional composite material

Abstract

The invention discloses a spinal cord injury repair multifunctional composite material and a preparation method and application thereof. The material is prepared from chitosan-loaded nano manganese dioxide, resveratrol and nerve growth factors. The method comprises the following steps: adding a dispersing agent and modified nano-manganese dioxide into a chitosan solution, and then adding a resveratrol solution and a diluted nerve growth factor solution into the chitosan solution to obtain a mixed solution; finally, the mixed solution is subjected to freeze drying treatment, and the spinal cord injury repairing multifunctional composite material is obtained. The spinal cord injury repair multifunctional composite material can be used as a medical stent material for treating spinal cord injury. Through the innovation of drug controlled release (pH response + slow release) and multi-mechanism cooperation (oxidation resistance / inflammation resistance / regeneration), the limitation of a single mode of spinal cord injury treatment is broken through, and a comprehensive strategy with a wide application prospect is provided for SCI repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of spinal cord injury implant materials, and particularly relates to a spinal cord injury repair multifunctional composite material and a preparation method and application thereof. BACKGROUND

[0002] Spinal cord injury (SCI) is a severe nervous system disease, which usually leads to motor, sensory and autonomic nervous dysfunction below the injury plane. At present, the clinical treatment methods (such as surgical decompression, hormone impact therapy, etc.) have limited effect, and it is difficult to achieve nerve regeneration and functional recovery. Therefore, developing new biomaterials to promote nerve repair and regulate the injury microenvironment has become a research hotspot.

[0003] The pathological process after spinal cord injury can be divided into primary injury (mechanical damage) and secondary injury (oxidative stress, inflammation, glial scar formation, etc.). Secondary injury involves the following key mechanisms: (1) oxidative stress: a large amount of reactive oxygen species (ROS) accumulates after injury, leading to neuronal apoptosis and lipid peroxidation; (2) neural inflammation: activated microglia release TNF-α, IL-6 and other pro-inflammatory factors, exacerbating tissue damage; (3) lack of neurotrophic factors: the decrease of endogenous NGF, BDNF and other factors affects axonal regeneration; (4) glial scar barrier: the proliferation of astrocytes forms a physical / chemical barrier, hindering nerve regeneration.

[0004] Traditional treatment methods (such as methylprednisolone) can only temporarily suppress inflammation and cannot promote nerve regeneration. Therefore, it is urgent to develop a spinal cord injury medical material that can simultaneously remove ROS, inhibit inflammation and promote axonal regeneration. SUMMARY

[0005] The present application provides a spinal cord injury repair multifunctional composite material and a preparation method and application thereof, to solve the problem that the existing spinal cord injury treatment methods cannot simultaneously remove ROS, inhibit inflammation and promote axonal regeneration.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A spinal cord injury repair multifunctional composite material, comprising chitosan as a cell-compatible carrier, and nano-manganese dioxide, resveratrol and nerve growth factor loaded by the chitosan.

[0007] A preparation method of the above-mentioned spinal cord injury repair multifunctional composite material, the process is as follows: Preparation of modified nano-manganese dioxide, and preparation of resveratrol solution and nerve growth factor solution; adding the dispersant, the modified nano manganese dioxide into the chitosan solution, and then adding the resveratrol solution and the diluted nerve growth factor solution into the chitosan solution, and then stirring uniformly, thereby obtaining a mixed solution; Finally, the mixed solution is subjected to freeze-drying treatment, so that the nano manganese dioxide, the resveratrol and the nerve growth factor are loaded on the chitosan, thereby obtaining the spinal cord injury repair multifunctional composite material.

[0008] Further, 1-4 mg of the modified nano manganese dioxide, 100-1000 μL of the resveratrol solution and 0.5-1 mL of the diluted nerve growth factor solution are added into 1 mL of the chitosan solution in terms of the volume of the chitosan solution.

[0009] Further, the modified nano manganese dioxide is obtained by modifying the nano manganese dioxide with sodium citrate.

[0010] Further, the solute in the resveratrol solution is resveratrol, and the solvent is methanol; 0.05-0.2 g of resveratrol is added into 1 mL of methanol in terms of the volume of the methanol, thereby obtaining the resveratrol solution.

[0011] Further, the solute in the nerve growth factor solution is nerve growth factor, and the solvent is a phosphate buffer solution, wherein the solute in the phosphate buffer solution is PBS powder, and the solvent is deionized water; the concentration of the phosphate buffer solution is 9.35x10 3 g / mL; 1 mg of nerve growth factor is added into 1 mL of the phosphate buffer solution in terms of the volume of the phosphate buffer solution, thereby obtaining the nerve growth factor solution.

[0012] Further, the concentration of the nerve growth factor solution is diluted to 0.25-0.5 mg / mL, and then added into the chitosan solution in which the modified nano manganese dioxide is dispersed.

[0013] Further, in the aqueous acetic acid solution, the volume fraction percentage of ethanol is 1-2 v / v %; and in the chitosan solution, the mass volume percentage of chitosan is 1-2 wt / v %.

[0014] Further, when freeze-drying, the mixed solution is poured into a mold, pre-frozen at a temperature of -80 °C for 48-72 h, and then freeze-dried at a temperature of -50 °C and a pressure of 0.1-0.2 mbar for 48-72 h, thereby obtaining the spinal cord injury repair multifunctional composite material.

[0015] The application of the above-mentioned multifunctional composite material for repairing spinal cord injury as a medical stent material for treating spinal cord injury.

[0016] In the present application, chitosan (CS) is a natural cationic polysaccharide, which has the following advantages: (1) biocompatibility: degradable and non-immunogenic, which has been used in nerve conduits, drug carriers, etc.; (2) structural support: can form a three-dimensional porous scaffold (pore size 50-100 μm), guiding the directional growth of axons; (3) drug release: the protonation of its amino group (pH < 6.5) can achieve pH-responsive drug release, adapting to the acidic microenvironment of the injury area; (4) antibacterial / hemostatic: the positive charged surface promotes platelet aggregation, reducing the risk of infection.

[0017] However, the single CS scaffold lacks mechanical strength and lacks antioxidant / anti-inflammatory activity, so it needs to be combined with other functional materials for optimization, therefore, the present application adds modified nano manganese dioxide and resveratrol (RES), wherein: Nano manganese dioxide has the following unique advantages in spinal cord repair: (1) ROS scavenging: reducing oxidative stress by catalyzing the decomposition of H2O2; (2) local oxygen supply: the generated O2 alleviates the hypoxia in the injury center and inhibits neuronal apoptosis. Therefore, the present application adds modified nano manganese dioxide to the chitosan solution, which not only makes the final material have good mechanical strength, but also can alleviate oxidative stress and improve the local hypoxic microenvironment.

[0018] The neuroprotective mechanism of resveratrol is: (1) anti-inflammatory effect: inhibiting the NF-κB pathway and reducing the release of pro-inflammatory factors TNF-α and IL-6; (2) antioxidant: activating the Nrf2 / ARE pathway and up-regulating the enzyme activity of SOD, GSH-Px, etc. Therefore, the present application adds modified nano manganese dioxide and resveratrol solution to the chitosan solution, which improves the delivery efficiency of resveratrol through the CS-MnO2 carrier, thereby avoiding the problems of poor water solubility and short half-life of resveratrol.

[0019] In addition, in the present application, nerve growth factor (NGF) is also added to the chitosan solution, which loads NGF through chitosan, promotes neuronal survival, inhibits the expression of apoptosis proteins (such as Bax), activates regeneration-related genes such as GAP-43 to make axons grow, and promotes the differentiation of neural stem cells to mature neuron phenotype. Considering that NGF is easily degraded in vivo, therefore, the present application finally realizes long-acting sustained release by freeze-drying to fix NGF on the CS-MnO2 scaffold.

[0020] Therefore, when the present application is used as a medical material for repairing spinal cord injury, it can achieve nerve regeneration and repair spinal cord injury, and has anti-inflammatory and antioxidant functions. Compared with the prior art, the present application has the following advantages: Three-effect synergistic effect MnO2: Catalyzes the decomposition of H2O2, clears ROS, and alleviates local hypoxia; RES: Reduces the expression of inflammatory factors such as TNF-α and IL-6 by inhibiting the NF-κB pathway; NGF: Activates the TrkA / PI3K / Akt pathway and promotes axonal regeneration. Synergistic effect: The oxidative microenvironment improved by MnO2 provides a basis for the anti-inflammatory effect of RES, and the two work together to optimize the microenvironment to enhance the neuroregenerative effect of NGF.

[0021] (2) Intelligent responsive drug release pH-responsive RES release: In the acidic environment of the damaged area (pH = 5.5-6.5), chitosan protonation (-NH3⁺) promotes rapid RES release; release is slow in normal tissue (pH = 7.4). Hypoxia-activated MnO2: Under hypoxic conditions, the catalytic activity of MnO2 is enhanced, achieving "on-demand oxygen supply". Long-term sustained release of NGF: Freeze-drying fixation technology extends the NGF sustained-release period, avoiding frequent dosing.

[0022] (3) Three-dimensional porous scaffold Chitosan promotes cell migration and nutrient exchange; its amino groups promote neuronal adhesion; and MnO2 enhances drug loading stability.

[0023] (4) Excellent biocompatibility and biodegradability Chitosan has good biocompatibility and degradation properties, while MnO2 decomposes into Mn²⁺ (a normal trace element in the body). A small amount of manganese dioxide is harmless to cells. Attached Figure Description

[0024] Figure 1 The above is an in vitro free radical capture diagram of Example 2 of the present invention, wherein: (a) is the UV-Vis spectrum of DPPH, CS, CM, and CM-Res, (b) is the UV-Vis spectrum of H2O2, CS, CM, and CM-Res, (c) is the UV-Vis spectrum of ·OH, CS, CM, and CM-Res, and (d) is the DPPH, H2O2, and ·OH scavenging rate of CS, CM, and CM-Res.

[0025] Figure 2 This is a cell compatibility diagram of CS, CM, CM-Res, and CM-Res-NGF in Example 2 of the present invention.

[0026] Figure 3 The diagram shows the ROS scavenging ability of PC-12 cells in Example 2 of this invention, where: (a) is the ROS fluorescence diagram of PC-12 cells co-cultured with CS, CM, CM-Res, and CM-Res-NGF for 6 and 12 h, and (b) is the relative fluorescence intensity of ROS of PC-12 cells co-cultured with CS, CM, CM-Res, and CM-Res-NGF for 6 and 12 h.

[0027] Figure 4 This is a cell differentiation diagram from Example 2 of the present invention, showing the fluorescence of PC-12 cells and cells co-cultured with LPS, CM-Res-NGF (LPS), CM-Res-NGF (LPS-PR), and CM-Res-NGF for 3 days. CM-Res-NGF (LPS-PR) is a CM-Res-NGF scaffold co-cultured with PC-12 and Raw246.7 cells under LPS stimulation.

[0028] Figure 5 The image shows the anti-inflammatory ability of Raw246.7 cells co-cultured with LPS, CM-Res-NGF (LPS), and CM (LPS) for 24 hours, along with DAPI, iNOS, and ARG-1 fluorescence. Detailed Implementation

[0029] Example 1

[0030] This embodiment discloses a multifunctional composite material for spinal cord injury repair, including chitosan (CS) as a cell compatibility carrier. The chitosan is loaded with nano-manganese dioxide, resveratrol (RES) and nerve growth factor (NGF) through freeze-drying technology.

[0031] The multifunctional composite material for spinal cord injury repair disclosed in this embodiment can simulate the biomimetic natural extracellular matrix (ECM) tissue structure to fill the cavity in the spinal cord injury area and guide tissue regeneration. At the same time, it can be used as a carrier for drugs, cells or bioactive factors to treat spinal cord injury through controlled drug release (pH response + sustained release) and multi-mechanism synergy (antioxidant / anti-inflammatory / regeneration).

[0032] Therefore, the multifunctional composite material for spinal cord injury repair disclosed in this embodiment can be used as a medical scaffold material for treating spinal cord injuries. Specifically, CS acts as a biodegradable carrier, forming a three-dimensional porous scaffold; MnO2 is uniformly dispersed in the chitosan matrix to catalyze the decomposition of reactive oxygen species (ROS) and improve local hypoxia; RES is loaded into the CS-MnO2 system to exert antioxidant, anti-inflammatory, and anti-apoptotic effects, and achieves pH-responsive release in an acidic microenvironment; NGF is fixed inside the scaffold by freeze-drying to achieve long-term sustained release and promote nerve regeneration.

[0033] Example 2 This embodiment discloses a method for preparing the multifunctional composite material for spinal cord injury repair described in Embodiment 1, the process of which is as follows: Step 1: Preparation of nano-manganese dioxide Weigh out potassium permanganate and add it to deionized water. Stir until the potassium permanganate dissolves to prepare a potassium permanganate solution. The mass-volume percentage of potassium permanganate is 1 wt / v.

[0034] Oleic acid is added to potassium permanganate solution to carry out the reaction. The volume ratio of oleic acid to potassium permanganate solution is 2~5 v / v. In this example, the amount of oleic acid used is 2%.

[0035] After the reaction is complete, the reaction product is washed 3-5 times with ethanol, then placed in a vacuum chamber and dried at 50-60°C for 12-24 hours. Finally, the reaction product is ground to obtain nano-manganese dioxide. In this example, the optimal number of washes is 5, the optimal temperature is 50°C, and the optimal drying time is 24 hours.

[0036] Step 2: Preparation of modified nano-manganese dioxide Add 3.842g of sodium citrate to every 200mL of deionized water to prepare a sodium citrate solution.

[0037] Add 1 g of nano-manganese dioxide to every 100 mL of sodium citrate solution. Weigh the nano-manganese dioxide obtained in step 1 and add it to the sodium citrate solution. Heat the mixture in a water bath at 80℃~100℃ for 6~12 h with stirring. Then wash the reaction product 3~5 times and dry it at 50℃~60℃ for 12~24 h to obtain nano-manganese dioxide modified with sodium citrate.

[0038] In this embodiment, the optimal temperature for the water bath is 80°C, the optimal heating reaction time is 12 hours, the optimal number of washing cycles is 5, the optimal drying temperature is 50°C, and the optimal drying time is 24 hours.

[0039] Step 3: Prepare nerve growth factor solution Based on the volume of phosphate buffer solution, 1 mg of nerve growth factor was added to every 1 mL of phosphate buffer solution. Nerve growth factor powder was added to the phosphate buffer solution to prepare a nerve growth factor solution, which was then stored at -20°C. The solute in the phosphate buffer solution was PBS powder, the solvent was deionized water, and the concentration of the phosphate buffer solution was 9.35 × 10⁻⁶ mg / mL. 3 g / mL.

[0040] Then, the concentration of the nerve growth factor solution was diluted to 0.5 mg / mL.

[0041] Step 4: Prepare resveratrol solution Add 0.05g of resveratrol to every 1mL of methanol. Weigh out the resveratrol and add it to the methanol. Shake for 30 minutes to fully dissolve the resveratrol in the methanol to prepare a resveratrol solution.

[0042] Step 5: Prepare an acetic acid solution by adding 1 mL of acetic acid to every 49 mL of deionized water, and prepare a chitosan solution by adding 0.1 g of chitosan to every 50 mL of acetic acid solution.

[0043] A dispersant and modified nano-manganese dioxide were added to the chitosan solution, and the mixture was shaken for 30 minutes and then stirred to ensure that the modified nano-manganese dioxide was uniformly dispersed in the chitosan solution. Next, resveratrol solution and nerve growth factor solution diluted to 0.5 mg / mL were added to the chitosan solution containing the modified nano-manganese dioxide, and then stirred until homogeneous to obtain a mixed solution.

[0044] In this embodiment, based on the volume of the chitosan solution, 0.002 g of modified nano-manganese dioxide, 0.02 mL of resveratrol solution, and 0.01 mL of diluted nerve growth factor solution are added to every 1 mL of chitosan solution.

[0045] In this embodiment, the dispersant is Tween 80, and 0.02 mL of dispersant is added to every 1 mL of chitosan solution.

[0046] Step 6: Freeze-dry the mixed solution obtained in Step 5 to load nano-manganese dioxide, resveratrol and nerve growth factor onto chitosan, thereby obtaining a multifunctional composite material for spinal cord injury repair.

[0047] During freeze-drying, the mixed solution was poured into a mold, which was a 24-well plate, and then pre-frozen at -80℃ for 48~72h. Then it was transferred to a freeze dryer and freeze-dried at -50℃ and 0.1-0.2mbar for 48~72h. This yielded a multifunctional composite material for spinal cord injury repair, in which chitosan (CS) encapsulated nano-modified manganese dioxide, resveratrol (RES), and nerve growth factor (NGF).

[0048] Example 3 This embodiment discloses a method for preparing the multifunctional composite material for spinal cord injury repair described in Embodiment 1, the process of which is as follows: Step 1: Preparation of nano-manganese dioxide Weigh out potassium permanganate and add it to deionized water. Stir until the potassium permanganate dissolves to prepare a potassium permanganate solution. The mass-volume percentage of potassium permanganate is 1 wt / v.

[0049] Oleic acid is added to potassium permanganate solution to carry out the reaction. The volume ratio of oleic acid to potassium permanganate solution is 2~5 v / v. In this example, the amount of oleic acid used is 2%.

[0050] After the reaction is complete, the reaction product is washed 3-5 times with ethanol, then placed in a vacuum chamber and dried at 50-60°C for 12-24 hours. Finally, the reaction product is ground to obtain nano-manganese dioxide. In this example, the optimal number of washes is 5, the optimal temperature is 50°C, and the optimal drying time is 24 hours.

[0051] Step 2: Preparation of modified nano-manganese dioxide Add 3.842g of sodium citrate to every 200mL of deionized water to prepare a sodium citrate solution.

[0052] Add 1 g of nano-manganese dioxide to every 100 mL of sodium citrate solution. Weigh the nano-manganese dioxide obtained in step 1 and add it to the sodium citrate solution. Heat the mixture in a water bath at 80℃~100℃ for 6~12 h with stirring. Then wash the reaction product 3~5 times and dry it at 50℃~60℃ for 12~24 h to obtain nano-manganese dioxide modified with sodium citrate.

[0053] In this embodiment, the optimal temperature for the water bath is 80°C, the optimal heating reaction time is 12 hours, the optimal number of washing cycles is 5, the optimal drying temperature is 50°C, and the optimal drying time is 24 hours.

[0054] Step 3: Prepare nerve growth factor solution Based on the volume of phosphate buffer solution, 1 mg of nerve growth factor was added to every 1 mL of phosphate buffer solution. Nerve growth factor powder was added to the phosphate buffer solution to prepare a nerve growth factor solution, which was then stored at -20°C. The solute in the phosphate buffer solution was PBS powder, the solvent was deionized water, and the concentration of the phosphate buffer solution was 9.35 × 10⁻⁶ mg / mL. 3 g / mL.

[0055] Then, the concentration of the nerve growth factor solution was diluted to 0.5 mg / mL.

[0056] Step 4: Prepare resveratrol solution Add 0.1g of resveratrol to every 1mL of methanol. Weigh out the resveratrol and add it to the methanol. Shake for 30 minutes to fully dissolve the resveratrol in the methanol to prepare a resveratrol solution.

[0057] Step 5: Prepare an acetic acid solution by adding 1 mL of acetic acid to every 49 mL of deionized water, and prepare a chitosan solution by adding 0.1 g of chitosan to every 50 mL of acetic acid solution.

[0058] A dispersant and modified nano-manganese dioxide were added to the chitosan solution, and the mixture was shaken for 30 minutes and then stirred to ensure that the modified nano-manganese dioxide was uniformly dispersed in the chitosan solution. Next, resveratrol solution and nerve growth factor solution diluted to 0.5 mg / mL were added to the chitosan solution containing the modified nano-manganese dioxide, and then stirred until homogeneous to obtain a mixed solution.

[0059] In this embodiment, based on the volume of the chitosan solution, 0.002 g of modified nano-manganese dioxide, 0.01 mL of resveratrol solution, and 0.01 mL of diluted nerve growth factor solution are added to every 1 mL of chitosan solution.

[0060] In this embodiment, the dispersant is Tween 80, and 0.02 mL of dispersant is added to every 1 mL of chitosan solution.

[0061] Step 6: Freeze-dry the mixed solution obtained in Step 5 to load nano-manganese dioxide, resveratrol and nerve growth factor onto chitosan, thereby obtaining a multifunctional composite material for spinal cord injury repair.

[0062] During freeze-drying, the mixed solution was poured into a mold, which was a 24-well plate, and then pre-frozen at -80℃ for 48~72h. Then it was transferred to a freeze dryer and freeze-dried at -50℃ and 0.1-0.2mbar for 48~72h. This yielded a multifunctional composite material for spinal cord injury repair, in which chitosan (CS) encapsulated nano-modified manganese dioxide, resveratrol (RES), and nerve growth factor (NGF).

[0063] Example 4 This embodiment discloses a method for preparing the multifunctional composite material for spinal cord injury repair described in Embodiment 1, the process of which is as follows: Step 1: Preparation of nano-manganese dioxide Weigh out potassium permanganate and add it to deionized water. Stir until the potassium permanganate dissolves to prepare a potassium permanganate solution. The concentration of potassium permanganate in deionized water is 1 wt / v.

[0064] Oleic acid is added to potassium permanganate solution to carry out the reaction. The volume ratio of oleic acid to potassium permanganate solution is 2~5 v / v. In this example, the amount of oleic acid used is 2%.

[0065] After the reaction is complete, the reaction product is washed 3-5 times with ethanol, then placed in a vacuum chamber and dried at 50-60°C for 12-24 hours. Finally, the reaction product is ground to obtain nano-manganese dioxide. In this example, the optimal number of washes is 5, the optimal temperature is 50°C, and the optimal drying time is 24 hours.

[0066] Step 2: Preparation of modified nano-manganese dioxide Add 3.842g of sodium citrate to every 200mL of deionized water to prepare a sodium citrate solution.

[0067] Add 1 g of nano-manganese dioxide to every 100 mL of sodium citrate solution. Weigh the nano-manganese dioxide obtained in step 1 and add it to the sodium citrate solution. Heat the mixture in a water bath at 80℃~100℃ for 6~12 h with stirring. Then wash the reaction product 3~5 times and dry it at 50℃~60℃ for 12~24 h to obtain nano-manganese dioxide modified with sodium citrate.

[0068] In this embodiment, the optimal temperature for the water bath is 80°C, the optimal heating reaction time is 12 hours, the optimal number of washing cycles is 5, the optimal drying temperature is 50°C, and the optimal drying time is 24 hours.

[0069] Step 3: Prepare nerve growth factor solution Based on the volume of phosphate buffer solution, 1 mg of nerve growth factor was added to every 1 mL of phosphate buffer solution. Nerve growth factor powder was added to the phosphate buffer solution to prepare a nerve growth factor solution, which was then stored at -20°C. The solute in the phosphate buffer solution was PBS powder, the solvent was deionized water, and the concentration of the phosphate buffer solution was 9.35 x 10⁻⁶ mg / mL. 3 g / mL.

[0070] Then, the concentration of the nerve growth factor solution was diluted to 0.5 mg / mL.

[0071] Step 4: Prepare resveratrol solution Add 0.2g of resveratrol to every 1mL of methanol. Weigh out the resveratrol and add it to the methanol. Shake for 30 minutes to fully dissolve the resveratrol in the methanol to prepare a resveratrol solution.

[0072] Step 5: Prepare an acetic acid solution by adding 1 mL of acetic acid to every 49 mL of deionized water, and prepare a chitosan solution by adding 0.1 g of chitosan to every 50 mL of acetic acid solution.

[0073] A dispersant and modified nano-manganese dioxide were added to the chitosan solution, and the mixture was shaken for 30 minutes and then stirred to ensure that the modified nano-manganese dioxide was uniformly dispersed in the chitosan solution. Next, resveratrol solution and nerve growth factor solution diluted to 0.5 mg / mL were added to the chitosan solution containing the modified nano-manganese dioxide, and then stirred until homogeneous to obtain a mixed solution.

[0074] In this embodiment, based on the volume of the chitosan solution, 0g of modified nano-manganese dioxide, 0.02mL of resveratrol solution, and 0mL of diluted nerve growth factor solution were added to every 1mL of chitosan solution.

[0075] In this embodiment, the dispersant is Tween 80, and 0.02 mL of dispersant is added to every 1 mL of chitosan solution.

[0076] Step 6: Freeze-dry the mixed solution obtained in Step 5 to load nano-manganese dioxide, resveratrol and nerve growth factor onto chitosan, thereby obtaining a multifunctional composite material for spinal cord injury repair.

[0077] During freeze-drying, the mixed solution was poured into a mold, which was a 24-well plate, and then pre-frozen at -80℃ for 48~72h. Then it was transferred to a freeze dryer and freeze-dried at -50℃ and 0.1-0.2mbar for 48~72h. This yielded a multifunctional composite material for spinal cord injury repair, in which chitosan (CS) encapsulated nano-modified manganese dioxide, resveratrol (RES), and nerve growth factor (NGF).

[0078] In the above embodiments, the final multifunctional composite material for spinal cord injury repair prepared in Example 2 exhibits the best performance. The performance of the CM-Res-NGF prepared in Example 2 is described below with reference to the performance diagram of the multifunctional composite material for spinal cord injury repair (named CM-Res-NGF).

[0079] Figure 1 This study evaluated the antioxidant capacity of CS, CM, and CM-Res scaffolds. The results confirmed that both CM and CM-Res scaffolds effectively captured free radicals. The CS-Res scaffold achieved scavenging rates of 95.57%, 87.45%, and 86.58% for DPPH free radicals, H2O2, and ·OH, respectively. The CM-Res scaffold achieved scavenging rates of 78.99%, 75.74%, and 76.25% for DPPH free radicals, H2O2, and ·OH, respectively. The free radical scavenging capacity of pure CS was significantly lower than that of CM and CM-Res scaffolds, indicating that manganese dioxide and the drug resveratrol (Res) play important roles in free radical scavenging.

[0080] Figure 2 It can be seen that the cell viability of CS, CM, CM-Res, and CM-Res-NGF is greater than 80%, indicating good cell compatibility.

[0081] Figure 3 As shown in the fluorescence micrograph of a, CM, CM-Res, and CM-Res-NGF can all significantly reduce intracellular ROS levels. Figure 3The relative fluorescence intensity shown in b further confirms the ROS scavenging ability of manganese dioxide and Res, and the scavenging effect of ROS is more significant after the addition of Res.

[0082] Figure 4 The fluorescence microscopy images show that NGF can induce differentiation of PC-12 cells and inhibit differentiation under LPS stimulation. The CM-Res-NGF scaffold, when co-cultured with PC-12 and Raw246.7 cells under LPS stimulation, showed more significant cell differentiation than when co-cultured with PC-12 cells alone under LPS stimulation. This may be because macrophages release certain factors that promote differentiation during the anti-inflammatory process.

[0083] Figure 5 The fluorescence images show that Raw246.7 cells transform into M1 under LPS stimulation. The marker iNOS of M1 type emits obvious green fluorescence, while the M2 type ARG-1 does not fluoresce. Under LPS stimulation, the CM-Res-NGF material can effectively reduce inflammation, causing Raw246.7 cells to transform from M1 type to M2 type.

[0084] The preferred embodiments of the present invention have been described above. These embodiments are merely descriptions of preferred embodiments and are not intended to limit the scope or concept of the invention. The specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. Such combinations, as long as they do not violate the spirit of the present invention, should also be considered as part of this disclosure. To avoid unnecessary repetition, the present invention will not further describe the various possible combinations.

[0085] This invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this invention and without departing from the design idea of ​​this invention, all modifications and improvements made by those skilled in the art to the technical solutions of this invention should fall within the protection scope of this invention. The technical content for which protection is sought in this invention has been fully described in the claims.

Claims

1. A multifunctional composite material for spinal cord injury repair, characterized in that, This includes chitosan as a cell compatibility carrier, as well as nano-manganese dioxide, resveratrol, and nerve growth factor loaded with chitosan.

2. A method for preparing the multifunctional composite material for spinal cord injury repair as described in claim 1, characterized in that, The process is as follows: Modified nano-manganese dioxide was prepared, and resveratrol solution and nerve growth factor solution were prepared. Chitosan is added to an aqueous acetic acid solution to form a chitosan solution; then a dispersant and modified nano-manganese dioxide are added to the chitosan solution to make the modified nano-manganese dioxide uniformly dispersed in the chitosan solution. Then, resveratrol solution and diluted nerve growth factor solution are added to the chitosan solution and stirred evenly to obtain a mixed solution. Finally, the mixed solution was freeze-dried to load nano-manganese dioxide, resveratrol, and nerve growth factor onto chitosan, thereby obtaining a multifunctional composite material for spinal cord injury repair.

3. The method for preparing the multifunctional composite material for spinal cord injury repair according to claim 2, characterized in that, Based on the volume of the chitosan solution, add 1-4 mg of modified nano-manganese dioxide, 100-1000 μL of resveratrol solution, and 0.5-1 mL of diluted nerve growth factor solution to each 1 mL of chitosan solution.

4. The method for preparing the multifunctional composite material for spinal cord injury repair according to claim 3, characterized in that, The modified nano-manganese dioxide was obtained by modifying nano-manganese dioxide with sodium citrate.

5. The method for preparing the multifunctional composite material for spinal cord injury repair according to claim 3, characterized in that, The solute in the resveratrol solution is resveratrol, and the solvent is methanol. 0.05~0.2g of resveratrol is added to every 1mL of methanol to prepare the resveratrol solution.

6. The method for preparing the multifunctional composite material for spinal cord injury repair according to claim 3, characterized in that, The solute of the nerve growth factor solution is nerve growth factor, and the solvent is a phosphate buffer solution. The solute in the phosphate buffer solution is PBS powder, and the solvent is deionized water. The concentration of the phosphate buffer solution is 9.35 x 10⁻⁶. 3 g / mL; A nerve growth factor solution was prepared by adding 1 mg of nerve growth factor to every 1 mL of phosphate buffer solution.

7. The method for preparing the multifunctional composite material for spinal cord injury repair according to claim 6, characterized in that, The concentration of the nerve growth factor solution was diluted to 0.25~0.5 mg / mL and then added to the chitosan solution containing modified nano-manganese dioxide.

8. The method for preparing the multifunctional composite material for spinal cord injury repair according to claim 2, characterized in that, In the acetic acid aqueous solution, the volume fraction of ethanol is 1~2 v / v; in the chitosan solution, the mass volume percentage of chitosan is 1~2 wt / v.

9. The method for preparing the multifunctional composite material for spinal cord injury repair according to claim 2, characterized in that, During freeze-drying, the mixed solution is poured into a mold and pre-frozen at -80℃ for 48~72h, and then freeze-dried at -50℃ and 0.1-0.2mbar for 48~72h, thereby obtaining a multifunctional composite material for spinal cord injury repair.

10. The application of the multifunctional composite material for spinal cord injury repair as described in claim 1 as a medical scaffold material for treating spinal cord injury.

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