Repair material for trigeminal nerve injury as well as preparation method and application of repair material

Abstract

The invention belongs to the technical field of medical biological materials, and particularly relates to a repair material for trigeminal nerve injury as well as a preparation method and application of the repair material. The repairing material is prepared from the following raw materials in parts by mass: 0.5 to 5 parts of nervous tissue acellular matrix micro powder, 0.5 to 5 parts of oxidized sodium alginate, 0.005 to 0.02 part of calcium dobesilate, 0.4 to 1 part of Ca-EDTA (Ethylene Diamine Tetraacetic Acid) and 0.4 to 1 part of D-glucolactone. The repairing material is directly injected to the exposed nerve part, damaged nerves are wrapped, the protection effect is achieved, external environment irritation is prevented, the components of the repairing material have the nerve nourishing effect, repairing of the damaged nerves can be promoted, permanent injury of trigeminal nerves is avoided, the duration time of related symptoms is shortened, and the repairing material has a good application prospect. Therefore, the method has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical biomaterials, and particularly relates to a repair material for trigeminal nerve injury and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission of prior art against anyone, but is intended solely to elicit the general background of the application.

[0003] Trigeminal neuralgia is a common nervous system disease, mainly manifested as repeated paroxysmal severe neuralgia in the trigeminal nerve distribution area of the face. Although this pain does not endanger life, it can seriously affect the normal life of patients. Clinically, trigeminal neuralgia is divided into primary and secondary. Primary neuralgia is usually caused by vascular compression of the trigeminal nerve root, without clear structural lesions. Secondary neuralgia is related to tumor, multiple sclerosis, inflammation and other organic lesions invading the trigeminal nerve.

[0004] Both types of trigeminal neuralgia are accompanied by some degree of nerve injury. Primary neuralgia is mainly caused by compression of the trigeminal nerve root by adjacent blood vessels, leading to local demyelination of the nerve and abnormal electrical signal transmission. Its injury is reversible and belongs to functional nerve injury. Secondary neuralgia is usually caused by structural nerve injury caused by organic lesions, which is usually accompanied by nerve fiber rupture, axon compression necrosis or perineurium fibrosis. These injuries are mostly irreversible and have poor self-repairing ability. At present, the treatment methods for the above trigeminal neuralgia mainly include drug therapy, nerve block / regulation therapy and surgical treatment. Among them, drug therapy (such as carbamazepine) can only temporarily relieve pain by inhibiting abnormal nerve discharge, and cannot solve the root problems such as vascular compression and nerve injury. Nerve block / regulation therapy can only play a role by improving local blood circulation or slightly activating nerve endings, and has little effect on promoting axon regeneration for structural nerve injury. Surgical treatment includes methods such as using microvascular decompression, radiofrequency ablation and balloon compression to relieve the compression of blood vessels on the nerve, destroy the nerve fibers that transmit pain, and compress the trigeminal nerve ganglion to relieve pain. However, it has problems such as insufficient stability of treatment effect and limited scope of surgery. Moreover, structural nerve injury accompanied by secondary neuralgia usually needs to rely on nerve suture, transplantation or release surgery to promote nerve repair after lesion resection, but these treatment methods have problems such as poor nerve regeneration efficiency and functional recovery. Generally, it is difficult to reverse the neuronal degeneration caused by structural damage. Therefore, it is of great significance to develop a convenient and effective means for the treatment and prevention of secondary degeneration of trigeminal nerve injury. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a repair material for trigeminal nerve injury and a preparation method and application thereof. Specifically, the repair material is directly injected into the exposed site of the nerve to form a package for the damaged nerve, thereby playing a protective role and preventing external environmental irritation. Moreover, the components of the repair material have a nerve-nourishing effect, which can promote the repair of the damaged nerve and avoid permanent damage to the trigeminal nerve. Based on the above research results, the present application is completed.

[0006] The present application is realized by the following technical solutions: In a first aspect, the present application provides a repair material for trigeminal nerve injury, which comprises the following raw materials in mass fractions: 0.5-5 parts of nerve tissue acellular matrix micro powder, 0.5-5 parts of oxidized sodium alginate, 0.005-0.02 parts of calcium dobesilate, 0.4-1 parts of Ca-EDTA, and 0.4-1 parts of D-gluconolactone.

[0007] The nerve tissue acellular matrix micro powder (NEP) can be obtained by freeze-drying and pulverizing the acellular nerve tissue, and the particle size is controlled to be 50-200 pm. The acellular nerve tissue can be obtained by removing cells from non-human mammalian nerve tissue. Further, the acellular nerve tissue can be prepared by the method provided in CN113304321A, which will not be described here.

[0008] Further, the repair material is a hydrogel preparation.

[0009] In a second aspect, the present application provides a preparation method of the above-mentioned repair material, which comprises: The oxidized sodium alginate (OSA) and the nerve tissue acellular matrix micro powder containing calcium dobesilate are mixed and reacted in a liquid phase environment to obtain a NEP / OSA mixed system. Ca-EDTA and D-gluconolactone are added to the above-mentioned system to obtain the repair material.

[0010] In a third aspect, the present application provides the use of the above-mentioned repair material in the preparation of a trigeminal nerve injury repair product.

[0011] The product can be a medical device.

[0012] In a fourth aspect, the present application provides a trigeminal nerve injury repair product, which comprises at least the above-mentioned repair material.

[0013] The above-mentioned one or more technical solutions have the following beneficial technical effects: (1) The above technical solution uses sodium periodate as an oxidizing agent to modify sodium alginate, and the aldehyde group forms an imine bond with the amino group in NEP to form a Schiff base structure, which reduces the potential physiological toxicity of the aldehyde group, and because the imine bond is a dynamic covalent bond, it gives the hydrogel self-healing properties. Its mechanical properties are gradually enhanced, and the hydrogel structure is more stable.

[0014] (2) The hydrogel of the above technical solution has a double network structure, and the aldehyde group in the oxidized sodium alginate is crosslinked with the amino group in NEP, and the unoxidized sodium alginate is uniformly in-situ crosslinked with calcium ions under the action of D-gluconic acid lactone, the structure is more stable, the mechanical properties are stronger, and the adhesion to the contacted tissue can be realized, and it is not easy to slip.

[0015] (3) The NEP in the repair hydrogel of the above technical solution contains a variety of nerve growth factors, which can be slowly released with the degradation of the matrix powder, have a nutritional effect on the possibly damaged nerves, promote the repair and regeneration of the damaged nerves; at the same time, with the release of calcium dobesilate with the degradation of the hydrogel, the damaged nervous system is positively repaired, and the local microcirculation disorder is improved through multiple pathways, the oxidative defense function is enhanced, thereby improving the recovery of vascular function of the body, promoting the repair of damaged tissues, avoiding the continuous development and deterioration into permanent damage, and shortening the duration of related trigeminal nerve injury symptoms.

[0016] (4) The repair hydrogel of the above technical solution can be injected into the exposed nerve site through a pre-filled syringe, and a high-strength gel can be formed in-situ after shaping to wrap the damaged nerve and prevent external environmental irritation from causing further nerve damage. BRIEF DESCRIPTION OF DRAWINGS

[0017] The drawings accompanying the specification of this application form a part thereof, serve to provide further understanding of the application, and together with the description of the exemplary embodiments of the application given below, explain the application, and do not constitute an improper limitation of the application.

[0018] Figure 1 Self-healing situation of sample 1 and sample 2 prepared by the application after being extruded into a vial for 1 hour A: sample 1 presents a gel state, B: sample 2 maintains the state when extruded.

[0019] Figure 2 The storage modulus (G') and loss modulus (G'') of the hydrogel prepared by the application change with the frequency.

[0020] Figure 3 Detection of the expression level of inflammatory cytokines in each group of RAW 264.7 after being stimulated as an inflammatory stimulant by the ELISA method of the application. S. aureus

[0021] Figure 4 ​The in vitro degradation of each experimental group of the present application is shown in Table 1. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed description is illustrative only, and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0023] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the inventive subject matter. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0024] As mentioned above, the current treatment of trigeminal nerve injury is not ideal. In view of this, the present application provides a repair material for trigeminal nerve injury. The repair material is directly injected into the exposed site of the nerve to form a package for the damaged nerve, thereby playing a protective role. Meanwhile, the active ingredients in the repair material have the effect of nourishing the nerve, and can promote the repair and regeneration of the damaged nerve.

[0025] The inventors have found that the degradation rate of sodium alginate (SA) single network hydrogel in a physiological environment is too fast, the stability is poor, and it is difficult to maintain high mechanical properties. Studies have shown that the double network structure obtained by chemical modification of SA or compounding with other materials has great advantages in tissue repair.

[0026] The ortho-hydroxyl group in the SA structural unit can be oxidized to a dialdehyde group by sodium periodate, which can react with the amino group in the nerve tissue acellular matrix powder to form a Schiff base, and the unreacted sodium alginate is crosslinked with Ca 2+ to form nerve tissue acellular matrix powder / calcium alginate (NEP / OCA) double network hydrogel. The present application uses sodium periodate as an oxidizing agent to modify SA, reacts with NEP to prepare a hydrogel system; and adds calcium dobesilate (CaD) and Ca-EDTA as a calcium source in the system, and uses D-gluconic acid lactone (GDL) to promote the slow release of Ca 2+ and crosslinking with SA to form a double crosslinked network structure, further improving the mechanical properties of the hydrogel.

[0027] Further, calcium dobesilate is a new type of microvascular protective agent, which mainly reduces capillary permeability, inhibits platelet aggregation response, reduces blood viscosity, improves microcirculation disorder, and has strong reducing property, and is a recognized strong antioxidant. On the basis of protecting damaged nerves by using nerve tissue acellular matrix micro powder, calcium dobesilate is used for positive repair of damaged nervous system, improves local microcirculation disorder through multiple pathways, reduces the generation of active oxygen species and excessive collagen formation in the body by inhibiting vasoactive mediators such as 5-hydroxytryptamine, slow-release peptides and thromboxane B2, and enhances oxidative defense function, thereby improving the recovery of vascular function of the body and promoting the repair of damaged tissues.

[0028] Therefore, in one typical embodiment of the present application, a repair material for trigeminal nerve injury is provided, which comprises the following raw materials in mass fraction: Nerve tissue acellular matrix micro powder 0.5-5 parts, oxidized sodium alginate 0.5-5 parts, calcium dobesilate 0.005-0.02 parts, Ca-EDTA 0.4-1 parts, and D-gluconolactone 0.4-1 parts.

[0029] The nerve tissue acellular matrix micro powder can be obtained by freeze-drying and crushing of acellular nerve tissue, and the particle size is controlled to be 50-200 μm. The acellular nerve tissue can be obtained by removing cells from non-human mammalian nerve tissue (further, it can be non-human mammalian peripheral nerve tissue such as pig sciatic nerve), and further, the acellular nerve tissue can be prepared by the method provided in CN113304321A, which will not be described here.

[0030] Further, the repair material is a hydrogel preparation.

[0031] In a second aspect of the present application, a preparation method of the above-mentioned repair material is provided, which comprises: The oxidized sodium alginate and the nerve tissue acellular matrix micro powder containing calcium dobesilate are mixed and reacted in a liquid phase environment to obtain a NEP / OSA mixed system; Ca-EDTA and D-gluconolactone are added to the above-mentioned system to obtain the repair material.

[0032] In the above-mentioned preparation method, CaD and Ca-EDTA are used as calcium sources, and H + Ca is released from the chelating system 2+ SA is crosslinked, and after incubation, a NEP / OCA hydrogel with a double crosslinking network structure is formed.

[0033] The liquid phase environment can be an aqueous phase.

[0034] The oxidized sodium alginate can be obtained by a commercial method. In one specific embodiment of the present application, the oxidized sodium alginate is obtained by modifying sodium alginate with sodium periodate, and the oxidation degree of the oxidized sodium alginate is controlled to be 30%-80%, preferably about 50%.

[0035] Further, the oxidized sodium alginate is prepared by the following method: Sodium alginate is dissolved in ethanol to obtain a suspension A, and sodium periodate is dissolved in water to obtain a solution B; the solution B is added to the suspension A, and the reaction is carried out under dark condition for 0.5-6 hours, then the unreacted sodium periodate is neutralized, the reaction mixture is added to anhydrous ethanol for precipitation, the precipitate is dissolved in water, and then precipitated with anhydrous ethanol, and the operation is repeated for 2-4 times, and the white flocculent oxidized sodium alginate is obtained after drying.

[0036] The mass ratio of the sodium alginate to the sodium periodate is 1: (0.3-0.8).

[0037] In a third aspect of the present application, the above-mentioned repair material is provided for use in preparing a trigeminal nerve injury repair product.

[0038] Further, the product has at least the following uses: (a) physically wrapping the damaged nerve to prevent further nerve damage caused by external environment irritation; (b) nourishing the damaged nerve to promote the repair of the damaged nerve; (c) enhancing the oxidative defense function, improving the recovery of the vascular function of the body, promoting the repair of the damaged tissue, and shortening the duration of the trigeminal nerve injury symptoms.

[0039] Further, the product can be a medical device.

[0040] In a fourth aspect of the present application, a trigeminal nerve injury repair product is provided, which comprises at least the above-mentioned repair material.

[0041] In another specific embodiment of the present application, the product can be a medical device.

[0042] In another specific embodiment of the present application, when the product is a medical device, it can be a drug-device combination product, which refers to a product composed of a drug and a medical device and produced as a single entity. One specific embodiment of the drug-device combination product can be the above-mentioned repair material, which can obviously be used as a hydrogel dressing medical device.

[0043] In another specific embodiment of the present application, the drug can further comprise at least one non-drug active ingredient, which can include pharmaceutically acceptable excipients, carriers, etc., which are not specifically limited herein.

[0044] In summary, the repair material (i.e., hydrogel material) of the present invention can be used to repair nerve damage caused by primary trigeminal neuralgia and secondary trigeminal neuralgia. The material can act as a physical barrier and promote the repair and regeneration of damaged nerves in the repair of both types of trigeminal nerve injuries.

[0045] (1) For primary trigeminal neuralgia caused by compression of the trigeminal nerve root by adjacent blood vessels, this repair material can be directly injected into the compressed nerve site and solidified in situ to form a high-strength gel, acting as a pad between the damaged trigeminal nerve and the compressing blood vessel, physically isolating the two and terminating further nerve damage. At the same time, the active ingredients in the hydrogel material have a nourishing effect on the functionally damaged trigeminal nerve, promoting its repair. The gel repair material of the present invention is suitable for primary trigeminal neuralgia with mild symptoms and requiring only short-term physical blockage.

[0046] (2) For secondary trigeminal neuralgia caused by organic lesions, the trigeminal nerve is structurally damaged. Such damage exceeds its self-repair ability and requires active intervention to repair it. After the lesion is removed, the repair material can be directly injected into the damaged trigeminal nerve and form a high-strength gel in situ, which physically blocks external environmental irritation and plays a protective role. The active ingredients in the repair material can effectively regulate the microenvironment of nerve regeneration, promote the repair and regeneration of damaged nerve tissue, and avoid secondary damage that may be caused by nerve suturing.

[0047] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0048] Example 1 1. Preparation of decellularized matrix micropowder (NEP) for neural tissue The NEP preparation method adopts CN113304321A, the method for preparing decellularized matrix from neural tissue, as detailed below: (1) Take fresh pig sciatic nerve, remove the fascia, fat and other substances attached to the nerve tissue, cut it into small pieces, wash it 3 times with deionized water, place it in a -70℃ ultra-low temperature freezer, freeze for 24 hours, thaw at 25℃, and cycle the freeze-thaw cycle 3 times.

[0049] (2) The substrate was rinsed with 2% (w / v) Triton X-100 solution for 2 h, 0.05% (v / v) peracetic acid solution for 1 h, and 10 times PBS solution until the pH value was neutral. After draining the water, 20 g of decellularized matrix was weighed and added to 100 mL of PBS solution. The solution was homogenized at high speed and poured into a stainless steel mold. After pre-freezing at -20℃ for 24 h, it was placed in a freeze dryer for freeze drying for 48 h. After sterilization by cobalt-60 irradiation, the micro powder was obtained by pulverizing and sieving. The micro powder with a particle size of 75~150 μm was obtained by passing it through 100 mesh and 200 mesh sieves respectively.

[0050] 2. Preparation of NEP / OCA hydrogel (1) Preparation of oxidized sodium alginate (OSA) Weigh 20g of sodium alginate powder and disperse it in 100mL of ethanol to obtain suspension A; Dissolve 13g of sodium periodate in 100mL of deionized water to obtain solution B; Solution B was added to suspension A and reacted for 4 hours in the dark. 12 mL of ethylene glycol was added and reacted for 1 hour to neutralize unreacted sodium periodate. The reaction mixture was then added to 800 mL of vigorously stirred anhydrous ethanol, filtered, dissolved in deionized water, and precipitated with 250 mL of anhydrous ethanol. This process was repeated three times. After vacuum drying at 50°C for 24 hours, white flocculent OSA was obtained. The mass ratio of sodium alginate to sodium periodate was 1:0.65, and the oxidation degree was 52.3%.

[0051] (2) Preparation of NEP / OCA hydrogel Add 20 mL of 1.5 mg / mL CaD deionized water solution to 80 mL of NEP deionized water dispersion, mix well to obtain suspension C, the NEP concentration in suspension C is 2% (w / v), and swell at 37℃ for 2 h; Prepare 10 mL of deionized water containing 8 mmol Ca-EDTA and freshly prepared 16 mmol D-gluconolactone (GDL) to obtain solution D; 4g of OSA was dissolved in 90mL of deionized water, and suspension C was added with stirring. After ultrasonic mixing, solution D was added, and the mixture was pre-filled into syringes. The solution was incubated at 37℃ for 1 hour to obtain a hydrogel. After irradiation sterilization, the hydrogel repair material sample 1 of the present invention was obtained. The FTIR spectrum showed that sample 1 exhibited an imine stretching vibration absorption peak.

[0052] Comparative Example 1 Sodium alginate was not oxidized and hydrogel sample 2 was prepared from SA, NEP prepared in Example 1, CaD, and Ca-EDTA according to the steps in Example 1.

[0053] 1. Preparation of decellularized neural tissue matrix micropowder (NEP) (same as Example 1) (1) Take fresh pig sciatic nerve, remove the fascia, fat and other substances attached to the nerve tissue, cut it into small pieces, wash it 3 times with deionized water, place it in a -70℃ ultra-low temperature freezer, freeze for 24 hours, thaw at 25℃, and cycle the freeze-thaw cycle 3 times.

[0054] (2) The substrate was rinsed with 2% (w / v) Triton X-100 solution for 2 h, 0.05% (v / v) peracetic acid solution for 1 h, and 10 times PBS solution until the pH value was neutral. After draining the water, 20 g of decellularized matrix was weighed and added to 100 mL of PBS solution. The solution was homogenized at high speed and poured into a stainless steel mold. After pre-freezing at -20℃ for 24 h, it was placed in a freeze dryer for freeze drying for 48 h. After sterilization by cobalt-60 irradiation, the micro powder was obtained by pulverizing and sieving. The micro powder with a particle size of 75~150 μm was obtained by passing it through 100 mesh and 200 mesh sieves respectively.

[0055] 2. Preparation of NEP / CA hydrogel Add 20 mL of 1.5 mg / mL CaD deionized water solution to 80 mL of NEP dispersion, mix well to obtain suspension C, the NEP concentration in suspension C is 2% (w / v), and swell at 37℃ for 2 h; Prepare 10 mL of deionized water containing 8 mmol Ca-EDTA and freshly prepared 16 mmol D-gluconolactone (GDL) to obtain solution D; 4g of SA was dissolved in 90mL of deionized water, and suspension C was added with stirring. After ultrasonic mixing, solution D was added, and the mixture was pre-filled into syringes. The solution was incubated at 37℃ for 1 hour to obtain a hydrogel. After irradiation sterilization, hydrogel repair material sample 2 was obtained. The FTIR spectrum showed that sample 2 did not show an imine stretching vibration absorption peak, suggesting that it has no self-healing ability.

[0056] Comparative Example 2 Suspension C was a 100 mL NEP aqueous dispersion with a NEP concentration of 2% (w / v) and no CaD was added. Other steps were the same as in Example 1, resulting in hydrogel repair material sample 3. The FTIR spectrum showed that sample 3 exhibited an imine stretching vibration absorption peak.

[0057] Comparative Example 3 Without adding D-gluconolactone (GDL), the other steps are the same as in Example 1.

[0058] 1. Preparation of decellularized matrix micropowder (NEP) for neural tissue (1) Take fresh pig sciatic nerve, remove the fascia, fat and other substances attached to the nerve tissue, cut it into small pieces, wash it 3 times with deionized water, place it in a -70℃ ultra-low temperature freezer, freeze for 24 hours, thaw at 25℃, and cycle the freeze-thaw cycle 3 times.

[0059] (2) Rinse with 2% (w / v) Triton X-100 solution for 2h, rinse with 0.05% (v / v) peracetic acid solution for 1h, rinse with 10 times PBS solution until pH is neutral, drain the water, weigh 20g and put it into 100mL PBS solution, homogenize at high speed to form a solution, pour into a stainless steel mold, pre-freeze at -20℃ for 24h, put it into a freeze dryer for freeze drying for 48h, sterilize with cobalt-60 irradiation, pulverize and sieve to obtain micro powder, pass through 100 mesh and 200 mesh sieves respectively to obtain micro powder with a particle size of 75~150μm.

[0060] 2. Preparation of NEP / OCA hydrogel (1) Preparation of oxidized sodium alginate (OSA) Weigh 20g of sodium alginate (SA) powder and disperse it in 100mL of ethanol to obtain suspension A; Dissolve 13g of sodium periodate in 100mL of deionized water to obtain solution B; Solution B was added to suspension A and reacted for 4 hours in the dark. Then, 12 mL of ethylene glycol was added and reacted for 1 hour to neutralize unreacted sodium periodate. The reaction mixture was then added to 800 mL of vigorously stirred anhydrous ethanol, filtered, dissolved in deionized water, and precipitated with 250 mL of anhydrous ethanol. This process was repeated three times. After vacuum drying at 50°C for 24 hours, white flocculent OSA was obtained. The oxidation degree was 52.3%.

[0061] (2) Preparation of NEP / OCA hydrogel Add 20 mL of 1.5 mg / mL CaD deionized water solution to 80 mL of NEP dispersion, mix well to obtain suspension C, the NEP concentration in suspension C is 2% (w / v), and swell at 37℃ for 2 h; Prepare 10 mL of a deionized aqueous solution containing 8 mmol Ca-EDTA to obtain solution D; 4g of OSA was dissolved in 90mL of deionized water, and suspension C was added with stirring. After ultrasonic mixing, solution D was added, and the mixture was pre-filled into syringes. The solution was incubated at 37℃ for 1h to obtain a hydrogel. After irradiation sterilization, the hydrogel repair material sample 4 of this invention was obtained. The FTIR spectrum showed that sample 4 exhibited an imine stretching vibration absorption peak.

[0062] Comparative Example 4 Adding 40 mmol of D-gluconolactone (GDL) and following the same steps as in Example 1, hydrogel repair material sample 5 was obtained. The FTIR spectrum showed that sample 5 exhibited an imine stretching vibration absorption peak.

[0063] Experimental Section 1. Hydrogel gelation time and compressive strength Following the methods of Example 1 and Comparative Examples 1-4, the components were mixed sequentially to obtain a mixed system. 2 mL of each mixture was poured into a centrifuge tube, and the centrifuge tube was tilted every 0.5 min until the liquid level of the mixture no longer changed. The time of adding solution D was recorded as t0, and the time during which the liquid level of the mixture did not change was recorded as t1. The gelation time of the composite hydrogel was t = t1 - t0.

[0064] The above mixture was slowly poured into a cylindrical (H=10mm, φ=15mm) polytetrafluoroethylene mold, incubated at 37℃, and then demolded to obtain the composite hydrogel. The compressive strength of the composite hydrogel in the wet state was tested using an electronic universal testing machine. The testing speed was set to 5mm / min, and each sample was tested three times. The average value was taken as the compressive strength of the composite hydrogel.

[0065] Table 1. Time and compressive strength of each component adhesive

[0066] As shown in Table 1, with the same calcium source and SA, adding GDL can shorten the gelation time, and the hydrogel forms a uniform network structure with good compressive strength. Excessive addition of GDL will lead to excessive decomposition of calcium source, resulting in an incomplete network and reduced compressive strength. The addition of the second network makes the porous network structure denser and improves the system's resistance to deformation.

[0067] 2. Self-healing experiment Sample 1 prepared in Example 1 and Sample 2 prepared in Comparative Example 1 were squeezed from the syringe into a circular container and allowed to repair for 10 minutes without any external interference. The repair effect was observed after 10 minutes. Figure 1 As shown. Sample 1 ( Figure 1 Sample A) showed no obvious splicing marks after 10 minutes of repair, exhibiting a uniform and integral appearance. Furthermore, it did not separate under tension and bending, indicating that the material obtained in Example 1 possesses good self-healing properties. Sample 2 (… Figure 1 (B) After 10 minutes in the mold, it was still in the extrusion state and did not fuse together at the contact point, confirming that the material obtained in Comparative Example 1 has no imine bonds and does not have self-healing properties.

[0068] 3. Cytotoxicity test The in vitro cytotoxicity of the hydrogel materials prepared in this invention was evaluated using the CCK-8 assay. 96-well cell culture plates were coated with samples prepared in each example and comparative example, and L929 cells were seeded at a density of approximately 1000 cells / well, with 5 parallel wells. Cell-free DMEM medium was used as a blank control. After culturing in an incubator (37℃, 5% CO2) for 48 h, 10 µL of CCK-8 solution was added to each well, and the plates were incubated at 37℃ for another 4 h. Then, 80 µL of the solution was added to a new 96-well plate, and the absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0069] As shown in Table 2, the cell viability of each group was above 100%, the toxicity level was 0, and it had excellent biocompatibility.

[0070] Table 2. Cytotoxicity of materials in each group

[0071] 4. Rheological property testing A 1.0 mm thick hydrogel sample was placed between the plates of an RS6000 rotational rheometer to test the changes in the storage modulus (G') and loss modulus (G'') of the hydrogel with frequency (f) at 37 °C. The test conditions were a frequency strain of 1%.

[0072] Rheological tests show that ( Figure 2 Compared with the single network (sample 2 prepared in Comparative Example 1), the dual network (sample 1 prepared in Example 1) has an increased storage modulus G' of about 1000 Pa at low strain, and better shear resistance.

[0073] 5. In vitro cell anti-inflammatory experiment Will S. aureus As an inflammatory stimulus, RAW 264.7 was stimulated to simulate an in vitro inflammation model. The experimental groups were as follows: 1) Blank control group: Cells were not treated with any stimulating agents; 2) Negative control group: Cells were treated with only 1 μg / mL S. aureus Stimulate; 3) Sample Group 1: Cells were treated with 1 μg / mL S. aureus After stimulation, incubate on sterile sample 1; 4) Sample group 3: Cells were treated with 1 μg / mL S. aureus After stimulation, it was incubated on sterile sample 3.

[0074] The expression of inflammatory factors TNF-α, IL-6, and IL-8 on the surface of RAW264.7 hydrogel was analyzed using qRT-PCR.

[0075] likeFigure 3 As shown, S. aureus After in vitro stimulation of macrophages, compared with the negative control group, sample 1 inhibited the release of RAW264.7 inflammatory cytokines TNF-α, IL-6, and IL-8, while sample 3 showed a much lower inhibitory effect on inflammation than sample 1.

[0076] 6. In vitro degradation experiment Equal amounts of material samples from each embodiment and comparative example were placed in reagent bottles containing simulated body fluid (SBF solution). Degradation was observed at 20, 40, 60, and 80 days, and the mass loss rate was calculated. Mass loss rate (%) = (m0 - mt) / m0 × 100%, where m0 is the initial experimental mass and mt is the mass after degradation time t.

[0077] Depend on Figure 4 It was found that the simple calcium ion cross-linked single-network hydrogel degraded the fastest, and both the absence of GDL and excessive addition accelerated the degradation, presumably related to the uneven network structure. In contrast, Sample 1 prepared in Example 1 showed a mass loss rate of 78% on day 80 in a simulated body fluid environment, meeting the expected requirements.

[0078] It should be noted that the above examples are only used to illustrate the technical solutions of this embodiment and are not intended to limit it. Although this embodiment has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of this embodiment as needed, without departing from the spirit and scope of the technical solutions of this embodiment.

Claims

1. A repair material for trigeminal nerve injury, characterized in that, The repair material comprises the following raw materials in parts by weight: The composition of the following components is as follows: 0.5-5 parts of decellularized matrix micropowder for nerve tissue, 0.5-5 parts of sodium alginate, 0.005-0.02 parts of calcium dobesilate, 0.4-1 parts of Ca-EDTA, and 0.4-1 parts of D-gluconolactone.

2. The repair material as described in claim 1, characterized in that, The decellularized matrix micropowder of the nerve tissue is obtained by freeze-drying and pulverizing decellularized nerve tissue, with a particle size controlled at 50~200μm. Furthermore, the decellularized neural tissue can be obtained by removing cells from non-human mammalian neural tissue.

3. The repair material as described in claim 1, characterized in that, The repair material is a hydrogel formulation.

4. The method for preparing the repair material according to any one of claims 1-3, characterized in that, The preparation method includes: The NEP / OSA mixed system was obtained by mixing sodium oxidized alginate with decellularized nerve tissue matrix microparticles containing calcium dobesilate in a liquid environment; Ca-EDTA and D-gluconolactone were added to the above system to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The liquid environment is an aqueous phase.

6. The preparation method according to claim 4, characterized in that, The oxidized sodium alginate is obtained by oxidizing sodium alginate with sodium periodate, and the degree of oxidation of the oxidized sodium alginate is controlled to be 30%-80%.

7. The preparation method according to claim 6, characterized in that, The oxidized sodium alginate was prepared by the following method: Sodium alginate was dissolved in ethanol to obtain suspension A, and sodium periodate was dissolved in water to obtain solution B. Solution B was added to suspension A and reacted under light-protected conditions for 0.5-6 hours. Unreacted sodium periodate was then neutralized. The mixture of reactants was added to anhydrous ethanol for precipitation. The precipitate was dissolved in water and then precipitated again with anhydrous ethanol. This process was repeated 2-4 times. After drying, white flocculent oxidized sodium alginate was obtained. Furthermore, the mass ratio of sodium alginate to sodium periodate is 1:(0.3-0.8).

8. The use of the repair material according to any one of claims 1-3 or the repair material prepared by the preparation method according to any one of claims 4-7 in the preparation of trigeminal nerve injury repair products; The product has at least the following uses: (a) Physically wrap the damaged nerve to prevent further nerve damage caused by external environmental irritation; (b) Nutritionally nourish damaged nerves and promote the repair of damaged nerves; (c) Enhances oxidative defense function, improves the body's vascular function recovery, promotes the repair of damaged tissues, and shortens the duration of trigeminal nerve injury symptoms.

9. The application as described in claim 8, characterized in that, The product in question is a medical device.

10. A trigeminal nerve injury repair product, characterized in that, The product comprises at least the repair material as described in any one of claims 1-3 or the repair material prepared by the preparation method described in any one of claims 4-7.

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