Compositions, methods of making and using the same

Abstract

The application discloses a composition, a preparation method and application thereof. The application provides a preparation method of the composition, the composition prepared through the preparation method can be applied to preparation of a nerve repair material, the application also provides application of the composition in reducing a hydrolysis speed of the nerve repair material and improving antioxidant capacity of the nerve repair material, the application further provides a preparation method of a tissue engineering scaffold based on the composition, the prepared tissue engineering scaffold and the aforementioned composition can be applied to a nerve repair medical device, and the application has a wide development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to compositions, their preparation methods, and applications. Background Technology

[0002] Nerve injury commonly occurs due to various traumas and other conditions, resulting in partial or complete damage to nerve cells. It is one of the most common and frequently occurring types of injury in clinical practice, often leading to sensory and motor dysfunction or loss, severely impacting patients' quality of life and work efficiency. Peripheral nerve injury is a common surgical condition. Compared to the central nervous system, peripheral nerves have a certain regenerative capacity, which is the basis for functional recovery after peripheral nerve injury repair. Establishing effective connections between proximal and distal nerves and target organs after repair is a prerequisite for nerve function recovery. Although modern microsurgical repair techniques have continuously improved, the precise localization of repair remains unattainable. Many regenerated axons of proximal motor and sensory nerves fail to regenerate precisely to the corresponding distal endoneurotic canals, thus failing to reach the corresponding target organs. This leads to loss of corresponding nerve function. Mismatched regeneration between motor nerves can cause nerve antagonism, and mismatched regeneration between sensory nerves can lead to sensory innervation disorder. Therefore, improving the precision and efficiency of nerve regeneration, and ultimately enhancing clinical surgical outcomes, is of great significance.

[0003] Currently available neural repair materials can be categorized into degradable and non-degradable materials based on their biodegradability. Degradable materials mainly include naturally derived materials and synthetic materials. Naturally derived materials, such as extracellular matrix (ECM), gelatin, chitosan, alginate, cellulose, hyaluronic acid, liposomes, and silk fibroin, exhibit good tissue compatibility and low antigenicity. ECM materials, for example, possess a natural porous structure system that facilitates cell adhesion. However, they typically have poor biomechanical properties and lack sufficient physical strength to protect damaged sites. Synthetic materials, usually polymers such as PGA, PLA, and PLGA, generally possess better physical properties, including certain mechanical strength, flexibility, biodegradability, and permeability. However, synthetic materials exhibit some cytotoxicity and poor biocompatibility, leading to strong inflammatory responses in the host. Some synthetic materials, while achieving immune tolerance in the host, have poor cell compatibility, hindering cell adhesion and tissue repair. Therefore, finding a nerve repair material with excellent properties in all aspects and no fatal shortcomings is the direction of scientific research in the field of nerve repair. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solution:

[0005] The first aspect of the present invention provides a method for preparing a composition, the method comprising:

[0006] 1) Dissolve gelatin in water to obtain a gelatin aqueous solution;

[0007] 2) Add AMB to the gelatin aqueous solution and stir;

[0008] 3) Add ammonium persulfate, stir and mix well, and after 24 hours, the first product is obtained. The first product is dialyzed in DI water and freeze-dried for 2 days to obtain the second product.

[0009] 4) Add PBS solution to the second product from step 3), dissolve at 50°C to achieve a final solution concentration of 20%, and adjust the pH to 7.0-7.2;

[0010] 5) Add Silma gel to the LAP solution and let it stand to dissolve;

[0011] 6) Add the solution described in step 4) to the solution in step 5) to obtain the third product, which is then wrapped in tin foil;

[0012] 7) Irradiate the third product with a light source to make it a gel-like substance, completing the first cross-linking and obtaining the fourth product;

[0013] 8) The fourth product was soaked in NHS and EDC solution overnight to complete the second crosslinking and obtain the composition.

[0014] Furthermore, in step 1) of the preparation method, the gelatin in the gelatin aqueous solution has a mass-volume ratio of 40%, and its unit is (Wt).

[0015] In some embodiments, the gelatin with a mass-to-volume ratio of 40% means that 100 mL of solution contains 40 g of gelatin.

[0016] Furthermore, the water in step 1) is deionized water.

[0017] Furthermore, the mass ratio of AMB in step 2) to ammonium persulfate in step 3) is 200:273.

[0018] Furthermore, the stirring method in step 2) is mechanical stirring.

[0019] Furthermore, in step 3), the first product is an aqueous solution of PAMBG.

[0020] Furthermore, the second product in step 3) is solid PAMBG.

[0021] Furthermore, in step 5), the ratio of Silma gel to LAP solution is 25-27.5 mg: 50 μL.

[0022] In some specific implementations, the ratio of Silma gel to LAP solution in step 5) is 25 mg: 50 μL, 25.5 mg: 50 μL, 26 mg: 50 μL, 26.5 mg: 50 μL, 27 mg: 50 μL, and 27.5 mg: 50 μL.

[0023] The term "Silma" refers to methacrylamide silk fibroin. As used herein, the term "comprising" has the broad standard meaning of "including," "covering," or "containing." It includes one or more elements explicitly listed, and also allows, but is not required, the presence of one or more other elements not cited. In addition to this broad meaning as used herein, the term "comprising" also encompasses the restrictive meaning of "consisting of," according to which only the one or more elements explicitly listed are present, and no other one or more elements are present. Furthermore, the term "comprising" also includes the meaning of "substantially consisting of," which means that one or more additional elements may be present in addition to those explicitly listed, provided that the presence of the additional one or more elements does not alter the technical effect achieved by the one or more elements explicitly listed.

[0024] Furthermore, in step 5), the weight ratio of the LAP solution is 0.5%, and the LAP solution is a DI aqueous solution.

[0025] In one embodiment, the weight ratio of 0.5% refers to a mass ratio (Wt), i.e., 0.05g of LAP powder added to 10mL of solvent.

[0026] Furthermore, in step 5), before allowing the mixture to stand and dissolve, a step of high-speed centrifugation at room temperature can be added to remove the flocculent precipitate.

[0027] Furthermore, in step 6), the mass ratio of Silma gel in the solution of step 5) to PAMBG in the solution of step 4) is 1:5-11.

[0028] Furthermore, in step 6), the mass ratio of Silma gel in the solution of step 5) to PAMBG in the solution of step 4) is 1:5 or 1:11.

[0029] In some specific implementations, the mass ratio of Silma gel in the solution of step 5) to PAMBG in the solution of step 4) added in step 6) is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or 1:11.

[0030] Furthermore, in step 6), the third product is cured by irradiation with high ultraviolet light or ultraviolet light source.

[0031] Furthermore, the ultraviolet light source includes vacuum ultraviolet UVD, short-wave ultraviolet UVC, medium-wave ultraviolet UVB, and long-wave ultraviolet UVA.

[0032] In some specific implementations, the high ultraviolet light wavelength range is 405-410nm, the vacuum ultraviolet light wavelength range is 100-200nm, the short-wave ultraviolet light wavelength range is 200-280nm, the medium-wave ultraviolet light wavelength range is 280-320nm, and the long-wave ultraviolet light wavelength range is 320-400nm.

[0033] Furthermore, in step 6), the third product is cured using a 405nm light source.

[0034] Furthermore, in step 7), the molar concentration of NHS is 2M.

[0035] Furthermore, in step 7), the molar concentration of EDC is 4M.

[0036] In some specific embodiments, the preparation of the composition can begin directly from step 5), by adding 20% ​​PAMBG solution, followed by the foil wrapping and step 7) operations in the subsequent composition preparation process.

[0037] A second aspect of the present invention provides a composition prepared according to the preparation method described above.

[0038] Furthermore, the mass-volume ratio of PAMB-G-Silma in the composition ranges from 25% to 27.5%.

[0039] In some specific embodiments, the mass-to-volume ratio of PAMB-G-Silma in the composition is 25%, 25.5%, 26%, 26.5%, 27%, and 27.5%.

[0040] As used herein, the term "composition" covers and discloses any physical entity comprising (or consisting of or substantially consisting of) the respective listed substances. The physical form of the composition is not limited. For example, the term "composition" covers and discloses a powder in which each of the listed substances exists in powder form. For example, the term "composition" discloses a gel in which the listed substances exist in the form of a hydrogel after crosslinking.

[0041] The third aspect of the present invention provides the application of the preparation method described in the first aspect or the composition described in the second aspect in the preparation of nerve repair materials.

[0042] Furthermore, the nerve repair material includes peripheral nerve repair material.

[0043] Furthermore, the peripheral nerve repair material includes ganglion repair material, nerve trunk repair material, nerve plexus repair material, and nerve terminal repair material.

[0044] In this specification, when a part is referred to as "including" a component, unless otherwise specified, this does not mean that other components are excluded, but rather that other components may also be included.

[0045] In some specific embodiments, the neural repair material can induce changes in the expression of PC12 differentiation-related neural factors. In one specific embodiment, the material induces axon growth in neurons during nerve repair.

[0046] Furthermore, the neural repair material is a non-conductive material.

[0047] Furthermore, the neural repair material may also contain added cytokines.

[0048] In some specific implementations, the neural repair material may be supplemented with cytokines, such as growth factors and adhesion factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). By releasing or controlling the release of cytokines, the extension and regeneration of damaged neuronal axons can be promoted, which is beneficial to the regeneration of nerve tissue and the recovery of function, such as the restoration of nerve conduction velocity and the normalization of compound muscle action potential (CMAP).

[0049] The terms "neural repair" and "neural regeneration" are synonymous, both referring to the regeneration or repair of neural tissue, cells, or cellular products. This mechanism can include the generation of new neurons, glial cells, axons, myelin, or synapses. The difference between neural regeneration in the peripheral nervous system (PNS) and the central nervous system (CNS) lies in the functional mechanisms involved, particularly the extent and speed of repair. The peripheral regeneration process can be divided into the following main events: Wallerian degeneration, axonal regeneration / growth, and reinnervation. When an axon is damaged, the distal segment undergoes Wallerian degeneration and loses its myelin sheath. The proximal segment may die due to apoptosis or undergo a staining reaction, i.e., the dissolution and breakage of the neuronal's staining material (such as chromatin), which is an attempt at repair. Events occurring in peripheral regeneration occur relative to the axis of nerve injury. The proximal stump refers to the end of the injured neuron that remains attached to the neuronal cell body; the proximal stump is the regenerated portion. The distal stump refers to the damaged neuron tip that is still attached to the axon terminal; it is the degenerated part of the neuron, but the distal stump is still preserved in the area where the regenerating axon grows toward it.

[0050] The fourth aspect of the present invention provides the application of the preparation method described in the first aspect or the composition described in the second aspect in reducing the hydrolysis rate of nerve repair materials and improving the antioxidant capacity of nerve repair materials.

[0051] The fifth aspect of the present invention provides a method for preparing a tissue engineering scaffold, the method comprising the step of preparing a tissue engineering scaffold by 3D printing using the composition described in the second aspect.

[0052] Furthermore, the preparation method also includes the steps of using a 3D scanner to acquire three-dimensional data of the damaged area and using a 3D printer to manufacture a mold for tissue engineering scaffolds.

[0053] As used herein, the term "tissue engineering scaffold" refers to a structure used to enhance or promote cell growth and / or tissue formation. A tissue engineering scaffold can be a two-dimensional or three-dimensional porous structure that provides a template for cell growth. Tissue engineering scaffolds can be perfused, coated, or otherwise comprised of cells, growth factors, or other bioactive agents to promote cell growth.

[0054] In some specific embodiments, the method for preparing the tissue engineering scaffold includes the following steps: 1) preparing the composition using the method for preparing the composition provided in the first aspect of the present invention, or directly using the composition provided in the second aspect of the present invention; 2) scanning the three-dimensional data of the damaged site using a 3D scanner, and manufacturing a mold for the tissue engineering scaffold using a 3D printer; 3) using the composition in step 1) and the mold in step 2) to 3D print and prepare the tissue engineering scaffold.

[0055] The sixth aspect of the present invention provides a tissue engineering scaffold prepared according to the preparation method described in the fifth aspect above.

[0056] The seventh aspect of the present invention provides the application of the preparation method described in the first aspect, the composition described in the second aspect, the preparation method described in the fifth aspect, or the tissue engineering scaffold described in the sixth aspect in a nerve repair medical device.

[0057] Furthermore, the nerve repair medical device includes isolation and protection for nerve damage and bridging of nerve defects.

[0058] Furthermore, the nerves include the central nervous system and peripheral nerves.

[0059] As used herein, the term "central nervous system" refers to the cranial nerves and spinal nerves. As used herein, the term "peripheral nervous system" refers to one of several nerves that branch from the spinal cord and extend to all parts of the body. Unlike the central nervous system, nerve regeneration is more common in the peripheral nervous system.

[0060] As used in this article, the term "medical device" refers to any tool used in a medical setting for the diagnosis or treatment of a patient, such as surgical tools like scalpels and forceps, scissors, and sutures. The term "medical device" as used in this article includes neuroreparative medical devices.

[0061] As used herein, the term "peripheral nerve injury" refers to damage to one or more pairs of motor and sensory nerves in the peripheral nervous system that connects the brain and spinal cord (central nervous system) to the whole body. Peripheral nerve injuries are commonly caused by lacerations (cuts or tears in nerve tissue); severe bruising (contusions); damage to surrounding tissues during surgery; stretching (traction); drug injection injuries; and electrical injuries. Symptoms of peripheral nerve injuries depend on the type of nerve damaged—motor, sensory, or autonomic. Motor nerves control all muscle movements under conscious control, such as the muscles used for walking, grasping, or speaking. Sensory nerves transmit information such as the sensation of light touch, temperature, or pain from cutting. Autonomic nerves control organs and regulate unconscious activities in animals, such as breathing, digesting food, and the function of the heart and glands. Peripheral nerves control sensory, motor, and motor coordination functions. They are fragile and easily injured. Common, non-limiting examples of peripheral nerve injuries include damage to the brachial plexus, sciatic nerve, peroneal nerve, and spinal accessory nerves.

[0062] The eighth aspect of the present invention provides the application of the preparation method described in the first aspect, the composition described in the second aspect, the preparation method described in the fifth aspect, or the tissue engineering scaffold described in the sixth aspect in constructing a 3D model system or device.

[0063] Furthermore, the 3D model building system or apparatus described in the eighth aspect includes one or more processors and a memory for storing one or more computer programs.

[0064] The terms “system” and “component” as well as “module” refer to computer-related entities (hardware, a combination of hardware and software, software, or software in execution). For example, a component can be, but is not limited to, a process running on a processor, a processor, a hard disk drive, multiple storage drives (of optical and / or magnetic storage media), an object, an executable file, a thread of execution, a program, and / or the computer itself. By way of illustration, both an application running on a server and the server itself can be components.

[0065] The term "device" as used in this invention is not limited to one or a specific number of physical objects. As used herein, a device can be any medical or electronic component having multiple parts that can implement at least some portions of this disclosure. Although the term "device" is used in the following description and examples to describe certain aspects of this disclosure, the term "device" is not limited to a particular configuration, type, or number of objects.

[0066] The terms "processor" or "memory" in this invention include computing devices having one processor or one memory, as well as devices having multiple processors or multiple memories, that can be used to perform some or all of the steps described. "Processor" can include more than one processor, for example, a multi-core design or multiple processors each having a multi-core design.

[0067] The term "processor" refers to any type of processor, such as a microprocessor, embedded processor, digital signal processor (DSP), network processor, or other device for executing code, and may include more than one processor, such as a multi-core design or multiple processors, each having a multi-core design. A processor may be configured to execute a sequence of computer program instructions, such as those stored in memory, to perform various operations, processes, and methods according to exemplary embodiments of the content of this invention.

[0068] The term "memory" refers to any type of long-term, short-term, volatile, non-volatile, or other memory, and should not be limited to any particular type of memory or any particular number of memories or the type of media for storing memory. The term "memory" can be any suitable memory element (e.g., random access memory (RAM), read-only memory (ROM), erasable programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), application-specific integrated circuit (ASIC), etc.), software, hardware, firmware, or any other suitable component, device, element, or object where appropriate and based on a particular need. Attached Figure Description

[0069] Figure 1 These are morphological images of PAMB-G-Silma hydrogels after photocrosslinking at different concentrations;

[0070] Figure 2 These are electron micrographs of different hydrogels; where A is Silma hydrogel, B is PAMB-G, and C is 27.5% hydrogel.

[0071] PAMB-G-Silma gel, D is 25% PAMB-G-Silma gel;

[0072] Figure 3 This is a graph showing the photocrosslinking properties of PAMB-G-Silma gel;

[0073] Figure 4 This is a 3D printing image of PAMB-G-Silma hydrogel;

[0074] Figure 5 This is a deformation test diagram of PAMB-G-Silma gel catheter;

[0075] Figure 6 This is the CV cycling curve of PAMB-G-Silma gel;

[0076] Figure 7 This is an electron microscope image of a PAMB-G-Silma gel catheter;

[0077] Figure 8 The graph shows the degradation curves of hydrogel systems with different concentrations of Silma and PAMB-G in mice.

[0078] Figure 9 These are images showing the degradation of different hydrogel systems on mouse skin.

[0079] Figure 10 This is a HE staining image of mouse subcutaneous hydrogel degradation;

[0080] Figure 11 This is a statistical graph showing the expression of different concentrations of PAMB-G-Silma-induced PC12 differentiation-related neural factors.

[0081] Figure 12 These are actual images of DCFH fluorescence intensity detection after LPS-induced cells were co-cultured on different hydrogels; where A is the positive control, B is a 25% PAMB-G-Silma gel, C is a 27.5% PAMB-G-Silma gel, and D is the negative control.

[0082] Figure 13 This is a statistical graph of DCFH fluorescence intensity detection data after LPS-induced cells were co-cultured on different hydrogels;

[0083] Figure 14 These are electron micrographs of PC12 cells co-cultured with different concentrations of PAMB-G-Silma hydrogel; where A is the control, B is Silma hydrogel, C is 27.5% PAMB-G-Silma gel, and D is 25% PAMB-G-Silma gel. Detailed Implementation

[0084] Hereinafter, in order to specifically illustrate the present invention, embodiments are provided for detailed description. However, embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not to be construed as limited to the embodiments described below. The embodiments provided in this specification are intended to illustrate the present invention more completely to those skilled in the art.

[0085] Example 1

[0086] 1. Experimental materials

[0087] Gelatin (Vetec), APS (SIGMA-ALDRICH), DI water (Hetai), PBS solution (Biosharp), AMB (Alfa Esa (China) Chemical Co., Ltd.), Silma gel (EFL Suzhou), LAP (EFL Suzhou), NHS, EDC (Maclean's).

[0088] 2. Experimental Methods

[0089] 1) Prepare a 40% gelatin aqueous solution by dissolving the required amount of gelatin powder in deionized water.

[0090] 2) Add 200 mg of AMB to the gelatin aqueous solution and stir mechanically.

[0091] 3) 273 mg of ammonium persulfate (APS) was added as a catalyst for oxidative polymerization to polymerize AMB monomers and graft them onto the gelatin backbone. After 24 hours, the resulting PAMBG copolymer was dialyzed in DI water and freeze-dried for two days.

[0092] 4) Add a certain volume of PBS solution and dissolve at 50°C to make the final PAMBG mass ratio 20% Wt, and adjust the pH value to 7.0-7.2.

[0093] 5) Weigh out 25 mg and 27.5 mg of Silma gel respectively and dissolve them in 50 μL of 0.5% Wt LAP DI solution as experimental group 1 and experimental group 2. Let them stand until fully dissolved. The presence of flocculent precipitate is normal. Avoid heating and centrifuge at high speed. Dissolve 30 mg of Silma gel in 100 μL of 0.25% Wt LAP DI solution as the control group.

[0094] 6) Add 25 μL of the above PAMB-G solution and 25 μL of PBS solution to experimental group 1. Add 12.5 μL of the above PAMB-G solution and 37.5 μL of PBS solution to experimental group 2. Obtain 3D printing inks (hydrogels) with PAMB-G:Silma ratios of 1:5 and 1:11, wrap them in aluminum foil for preservation to prevent curing. Curing was carried out using a 405 nm light source, depending on the shape of the hydrogels to be produced.

[0095] 7) Immerse the obtained 3D printing ink in a solution of 2M NHS and 4M EDC overnight to obtain a double crosslinked hydrogel.

[0096] 8) Based on the characteristics of 3D printing ink, nerve repair materials of different shapes can be made as needed and applied to the repair of peripheral nerve injuries.

[0097] Example 2 Performance Test

[0098] 1. The photocrosslinking plasticity experiment was conducted on the hydrogel 3D printing ink prepared by the method in Example 1, and the results are as follows: Figure 1 As shown, different concentrations of PAMB-G-Silma were used in the experiment, and the results proved that hydrogel 3D printing inks of different concentrations after photocrosslinking can be molded into arbitrary shapes.

[0099] 2. The microstructure of Silma hydrogel, PAMB-G, 27.5% PAMB-G-Silma, and 25% PAMB-G-Silma gel was observed using electron microscopy. The results are as follows: Figure 2 As shown, the results indicate that Silma hydrogels of different concentrations, PAMB-G, 27.5% PAMB-G-Silma, and 25% PAMB-G-Silma gels all exhibited porous structures under electron microscopy, suggesting that the pores of the synthesized hydrogel system meet the basic material exchange requirements in organisms.

[0100] 3. Experimental analysis was conducted on the photocrosslinking properties of PAMB-G-Silma gel, and the results are as follows: Figure 3 As shown, the results indicate that PAMB-G doping does not affect the photocrosslinking properties of Silma. The PAMB-G-Silma gel still exhibits good photocrosslinking characteristics.

[0101] 4. 3D printing was performed using PAMB-G-Silma hydrogel, and the printing results are as follows: Figure 4 As shown in the figure, the images demonstrate that PAMB-G-Silma inks of different concentrations can all be used for 3D printing, indicating that PAMB-G-Silma inks of different concentrations have good 3D printability.

[0102] 5. The deformation of the PAMB-G-Silma gel catheter was tested and analyzed, and the results are as follows: Figure 5 As shown, the results indicate that the prepared PAMB-G-Silma gel catheter has a certain deformation capability. By connecting it to a multimeter, it was found that it has a certain conductivity (this result is a rough test, and the catheter resistance is affected by its shape and size).

[0103] 6. Cyclic voltammetry (CV) curves were analyzed. The CV curves of Silma hydrogel, PAMB-G, 27.5% PAMB-G-Silma, and 25% PAMB-G-Silma gels were tested respectively. The results are as follows: Figure 6As shown, the results indicate that the CV cycle closure area of ​​Silma hydrogel, 27.5% PAMB-G-Silma, 25% PAMB-G-Silma, and PAMB-G gel gradually increases, suggesting that the capacitance of the gel gradually increases with the increase of PAMB-G gel concentration, resulting in better conductivity.

[0104] 7. Electron microscopy was performed on the catheters prepared with PAMB-G-Silma gel, and the results are as follows: Figure 7 As shown, the results indicate that the catheter has a good morphology and a loose, porous structure, which meets the needs of free entry and exit of tissue fluid in the body for substance exchange.

[0105] 8. The degradation rate of hydrogels in vivo is an important indicator of their properties. Therefore, the degradation rates of SF, S27.5F&P, S25 F&P, D27.5 F&P, D25 F&P, and PAMB-G were statistically analyzed. S27.5F&P and S25 F&P were obtained by irradiating 27.5% PAMB-G-Silma and 25% PAMB-G-Silma products with only a 405nm light source. D27.5F&P and D25 F&P were obtained by irradiating 27.5% PAMB-G-Silma and 25% PAMB-G-Silma products with a 405nm light source, followed by overnight immersion in 2M NHS and 4M EDC solutions to obtain bi-crosslinked hydrogels. The results are as follows: Figure 8 As shown, the results indicate that the PAMB-G hydrogel alone degraded the fastest, while the SF(Silma) hydrogel degraded the slowest. The 27.5% PAMB-G-Silma and 25% PAMB-G-Silma hydrogels exhibited moderate degradation rates, meeting the basic requirements for nerve regeneration. Actual images of the hydrogel systems degrading under mouse skin are shown below. Figure 9 As shown in the figure. To gain a more intuitive understanding of the microstructure of hydrogel degradation, the hydrogel degraded from mouse skin on day 45 was stained with hematoxylin and eosin (HE). The results are shown in the figure. Figure 10 As shown, the results indicate that after 45 days, the hydrogel was replaced by a large number of tissue cells, suggesting that hydrogels with different components have good biocompatibility and degradability.

[0106] Example 3: The role of hydrogels in nerve repair

[0107] 1. To demonstrate that PAMB-G-Silma hydrogel promotes nerve repair, the expression of different concentrations of PAMB-G-Silma hydrogel and PC12 differentiation-related neurotrophic factors BDNF and caspase-3 was analyzed. The results are as follows: Figure 11As shown, the results indicated that different concentrations of PAMB-G-Silma hydrogel had varying effects on the expression of neurotrophic factors. For BDNF, the expression in the 25% PAMB-G-Silma group was significantly higher than that in the control group and the 27.5% PAMB-G-Silma group, and also higher than that in the SF group alone, with statistically significant differences. For the apoptosis-related factor Caspase-3, the expression in the 27.5% PAMB-G-Silma group was significantly higher than that in the SF group. C represents the blank control, and SF represents Silma hydrogel. The results suggest that both Silma hydrogel and 25% PAMB-G-Silma contain factors that promote neuronal differentiation. The 25% PAMB-G-Silma group showed better neurotrophic factor expression ability compared to Silma hydrogel. Caspase-3 results showed that pc12 cells co-cultured with Silma hydrogel and 25% PAMB-G-Silma did not undergo apoptosis. The cells exhibit good biocompatibility.

[0108] 2. Fluorescence intensity was measured in the positive control, 25% PAMB-G-Silma hydrogel, 27.5% PAMB-G-Silma hydrogel, and negative control. Higher fluorescence intensity indicated higher DCFH and ROS, suggesting weaker antioxidant capacity. Results are as follows: Figure 12 As shown in the figure, the low fluorescence intensity of 25% PAMB-G-Silma indicates its good antioxidant capacity. The statistical results of its fluorescence intensity are as follows: Figure 13 As shown.

[0109] 3. PC12 cells were co-cultured with different concentrations of PAMB-G-Silma hydrogels, and their microstructure was observed using electron microscopy. The results are as follows: Figure 14 As shown in the figure, A is the control, B is Silma hydrogel, C is 27.5% PAMB-G-Silma, and D is 25% PAMB-G-Silma. By co-culturing PC12 cells with hydrogels of different concentrations, the results showed that PC12 cells could adhere to the surface of the hydrogel, suggesting that nerve cells may adhere and grow on ducts in vivo.

[0110] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method of preparing a composition, characterized by, The preparation method is: 1) dissolving gelatin in water to obtain a gelatin aqueous solution; 2) adding AMB to the gelatin aqueous solution and stirring; 3) adding ammonium persulfate and stirring to mix evenly, and obtaining a first product after 24 h, which is dialyzed in DI water and freeze-dried for 2 d to obtain a second product; 4) adding a PBS solution to the second product of step 3), dissolving at 50°C to obtain a solution with a final concentration of 20%, and adjusting the pH value to 7.0-7.2; 5) adding Silma gel to the LAP solution and standing to dissolve; 6) adding the solution of step 4) to the solution of step 5) to obtain a third product, which is wrapped with tin foil; 7) irradiating the third product with a light source to make it into a gel-like substance, completing the first cross-linking to obtain a fourth product; 8) soaking the fourth product in a NHS and EDC solution overnight to complete the second cross-linking, and obtaining a composition; The mass ratio of AMB in step 2) to ammonium persulfate in step 3) is 200:273; The first product in step 3) is a PAMBG aqueous solution; The second product in step 3) is a PAMBG solid; The mass ratio of the LAP solution in step 5) is 0.5%, and the LAP solution is a DI water solution; The ratio of Silma gel to LAP solution in step 5) is 25-27.5 mg: 50 μL; The mass ratio of Silma gel in the solution of step 5) to PAMBG in the solution of step 4) in step 6) is 1:5-11.

2. The production method according to claim 1, characterized by, The mass / volume ratio of gelatin in the gelatin aqueous solution in step 1) of the preparation method is 40%.

3. The preparation method of claim 1, wherein the water in step 1) is deionized water.

4. The preparation method of claim 1, wherein the stirring mode in step 2) is mechanical stirring.

5. The preparation method of claim 1, wherein before standing to dissolve in step 5), a step of removing flocculent precipitates by high-speed centrifugation at room temperature can be further added.

6. The preparation method of claim 1, wherein the third product solidification in step 6) uses ultraviolet light source irradiation.

7. The preparation method of claim 6, wherein the ultraviolet light source includes vacuum ultraviolet UVD, short-wave ultraviolet UVC, medium-wave ultraviolet UVB, or long-wave ultraviolet UVA.

8. The preparation method of claim 1, wherein the third product solidification in step 6) uses 405 nm light source irradiation.

9. The preparation method of claim 1, wherein the molar concentration of NHS in step 8) is 2 M.

10. The preparation method of claim 1, wherein the molar concentration of EDC in step 8) is 4 M.

11. A composition prepared by the preparation method of any one of claims 1-10; the mass / volume ratio of PAMB-G-Silma in the composition ranges from 25% to 27.5%.

12. Use of the preparation method of any one of claims 1-10 or the composition of claim 11 in the preparation of a nerve repair material.

13. The use of claim 12, wherein the nerve repair material includes a peripheral nerve repair material.

14. The use according to claim 13, wherein the peripheral nerve repair material comprises a ganglion repair material, a nerve trunk repair material, a nerve plexus repair material, or a nerve terminal repair material.

15. Use of the preparation method of any one of claims 1-10 or the composition of claim 11 for reducing the hydrolysis rate of a nerve repair material and / or increasing the antioxidant capacity of a nerve repair material.

16. A method of preparing a tissue engineering scaffold, characterized by, The preparation method comprises a step of preparing a tissue engineering scaffold by 3D printing using the composition of claim 11.

17. The method of claim 16, wherein, The preparation method further comprises a step of obtaining three-dimensional data of the damaged site using a 3D scanner and manufacturing a mold for the tissue engineering scaffold using a 3D printer.

18. A tissue engineering scaffold prepared according to the preparation method of claim 16 or 17.

19. Use of the preparation method of any one of claims 1-10, the composition of claim 11, the preparation method of claim 16 or 17, or the tissue engineering scaffold of claim 18 for preparing a nerve repair medical device.

20. The use according to claim 19, wherein the nerve repair medical device comprises an isolation protection device for nerve injury or a bridging device for nerve defect.

21. Use of the preparation method of any one of claims 1-10, the composition of claim 11, the preparation method of claim 16 or 17, or the tissue engineering scaffold of claim 18 for constructing a 3D model system.

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