A method for preparing a chitosan-based injectable self-healing hydrogel

Abstract

The application discloses a preparation method of a chitosan-based injectable self-healing hydrogel, and the method comprises the following steps: adding a sodium hyaluronate solution or a mixed solution of sodium hyaluronate and sodium bisulfate into a chitosan acetic acid solution drop by drop while stirring, performing suction filtration and flushing after the dropping is completed, and obtaining an initial hydrogel; pre-freezing the initial hydrogel, freeze-drying the initial hydrogel, grinding the freeze-dried hydrogel to obtain a freeze-dried gel powder; adding the freeze-dried gel powder into a solvent to prepare a sol, and obtaining a secondary hydrogel product after standing; the freeze-dried gel powder has good injectability in the secondary gelation process, and the obtained secondary hydrogel has self-healing property; the application solves the storage difficulty of general hydrogels, the prepared gel powder can be stored at room temperature for a long time, the secondary gelation process is simple to operate, the hydrogel has high biocompatibility and safety, and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a method for preparing a chitosan-based injectable self-healing hydrogel in two steps. BACKGROUND

[0002] Hydrogel is a kind of polymer material with three-dimensional network structure, which can absorb a large amount of water and biological liquid. Hydrogel is considered as an ideal soft tissue scaffold material due to its similar structure to extracellular matrix, and can be used in the fields of cartilage regeneration, wound dressing, skin scaffold, drug delivery, etc. Natural polysaccharides such as chitosan and hyaluronic acid have good biocompatibility, biodegradability and low immunogenicity, and are considered as ideal materials for delivering small molecules, bioactive ingredients and therapeutic cells. Natural polysaccharide hydrogel has wide application prospect, especially the polysaccharide hydrogel mainly composed of chitosan, hyaluronic acid, cellulose, konjac glucomannan and sodium alginate is widely used in the fields of biological engineering and pharmaceuticals. Compared with traditional hydrogel, injectable hydrogel can be injected directly into the target lesion area by using a small needle through minimally invasive technology, which greatly reduces the operation difficulty compared with implantation surgery and avoids further tissue damage and loss of function. The self-healing performance of hydrogel is that the hydrogel can partially or completely restore to the original state within a few seconds to a few hours after the removal of external force after being damaged by external force. This self-healing performance helps to prolong the service life of the material and restore and preserve its original properties.

[0003] At present, there are still some deficiencies in the injectable self-healing hydrogel, for example, toxic cross-linking agents such as glutaraldehyde are used in the preparation process, which has certain cytotoxicity and limits its application in biomedical materials; most of the injectable self-healing hydrogels need to modify the structure of the raw materials to introduce dynamic bonds or temperature-sensitive structures, which is usually a long and complex process and also affects the biocompatibility of the hydrogel. At present, most of the researches on injectable hydrogel are temperature-sensitive hydrogel, which is greatly affected by temperature. The hydrogel will become gel at high temperature and needs to be stored at low temperature, which increases the demand and cost of transportation and storage. SUMMARY

[0004] The present application solves the problems of the above-mentioned injectable self-healing hydrogel, such as the influence of toxic cross-linking agent on biocompatibility, the complexity of raw material modification process and storage difficulty, and provides a preparation method of chitosan-based injectable self-healing hydrogel, which is prepared by forming a polyelectrolyte complex between chitosan and sodium hyaluronate, and realizes self-healing through hydrogen bonding and electrostatic interaction, and has the advantages of simple preparation method, no use of toxic cross-linking agent in the preparation process of the hydrogel, no modification of raw materials, long storage time of the prepared freeze-dried gel powder at room temperature, convenient transportation, simplified preparation process before operation through the secondary gel method, simple operation, and application in filling irregular defects, cartilage repair, skin repair and the like.

[0005] The technical scheme of the present application is:

[0006] A preparation method of chitosan-based injectable self-healing hydrogel, comprising the following steps:

[0007] (1) Preparation of initial hydrogel: sodium hyaluronate is added to water to prepare a sodium hyaluronate solution, chitosan is added to an acetic acid aqueous solution to prepare a chitosan acetic acid solution, the sodium hyaluronate solution is added dropwise to the chitosan acetic acid solution using a peristaltic pump, and stirring is performed during the dropwise addition, white granular polyelectrolyte complexes are formed in the mixed solution, and the solution is subjected to suction filtration and ultrapure water washing after the dropwise addition is completed, to obtain an initial hydrogel;

[0008] (2) Preparation of freeze-dried gel powder: the initial hydrogel is pre-frozen in a refrigerator, and then is placed in a vacuum freeze-drying box for freeze-drying, to obtain freeze-dried hydrogel, and the freeze-dried hydrogel is ground to obtain freeze-dried gel powder;

[0009] (3) Preparation of secondary gel: the freeze-dried gel powder is added to a cross-linking solvent to prepare a sol, and the sol becomes a hydrogel after standing for 6-30 min, to obtain a secondary hydrogel product.

[0010] Further, the concentration of the sodium hyaluronate solution and the chitosan acetic acid aqueous solution in step (1) is 1-5 mg / mL, the mass fraction of the acetic acid aqueous solution is 0.5%-3%, and the mass ratio of chitosan to sodium hyaluronate is 1:1-2.5:1 when the sodium hyaluronate solution and the chitosan acetic acid aqueous solution are mixed.

[0011] Further, step (1) is replaced by: adding sodium hyaluronate into water to prepare a sodium hyaluronate solution, adding sodium bisulfate into the sodium hyaluronate solution to form a sodium hyaluronate solution containing sodium bisulfate, adding chitosan into an acetic acid aqueous solution to prepare a chitosan acetic acid solution, and adding the sodium hyaluronate solution containing sodium bisulfate into the chitosan acetic acid solution dropwise while stirring, and then performing suction filtration and washing with ultrapure water after the dropping is completed to obtain an initial hydrogel; wherein the mass fraction of the acetic acid aqueous solution is 0.5% to 3%, the concentration of the sodium hyaluronate solution containing sodium bisulfate and the chitosan acetic acid solution is 1 mg / mL to 5 mg / mL, and the molar ratio of the positive ion (amino group) of chitosan to the sum of the negative ions (carboxyl group and bisulfate) of sodium hyaluronate and sodium bisulfate in the chitosan acetic acid solution and the sodium hyaluronate solution containing sodium bisulfate is 1:1 to 2.5:1.

[0012] Further, in step (1), the sodium hyaluronate solution or the sodium hyaluronate solution containing sodium bisulfate is added into the chitosan acetic acid solution by using a peristaltic pump, and the dropping rate is 1 to 10 r / min.

[0013] Further, in step (2), the pre-freezing temperature is -40 to -60°C, and the time is 6 to 16 h; and the freeze-drying time is 12 to 36 h.

[0014] Further, in step (3), the cross-linking solvent is a calcium chloride solution or a mixed solution of calcium chloride and glycine, the concentration of the calcium chloride solution is 0.05 M to 0.5 M, the concentration of the mixed solution of calcium chloride and glycine is 0.05 M to 0.5 M, the molar ratio of calcium chloride to glycine is 1:1 to 1:3, and the mass fraction of the freeze-dried gel powder in the cross-linking solvent is 5 wt% to 10 wt%.

[0015] Advantages of the present application:

[0016] (1) The initial hydrogel of the present application is formed only by the interaction between positive and negative ions, does not need a cross-linking agent, and mainly uses chitosan and sodium hyaluronate or a mixture of sodium bisulfate and sodium hyaluronate, so that the modification of chitosan and sodium hyaluronate is not needed, the obtained hydrogel has good biocompatibility, and the initial hydrogel has certain viscosity and antibacterial property.

[0017] (2) The freeze-dried gel powder prepared by the present application can be stored for a long time at room temperature, is convenient to transport, and reduces the transportation cost, and the long-time storage at room temperature has little effect on the performance of the reformed hydrogel.

[0018] (3) The injectability and self-healing property of the hydrogel are realized by the twice gelation method of the present application, the operation for preparing the secondary gel is simple, the gelation time is moderate, and the preparation process before the operation can be simplified, which is convenient for the operation.

[0019] (3) The preparation process of this invention is simple, green and environmentally friendly, safe and efficient, and has a wide range of application prospects. It can be used for cartilage repair, skin defect repair, wound dressing, filling irregular defects, etc. Attached Figure Description

[0020] Figure 1 Image of the initial hydrogel freeze-dried in Example 3;

[0021] Figure 2 The injection performance diagram is shown in Example 3;

[0022] Figure 3 This is a secondary hydrogel diagram of Example 3;

[0023] Figure 4 The self-healing performance diagrams for Example 3 are shown below: (a) secondary hydrogel diagram, (b) secondary hydrogel cut-out diagram, (c) 0-minute self-healing diagram, (d) 16-hour self-healing diagram (positioned in the vertical direction).

[0024] Figure 5 The images show the FT-IR spectra of chitosan, sodium hyaluronate, the initial hydrogel, and the secondary hydrogel in Example 3. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0026] Example 1

[0027] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0028] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution was slowly added to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. At this time, the mass ratio of chitosan to sodium hyaluronate was 1:1, and the molar ratio of the positive ion (amino) of chitosan to the negative ion (carboxyl) of sodium hyaluronate solution was 2.5:1. The solution was mixed while being added dropwise and stirred. A white granular polyelectrolyte complex was formed. After the addition was completed, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0029] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0030] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.05M calcium chloride solution to obtain a sol. The mass fraction of the lyophilized gel powder in the calcium chloride solution was 8wt%. After standing for 30 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0031] Example 2

[0032] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0033] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution was slowly added to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. At this time, the mass ratio of chitosan to sodium hyaluronate was 1:1, and the molar ratio of the positive ion (amino) of chitosan to the negative ion (carboxyl) of sodium hyaluronate solution was 2.5:1. The solution was mixed while being added dropwise and stirred. A white granular polyelectrolyte complex was formed. After the addition was completed, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0034] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0035] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.1M calcium chloride solution to obtain a sol. The lyophilized gel powder accounted for 8wt% of the calcium chloride solution. After standing for 17 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0036] Example 3

[0037] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0038] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution was slowly added to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. At this time, the mass ratio of chitosan to sodium hyaluronate was 1:1, and the molar ratio of the positive ion (amino) of chitosan to the negative ion (carboxyl) of sodium hyaluronate solution was 2.5:1. The solution was mixed while being added dropwise and stirred. A white granular polyelectrolyte complex was formed. After the addition was completed, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0039] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0040] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.13M calcium chloride solution to obtain a sol. The lyophilized gel powder accounted for 8 wt% of the calcium chloride solution. After standing for 8 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0041] Example 4

[0042] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0043] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution was slowly added to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. At this time, the mass ratio of chitosan to sodium hyaluronate was 1:1, and the molar ratio of the positive ion (amino) of chitosan to the negative ion (carboxyl) of sodium hyaluronate solution was 2.5:1. The solution was mixed while being added dropwise and stirred. A white granular polyelectrolyte complex was formed. After the addition was completed, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0044] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0045] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.15M calcium chloride solution to obtain a sol. The lyophilized gel powder accounted for 8 wt% of the calcium chloride solution. After standing for 7 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0046] Example 5

[0047] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0048] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution was slowly added to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. At this time, the mass ratio of chitosan to sodium hyaluronate was 1:1, and the molar ratio of the positive ion (amino) of chitosan to the negative ion (carboxyl) of sodium hyaluronate solution was 2.5:1. The solution was mixed while being added dropwise and stirred. A white granular polyelectrolyte complex was formed. After the addition was completed, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0049] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0050] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.13M calcium chloride solution to obtain a sol. The mass fraction of the lyophilized gel powder in the calcium chloride solution was 10wt%. After standing for 6 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0051] Example 6

[0052] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0053] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 1% to prepare a chitosan acetic acid solution with a concentration of 2.5 mg / mL. An equal volume of sodium hyaluronate solution was slowly added to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. At this time, the mass ratio of chitosan to sodium hyaluronate was 2.5:1, and the molar ratio of the positive ion (amino) of chitosan to the negative ion (carboxyl) of sodium hyaluronate solution was 6.2:1. The solution was mixed while being added dropwise and stirred. A white granular polyelectrolyte complex was formed. After the addition was completed, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0054] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 16h, and then freeze-dried in a vacuum freeze-drying oven for 36h to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0055] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in a 0.15M mixed solution of calcium chloride and glycine to obtain a sol, wherein the molar ratio of calcium chloride to glycine was 1:1, and the mass fraction of the lyophilized gel powder in the mixed solution of calcium chloride and glycine was 10wt%. After standing for 7 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0056] Example 7

[0057] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0058] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution was slowly added to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. At this time, the mass ratio of chitosan to sodium hyaluronate was 1:1, and the molar ratio of the positive ion (amino) of chitosan to the negative ion (carboxyl) of sodium hyaluronate solution was 2.5:1. The solution was mixed while being added dropwise and stirred. A white granular polyelectrolyte complex was formed. After the addition was completed, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0059] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 16h, and then freeze-dried in a vacuum freeze-drying oven for 24h to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0060] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.13M calcium chloride solution to obtain a sol. The lyophilized gel powder accounted for 5 wt% of the calcium chloride solution. After standing for 24 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0061] Example 8

[0062] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0063] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Sodium bisulfate was added to the sodium hyaluronate solution to obtain a sodium hyaluronate solution containing sodium bisulfate. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution containing sodium bisulfate was slowly added dropwise to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. The molar ratio of the sum of the positive ions (amino groups) in the chitosan acetic acid solution and the negative ions (carboxyl groups of sodium hyaluronate and hydrogen sulfate groups of sodium bisulfate) in the sodium hyaluronate solution containing sodium bisulfate was 1:1. While adding the solution dropwise and stirring, a white granular polyelectrolyte complex was formed. After the solution was added dropwise, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0064] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0065] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.13M calcium chloride solution to obtain a sol. The lyophilized gel powder accounted for 8 wt% of the calcium chloride solution. After standing for 11 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0066] Example 9

[0067] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0068] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 1 mg / mL. Sodium bisulfate was added to the sodium hyaluronate solution to obtain a sodium hyaluronate solution containing sodium bisulfate. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 1 mg / mL. An equal volume of sodium hyaluronate solution containing sodium bisulfate was slowly added dropwise to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. The molar ratio of the sum of the positive ions (amino groups) in the chitosan acetic acid solution and the negative ions (carboxyl groups of sodium hyaluronate and hydrogen sulfate groups of sodium bisulfate) in the sodium hyaluronate solution containing sodium bisulfate was 1.6:1. While adding the solution dropwise and stirring, a white granular polyelectrolyte complex was formed. After the solution was added dropwise, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0069] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0070] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in 0.13M calcium chloride solution to obtain a sol. The lyophilized gel powder accounted for 8 wt% of the calcium chloride solution. After standing for 12 minutes, the sol turned into a hydrogel, thus obtaining a secondary gel.

[0071] Example 10

[0072] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0073] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 2 mg / mL. Sodium bisulfate was added to the sodium hyaluronate solution to obtain a sodium hyaluronate solution containing sodium bisulfate. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 0.5% to prepare a chitosan acetic acid solution with a concentration of 2 mg / mL. An equal volume of sodium hyaluronate solution containing sodium bisulfate was slowly added dropwise to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. The molar ratio of the sum of the positive ions (amino groups) in the chitosan acetic acid solution and the negative ions (carboxyl groups of sodium hyaluronate and hydrogen sulfate groups of sodium bisulfate) in the sodium hyaluronate solution containing sodium bisulfate was 2:1. While adding the solution dropwise and stirring, a white granular polyelectrolyte complex was formed. After the solution was added dropwise, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0074] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 6 hours, and then freeze-dried in a vacuum freeze-drying oven for 24 hours to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0075] (3) Preparation of secondary gel: The freeze-dried gel powder was dissolved in 0.5M calcium chloride sol. The freeze-dried gel powder accounted for 8wt% of the calcium chloride solution. After standing for 6 minutes, the sol turned into a hydrogel, thus obtaining a secondary gel.

[0076] Example 11

[0077] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0078] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 2.5 mg / mL. Sodium bisulfate was added to the sodium hyaluronate solution to obtain a sodium hyaluronate solution containing sodium bisulfate. Chitosan was added to an acetic acid aqueous solution with a mass fraction of 1.6% to prepare a chitosan acetate solution with a concentration of 2.5 mg / mL. An equal volume of sodium hyaluronate solution containing sodium bisulfate was slowly added dropwise to the chitosan acetate solution at a rate of 1 r / min using a peristaltic pump. The molar ratio of the sum of the positive ions (amino groups) in the chitosan acetate solution to the negative ions (carboxyl groups of sodium hyaluronate and bisulfate groups of sodium bisulfate) in the sodium hyaluronate solution containing sodium bisulfate was 1:1. While adding the solution dropwise, the mixture was stirred to form a white granular polyelectrolyte complex. After the solution was added dropwise, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0079] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -40℃ freezer for 12h, and then freeze-dried in a vacuum freeze-drying oven for 12h to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0080] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in a 0.05M calcium chloride and glycine mixed solution to obtain a sol. The mass fraction of the lyophilized gel powder in the calcium chloride and glycine mixed solution was 5wt%, and the molar ratio of calcium chloride to glycine was 1:2. After standing for 25 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0081] Example 12

[0082] A method for preparing a chitosan-based injectable self-healing hydrogel includes the following steps:

[0083] (1) Preparation of initial hydrogel: Sodium hyaluronate was added to water to prepare a sodium hyaluronate solution with a concentration of 5 mg / mL. Sodium bisulfate was added to the sodium hyaluronate solution to obtain a sodium hyaluronate solution containing sodium bisulfate. Chitosan was added to a 3% acetic acid aqueous solution to prepare a chitosan acetic acid solution with a concentration of 5 mg / mL. An equal volume of sodium hyaluronate solution containing sodium bisulfate was slowly added dropwise to the chitosan acetic acid solution at a rate of 5 r / min using a peristaltic pump. The molar ratio of the sum of the positive ions (amino groups) in the chitosan acetic acid solution and the negative ions (carboxyl groups of sodium hyaluronate and hydrogen sulfate groups of sodium bisulfate) in the sodium hyaluronate solution containing sodium bisulfate was 1:1. While adding the solution dropwise, the mixture was stirred to form a polyelectrolyte complex. After the solution was added dropwise, the complex solution was filtered and rinsed with ultrapure water to obtain the initial hydrogel.

[0084] (2) Preparation of freeze-dried gel powder: The initial hydrogel was pre-frozen in a -60℃ freezer for 12h, and then freeze-dried in a vacuum freeze-drying oven for 12h to obtain freeze-dried hydrogel. The freeze-dried hydrogel was then ground to obtain freeze-dried gel powder.

[0085] (3) Preparation of secondary gel: The lyophilized gel powder was dissolved in a 0.5M calcium chloride and glycine mixed solution to obtain a sol. The mass fraction of the lyophilized gel powder in the calcium chloride and glycine mixed solution was 5wt%, and the molar ratio of calcium chloride to glycine was 1:3. After standing for 18 minutes, the sol will turn into a hydrogel, thus obtaining a secondary gel.

[0086] As can be seen from Examples 1, 2, 3, and 4, when the content of lyophilized gel powder is the same, increasing the concentration of calcium chloride solution can increase the secondary gelation rate. When the calcium chloride concentration increases from 0.05M to 0.13M, the gelation time decreases significantly, from 30 minutes to 8 minutes. When the calcium chloride concentration increases from 0.13M to 0.15M, the gelation time only decreases by 1 minute. Excessive calcium chloride solution concentration can negatively affect cell number and cell integrity, thereby affecting cell proliferation and differentiation. It can also increase extracellular osmotic pressure, leading to cell shrinkage. Considering these factors, when the gelation time is not significantly different, the concentration of calcium chloride should be minimized.

[0087] In Examples 7, 3, and 5, the concentration of calcium chloride solution was 0.13 M, the content of lyophilized gel powder was 5%, 8%, and 10%, respectively, and the gelation time was 24 min, 8 min, and 6 min, respectively. As the content of gel powder increased, the gelation time increased significantly. However, as the content of gel powder increased, the viscosity in the sol state also increased. The increase in viscosity would affect the injection performance and the structural uniformity of the formed hydrogel.

[0088] Different amounts of sodium bisulfate were added in Examples 3, 9, and 8. The compressive strengths at 60% deformation were 0.0178 MPa, 0.0276 MPa, and 0.0298 MPa, respectively. As the amount of sodium bisulfate added increased, the anion content gradually increased, the molar ratio of positive to negative ions decreased, and the compressive strength gradually increased. The reason for this result may be that the bisulfate ions can react with the amino groups of chitosan to form new cross-linking sites, thereby improving the mechanical strength of the hydrogel.

[0089] Figure 1 This is a morphology image of the initial hydrogel after freeze-drying in Example 3. It can be seen that small particles are aggregated together. The reason for the aggregation is that the initial hydrogel itself has a certain viscosity, and the filtration process causes it to stick together.

[0090] Figure 2 The figure shows the injection performance of Example 3. As can be seen from the figure, the hydrogel has good injection performance during the secondary gelation process. It can be injected with a syringe to form relatively complex shapes. After injection, the hydrogel can be formed quickly. The difference in the thickness of the strokes in the figure is due to manual injection. The injection rate cannot be kept completely consistent, resulting in uneven thickness of the strokes.

[0091] Figure 3 The image shows the morphology of the secondary hydrogel prepared in Example 3. The secondary hydrogel has no obvious particles inside and forms a uniform gel, which has good flowability during the filling process into the mold.

[0092] Figure 4 The diagram shows the self-healing performance of Example 3. For easy comparison, 0.05g of methyl orange powder was added to calcium chloride solvent in Example 3, while keeping other conditions unchanged, to obtain an orange hydrogel. Two hydrogels of different colors, the one prepared according to the method of Example 3 and the one orange hydrogel, were cut in half. Half of each half was then taken and joined together. When picked up with tweezers, it can be seen that the hydrogels did not separate at the joint. Figure 4 As shown in (a) secondary hydrogel image, (b) secondary hydrogel cut-out image, (c) 0-minute self-healing image, and (d) 16-hour self-healing image, after 16 hours, the white half of the hydrogel can be seen to turn orange, and the seam at the joint becomes blurred.

[0093] Figure 5 The figures show the FT-IR spectra of chitosan CS, sodium hyaluronate SH, the lyophilized gel powder CS / SH from step (2), and the secondary hydrogel CS / SH-CaCl2 from Example 3. As can be seen from the figures, the characteristic peak of CS is at 3415 cm⁻¹. -1 The peak at 1617 cm⁻¹ represents the stretching vibration peaks of NH and OH. -1The peak at 1383 cm⁻¹ is an overlap of the C=O stretching vibration of amide I and the NH bending vibration of amide II. -1 The peak at 1156 cm⁻¹ is the symmetrical vibration peak of -CH₃. -1 and 1085cm -1 The peak at 3423 cm⁻¹ represents the stretching vibration of COC; in the FT-IR spectrum of SH, a peak at 3423 cm⁻¹ is observed. -1 The absorption band at 1636 cm⁻¹ is the stretching vibration peak of -OH. -1 The peak at 1153 cm⁻¹ is the C=O stretching vibration peak of the carboxyl group. -1 and 1077cm -1 The peak at 1617 cm⁻¹ corresponds to the stretching vibration of COC; however, in the FT-IR spectrum of CS / SH, the absorption peak corresponding to C=O in CS and SH (1617 cm⁻¹) is actually the peak at 1617 cm⁻¹. -1 and 1636cm -1 Blue shift occurred (1644cm) -1 The presence of this group indicates greater stability, which can be explained by the electrostatic interaction between the carboxyl group of SH and the amino group of CS, forming a stable polyelectrolyte complex. In the FT-IR spectrum of CS / SH-CaCl2, the 1644 cm⁻¹ of CS / SH... -1 The peak redshifted to 1640cm. -1 At this point, grinding may have disrupted some of the bond between CS and SH, and the secondary gelation formed new hydrogen bonds.

Claims

1. A process for the preparation of chitosan-based injectable self-healing hydrogel, characterized by, The method comprises the following steps: (1) preparing a sodium hyaluronate solution by adding sodium hyaluronate into water, preparing a chitosan acetic acid solution by adding chitosan into an acetic acid aqueous solution, adding the sodium hyaluronate solution into the chitosan acetic acid solution dropwise while stirring, and then performing suction filtration and ultrapure water washing after the dropping is completed to obtain an initial hydrogel; (2) pre-freezing the initial hydrogel, freeze-drying the initial hydrogel to obtain a freeze-dried hydrogel, and grinding the freeze-dried hydrogel to obtain a freeze-dried gel powder; (3) adding the freeze-dried gel powder into a crosslinking solvent to prepare a sol, and then standing for 6-30 min to obtain a secondary hydrogel product. The crosslinking solvent is a calcium chloride solution or a mixed solution of calcium chloride and glycine, the concentration of the calcium chloride solution is 0.05-0.5 mol / L, and the concentration of the mixed solution of calcium chloride and glycine is 0.05-0.5 mol / L, wherein the molar ratio of calcium chloride to glycine is 1:1-1:

3.

2. The process for the preparation of chitosan based injectable self-healing hydrogel as claimed in claim 1, wherein, In step (1), the concentration of the sodium hyaluronate solution and the chitosan acetic acid aqueous solution is 1 mg / mL-5 mg / mL, the mass fraction of the acetic acid solution is 0.5%-3%, and the mass ratio of chitosan to sodium hyaluronate is 1:1-2.5:

1.

3. The process for the preparation of chitosan based injectable self-healing hydrogel as claimed in claim 1, wherein, In step (1), the sodium hyaluronate is added into water to prepare a sodium hyaluronate solution, sodium bisulfate is further added into the sodium hyaluronate solution to form a sodium hyaluronate solution containing sodium bisulfate, chitosan is added into an acetic acid aqueous solution to prepare a chitosan acetic acid solution, the sodium hyaluronate solution containing sodium bisulfate is slowly added into the chitosan acetic acid solution while stirring, suction filtration and ultrapure water washing are performed after the dropping is completed to obtain an initial hydrogel, wherein the concentration of the sodium hyaluronate solution and the chitosan acetic acid solution is 1 mg / mL-5 mg / mL, the mass fraction of the acetic acid aqueous solution is 0.5%-3%, and the molar ratio of the amino groups in the chitosan acetic acid solution to the sum of the carboxyl groups and the sodium bisulfate groups in the sodium hyaluronate and sodium bisulfate is 1:1-2.5:

1.

4. The method for preparing the injectable self-healing hydrogel based on chitosan according to claim 2 or 3, characterized in that, In step (1), the sodium hyaluronate solution or the sodium hyaluronate solution containing sodium bisulfate is added into the chitosan acetic acid solution by using a peristaltic pump, and the dropping rate is 1-10 r / min.

5. The process for the preparation of chitosan based injectable self-healing hydrogel as claimed in claim 1 wherein, In step (2), the pre-freezing temperature is-40--60℃, and the pre-freezing time is 6-16 h; the freeze-drying time is 12-36 h.

6. The process for the preparation of chitosan based injectable self-healing hydrogel as claimed in claim 1, wherein, In step (3), the mass fraction of the freeze-dried gel powder in the crosslinking solvent is 5%-10%.

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