Three-layer gradient self-healing hydrogel and preparation method and application thereof

Abstract

The application belongs to the technical field of polymer materials, and particularly relates to a three-layer gradient self-healing hydrogel as well as a preparation method and application thereof. The application provides a composition for preparing the three-layer gradient self-healing hydrogel, which comprises composition A, composition B and / or composition C; the composition A and the composition B are both composed of modified chondroitin sulfate, carboxymethyl chitosan and tannic acid, and the difference lies in that the tannic acid concentration in the composition A is 0.5-1.5% w / v, and the tannic acid concentration in the composition B is 2.5-3.5% w / v; the composition C comprises modified chondroitin sulfate, carboxymethyl chitosan, tannic acid and nano-hydroxyapatite, and the tannic acid concentration is 2.5-3.5% w / v. The three-layer gradient self-healing hydrogel has good self-healing performance, mechanical property, tissue adhesion performance and antioxidant property, and can be applied to integrated repair of joint cartilage-bone tissue.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of high polymer materials, and particularly relates to a three-layer gradient self-healing hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Articular cartilage is a layer of connective tissue covering the articular surface. The normal cartilage surface is smooth and elastic, which can reduce the vibration and impact during movement. When the articular cartilage is damaged or degenerated, it will cause pain and movement limitation. Since the cartilage tissue has highly differentiated blood vessels, once the cartilage tissue is damaged, its self-repairing ability is very limited. And the damage of articular cartilage is often accompanied by the damage of the integrated subchondral bone. Existing studies have shown that a single treatment strategy for articular cartilage often has limited effect, while a comprehensive treatment combining cartilage and subchondral bone can achieve better effect. Therefore, it is of great significance to combine articular cartilage and bone tissue engineering to construct an integrated cartilage-bone tissue repair material for the integrated repair of cartilage-bone.

[0003] Hydrogel is the first choice for cartilage repair material due to its similar structure to natural cartilage extracellular matrix (ECM), good biocompatibility and mechanical properties. Among them, self-healing hydrogel has become a research hotspot in the field of cartilage repair due to its good self-repairing function. Self-healing hydrogel is a new type of hydrogel which can automatically restore its integrity and functionality without any external force after being damaged. The articular cartilage tissue has a three-layer gradient structure from top to bottom, so it is expected to realize cartilage repair by constructing a layered gradient self-healing hydrogel material with similar structure and natural tissue from the perspective of tissue engineering.

[0004] In the prior art, CN103721293A patent reports a light crosslinking multi-layer gradient hydrogel with controllable release of active factors and a preparation method. The gel structure is three layers, and the main components of each layer are composed of modified gelatin and modified carboxymethyl chitosan. The preparation method comprises: modifying the double bonds of gelatin and carboxymethyl chitosan with glycidyl methacrylate; modifying the double bond of the modified gelatin with heparin; preparing three reaction liquids of three kinds of modified gelatin and modified carboxymethyl chitosan and crosslinking agent PEGDMA and photoinitiator I2959, respectively, and continuously injecting the three reaction liquids into the mold in sequence, and then light crosslinking to prepare a multi-layer gradient hydrogel with controllable release of active factors for gradient distribution of articular cartilage tissue repair.

[0005] At present, there is a lack of effective method for repairing damaged articular cartilage in clinic. Therefore, it is necessary to prepare a layered gradient self-healing hydrogel material with similar structure and natural tissue for the integrated repair of cartilage-bone. SUMMARY

[0006] In view of the above, the present application provides a three-layer gradient self-healing hydrogel for articular cartilage-bone integration repair, a composition, a preparation method and an application thereof. The present application uses carboxymethyl chitosan which is soluble in water and contains a large number of amino groups, and chondroitin sulfate which is the main component of cartilage tissue as raw materials, prepares chondroitin sulfate / chitosan self-healing hydrogel through dynamic Schiff base reaction, and introduces TA to enhance the mechanical properties of the self-healing hydrogel, endow it with adhesion and antioxidant properties. At the same time, based on the layered gradient structure of natural cartilage-bone, a three-layer gradient self-healing hydrogel is prepared by optimizing the preparation conditions and adding hydroxyapatite which can promote bone formation. The present application provides a basis for preparing a new type of gradient self-healing hydrogel for repairing cartilage-bone tissue.

[0007] One of the purposes of the present application is to provide a composition for preparing a three-layer gradient self-healing hydrogel.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0009] The composition for preparing a three-layer gradient self-healing hydrogel comprises composition A, composition B and / or composition C; the composition A comprises modified chondroitin sulfate, carboxymethyl chitosan and tannic acid, the concentration of tannic acid in the composition A is 0.5% w / v-1.5% w / v, and the volume ratio of modified chondroitin sulfate to carboxymethyl chitosan is 1:5-1:7; the composition B comprises modified chondroitin sulfate, carboxymethyl chitosan and tannic acid, the concentration of tannic acid in the composition B is 2.5% w / v-3.5% w / v, and the volume ratio of modified chondroitin sulfate to carboxymethyl chitosan is 1:5-1:7; the composition C comprises modified chondroitin sulfate, carboxymethyl chitosan, tannic acid and nano-hydroxyapatite, the concentration of tannic acid in the composition C is 2.5% w / v-3.5% w / v, and the volume ratio of modified chondroitin sulfate to carboxymethyl chitosan is 1:5-1:7.

[0010] Further, in the composition A, the composition B and the composition C, the concentration of modified chondroitin sulfate is 18% w / v-22% w / v; in the composition A, the composition B and the composition C, the concentration of carboxymethyl chitosan is 3% w / v-5% w / v; and in the composition C, the concentration of nano-hydroxyapatite is 5% w / v-7% w / v.

[0011] Further, the modified chondroitin sulfate is aldehyde-modified chondroitin sulfate, wherein the content of aldehyde groups is 1.0 mmol / g-1.5 mmol / g.

[0012] Further, the tannic acid concentration in the composition A is 1% w / v, the volume ratio of modified chondroitin sulfate and carboxymethyl chitosan is 1:6; the tannic acid concentration in the composition B is 3% w / v, the volume ratio of modified chondroitin sulfate and carboxymethyl chitosan is 1:6; the tannic acid concentration in the composition C is 3%, the volume ratio of modified chondroitin sulfate and carboxymethyl chitosan is 1:6; the modified chondroitin sulfate is aldehyde-modified chondroitin sulfate with a concentration of 20% w / v, wherein the aldehyde group content is 1.2 mmol / g; the concentration of carboxymethyl chitosan is 4% w / v; and the concentration of nano-hydroxyapatite is 6% w / v.

[0013] The second object of the present application is to provide a preparation method of the aforementioned three-layer gradient self-healing hydrogel.

[0014] To achieve the above object, the present application adopts the following technical solutions:

[0015] The preparation method of the aforementioned three-layer gradient self-healing hydrogel comprises the following steps:

[0016] (1) Preparation of OCS1-CMCS-TA1 hydrogel: mixing modified chondroitin sulfate solution and carboxymethyl chitosan solution to prepare chondroitin sulfate / chitosan self-healing hydrogel by dynamic Schiff base reaction; soaking the chondroitin sulfate / chitosan self-healing hydrogel in a tannic acid solution with a concentration of 0.5% w / v to 1.5% w / v to obtain the OCS1-CMCS-TA1 hydrogel;

[0017] (2) Preparation of OCS1-CMCS-TA3 hydrogel: mixing modified chondroitin sulfate solution and carboxymethyl chitosan solution to prepare chondroitin sulfate / chitosan self-healing hydrogel by dynamic Schiff base reaction; soaking the chondroitin sulfate / chitosan self-healing hydrogel in a tannic acid solution with a concentration of 2.5% w / v to 3.5% w / v to obtain the OCS1-CMCS-TA3 hydrogel;

[0018] (3) Preparation of OCS1-CMCS / HAp6-TA3 hydrogel: adding nano-hydroxyapatite to carboxymethyl chitosan solution to obtain CMCS / HAp solution; mixing the CMCS / HAp solution with modified chondroitin sulfate solution to obtain OCS1-CMCS / HAp6 hydrogel; soaking the OCS1-CMCS / HAp6 hydrogel in a tannic acid solution with a concentration of 2.5% w / v to 3.5% w / v to obtain the OCS1-CMCS / HAp6-TA3 hydrogel;

[0019] (4) Preparation of the three-layer gradient self-healing hydrogel: stack the OCS1-CMCS-TA1 hydrogel obtained in step (1), the OCS1-CMCS-TA3 hydrogel obtained in step (2), and the OCS1-CMCS / HAp6-TA3 hydrogel obtained in step (3) from top to bottom to obtain the three-layer gradient self-healing hydrogel.

[0020] Further, in step (1), the concentration of the tannic acid solution is 1% w / v; in step (2) and step (3), the concentration of the tannic acid solution is 3% w / v.

[0021] Further, in step (1) to step (3), the concentration of the modified chondroitin sulfate solution is 20% w / v.

[0022] Further, in step (1) to step (3), the concentration of the carboxymethyl chitosan solution is 4% w / v.

[0023] Further, in step (1) to step (3), the volume ratio of the modified chondroitin sulfate solution to the carboxymethyl chitosan solution is 1:6.

[0024] Further, in step (3), the concentration of the nano-hydroxyapatite is 6% w / v.

[0025] Further, the preparation solvents of the modified chondroitin sulfate solution, the carboxymethyl chitosan solution, and the tannic acid solution are all ultrapure water.

[0026] Further, in step (1) to step (3), the soaking time of the hydrogel in the tannic acid solution is 30 min.

[0027] Further, after soaking, the surface of the hydrogel is rinsed with ultrapure water to remove excess tannic acid solution.

[0028] Further, the aldehyde group content of the modified chondroitin sulfate is 1.0 mmol / g to 1.5 mmol / g; the modified chondroitin sulfate is prepared by the following method: after dissolving chondroitin sulfate sodium salt, sodium periodate is added for oxidation reaction; after the reaction is completed, an ethanol solution is added and stirred, and the obtained product is precipitated, dialyzed, and freeze-dried to obtain aldehyde-modified chondroitin sulfate.

[0029] As a preference, the oxidation reaction time is 6 h.

[0030] As a preference, the precipitation is carried out at 4°C for 4 h.

[0031] As a preference, the dialysis time is 3 days.

[0032] As the most preferred, the modified chondroitin sulfate is prepared by the following method:

[0033] After dissolving the chondroitin sulfate sodium salt, sodium periodate is added to react for 6 hours; then an ethanol solution is added to stir in the dark for 15 minutes, and the precipitate is collected after precipitation at 4 DEG C for 4 hours; the precipitate is dialyzed in a dialysis bag with a MWCO of 300 for 3 days, and is freeze-dried to obtain the aldehyde-modified chondroitin sulfate; the aldehyde group content in the aldehyde-modified chondroitin sulfate is 1.2 mmol / g.

[0034] Further, in step (4), the three-layer gradient self-healing hydrogel is prepared by using the self-healing property and adhesion between OCS1-CMCS-TA1 hydrogel, OCS1-CMCS-TA3 hydrogel and OCS1-CMCS / HAp6-TA3 hydrogel.

[0035] A third object of the present application is to provide a three-layer gradient self-healing hydrogel prepared by the above preparation method.

[0036] To achieve the above object, the present application adopts the following technical solution:

[0037] The three-layer gradient self-healing hydrogel is prepared by the above preparation method.

[0038] Further, the OCS1-CMCS-TA1 hydrogel, OCS1-CMCS-TA3 hydrogel and OCS1-CMCS / HAp6-TA3 hydrogel in the three-layer gradient self-healing hydrogel are all double-network structures, the aldehyde group of the modified chondroitin sulfate reacts with the amino group of the carboxymethyl chitosan to form a Schiff base, which constitutes the first cross-linking network structure of the hydrogel; the hydrogen bond of tannic acid itself and the hydrogen bond formed between tannic acid and the modified chondroitin sulfate and the carboxymethyl chitosan constitute the second cross-linking network structure of the hydrogel.

[0039] A fourth object of the present application is to provide an application of the above composition and / or the above three-layer gradient self-healing hydrogel in the preparation of an integrated repair product for articular cartilage-bone tissue.

[0040] The present application has the following beneficial effects:

[0041] 1. The present application uses chondroitin sulfate and carboxymethyl chitosan as raw materials, and prepares chondroitin sulfate / chitosan self-healing hydrogel through dynamic Schiff base reaction, and introduces tannic acid into the system to enhance the mechanical properties of the self-healing hydrogel, and endows it with adhesion and antioxidant properties. At the same time, from the perspective of bionics, by changing the preparation conditions and adding hydroxyapatite which can promote bone formation, a three-layer gradient self-healing hydrogel material similar to the structure of natural cartilage tissue is prepared, which is of great significance for the integrated repair of articular cartilage-bone tissue.

[0042] 2. The application provides a preparation method of a three-layer gradient self-healing hydrogel, and explores the influence of the concentration, ratio and different tannic acid concentrations of each raw material on the mechanical properties, adhesion, antioxidant properties and other properties of the self-healing hydrogel, and finally obtains the optimal preparation conditions: the first layer is OCS1-CMCS-TA1 hydrogel, OCS1:CMCS=1:6, the TA concentration is 1% w / v, the OCS1 concentration is 20% w / v, and the CMCS concentration is 4% w / v; the second layer is OCS1-CMCS-TA3 hydrogel, OCS1:CMCS=1:6, the TA concentration is 3% w / v, the OCS1 concentration is 20% w / v, and the CMCS concentration is 4% w / v; and the third layer is OCS1-CMCS / HAp-TA3 hydrogel, OCS1:CMCS=1:6, the TA concentration is 3% w / v, the HAp concentration is 6% w / v, the OCS1 concentration is 20% w / v, and the CMCS concentration is 4% w / v. The preparation method provided by the application adopts Schiff base reaction, has simple and mild conditions, and can prepare a hydrogel material with self-repairing function without external stimulation. The application provides a basis for preparing a new type of gradient self-healing hydrogel that can self-repair and repair cartilage-bone tissue.

[0043] 3. The three-layer gradient self-healing hydrogel of the application has super strong self-healing performance, two pieces of hydrogel are stacked, the hydrogel gap disappears at 1 h, the hydrogel is completely self-healed at 3 h, and the self-healing gap cannot be seen at all and the color is uniform at 5 h.

[0044] 4. The three-layer gradient self-healing hydrogel of the application has excellent tissue adhesion, and the elastic modulus of the first layer to the third layer of the hydrogel is 282 KPa, 328 KPa, 411 KPa, respectively, and the adhesion strength is 5.7 KPa, 9.3 KPa, 9.1 KPa, respectively.

[0045] 5. The three-layer gradient self-healing hydrogel of the application has excellent swelling performance, and the swelling rates of the first layer to the third layer of the hydrogel are 1083%, 1077% and 954% respectively after absorbing water for 4 hours.

[0046] 6. The three-layer gradient self-healing hydrogel of the application has excellent antioxidant performance, the DPPH clearance rate of the first layer OCS-CMCS-TA1 hydrogel is 68%, the DPPH clearance rate of the second layer OCS-CMCS-TA3 hydrogel reaches 81%, and the DPPH clearance rate of the third layer OCS-CMCS / HAp-TA3 hydrogel reaches 80%. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The aldehyde-modified nuclear magnetic resonance hydrogen spectrum of chondroitin sulfate;

[0048] Figure 2The gelation diagram of OCS1-CMCS-TA self-healing hydrogel and OCS1-CMCS / HAp-TA self-healing hydrogel;

[0049] Figure 3 The stress-strain curve diagram of OCS1-CMCS single network self-healing hydrogel;

[0050] Figure 4 The elastic modulus diagram of OCS1-CMCS single network self-healing hydrogel;

[0051] Figure 5 The stress-strain curve diagram of OCS2-CMCS single network self-healing hydrogel;

[0052] Figure 6 The elastic modulus diagram of OCS2-CMCS single network self-healing hydrogel;

[0053] Figure 7 The stress-strain curve diagram of OCS1-CMCS-TA double network self-healing hydrogel with different TA concentrations;

[0054] Figure 8 The elastic modulus diagram of OCS1-CMCS-TA double network self-healing hydrogel with different TA concentrations;

[0055] Figure 9 The stress-strain curve diagram of OCS1-CMCS / HAp-TA double network self-healing hydrogel loaded with different amounts of HAp;

[0056] Figure 10 The elastic modulus diagram of OCS1-CMCS / HAp-TA double network self-healing hydrogel loaded with different amounts of HAp;

[0057] Figure 11 The schematic diagram of adhesion test;

[0058] Figure 12 The adhesion performance test result diagram of OCS-CMCS-TA double network self-healing hydrogel; wherein, Figure 12 A and Figure 12 B is a display diagram of the hydrogel adhering to a plastic glove and bearing a certain pulling force; Figure 12 C is a display diagram of the hydrogel adhering to the skin; Figure 12 D is a display diagram of the hydrogel adhering to rubber; Figure 12 E is a display diagram of the hydrogel adhering to a plastic tube; Figure 12 F is a display diagram of the hydrogel adhering to a plastic glove;

[0059] Figure 13 The force-displacement curve diagram of OCS-CMCS-TA hydrogel with different TA concentrations;

[0060] Figure 14 Adhesion strength graph of OCS-CMCS-TA hydrogels with different TA concentrations;

[0061] Figure 15 Force displacement curve graph of OCS1-CMCS / HAp-TA3 hydrogel loaded with hydroxyapatite nanoparticles;

[0062] Figure 16 Adhesion strength graph of OCS1-CMCS / HAp-TA3 hydrogel loaded with hydroxyapatite nanoparticles;

[0063] Figure 17 Schematic diagram of gradient self-healing hydrogel;

[0064] Figure 18 SEM image of hydrogel after freeze-drying, wherein, Figure 18 -A is the SEM image of OCS1-CMCS-TA1 hydrogel with magnification × 100; Figure 18 -B is the SEM image of OCS1-CMCS-TA3 hydrogel with magnification × 100; Figure 18 -C is the SEM image of OCS1-CMCS / HAp-TA3 hydrogel with magnification × 100; Figure 18 -D is the SEM image of OCS1-CMCS / HAp-TA3 hydrogel with magnification × 2000;

[0065] Figure 19 Swelling rate graph of OCS-CMCS-TA1, OCS-CMCS-TA3 and OCS-CMCS / HAp-TA3 hydrogels;

[0066] Figure 20 Graph of hydrogel self-healing behavior, wherein, Figure 20 -A is the graph of micro-observation of hydrogel self-healing behavior, Figure 20 -B is the graph of macro-observation of hydrogel self-healing behavior;

[0067] Figure 21 Graph of absorbance change of different hydrogels after reaction with DPPH;

[0068] Figure 22 Statistical graph of DPPH scavenging rate of different hydrogels. DETAILED DESCRIPTION

[0069] The technical solutions of the present application will be described further and more clearly in combination with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments in the present application without creative labor are within the protection scope of the present application.

[0070] In the present application, chondroitin sulfate (CS) is a sulfated glycosaminoglycan composed of alternating units of beta-1, 4-linked glucuronic acid and beta-1, 3-N-acetyl-D-glucosamine. Chondroitin sulfate is almost distributed on the surface of all cells and is the main component of extracellular matrix, playing an important role in regulating cell migration, receptor binding and other cell functions. In addition to having anti-inflammatory, anti-tumor, promoting cell regeneration and other biological activities, chondroitin sulfate is also widely used in biological scaffolds to participate in bone, cartilage, skin and other tissue engineering fields.

[0071] In the present application, chitosan (CS) is a mucopolysaccharide widely existing in nature and is also a potential material for cartilage repair. As a product of chitin, chitosan is non-toxic, degradable, and has good adhesion. A large number of positively charged free amino groups in chitosan molecules can react with polyvalent anions to form hydrogel, which is then used in the fields of biology, medicine, food and the like. In addition, chitosan is also the main component of connective tissue, and has the properties of anti-inflammatory and antibacterial effects, antioxidant properties and the like. Carboxymethyl chitosan (CMCS) is a derivative of chitosan, which is prepared by introducing a carboxymethyl functional group into the chitosan molecule. Carboxymethyl chitosan not only maintains the biocompatibility and biodegradability of chitosan, but also has better water solubility, film forming property, moisture retention property, antibacterial property and heavy metal chelation due to the introduction of carboxymethyl. Therefore, carboxymethyl chitosan is widely used in the field of medicine.

[0072] In the present application, tannic acid (TA) is a natural polyphenolic compound rich in o-benzene triol and o-benzene diol, and has various interactions. Existing research shows that TA can be used as a universal gelling agent to cross-link polymers into hydrogel; at the same time, tannic acid can also be used as a cross-linking agent for the modification of biological materials. And tannic acid has strong antioxidant property, which is because tannic acid is rich in o-benzene triol and o-benzene diol, and the o-benzene triol and o-benzene diol hydroxyl groups can effectively scavenge oxygen free radicals, thereby blocking the continuous reaction of free radicals. The antioxidant property of tannic acid mainly reflects in two aspects: 1) tannic acid can reduce the oxygen content in the environment through reduction reaction; 2) tannic acid can act as a hydrogen donor to release hydrogen and combine with free radicals in the environment, preventing the chain reaction initiated by free radicals, thereby preventing the continuation and transmission of the oxidation process.

[0073] In the embodiment of the present application, the main experimental instruments are shown in Table 1.

[0074] Table 1. List of experimental instruments

[0075] Equipment name Equipment model Use Ultrasonic cleaner SB-5200DTDN Dispersion UV spectrophotometer UV755B Analysis Constant-temperature heating magnetic stirrer DF-101S Stirring Electric heating constant-temperature drying box DGG-9076A Drying Freeze dryer Lab-1D-80 Freeze drying Mechanical tester Sansitejie Measurement Scanning electron microscope DX-7000 Analysis

[0076] In the embodiment of the present application, the main experimental raw materials are shown in Table 2.

[0077] Table 2. List of experimental raw materials

[0078]

[0079]

[0080] Example 1

[0081] (1) Chondroitin sulfate modification

[0082] Dissolve 1 g of chondroitin sulfate sodium salt in 20 mL of ultrapure water, fully dissolve, then add 1.5 g and 0.7 g of sodium periodate, respectively, react for 6 h, then add 40 mL of ethanol solution and continue stirring for 15 min (avoiding light), precipitate at 4℃ for 4 h to collect the precipitated OCS, then dialyze the precipitate with a MWCO 300 dialysis bag for 3 d, then freeze-dry to obtain modified chondroitin sulfate, which are named as OCS1 and OCS2, respectively.

[0083] (2) Preparation of single-network OCS-CMCS self-healing hydrogel

[0084] 1) Weigh 2 g of OCS1 and 2 g of OCS2 and dissolve them in 10 mL of ultrapure water, respectively, to prepare 20% w / v OCS1 and OCS2 solutions.

[0085] 2) Weigh 0.4 g of carboxymethyl chitosan and dissolve it in 10 mL of ultrapure water, stir for 1 h to form a transparent and uniform solution, to obtain a 4% w / v CMCS solution.

[0086] 3) According to the test grouping shown in Table 3, mix the OCS solution obtained in step 1) and the CMCS solution obtained in step 2) in a syringe according to a certain volume ratio, and place it at room temperature to form an OCS-CMCS single-network hydrogel.

[0087] Table 3. Experimental grouping table of single-network OCS-CMCS self-healing hydrogel

[0088] Sample number Proportion 1 OCS1:CMCS=1:4 2 OCS1:CMCS=1:6 3 OCS1:CMCS=1:8 4 OCS2:CMCS=1:4 5 OCS2:CMCS=1:6 6 OCS2:CMCS=1:8

[0089] (3) Preparation of double-network OCS1-CMCS-TA self-healing hydrogel

[0090] 1) Weigh 2 g of OCS1 and dissolve it in 10 mL of ultrapure water to prepare a 20% w / v OCS1 solution.

[0091] 2) Weigh 0.4 g of carboxymethyl chitosan and dissolve it in 10 mL of ultrapure water to form a transparent and uniform solution after stirring for 1 h, obtaining a 4% w / v CMCS solution.

[0092] 3) Mix the OCS1 solution obtained in step 1) and the CMCS solution obtained in step 2) in a syringe at a volume ratio of 1:6, and place it at room temperature to form an OCS-CMCS single-network wet hydrogel; after freeze-drying, a dry hydrogel is obtained.

[0093] 4) Weigh 0.1 g, 0.3 g, 0.5 g, and 1 g of tannic acid powder respectively and dissolve it in 10 mL of ultrapure water to form 1% w / v, 3% w / v, 5% w / v, and 10% w / v transparent and uniform solutions after stirring for 10 min.

[0094] 5) This experiment is divided into 4 groups, as shown in Table 4. Soak the wet hydrogel prepared in step 3) and the dry hydrogel after freeze-drying in 5 mL of TA solution with different concentrations for 30 min, and then rinse the surface of the hydrogel with excess TA solution with ultrapure water. The volume of the hydrogel is 4 × 10 -6 m 3 . Obtain a double-network OCS1-CMCS-TA self-healing hydrogel.

[0095] Table 4. Experimental grouping table of double-network OCS1-CMCS-TA self-healing hydrogel

[0096] Sample number Proportion 7 OCS1:CMCS-TA1 8 OCS1:CMCS-TA3 9 OCS1:CMCS-TA5 10 OCS1:CMCS-TA10

[0097] (4) Preparation of double-network OCS1-CMCS / HAp-TA self-healing hydrogel

[0098] 1) Weigh 2 g of OCS1 and dissolve it in 10 mL of ultrapure water to prepare a 20% w / v OCS1 solution.

[0099] 2) Weigh 0.4 g of carboxymethyl chitosan and dissolve it in 10 mL of ultrapure water to form a transparent and uniform solution after stirring for 1 h, then add 0.2 g, 0.4 g, 0.6 g, 0.8 g, and 1.0 g of nano-hydroxyapatite (HAp) respectively, and stir magnetically for 30 min to fully mix the HAp in the solution to form a milky white transparent and uniform CMCS / HAp solution.

[0100] 3) Mix the OCS1 solution obtained in step 1) and the CMCS / HAp solution obtained in step 2) in a syringe at a volume ratio of 1:6, and place it at room temperature to form an OCS-CMCS / HAp single-network hydrogel containing different amounts of HAp.

[0101] 4) Weigh 0.3 g of tannic acid powder and dissolve it in 10 mL of deionized water. Stir magnetically for 10 minutes to form a 3% w / v transparent homogeneous solution.

[0102] 5) The OCS-CMCS / HAp wet hydrogels containing different amounts of HAp prepared in step 3) were immersed in the 3% w / v TA solution prepared in step 4) above. After immersion, the excess TA solution on the surface of the hydrogel was rinsed with ultrapure water to obtain a double-network OCS1-CMCS / HAp-TA self-healing hydrogel.

[0103] Table 5. Experimental grouping of dual-network OCS1-CMCS / HAp-TA self-healing hydrogels

[0104] Sample number Proportion 11 OCS1:CMCS / HAp2-TA3 12 OCS1:CMCS / HAp4-TA3 13 OCS1:CMCS / HAp6-TA3 14 OCS1:CMCS / HAp8-TA3 15 OCS1:CMCS / HAp10-TA3

[0105] (5) Preparation of gradient self-healing hydrogel

[0106] The gradient self-healing hydrogel is fabricated by leveraging the self-healing and adhesion properties of OCS1-CMCS-TA and OCS1-CMCS / HAp-TA self-healing hydrogels. The first layer is OCS1-CMCS-TA1 hydrogel, the second is OCS1-CMCS-TA3 hydrogel, and the third is OCS1-CMCS / HAp6-TA3 hydrogel. The gradient self-healing hydrogel is formed by stacking the prepared OCS1-CMCS-T1A, OCS1-CMCS-TA3, and OCS1-CMCS / HAp6-TA3 hydrogels from top to bottom.

[0107] Example 2. Characterization of Chondroitin Sulfate Modification

[0108] (1) Nuclear magnetic resonance hydrogen spectrum test

[0109] H NMR spectroscopy ( 1 H NMR (H NMR) detects different absorption peaks and chemical shifts due to the different chemical environments of hydrogen atoms in a molecule. The magnitude of the chemical shift can be used to qualitatively determine whether the chondroitin sulfate modification is successful. Dissolve 0.8g of modified OCS in 500μL of deuterated water and inject it into an NMR tube for NMR analysis to verify the modification.

[0110] Results: The aldehyde modification of OCS was achieved by oxidation of the hydroxyl groups (-OH) on the CS backbone with sodium periodate. 1 H NMR Figure 1 As shown, the NMR of OCS is compared with that of CS. 1HNMR appeared a new C=0 characteristic peak at 5.18 ppm, indicating that the modification of aldehyde group of CS was successful.

[0111] (2) Test of aldehyde modification degree of chondroitin sulfate

[0112] The aldehyde group content of modified chondroitin sulfate was determined by hydroxylamine hydrochloride titration method, and the specific experimental steps included: 1) Preparation of hydroxylamine hydrochloride-methyl orange solution: weigh 4.35 g of hydroxylamine hydrochloride, dissolve with a small amount of deionized water, add 1.5 mL of 0.05% methyl orange aqueous solution, and dilute to 500 mL for standby. 2) Determination of aldehyde group of chondroitin sulfate: weigh 0.02 g of OCS, and record the mass as m; dissolve OCS in 5 mL of hydroxylamine hydrochloride-methyl orange solution, and stand for 3 h for standby. Titrate with 0.01 mol / L sodium hydroxide solution until the red color changes to yellow, and record the volume as V1; and make a blank control, and record the volume as V0. Calculate the aldehyde group content according to the following formula:

[0113] Aldehyde group content (mmol / g) = (V1-V0) x n NaOH / 2W

[0114] Wherein, V1 is the volume of sodium hydroxide consumed by the sample, V0 is the volume of sodium hydroxide consumed by the blank, n NaOH is the molar concentration of sodium hydroxide solution (mol / L), and W is the mass of OCS (g). Each group of experiments was repeated 3 times, and the average value was taken.

[0115] Results: The average volume of NaOH consumed by the blank group and OCS1 and OCS2, and the aldehyde group content of OCS are shown in Table 6. Hydroxylamine hydrochloride can react with aldehyde group to form oxime, and release HCl at the same time. The amount of HCl is titrated, and the aldehyde group content is calculated according to the above formula: the aldehyde group content of OCS1 with 1.5 g of sodium periodate is 1.2 mmol / g, and the aldehyde group content of OCS2 with 0.7 g of sodium periodate is 0.875 mmol / g.

[0116] Table 6. Aldehyde group content of OCS

[0117] Sample Average consumption of NaOH (mL) Aldehyde group content (mmol / g) Blank group 4.2 / OCS1 9 1.2 OCS2 7.7 0.875

[0118] (3) Gelation

[0119] The specific gelation of OCS1-CMCS-TA self-healing hydrogel and OCS1-CMCS / HAp-TA self-healing hydrogel was as follows: Figure 2The aldehyde groups of OCS1 react with the amino groups of CMCS to form Schiff base in the process of forming OCS-CMCS-TA self-healing hydrogel. This strong interaction constitutes the first crosslinking network structure of the hydrogel. However, the mechanical properties and adhesion properties of the single-network self-healing hydrogel prepared are weak. Therefore, TA is introduced to solve this problem. The hydrogen bonds between TA itself and other components of TA constitute the second crosslinking network structure of the hydrogel, which improves the mechanical properties and self-healing properties of the material, and the ortho-phenol of TA can endow the material with adhesion and antioxidant properties, and the hydroxyapatite can promote cartilage-bone formation.

[0120] Example 3. Mechanical property test

[0121] The hydrogels No. 1-15 in Example 1 were made into cylindrical samples with a diameter of 1 cm and a height of 6 mm. The compression test of the hydrogel was carried out using a mechanical tester at a rate of 5 mm / min, and the machine was stopped at the moment of crushing the gel. The elastic modulus of the hydrogel was obtained by calculating the slope of the straight line part of the stress-strain curve. Three parallel samples were made for each group of experiments, and the average value was taken.

[0122] The calculation formula of stress is as follows:

[0123] Stress = load / π x r 2 x 1000

[0124] Wherein, load is the weight value, unit N.

[0125] The calculation formula of strain is as follows:

[0126] Strain = position / h x 100

[0127] Wherein, position is displacement, unit mm.

[0128] The test results and analysis are as follows:

[0129] 1) The mechanical properties of single-network OCS-CMCS self-healing hydrogel: The stress-strain curves and elastic modulus of single-network self-healing hydrogel under different preparation conditions are shown in Figures 3-6 . Among them, Figures 3-4 are OCS1-CMCS single-network self-healing hydrogels with different proportions of OCS1 and CMCS with high degree of modification, and the elastic modulus reaches 93 KPa when OCS1:CMCS = 1:6 under the same gelation environment, while the elastic modulus of OCS1:CMCS = 1:4 and OCS1:CMCS = 1:8 is not as good as that of OCS1:CMCS = 1:6, because when OCS1:CMCS = 1:6, the aldehyde groups of OCS1 react completely with the amino groups of CMCS. Figures 5-6The OCS2-CMCS single network self-healing hydrogel with different proportions of OCS2 and CMCS with low modification degree can be seen that the elastic modulus reaches 86 KPa when OCS2:CMCS = 1:6 under the same gelation environment. It can be known by comparison that the mechanical properties of the hydrogel formed by high modification degree OCS1 are higher than those of the hydrogel formed by low modification degree OCS2, because in the same mass of OCS, the high modification degree OCS1 contains more aldehyde groups, and the mechanical properties of the OCS-CMCS hydrogel formed are stronger. According to the results, the high modification degree OCS1 is selected in the subsequent experiments under the condition of OCS1:CMCS = 1:6.

[0130] 2) Mechanical properties of the OCS-CMCS-TA double network self-healing hydrogel: In the process of forming the OCS-CMCS-TA self-healing hydrogel, the aldehyde groups of OCS1 react with the amino groups of CMCS to form Schiff base. This strong interaction constitutes the first cross-linked network structure of the hydrogel, but the mechanical properties and adhesion properties of the single network self-healing hydrogel prepared are weak, therefore, the present application solves this problem by introducing TA. TA will form hydrogen bond interaction with OCS1 and CMCS, constituting the second cross-linked network structure of the hydrogel, greatly improving the mechanical and adhesion properties of the hydrogel. The mechanical property detection results of the OCS1-CMCS-TA double network self-healing hydrogel prepared by immersing the single network self-healing hydrogel of OCS1:CMCS = 1:6 in TA solutions with different concentrations are shown in Table 2. Figures 7-8 As can be seen from the comparison of Table 1 and Table 2, Figures 7-8 , Figures 3-4 The mechanical properties of the double network self-healing hydrogel immersed in the TA solution are the highest, reaching 328 KPa, which is much higher than the 93 KPa of the single network self-healing hydrogel. This is because after immersing in the TA solution, a large number of hydrogen bonds are formed between TA and OCS1, thereby enhancing the mechanical properties of the hydrogel. And within a certain range, with the increase of the concentration of the TA solution, the mechanical properties of the OCS1-CMCS-TA double network self-healing hydrogel first increase and then decrease, and when the concentration of the TA solution is 3% w / v, the elastic modulus of the OCS1-CMCS-TA double network self-healing hydrogel reaches the highest value of 328 KPa. But when the concentration of the TA solution increases to 10% w / v, the mechanical properties of the OCS1-CMCS-TA double network self-healing hydrogel decrease to 220 KPa. This is because when the concentration of TA is too high, it will react rapidly and strongly with the surface layer of the hydrogel, forming a relatively hard shell layer, which leads to slow penetration of TA into the interior of the gel, thus causing the second cross-linked network of the hydrogel to be uneven, affecting the mechanical properties of the hydrogel.

[0131] During the experiment, the application also uses OCS1:CMCS=1:6 single network self-healing dry hydrogel soaked in different concentrations of TA solution to prepare OCS1-CMCS-TA double network self-healing hydrogel. However, the self-healing property and adhesion property of the dry hydrogel are poor in the subsequent test, so the wet hydrogel is finally selected for the experiment. According to the results, the application selects OCS1:CMCS=1:6 and TA concentration of 3% w / v for subsequent experiments.

[0132] 3) Network OCS1-CMCS / HAp-TA self-healing hydrogel mechanics: Hydroxyapatite can promote cartilage-bone formation, so hydroxyapatite is widely used in the field of bone cartilage tissue engineering scaffold materials. And studies have shown that adding a series of inorganic new nanoparticles to the hydrogel can be used as a hydrogel mechanics performance enhancer. Figures 9-10 The mechanical properties of OCS1-CMCS / HAp-TA self-healing hydrogel with different contents of hydroxyapatite nanoparticles added to the CMCS solution are shown in the figure. Comparison Figures 9-10 、 Figures 7-8 It can be seen that the elastic modulus of OCS1-CMCS / HAp-TA3 double network self-healing hydrogel is as high as 411KPa, which is higher than that of OCS1-CMCS-TA3 double network self-healing hydrogel. From Figures 9-10 It can be seen that when the concentration of HAp increases from 2% w / v to 6% w / v, the elastic modulus of the prepared OCS1-CMCS / HAp-TA3 hydrogel continues to rise, and when the concentration of HAp is 6% w / v, the maximum elastic modulus of the OCS1-CMCS / HAp-TA3 self-healing hydrogel is 411KPa, but when the concentration of nano-hydroxyapatite solution increases to 10% w / v, the mechanical properties of the OCS1-CMCS / HAp-TA3 self-healing hydrogel decrease to 318KPa. This is because the addition of excessive HAp causes HAp to not be completely dispersed in the hydrogel, HAp is not uniformly mixed, and thus the distribution of Hap nanoparticles in the hydrogel is not uniform. The stress is not uniform during the compression of the hydrogel, which makes the hydrogel more prone to be crushed.

[0133] Example 4. Adhesion property test

[0134] A small amount of OCS1-CMCS-TA hydrogel is added and fully contacted with the fingers, and the adhesion behavior is observed by stretching the fingers. In order to quantitatively analyze the adhesion strength of OCS1-CMCS-TA hydrogel, a glass slide is used for testing, first immerse the glass slide in a gelatin solution with a concentration of 5% for 0.5h, and then take it out and dry naturally. Then, the hydrogel is clamped between two glass slides to form a glass slide / hydrogel / glass slide sandwich structure, as shown in Figure 11The test was then performed on a mechanical testing machine at a speed of 5 mm / min. The maximum pulling force F required to separate the two glass slides was recorded.

[0135] The formula for calculating adhesion strength is: δ=F / A

[0136] Where A is the overlapping area, F is the maximum tensile stress, and each sample was measured three times in parallel.

[0137] Results: As Figure 12 As shown, Figure 12 -A~ Figure 12 -B shows that the hydrogel can adhere to the plastic gloves and can withstand a certain amount of tension without falling off. Figure 12 -C~ Figure 12 -E is the hydrogel's ability to adhere to various media, including skin, rubber, and plastic. Therefore, the OCS1-CMCS-TA hydrogel exhibits excellent tissue adhesion. This is because the present invention further introduces TA into the OCS-CMCS hydrogel, resulting in the hydrogel possessing a large number of catechol and pyrogallol groups. Consequently, the hydrogel exhibits mussel-like adhesion, capable of adhering to the surfaces of both hydrophilic and hydrophobic materials. The excellent tissue adhesion of the OCS1-CMCS-TA hydrogel not only supports cell adhesion, proliferation, and diffusion, but also maintains the phenotype of chondrocytes, which is of great significance for cartilage repair.

[0138] The results of quantitative measurement of the adhesion strength of OCS-CMCS-TA hydrogel by mechanical testing instrument are as follows Figures 13-14 As shown, the adhesion strength of OCS1-CMCS-T0 hydrogel is 1.5KPa, while the adhesion strength of OCS1-CMCS-T1 hydrogel is 5.7KPa. By comparison, it can be seen that the adhesion strength of the hydrogel is improved by soaking in TA solution. The improvement in the adhesion strength of the hydrogel is due to the introduction of TA. TA contains a large number of catechol groups, which causes hydrophobic and hydrogen bond interactions between the substrate and the catechol groups. As the TA concentration increases, the shear adhesion strength of the OCS1-CMCS-TA hydrogel gradually increases, but when the TA content continues to increase, the adhesion strength of the OCS-CMCS-TA hydrogel decreases instead, with a strength of 4.5KPa. This is because when the concentration of TA is too high, a harder shell will be formed on the surface of the hydrogel, so the uneven formation of the hydrogel affects the adhesion strength of the hydrogel. When OCS1-CMCS / HAp-TA3 hydrogel was prepared after adding hydroxyapatite to carboxymethyl chitosan, the shear adhesion strength test was carried out. The test results are shown in the figure. Figures 15-16 As shown in the figure, compared with OCS1-CMCS-T3 hydrogel, under the preparation conditions of a certain amount of HAp, the introduction of hydroxyapatite did not significantly change the adhesion strength of the hydrogel.

[0139] Based on the results of mechanical and adhesion performance tests, the gradient self-healing hydrogel is prepared by self-healing and adhesion between the hydrogels. Figure 17 As shown in FIG. 5, the preparation conditions of the three layers from top to bottom are as follows: the first layer is OCS1-CMCS-TA1 (OCS1:CMCS = 1:6, TA concentration is 1% w / v), the second layer is OCS1-CMCS-TA3 (OCS1:CMCS = 1:6, TA concentration is 3% w / v), and the third layer is OCS1-CMCS / HAp-TA3 (OCS1:CMCS = 1:6, TA concentration is 3% w / v, HAp concentration is 6% w / v). The elastic modulus of the three-layer hydrogel is 282 KPa, 328 KPa, and 411 KPa, respectively. The adhesion strength of the three-layer hydrogel is 5.7 KPa, 9.3 KPa, and 9.1 KPa, respectively.

[0140] Example 5. SEM observation

[0141] The morphology of the hydrogel is observed by SEM. SEM is a means of characterizing the surface morphology of materials, which uses different electron signals formed by the interaction of an electron beam with the surface of the sample to form a micro image. Its working principle is that the high-energy electron beam interacts with the sample surface to obtain characteristic signals, and the imaging function of the instrument forms a characteristic image. In addition, the gradient hydrogel is freeze-dried into three parts, the hydrogel sample is quenched with liquid nitrogen, and then the brittle fracture section is gold sprayed for 1 min, and the micro-porous structure of the three-layer hydrogel is observed.

[0142] Results: The morphology of the OCS1-CMCS-TA self-healing hydrogel and the OCS1-CMCS / HAp-TA self-healing hydrogel is shown in FIG. 6. Figure 18 The OCS1-CMCS-TA1 self-healing hydrogel has a uniform structure, a dense structure, and a very obvious pore structure. Further increasing the concentration of TA, the OCS1-CMCS-TA3 self-healing hydrogel has a smaller pore size and a more dense network structure compared with the OCS1-CMCS-TA1 self-healing hydrogel. This is because the increase of TA concentration enhances the hydrogen bonding between TA and OCS1 and CMCS, thereby increasing the crosslinking density of the OCS1-CMCS-TA3 self-healing hydrogel. When HAp is introduced, the OCS1-CMCS / HAp-TA3 self-healing hydrogel has a uniform structure, a more dense network structure, a reduced pore structure, and visible nano-hydroxyapatite particles on the pore wall.

[0143] Example 6. Swelling performance test

[0144] Swelling property is an important index to evaluate the performance of the scaffold. As a tissue engineering scaffold, its rapid swelling property is conducive to the transportation of nutrients, so that the cells embedded in the hydrogel will not die due to lack of nutrients for a long time. In order to more accurately evaluate the swelling property of the self-healing hydrogel, the self-healing hydrogel after freeze-drying is used for swelling property test. The gradient hydrogel is freeze-dried and cut into three parts, and each layer of hydrogel sample is weighed and measured before being soaked in 37℃ PBS buffer. The water-absorbed hydrogel is weighed (1h, 2h, 3h, 4h, 5h, 6h) within a predetermined time. The mass swelling rate (W SR ) of the hydrogel can be calculated according to the following formula:

[0145] W SR (%)=(m s -m0) / m0×100%

[0146] Wherein, m s and m0are the mass of the hydrogel before and after water absorption and swelling. Each sample is measured in triplicate.

[0147] Results: As shown in Figure 19 , in the first hour, OCS-CMCS-TA1 and OCS-CMCS-TA3 and OCS-CMCS / HAp-TA3 hydrogels rapidly absorb water, and by the fourth hour, the swelling rates of the three hydrogels are 1083%, 1077% and 954% respectively. Reach the swelling equilibrium. Comparing CS-CMCS-TA1 and OCS-CMCS-TA3 hydrogels, it is found that the swelling rates of the two are not much different, because the concentration of TA solution will not affect the network structure of the hydrogel. At the same time, the swelling rates of CS-CMCS-TA1 and OCS-CMCS-TA3 hydrogels are higher than that of OCS-CMCS / HAp-TA3 hydrogel, because the OCS-CMCS / HAp-TA3 hydrogel network contains Hap nanoparticles, which makes the network structure of the hydrogel more compact, and it is difficult for water molecules to enter the inside, so the equilibrium mass swelling rate is lower than that of OCS-CMCS-TA1 and OCS-CMCS-TA3 hydrogels.

[0148] Example 7. Self-healing property test

[0149] To study the self-healing properties of the hydrogel, the shear-healing method was used to study the self-repairing properties of the OCS-CMCS-TA self-healing hydrogel. Two identical self-healing hydrogels were prepared under the same conditions, one of which was dyed red with rhodamine, and the other was not dyed. Then the two hydrogels were cut into four pieces, and the two hydrogels of different colors were seamlessly placed on the broken surface, and allowed to self-heal at room temperature without any external pressure, and the microscopic healing was observed under a microscope. After a certain period of time, the healed hydrogel was stretched horizontally with tweezers to observe the healing.

[0150] The results are shown in Figure 20 Figure 20 In A, two pieces of OCS-CMCS-TA hydrogel of different colors were cut into four pieces, and the hydrogels of different colors were spliced together. It can be seen that the hydrogel formed by Schiff base bond and hydrogen bond has super strong self-healing properties. The gap of the hydrogel has disappeared in 1 h, and the hydrogel has completely self-healed in 3 h. From Figure 20 It can be seen from B that the entire hydrogel is red after 5 h, and the self-healing hydrogel after 5 h is stretched. It can be seen that the self-healing gap has completely disappeared and the color is uniform. The hydrogel cannot be pulled apart by the tweezers. The results show that the hydrogel has good self-healing effect.

[0151] Example 8. Antioxidant property test

[0152] In this example, the antioxidant capacity of OCS1-CMCS-TA hydrogel and OCS1-CMCS / HAp-TA hydrogel was tested by the ability to scavenge DPPH. DPPH has an absorption peak at 517 nm. The ethanol solution of DPPH is purple. When the hydrogen atoms in OCS1-CMCS-TA hydrogel and OCS1-CMCS / HAp-TA hydrogel are paired with electrons, reduced DPPH is generated, the absorption rate decreases, and the solution color is light yellow. The more electrons scavenged, the stronger the antioxidant capacity. Pure OCS1-CMCS hydrogel does not have the ability to scavenge DPPH free radicals. Each layer was prepared according to the gradient self-healing hydrogel three-layer preparation conditions, and the antioxidant test was carried out. The specific experimental steps are as follows: 100 mg of wet hydrogel is soaked in 6 mL of 80 μg / mL 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH) solution, so that the hydrogel and the solution react fully. The absorbance of the solution at 517 nm was measured by ultraviolet spectrophotometer, and the DPPH free radical scavenging rate was calculated according to the following formula:

[0153] Free radical scavenging rate = (D0-D i ) / D0×100%

[0154] ​D0 is the absorbance of the blank group, D is the absorbance of the experimental group, and D is the absorbance of the sample. i The absorbance of the experimental group is D. Each sample is measured in triplicate.

[0155] The results are shown in Table 1. Figures 21-22 As shown in Table 1, the introduction of TA, which contains abundant phenolic hydroxyl groups, can act as a terminator of hydroxyl radicals, reducing DPPH to diphenyl hydrazine. The DPPH scavenging rate of OCS-CMCS-TA1 hydrogel sample is 68%, and with the increase of TA content, the content of catechol and pyrogallol increases, the DPPH radical scavenging ability enhances, and the DPPH scavenging rate of OCS-CMCS-TA3 hydrogel sample reaches 81%, and the DPPH scavenging rate of OCS-CMCS / HAp-TA3 hydrogel sample reaches 80%. Reactive oxygen species (ROS) is the direct cause of cartilage cell damage, aging and apoptosis in osteoarthritis. TA has good antioxidant capacity and plays an important role in maintaining the oxidation balance of cells. Therefore, maintaining the stability of ROS level in cells is a feasible solution for the treatment of osteoarthritis.

[0156] From the experimental results of Examples 2-8 above, the following conclusions can be drawn:

[0157] (1) Based on the Schiff base reaction between OCS and CMCS, a single network self-healing hydrogel is prepared, and the mechanical properties gradually increase with the increase of OCS modification degree and the volume ratio of OCS / CMCS. The results show that when the aldehyde group modification degree of OCS is 1.2 mmol / g and the volume ratio of OCS:CMCS is 1:6, the elastic modulus is 93 KPa.

[0158] (2) In order to enhance the mechanical properties of the hydrogel, and endow the hydrogel with tissue adhesion and antioxidant properties, TA is introduced into the system. The preferred preparation conditions of OCS1-CMCS-TA double network self-healing hydrogel are (OCS1:CMCS=1:6, TA3%), and the highest elastic modulus is 328 KPa, and the adhesion strength is 9.3 KPa.

[0159] (3) The introduction of HAp not only enhances the mechanical properties of the hydrogel, but also helps bone formation. The preferred preparation conditions of OCS1-CMCS / HAp-TA3 double network self-healing hydrogel are (OCS1:CMCS=1:6, TA concentration is 3% w / v, and HAp concentration is 6% w / v), and the highest elastic modulus is 411 KPa.

[0160] (4) Based on the good self-healing and adhesion of the hydrogel, a three-layer gradient self-healing hydrogel is prepared, and the binding interface between the layers is firm. The gradient self-healing hydrogel has good mechanical properties, tissue adhesion, self-healing, swelling, and good antioxidant properties.

[0161] Based on the above, the optimal preparation conditions of the three-layer gradient self-healing hydrogel are as follows: the first layer is OCS1-CMCS-TA1 hydrogel, OCS1:CMCS = 1:6, the TA concentration is 1% w / v, the OCS1 concentration is 20% w / v, and the CMCS concentration is 4% w / v; the second layer is OCS1-CMCS-TA3 hydrogel, OCS1:CMCS = 1:6, the TA concentration is 3% w / v, the OCS1 concentration is 20% w / v, and the CMCS concentration is 4% w / v; and the third layer is OCS1-CMCS / HAp-TA3 hydrogel, OCS1:CMCS = 1:6, the TA concentration is 3% w / v, the HAp concentration is 6% w / v, the OCS1 concentration is 20% w / v, and the CMCS concentration is 4% w / v. The gradient self-healing hydrogel prepared under the conditions has good mechanical properties, tissue adhesion properties and antioxidant properties, and has the potential to be applied in cartilage-bone integrated repair.

Claims

1. A three-layer gradient self-healing hydrogel for articular cartilage-bone tissue integration repair, characterized in that, The three-layer gradient self-healing hydrogel is prepared by the following method: (1) The aldehyde-modified chondroitin sulfate solution and the carboxymethyl chitosan solution are mixed to prepare a chondroitin sulfate / chitosan self-healing hydrogel by a dynamic Schiff base reaction; the chondroitin sulfate / chitosan self-healing hydrogel is soaked in a tannic acid solution with a concentration of 0.5% w / v to 1.5% w / v to obtain an OCS1-CMCS-TA1 hydrogel; (2) The aldehyde-modified chondroitin sulfate solution and the carboxymethyl chitosan solution are mixed to prepare a chondroitin sulfate / chitosan self-healing hydrogel by a dynamic Schiff base reaction; the chondroitin sulfate / chitosan self-healing hydrogel is soaked in a tannic acid solution with a concentration of 2.5% w / v to 3.5% w / v to obtain an OCS1-CMCS-TA3 hydrogel; (3) The carboxymethyl chitosan solution is added with nano-hydroxyapatite to obtain a CMCS / HAp solution; the CMCS / HAp solution is mixed with the aldehyde-modified chondroitin sulfate solution to obtain an OCS1-CMCS / HAp6 hydrogel; the OCS1-CMCS / HAp6 hydrogel is soaked in a tannic acid solution with a concentration of 2.5% w / v to 3.5% w / v to obtain an OCS1-CMCS / HAp6-TA3 hydrogel; (4) The OCS1-CMCS-TA1 hydrogel obtained in step (1), the OCS1-CMCS-TA3 hydrogel obtained in step (2) and the OCS1-CMCS / HAp6-TA3 hydrogel obtained in step (3) are stacked from top to bottom to obtain the three-layer gradient self-healing hydrogel. In steps (1) to (3), the volume ratio of the aldehyde-modified chondroitin sulfate and the carboxymethyl chitosan is 1:5 to 1:

7.

2. The three-layer gradient self-healing hydrogel of claim 1, wherein, In the OCS1-CMCS-TA1 hydrogel, the OCS1-CMCS-TA3 hydrogel and the OCS1-CMCS / HAp6-TA3 hydrogel, the concentration of the aldehyde-modified chondroitin sulfate is 18% w / v to 22% w / v, and the concentration of the carboxymethyl chitosan is 3% w / v to 5% w / v; in the OCS1-CMCS / HAp6-TA3 hydrogel, the concentration of the nano-hydroxyapatite is 5% w / v to 7% w / v.

3. The tri-layer gradient self-healing hydrogel of claim 1, wherein, The aldehyde-modified chondroitin sulfate contains 1.0 mmol / g to 1.5 mmol / g of aldehyde groups.

4. The tri-layer gradient self-healing hydrogel of claim 1, wherein, The tannic acid concentration in the OCS1-CMCS-TA1 hydrogel is 1% w / v, and the volume ratio of aldehyde-modified chondroitin sulfate and carboxymethyl chitosan is 1:6; the tannic acid concentration in the OCS1-CMCS-TA3 hydrogel is 3% w / v, and the volume ratio of aldehyde-modified chondroitin sulfate and carboxymethyl chitosan is 1:6; the tannic acid concentration in the OCS1-CMCS / HAp6-TA3 hydrogel is 3%, and the volume ratio of aldehyde-modified chondroitin sulfate and carboxymethyl chitosan is 1:6; the concentration of aldehyde-modified chondroitin sulfate is 20% w / v, wherein the aldehyde group content is 1.2 mmol / g; the concentration of carboxymethyl chitosan is 4% w / v; and the concentration of nano-hydroxyapatite is 6% w / v.

5. The tri-layer gradient self-healing hydrogel of claim 1, wherein, The aldehyde-modified chondroitin sulfate is prepared by the following method: after dissolving chondroitin sulfate sodium salt, sodium periodate is added for oxidation reaction; after the reaction is completed, an ethanol solution is added and stirred, and the obtained product is precipitated, dialyzed, and freeze-dried to obtain aldehyde-modified chondroitin sulfate.

6. The tri-layer gradient self-healing hydrogel of claim 1, wherein, The OCS1-CMCS-TA1 hydrogel, the OCS1-CMCS-TA3 hydrogel, and the OCS1-CMCS / HAp6-TA3 hydrogel in the three-layer gradient self-healing hydrogel are all double-network structures, the aldehyde groups of the aldehyde-modified chondroitin sulfate react with the amino groups of the carboxymethyl chitosan to form Schiff bases, constituting the first cross-linking network structure of the hydrogel; the hydrogen bonds between tannic acid and the aldehyde-modified chondroitin sulfate and the carboxymethyl chitosan, and the hydrogen bonds of tannic acid itself, constitute the second cross-linking network structure of the hydrogel.

7. Use of the three-layer gradient self-healing hydrogel according to any one of claims 1-6 in the preparation of an integrated repair product for articular cartilage-bone tissue.

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