An injectable hydrogel for spinal cord injury repair, its preparation method and application

Abstract

The present invention discloses an injectable hydrogel for spinal cord injury repair, its preparation method and application. The injectable hydrogel comprises the following components in mass percentage: 1% to 10% of silk fibroin, 0.1% to 10% of magnesium manganese hydrotalcite, and the rest is water. The above injectable hydrogel is obtained by mixing an aqueous solution of silk fibroin and an aqueous solution of hydrotalcite and then standing. Due to the large number of hydroxyl groups and its layered structure on the surface of the hydrotalcite, the hydrogel network induced and formed by it has good structural stability; and the hydrogel has excellent shear thinning and self-healing properties, and can be in-situ injected into the injury site. On the one hand, it can effectively remove excessive reactive oxygen species (ROS) generated at the injury site, relieve the inflammatory reaction, and at the same time generate oxygen in the microenvironment, which can effectively improve the adverse condition of hypoxia at the injury site. In addition, the hydrogel can effectively promote the growth and differentiation of nerve cells, which is beneficial to the rapid repair of spinal cord injury.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation of biohydrogel materials, and particularly relates to an injectable hydrogel for spinal cord injury repair, a preparation method thereof, and an application thereof. Background Art

[0002] The spinal cord is one of the parts of the central nervous system that is vulnerable to injury. With the accelerating pace of modern life and the increasing development of transportation means, the rate of trauma accidents is rising day by day. For patients with spinal cord injury, the mild ones lose their working ability, and the severe ones lose their ability to take care of themselves due to immobility of the limbs and incontinence of urine and feces. This not only causes great physical and mental pain to the patients, but also brings a heavy economic burden to the country, society and family. Therefore, solving the problem of spinal cord injury repair has very important scientific significance and medical value.

[0003] Spinal cord injury is a complex and delicate process, which is divided into primary injury and secondary injury according to the pathological process of neuron injury. Among them, the severity of spinal cord injury is mainly determined in the secondary injury stage. This is mainly because during the secondary injury process, the microenvironment of spinal cord injury changes significantly, mainly including an increase in the level of reactive oxygen species, hypoxia caused by damaged blood vessels blocking the delivery of oxygen, an increase in inflammatory response, etc. Coupled with the poor self-repair ability of nerves themselves, spinal cord injury cannot regenerate naturally after injury. In recent years, based on this, researchers have developed a variety of treatment methods to prevent secondary injury. Considering that the spinal cord is a highly hydrated soft "material", hydrogels, especially injectable hydrogels, are ideal materials for spinal cord injury repair because of their good biocompatibility, high water content, characteristics similar to the extracellular matrix, and controllable three-dimensional network structure. However, the currently used hydrogel materials have relatively single functions, mostly concentrated on reactive oxygen species scavenging and inflammation inhibition, and their nerve repair ability is limited, and the overall treatment results are still very poor.

[0004] Therefore, there is a need to provide a multifunctional injectable hydrogel that can scavenge reactive oxygen species, inhibit inflammation, improve hypoxia, and effectively promote the repair and regeneration of neurons, thereby promoting the rapid repair of spinal cord injury. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an injectable hydrogel for spinal cord injury repair, a preparation method thereof, and an application thereof. By mixing silk fibroin and hydrotalcite in an aqueous solution and standing still, a hydrogel is obtained. The hydrogel has a stable structure and has shear thinning and self-healing properties. It can be in-situ injected into the injury site, effectively scavenge reactive oxygen species at the injury site and generate oxygen to improve the hypoxic microenvironment, and at the same time can significantly promote the growth of nerve cells, promote the repair and regeneration of neurons, thereby facilitating the rapid repair of the spinal cord injury site.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] In a first aspect of the present invention, there is provided an injectable hydrogel, which comprises the following components in mass percentage: 1% - 10% of silk fibroin, 0.1% - 10% of magnesium manganese hydrotalcite, and the balance being water.

[0008] In a second aspect of the present invention, there is provided a preparation method of the injectable hydrogel according to the first aspect, wherein silk fibroin, magnesium manganese hydrotalcite and water are uniformly mixed according to the formula ratio, and the injectable hydrogel is obtained by standing.

[0009] In the present invention, by introducing magnesium manganese hydrotalcite into the silk fibroin solution system, silk fibroin is negatively charged and magnesium manganese hydrotalcite is positively charged, and there is an electrostatic interaction between the two. Magnesium manganese hydrotalcite with a flaky intercalation structure can serve as a micro-skeleton to provide more adsorption sites for silk fibroin molecules. At the same time, a large number of hydroxyl groups existing on the surface of magnesium manganese hydrotalcite can form hydrogen bonds with silk fibroin molecules, thereby inducing the transformation of silk fibroin from an amorphous structure to a β-sheet structure and forming a hydrogel network with a stable structure.

[0010] Further, the silk fibroin is regenerated silk fibroin after degumming and treatment with lithium bromide.

[0011] Further, the preparation method of the regenerated silk fibroin is as follows: Raw silk is placed in a sodium carbonate solution for heat treatment, the sericin protein is removed by washing with water, and then the dried solid is dissolved in a lithium bromide solution, and after filtration, dialysis and centrifugation, a regenerated silk fibroin solution is obtained.

[0012] Further, the concentration of the sodium carbonate solution is 0.2% - 0.5% w / v.

[0013] Further, the heat treatment is specifically: Raw silk is placed in a sodium carbonate solution and heated to boiling, and the boiling time is 30 - 60 min.

[0014] In some preferred embodiments, the degummed solid is dissolved in a 9.3 mol / L lithium bromide solution, stirred at 55 - 65 °C for 1 - 4 h, and then a regenerated silk fibroin solution is obtained after filtration, dialysis and centrifugation.

[0015] Further, the preparation method of the magnesium manganese hydrotalcite is as follows: In an inert gas atmosphere, a magnesium salt, a manganese salt and an alkali solution are reacted at a pH of 9 - 11 to obtain the magnesium manganese hydrotalcite.

[0016] Further, the inert atmosphere is nitrogen or helium.

[0017] Further, the molar ratio of the magnesium salt to the manganese salt fed is 1 - 3:1.

[0018] Furthermore, the magnesium salt is one or more of magnesium nitrate, magnesium chloride, and magnesium acetate.

[0019] Furthermore, the manganese salt is one or more of manganese nitrate, manganese chloride, and manganese acetate.

[0020] Furthermore, the alkaline solution is sodium hydroxide solution and / or sodium carbonate solution, more preferably a mixed aqueous solution of sodium hydroxide and sodium carbonate. The molar ratio of sodium hydroxide to sodium carbonate in the mixed aqueous solution is preferably 1:0.01 - 0.05.

[0021] Furthermore, the temperature of the reaction is 60 - 80 °C, and the reaction time is 0.5 - 24 h.

[0022] Furthermore, dissolve magnesium nitrate and manganese nitrate in deionized water to obtain a metal salt solution. Drop the metal salt solution and the alkaline solution into the reaction vessel simultaneously, and react to obtain magnesium - manganese hydrotalcite.

[0023] The third aspect of the present invention provides an application of the injectable hydrogel described in the first aspect in the preparation of a drug for treating spinal cord injury.

[0024] The hydrogel prepared by the present invention has excellent shear - thinning and self - healing abilities, and can be in - situ injected into the injury site. The +3 - valent manganese ions that can be stably present in magnesium - manganese hydrotalcite have strong reducibility and can react with excessive reactive oxygen species (hydrogen peroxide, superoxide anion, hydroxyl radical, singlet oxygen, etc.) in the microenvironment after spinal cord injury, reducing the inflammatory response. At the same time, when the +3 - valent manganese ions react with hydrogen peroxide or superoxide anion in the microenvironment, oxygen is generated, which can improve the adverse condition of hypoxia in the microenvironment after spinal cord injury. In addition, under the synergistic effect of silk fibroin, magnesium - manganese hydrotalcite, and the multi - level network structure constructed by the two, the hydrogel exhibits a significant effect of promoting the growth and differentiation of nerve cells, thereby promoting the rapid repair and regeneration of neurons.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. The present invention provides an injectable hydrogel for spinal cord injury repair, which is prepared by mixing and standing silk fibroin and magnesium - manganese hydrotalcite in an aqueous solution. There is no need to add other coagulants or perform additional coagulation operations. The preparation method is simple, and the prepared hydrogel has good biocompatibility and biodegradability. The hydrogel prepared by the above method has good structural stability, and at the same time has excellent shear - thinning and self - repair properties, and can be used for in - situ injection into the spinal cord injury site.

[0027] 2. The hydrogel prepared by the present invention has good ability to capture and scavenge hydrogen peroxide, and at the same time can react with hydrogen peroxide and superoxide anions in the microenvironment to generate oxygen, so as to improve the hypoxic microenvironment of the damaged site; when the hydrogel is co-incubated with nerve cells, obvious neurite growth of nerve cells can be observed, which is significantly better than that of single silk fibroin hydrogel and silk fibroin hydrogel containing magnesium and manganese ions. It can be seen that the hydrogel formed by silk fibroin and magnesium-manganese hydrotalcite with a lamellar structure can effectively promote the growth and differentiation of nerve cells, thus promoting the repair and regeneration of neurons and being beneficial to the rapid recovery of spinal cord injury. Brief Description of the Drawings

[0028] Figure 1 Transmission electron microscope image of the magnesium-manganese hydrotalcite prepared in Example 1;

[0029] Figure 2 X-ray diffraction pattern of the magnesium-manganese hydrotalcite prepared in Example 1;

[0030] Figure 3 Sol-gel picture of the injectable hydrogel prepared in Example 1;

[0031] Figure 4 Stability test results of the injectable hydrogel prepared in Example 1;

[0032] Figure 5 Shear thinning performance test results of the injectable hydrogel prepared in Example 1;

[0033] Figure 6 Self-healing performance test results of the injectable hydrogel prepared in Example 1;

[0034] Figure 7 Characterization diagram of the hydrogen peroxide scavenging ability of the injectable hydrogel prepared in Example 1;

[0035] Figure 8 Characterization diagram of the oxygen generation ability of the injectable hydrogel prepared in Example 1 under hydrogen peroxide conditions;

[0036] Figure 9 Nerve cell growth and differentiation diagram of the injectable hydrogel prepared in Example 1;

[0037] Figure 10 Nerve cell growth and differentiation diagram of the silk fibroin hydrogel prepared in Comparative Example 2;

[0038] Figure 11 Nerve cell growth and differentiation diagram of the silk fibroin hydrogel containing magnesium and manganese ions prepared in Comparative Example 4. Detailed Description of the Invention

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items. The "including" or "comprising" described in the present invention means that in addition to the components described, other components may also be included or comprised. The "including" or "comprising" described in the present invention may also be replaced by the closed "consisting of" or "composed of".

[0040] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited are not intended to limit the present invention.

[0041] Example 1

[0042] This example relates to the preparation of an injectable hydrogel for spinal cord injury, specifically including the following steps:

[0043] (1) Preparation of silk fibroin: Boil raw silk in a 0.5% w / v sodium carbonate solution for 45 min, thoroughly wash with deionized water to remove sericin, and then place it in a room temperature for drying; dissolve the obtained solid after drying in a 9.3 mol / L lithium bromide solution and dissolve it at 60 °C for 1 h. After filtration, dialysis, and centrifugation, a regenerated silk fibroin solution with a mass percentage concentration of 10% is obtained;

[0044] (2) Preparation of magnesium manganese hydrotalcite: Dissolve magnesium nitrate and manganese nitrate solids with a molar ratio of 3:1 in deionized water to obtain a metal salt solution, denoted as solution A; weigh 1.6 g of sodium hydroxide and dissolve it in deionized water to obtain a sodium hydroxide solution, denoted as solution B. Under normal temperature conditions, slowly drop solution A and solution B into a three-necked flask at the same time, continuously stir during this process, and control the reaction pH to 10. After solution A is completely dropped, continue the reaction at normal temperature for 30 min and then transfer it to a reaction kettle and react at 60 °C for 12 h. After the reaction is complete, place it in a centrifuge and centrifuge at 8000 rpm, then wash it three times with deionized water and once with absolute ethanol, and finally disperse it in deionized water to obtain a magnesium manganese hydrotalcite with a mass percentage concentration of 2%;

[0045] Characterize the structure and properties of the magnesium manganese hydrotalcite prepared above, and the characterization results are as follows:

[0046] Figure 1 is the transmission electron microscope image of the magnesium manganese hydrotalcite prepared in this example. It can be seen from the figure that the prepared magnesium manganese hydrotalcite is a two-dimensional hexagonal sheet structure; Figure 2This is an XRD overlay of the magnesium-manganese hydrotalcite prepared in this example and the standard spectrum of hydrotalcite. It can be seen from the figure that the diffraction peaks of the magnesium-manganese hydrotalcite prepared above can all be attributed to hydrotalcite, which also shows that the magnesium-manganese hydrotalcite was successfully prepared.

[0047] (3) Preparation of injectable hydrogel: A 6% silk fibroin solution and a 0.5% magnesium-manganese hydrotalcite solution were uniformly mixed in the same volume and allowed to stand for a period of time to obtain an injectable hydrogel. Figure 3 shown.

[0048] Performance Research

[0049] (1) Stability

[0050] The injectable hydrogel prepared in this example was subjected to a frequency sweep test, and the results were as follows: Figure 4 As shown, at a shear rate of 0.1 to 100 s -1 Within the range, the storage modulus (G') and the loss modulus (G") are both parallel and the storage modulus (G') is much larger than the loss modulus (G"), which shows that the hydrogel prepared in this example has a stable structure.

[0051] (2) Shear thinning properties

[0052] The rheological properties of the injectable hydrogel prepared in this example were tested, and the results were as follows: Figure 5 As shown, the viscosity of the hydrogel decreases with increasing shear rate, showing excellent shear thinning ability and is easy to be squeezed out of the microneedle.

[0053] (3) Self-repair performance

[0054] The injectable hydrogel prepared in this example was subjected to amplitude sweep tests with applied strains of 1% and 300%, respectively. The results are shown in FIG. Figure 6 As shown in the figure, after recovering from high strain to low strain, the storage modulus (G') and loss modulus (G") of the hydrogel recovered stably and were close to the initial modulus. This phenomenon shows that the hydrogel prepared in this example has good self-healing ability.

[0055] (4) Hydrogen peroxide removal ability

[0056] The ability of the injectable hydrogel prepared in this example to scavenge hydrogen peroxide was further investigated as follows: the injectable hydrogel prepared in this example was mixed with 100 mM hydrogen peroxide for 10 seconds, then mixed with a titanium sulfate solution, and the absorbance peak at 415 nm was measured using a UV spectrophotometer. A control group was prepared without the hydrogel.

[0057] The results are as follows Figure 7As shown, compared with the control group, after adding the injectable hydrogel prepared in this example, the absorption peak at 415 nm decreased significantly, indicating that the hydrogel prepared in this example has good hydrogen peroxide scavenging ability.

[0058] (5) Oxygen generation ability under hydrogen peroxide conditions

[0059] To further study the oxygen generation ability of the injectable hydrogel prepared in this example under hydrogen peroxide conditions, the specific operation is as follows: Mix the injectable hydrogel prepared in this example with hydrogen peroxide at a concentration of 100 mM, and monitor the oxygen content generated with an oxygen probe. Without adding the hydrogel as the control group.

[0060] The results are as Figure 8 shown. Compared with the control group, after adding the injectable hydrogel prepared in this example, oxygen can be effectively generated in the mixed system.

[0061] (6) Promoting the growth and differentiation of nerve cells in an environment of excessive reactive oxygen species

[0062] Co-incubate the injectable hydrogel prepared in this example with nerve cells (PC12 cells) in an environment of excessive reactive oxygen species, and observe the growth of nerve cells. The specific operation is as follows: Seed PC12 cells at a density of 2×10 4 mL in a 24-well plate. After 12 h, add the injectable hydrogel prepared in this example and 25 μM hydrogen peroxide respectively and co-incubate for 12 h. Then observe the growth and differentiation of PC12 cells under a microscope.

[0063] The results are as Figure 9 shown. The nerve cells co-incubated with the injectable hydrogel showed obvious neurite outgrowth, indicating that the injectable hydrogel prepared in this example can effectively promote the growth and differentiation of nerve cells.

[0064] Example 2

[0065] This example relates to the preparation of an injectable hydrogel for spinal cord injury, which specifically includes the following steps:

[0066] (1) Preparation of silk fibroin: Boil raw silk in a 0.3% w / v sodium carbonate solution for 45 min, thoroughly wash it with deionized water to remove sericin, and then place it to dry at room temperature; dissolve the obtained solid after drying in a 9.3 mol / L lithium bromide solution and dissolve it at 60 °C for 4 h. After filtration, dialysis, and centrifugation, a regenerated silk fibroin solution with a mass percentage concentration of 8% is obtained;

[0067] (2) Preparation of magnesium manganese hydrotalcite: Magnesium nitrate and manganese nitrate solids with a molar ratio of 2:1 were dissolved in deionized water to obtain a metal salt solution, denoted as solution A; 1.6 g of sodium hydroxide was weighed and dissolved in deionized water to obtain a sodium hydroxide solution, denoted as solution B. Under normal temperature conditions, solution A and solution B were simultaneously and slowly dropped into a three-necked flask, and stirred continuously during this process while controlling the reaction pH to 9. After solution A was completely dropped, the reaction continued for 30 min at normal temperature and then transferred to a reaction kettle for reaction at 70 °C for 20 h. After the reaction was complete, it was centrifuged in a centrifuge at 8000 rpm, then washed three times with deionized water and once with absolute ethanol, and finally dispersed in deionized water to obtain magnesium manganese hydrotalcite with a mass percentage concentration of 1%;

[0068] (3) Preparation of injectable hydrogel: An 8% mass percentage concentration of silk fibroin solution and 0.3% magnesium manganese hydrotalcite with the same volume were uniformly mixed and allowed to stand for a period of time to obtain an injectable hydrogel.

[0069] Same as Example 1, the rheology, ability to scavenge hydrogen peroxide and generate oxygen, and the effect on the growth and differentiation of nerve cells under hydrogen peroxide conditions of the injectable hydrogel prepared in this example were studied. The research results showed that: the injectable hydrogel prepared in this example has good shear thinning performance, stability, and self-healing performance, and can effectively scavenge hydrogen peroxide and generate oxygen, and in the experiment of co-incubating with nerve cells under hydrogen peroxide conditions, it showed a significant ability to promote the growth and differentiation of nerve cells.

[0070] Example 3

[0071] This example relates to the preparation of an injectable hydrogel for spinal cord injury, which specifically includes the following steps:

[0072] (1) Preparation of silk fibroin: Raw silk was boiled in a 0.5% w / v sodium carbonate solution for 30 min, thoroughly washed with deionized water to remove sericin, and then placed in air-dry at room temperature; the solid obtained after drying was dissolved in a 9.3 mol / L lithium bromide solution and dissolved at 65 °C for 3 h. After filtration, dialysis, and centrifugation, a 8% mass percentage concentration of regenerated silk fibroin solution was obtained;

[0073] (2) Preparation of magnesium manganese hydrotalcite: Magnesium nitrate and manganese nitrate solids with a molar ratio of 3:1 were dissolved in deionized water to obtain a metal salt solution, denoted as solution A; 1.0 g of sodium hydroxide was weighed and dissolved in deionized water to obtain a sodium hydroxide solution, denoted as solution B. Under normal temperature conditions, solution A and solution B were simultaneously and slowly added dropwise into a three-necked flask, and continuously stirred during this process, while controlling the reaction pH to be 11. After solution A was completely added dropwise, the reaction continued for 30 min under normal temperature conditions and then transferred to a reaction kettle, and reacted at 60 °C for 12 h. After the reaction was complete, it was centrifuged in a centrifuge at 8000 rpm, then washed three times with deionized water and once with absolute ethanol, and finally dispersed in deionized water to obtain magnesium manganese hydrotalcite with a mass percentage concentration of 1.5%;

[0074] (3) Preparation of injectable hydrogel: A silk fibroin solution with a mass percentage concentration of 2% was uniformly mixed with magnesium manganese hydrotalcite with a mass percentage concentration of 1% in the same volume, and an injectable hydrogel was obtained after standing for a period of time.

[0075] Same as Example 1, the rheology, the ability to scavenge hydrogen peroxide and generate oxygen, and the effect on the growth and differentiation of nerve cells under hydrogen peroxide conditions of the injectable hydrogel prepared in this example were studied. The research results showed that: the injectable hydrogel prepared in this example has good shear thinning performance, stability and self-healing performance, and can effectively scavenge hydrogen peroxide and generate oxygen, and in the experiment of co-incubating with nerve cells under hydrogen peroxide conditions, it showed a significant ability to promote the growth and differentiation of nerve cells.

[0076] Example 4

[0077] This example relates to the preparation of an injectable hydrogel for spinal cord injury, which specifically includes the following steps:

[0078] (1) Preparation of silk fibroin: Raw silk was boiled in a 0.5% w / v sodium carbonate solution for 45 min, thoroughly washed with deionized water to remove sericin, and then placed in air-dry at room temperature; the solid obtained after drying was dissolved in a 9.3 mol / L lithium bromide solution and dissolved at 60 °C for 1 h. After filtration, dialysis and centrifugation, a regenerated silk fibroin solution with a mass percentage concentration of 10% was obtained;

[0079] (2) Preparation of magnesium-manganese hydrotalcite: Magnesium nitrate and manganese nitrate solids with a molar ratio of 1:1 were dissolved in deionized water to obtain a metal salt solution, denoted as solution A; 1.6 g of sodium hydroxide was weighed and dissolved in deionized water to obtain a sodium hydroxide solution, denoted as solution B. At room temperature, solution A and solution B were simultaneously and slowly added dropwise to a three-necked flask, and during this process, continuous stirring was carried out while controlling the reaction pH to 9. After solution A was completely added dropwise, the reaction continued at room temperature for 30 min and then was transferred to a reaction kettle, and the reaction was carried out at 80 °C for 6 h. After the reaction was complete, it was placed in a centrifuge and centrifuged at 8000 rpm, and then washed three times with deionized water and once with absolute ethanol, and finally dispersed in deionized water to obtain magnesium-manganese hydrotalcite with a mass percentage concentration of 2%;

[0080] (3) Preparation of injectable hydrogel: A silk fibroin solution with a mass percentage concentration of 5% and magnesium-manganese hydrotalcite with a mass percentage concentration of 0.2% were uniformly mixed in the same volume, and an injectable hydrogel was obtained after standing for a period of time.

[0081] Same as Example 1, the rheology, the ability to scavenge hydrogen peroxide and generate oxygen, and the effect on the growth and differentiation of nerve cells under hydrogen peroxide conditions of the injectable hydrogel prepared in this example were studied. The research results showed that: the injectable hydrogel prepared in this example had good shear-thinning performance, stability and self-healing performance, and could effectively scavenge hydrogen peroxide and generate oxygen, and in the experiment of co-incubating with nerve cells under hydrogen peroxide conditions, it showed a significant ability to promote the growth and differentiation of nerve cells.

[0082] Comparative Example 1

[0083] This comparative example relates to the preparation of a silk fibroin hydrogel, which specifically includes the following steps:

[0084] A silk fibroin solution with a mass percentage concentration of 0.5% obtained in Example 1 and magnesium-manganese hydrotalcite with a mass percentage concentration of 0.05% obtained in Example 1 were uniformly mixed in the same volume, and an injectable hydrogel could not be obtained after standing for the same time as in Example 1.

[0085] Comparative Example 2

[0086] This comparative example relates to the preparation of a silk fibroin hydrogel, which specifically includes the following steps:

[0087] A silk fibroin solution with a mass percentage concentration of 6% obtained in Example 1 was induced to form a pure silk fibroin hydrogel by ultrasonic treatment for 30 min.

[0088] Similar to Example 1, the rheology, hydrogen peroxide scavenging ability, oxygen generation ability, and the effects on the growth and differentiation of nerve cells under hydrogen peroxide conditions of the silk fibroin hydrogel prepared in this comparative example were studied. The research results showed that: the hydrogel obtained in this comparative example had poor shear thinning performance, stability, and self-healing ability, could not effectively scavenge hydrogen peroxide, and no oxygen generation was detected under hydrogen peroxide conditions.

[0089] Similar to Example 1, the silk fibroin hydrogel was used in the experiment of co-incubating with nerve cells under hydrogen peroxide conditions, and the results were as Figure 10 shown. The hydrogel had a poor effect on promoting the growth of nerve cells, and no obvious neurite growth was observed.

[0090] Comparative Example 3

[0091] This comparative example relates to the preparation of a silk fibroin hydrogel containing magnesium and manganese ions, which specifically includes the following steps:

[0092] The silk fibroin solution obtained in Example 1 with a mass percentage concentration of 6% was uniformly mixed with magnesium nitrate and manganese nitrate. The contents of magnesium ions and manganese ions in the mixed solution were the same as those in Example 1. After standing for the same time as in Example 1, an injectable hydrogel could not be obtained.

[0093] Comparative Example 4

[0094] This comparative example relates to the preparation of a silk fibroin hydrogel containing magnesium and manganese ions, which specifically includes the following steps:

[0095] The silk fibroin solution obtained in Example 1 with a mass percentage concentration of 6% was uniformly mixed with magnesium nitrate and manganese nitrate. The contents of magnesium ions and manganese ions in the mixed solution were the same as those in Example 1. A hydrogel was formed by ultrasonic induction for 30 min.

[0096] Similar to Example 1, the rheology, hydrogen peroxide scavenging ability, oxygen generation ability, and the effects on the growth and differentiation of nerve cells under hydrogen peroxide conditions of the silk fibroin hydrogel prepared in this comparative example were studied. The research results showed that: the hydrogel obtained in this comparative example had poor shear thinning performance, stability, and self-healing ability, could not effectively scavenge hydrogen peroxide, and no oxygen generation was detected under hydrogen peroxide conditions.

[0097] Similar to Example 1, the silk fibroin hydrogel containing magnesium and manganese ions was used in the experiment of co-incubating with nerve cells under hydrogen peroxide conditions, and the results were as Figure 11 shown. The hydrogel had a poor effect on promoting the growth of nerve cells. Not only was no obvious neurite growth observed, but the number of cells was significantly less than that of the cells cultured with the silk fibroin hydrogel.

[0098] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. An injectable hydrogel, characterized in that, The injectable hydrogel comprises the following components in mass percentage: 1% - 10% of silk fibroin, 0.1% - 10% of magnesium manganese hydrotalcite, and the balance being water; The silk fibroin is regenerated silk fibroin after degumming and lithium bromide treatment; The preparation method of the magnesium manganese hydrotalcite is as follows: in an inert gas atmosphere, a magnesium salt, a manganese salt and an alkali solution are reacted under the condition of pH 9 - 11 to obtain the magnesium manganese hydrotalcite; the feeding molar ratio of the magnesium salt to the manganese salt is 1 - 3:

1.

2. The injectable hydrogel according to claim 1, wherein The preparation method of the regenerated silk fibroin is as follows: raw silk is placed in a sodium carbonate solution for heat treatment, sericin protein is removed by washing with water, and then the dried solid is dissolved in a lithium bromide solution, and the regenerated silk fibroin solution is obtained after filtration, dialysis and centrifugation.

3. The injectable hydrogel according to claim 2, wherein The concentration of the sodium carbonate solution is 0.2% - 0.5% w / v; the heat treatment is specifically: raw silk is placed in the sodium carbonate solution and heated to boiling, and the boiling time is 30 - 60 min.

4. The injectable hydrogel according to claim 1, characterized in that, The magnesium salt is one or more of magnesium nitrate, magnesium chloride, magnesium acetate, and the manganese salt is one or more of manganese nitrate, manganese chloride, manganese acetate.

5. The injectable hydrogel according to claim 1, characterized in that, The alkali solution is sodium hydroxide solution and / or sodium carbonate solution.

6. The injectable hydrogel according to claim 1, wherein The temperature of the reaction is 60 - 80 °C, and the reaction time is 0.5 - 24 h.

7. A method for preparing the injectable hydrogel according to any one of claims 1-6, characterized in that, The silk fibroin, magnesium manganese hydrotalcite and water are uniformly mixed according to the formula ratio, and the injectable hydrogel is obtained by standing.

8. Use of the injectable hydrogel according to any one of claims 1 - 6 in the preparation of a drug for treating spinal cord injury.

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