An injectable and self-healing hydrogel for bone repair, its preparation method and application

Abstract

The present invention belongs to the field of biomedical materials, and relates to an injectable and self-healing hydrogel for bone repair, its preparation method and application. In the present invention, chitin / chitosan, a natural biomass waste with good biocompatibility and low cost, is modified, and at the same time, Schiff base bonds and acylhydrazone bonds are introduced into the natural polysaccharide network. On this basis, functional hydrogels with excellent properties are prepared by grafting adipic dihydrazide onto carboxymethyl chitin, grafting dodecane onto carboxymethyl chitosan, and grafting benzaldehyde onto carboxylated polyvinyl alcohol, solving the contradiction among the mechanical strength, injectability, biodegradability and self-healing ability of the hydrogel. The composite hydrogel of the present invention has excellent adhesion performance and a degradation rate matching bone tissue regeneration, thus solving the problems of poor adhesion of scaffold materials and hindrance to tissue regeneration in current clinical applications, and producing a biomedical scaffold with extremely broad application value and market prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials. Specifically, it relates to an injectable and self-healing hydrogel for bone repair, its preparation method and application. Background Art

[0002] In recent years, due to trauma, infection, and congenital malformations, the number of bone tissue defects has been increasing, and there is an urgent need for advanced biomedical materials to meet complex treatment requirements. Biomimetic scaffold materials not only serve as physical scaffolds in bone defect areas but also act as carriers for releasing growth factors to accelerate bone tissue reconstruction. So far, various materials, including allogeneic bone, xenogeneic bone, metal bone cement, bioglass, and polymers, have been used in experimental exploration of bone regeneration. However, allogeneic bone and xenogeneic bone have limited sources, high costs, poor adhesion, scarce donors, and poor immunogenicity, which limit their practical applications. Although prefabricated implant scaffolds have high strength, they have problems such as poor adaptability, insufficient biocompatibility, large surgical exposure area, mismatch between the degradation rate of the scaffold material and the growth rate of new bone tissue, increased bone loss, and secondary surgery, which increase the patient's recovery time and pain, thus limiting their practical applications. In contrast, dynamic hydrogels have good injectability and are considered the best carriers for tissue engineering growth factor delivery.

[0003] As is well known, self-healing behavior widely exists in many biological tissue structures, prompting scientists to spare no effort to explore the application of dynamic hydrogels with self-healing and injectability in biomedical engineering. It is worth noting that since the structure and function of hydrogels are highly similar to the extracellular matrix, they can effectively regulate the behavior and function of stem cells, including guiding and accelerating the migration and osteogenic differentiation of mesenchymal stem cells. Therefore, stem cell carrier hydrogels can be used in the field of rapid bone tissue regeneration. In particular, the mechanical strength of hydrogels has a positive regulatory effect on stem cell differentiation. However, high mechanical strength will result in a considerable sacrifice of the dynamic properties of hydrogels, attributed to the low exchange kinetics of stable gel network cross-linking points. While highly dynamic reversible cross-linking networks have rapid self-healing behavior, injectable behavior, etc., they will also weaken their gel mechanical strength. Under physiological conditions, hydrazone bonds tend to be kinetically "locked", which makes the hydrogel have a stable cross-linking network and mechanical strength but lack dynamic reversible properties and cannot self-heal without a catalyst. Although hydrazone bond-enhanced hydrogels can effectively stabilize the structure of bone defect areas, bone repair gels still need to have sufficient dynamic reversible properties to meet the requirements of rapid adaptation and filling at irregular defect sites and minimally invasive injection. Dynamic bonds such as hydrogen bonds, Schiff base bonds, and host-guest interactions endow hydrogels with self-healing and injectability, but their mechanical strength is relatively low. Currently, there are many reports on hydrogel materials for promoting bone repair, and there are also many reports on high-strength properties for promoting bone tissue regeneration. However, hydrogel engineering scaffolds with both macroscopic stability and microscopic dynamics are very limited. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention modifies natural biomass waste chitin / chitosan with good biocompatibility and low cost, and simultaneously introduces Schiff base bonds and acylhydrazone bonds into the natural polysaccharide network. On this basis, functional hydrogels with excellent performance are prepared by using carboxyethyl chitin grafted adipic dihydrazide (CECT-ADH), carboxyethyl chitosan grafted dodecane (CES-DOD), and carboxy polyvinyl alcohol grafted benzaldehyde (PVA-BA), solving the contradiction among the mechanical strength, injectability, biodegradability, and self-healing ability of the hydrogel. The strategy of combining multiple dynamic bonds is used to endow the hydrogel with appropriate mechanical strength, effectively simulate the extracellular matrix microenvironment, promote the differentiation of bone marrow mesenchymal stem cells, and thus promote bone tissue regeneration. The composite hydrogel of the present invention has excellent adhesion performance and a degradation rate matching bone tissue regeneration, thereby solving the problems of poor adhesion of scaffold materials and hindering tissue regeneration in current clinical applications, and producing a biomedical scaffold with extremely broad application value and market prospects.

[0005] In order to achieve the above object, the first aspect of the present invention provides a preparation method of an injectable and self-healing hydrogel for bone repair, comprising the following steps:

[0006] (1) Synthesis of carboxyethyl chitin grafted adipic dihydrazide CECT-ADH;

[0007] Dissolve carboxyethyl chitin in deionized water, add adipic dihydrazide, morpholineethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide to the above solution, stir the reaction in the dark, and obtain the product CECT-ADH after dialysis and freeze-drying;

[0008] (2) Synthesis of carboxyethyl chitosan grafted dodecanal CES-DOD;

[0009] Introduce morpholineethanesulfonic acid and sodium cyanoborohydride into the carboxyethyl chitosan solution. Subsequently, dissolve dodecanal in ethanol and drop it into the above solution under light-shielding conditions. React the obtained mixture, and obtain the product CES-DOD after dialysis and freeze-drying;

[0010] (3) Synthesis of polyvinyl alcohol grafted benzaldehyde PVA-BA;

[0011] Under a nitrogen atmosphere, add 4-hydroxybenzaldehyde, 4-formylbenzoic acid, and a catalytic amount of anhydrous pyridine to the polyvinyl alcohol dimethyl sulfoxide solution. After heating and stirring the reaction, add succinic anhydride, continue the reaction, then cool to room temperature, pour it into ice ether for reprecipitation, and obtain the product PVA-BA after redissolution, dialysis, and freeze-drying;

[0012] (4) Preparation of hydrogel;

[0013] Dissolve CECT-ADH, CES-DOD, and PVA-BA in PBS buffer respectively. Then, first mix the CECT-ADH solution and the CEC-DOD solution evenly, and then add the PVA-BA solution. After vortexing and quickly mixing the obtained mixture evenly, transfer it to a polytetrafluoroethylene mold and cure to obtain the CECT-ADH / PVA-BA / CES-DOD hydrogel.

[0014] According to a preferred embodiment of the present invention, in step (1), the molar ratio of carboxyethyl chitin, adipic dihydrazide, morpholineethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.8 - 2.2:4.5 - 5.5:0.2 - 0.3:0.2 - 0.3.

[0015] According to a preferred embodiment of the present invention, in step (1), the temperature of the light-shielded stirring reaction is 20 - 30 °C, and the time is 6 - 36 h.

[0016] According to a preferred embodiment of the present invention, in step (2), the molar ratio of dodecyl aldehyde, morpholineethanesulfonic acid, sodium cyanoborohydride, and carboxyethyl chitosan is 1:40 - 45:8 - 12:8 - 12.

[0017] According to a preferred embodiment of the present invention, in step (2), the dropping rate of the dodecyl aldehyde ethanol solution is 1 mL / 30 min - 1 mL / min, the temperature of the reaction is 20 - 30 °C, and the time is 12 - 48 h.

[0018] According to a preferred embodiment of the present invention, in step (3), the molar ratio of 4-hydroxybenzaldehyde, 4-formylbenzoic acid, polyvinyl alcohol, and succinic anhydride is 1:6 - 10:4 - 6:4 - 6.

[0019] According to a preferred embodiment of the present invention, in step (3), the temperature of the heating reaction is 75 - 85 °C, and the time is 2 - 4 h.

[0020] The time for continuing the reaction after adding succinic anhydride is 2 - 10 h.

[0021] According to a preferred embodiment of the present invention, in step (4), the pH value of the PBS buffer is 7.2 - 7.6.

[0022] The mixing of the PVA-BA solution with the CECT-ADH solution and the CES-DOD solution makes the amino group / aldehyde group ratio in the mixture 1.0 - 3.0.

[0023] The mixing of the PVA-BA solution with the CECT-ADH solution and the CES-DOD solution makes the solid content of the mixture 1.0 - 5.0 wt%; preferably, the concentration of the PVA-BA solution is 1.0 - 5.0 wt%, the concentration of the CECT-ADH solution is 1.0 - 5.0 wt%, and the concentration of the CES-DOD solution is 1.0 - 5.0 wt%, preferably 1.2 - 5.0 wt%.

[0024] The temperature of the aging is 37 °C and the time is 10 - 120 min.

[0025] The second aspect of the present invention provides a hydrogel prepared by the above preparation method.

[0026] The third aspect of the present invention provides the application of the above hydrogel in the preparation of bone repair materials.

[0027] The degradation rate of traditional scaffolds usually cannot be perfectly matched with the rate of new bone growth. Therefore, as the second most abundant natural polysaccharide in nature, chitin has attracted much attention due to its excellent biodegradability. Since lysozyme can act on the N-acetylamino group on the glucose ring in chitin, the degradation rate of pure chitin in vivo is too fast, which is not conducive to long-term bone reconstruction applications. Compared with chitin, chitosan obtained after deacetylation degrades slowly. Combining the principle that the degradation rate of the scaffold is highly matched with the growth rate of blood vessels and new bone, the hydrogel constructed by the composite of chitin and chitosan has good degradation behavior, effectively avoiding secondary surgery. Therefore, chitin / chitosan-based hydrogels can be used as ideal biomedical scaffolds due to their inherent biocompatibility and adhesiveness.

[0028] In the present invention, the raw material chitin is derivatized. Introducing hydrazide groups is beneficial to the construction of the hydrazone bond hydrogel network. Introducing dodecyl aldehyde and carboxyethyl onto the chitosan chain promotes water solubility while introducing adhesive groups, which helps the adhesion between the hydrogel and the surface of biological tissues. By regulating the ratio of chitin and chitosan, as well as the ratio of grafted groups, a high-strength, self-healing, injectable, and biodegradable dynamic hydrogel can be obtained, which can effectively simulate the extracellular matrix microenvironment, promote the differentiation of bone marrow mesenchymal stem cells, and show potential superior to commercial bone repair materials in the in vivo mouse cranial defect model, solving the problems of incompatibility between strength and self-adaptability, poor adhesion performance, and mismatch between degradation rate and bone tissue growth rate in current bone repair products on the market, and having broad application prospects and market value.

[0029] Compared with the prior art, the technical advantages of the present invention include:

[0030] (1) Most of the scaffold materials used in the prior art for bone defect repair, such as allogeneic bone, xenogeneic bone, metal scaffolds, bioactive glass, high-strength hydrogels, etc., are mostly prefabricated, with poor adaptability to the shape of the defect, poor immunogenicity, high cost, complex preparation process, troublesome operation, and insufficient biocompatibility, which affect the repair efficiency of bone defects. The present invention modifies and processes chitin from marine waste, which is rich in reserves and has excellent biocompatibility, to prepare a high-strength and self-adaptive bone repair scaffold, thereby accelerating the repair of bone defects.

[0031] (2) Although the hydrogel scaffold materials prepared by the prior art have high strength and can provide a biomimetic physical support for the defect site, most of them do not have dynamic properties. Therefore, during natural human activities, they do not fit tightly with the wound, have poor adhesion, and cannot provide a favorable environment for cell activities. The present invention can utilize the advantages of rich active groups of natural polysaccharides to solve the problems of poor adhesion and insufficient dynamic properties of current commercial bone repair hydrogel scaffolds, and prepare a hydrogel scaffold with an efficient dynamic network, which can effectively fit the bone defect site during movement, and effectively simulate the dynamic microenvironment of cells to promote the differentiation of stem cells into osteoblasts and promote the repair of bone defects.

[0032] (3) The bone repair hydrogel scaffolds prepared by the prior art have poor degradation performance in vivo, which hinders the growth rate and space of new tissues during bone repair. The present invention can solve the problem of mismatch between the degradation property of the existing scaffold and the growth rate of new tissues, and the obtained hydrogel scaffold has an appropriate degradation rate and biocompatibility, and will not cause problems such as inflammation, greatly improving the bone defect healing efficiency.

[0033] Other features and advantages of the present invention will be described in detail in the following specific implementation part. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] By describing the exemplary embodiments of the present invention in more detail in conjunction with the drawings, the above and other objects, features, and advantages of the present invention will become more apparent.

[0035] Figure 1 Shows the synthesis route of each component of the hydrogel scaffold of the present invention.

[0036] Figure 2 Shown is a solution of carboxyethyl chitin grafted with adipic dihydrazide (CECT-ADH).

[0037] Figure 3 Shown is a solution of carboxyethyl chitosan grafted with dodecanal (CES-DOD).

[0038] Figure 4 Shown is a solution of polyvinyl alcohol grafted with benzaldehyde (PVA-BA).

[0039] Figure 5 Mechanical strength images of an injectable and self-healing hydrogel for bone repair.

[0040] Figure 6 Healing images of an injectable and self-healing hydrogel for bone repair.

[0041] Figure 7 Injection images of an injectable and self-healing hydrogel for bone repair.

[0042] Figure 8 Adhesion images of an injectable and self-healing hydrogel for bone repair.

[0043] Figure 9 In vivo degradation images of an injectable and self-healing hydrogel for bone repair. Detailed implementation manners

[0044] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0045] Example 1

[0046] (1) Synthesis of carboxyethyl chitin grafted adipic dihydrazide (CECT-ADH)

[0047] The synthesis route of CECT-ADH is as shown in Figure 1 a of. Dissolve carboxyethyl chitin (CECT, 1.50 g, 5.98 mmol) in 300 mL of deionized water. Add adipic dihydrazide (ADH, 2.08 g, 11.95 mmol), morpholineethanesulfonic acid (MES, 639.8 mg, 30 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 286.4 mg, 1.49 mmol) and N-hydroxysuccinimide (NHS, 171.9 mg, 1.49 mmol) into the above solution, and stir the reaction in the dark at 25 °C for 10 hours. The final product CECT-ADH is obtained after dialysis and freeze-drying.

[0048] (2) Synthesis of carboxyethyl chitosan grafted dodecanal (CES-DOD)

[0049] The synthesis route of CES-DOD is as shown in Figure 1As shown in b of . MES (426.5 mg, 20 mmol) and sodium cyanoborohydride (NaCNBH3, 299.8 mg, 4.77 mmol) were introduced into 200 mL of carboxyethyl chitosan solution (CES, 1.00 g, 4.85 mmol). Subsequently, dodecanal (DOD, 88.5 mg, 0.48 mmol) was dissolved in ethanol and added dropwise into the above solution under light protection at a dropping rate of 1 mL / 15 min. After the mixture reacted at 25 °C for 24 h, it was loaded into a dialysis bag (MWCO 8k) and dialyzed against deionized water, and the product CES-DOD was obtained after freeze-drying.

[0050] (3) Synthesis of polyvinyl alcohol grafted benzaldehyde (PVA-BA)

[0051] The synthesis route of PVA-BA is as Figure 1 shown in c of . The detailed steps are as follows: Under a nitrogen atmosphere, 4-hydroxybenzaldehyde (0.68 g, 4.54 mmol), 4-formylbenzoic acid (DMAP, 4.80 g, 40 mmol), and a catalytic amount of anhydrous pyridine were added to 100 mL of polyvinyl alcohol (PVA, 1 g, 22.70 mmol) dimethyl sulfoxide solution. After stirring and reacting at 80 °C for 3 h, succinic anhydride (2 g, 22 mmol) was added. After reacting for 6 h, the temperature was lowered to room temperature, and it was poured into ice ether for reprecipitation. The precipitate was collected and redissolved in deionized water, loaded into a dialysis bag (MWCO 8k), and dialyzed against ethanol aqueous solution and deionized water respectively, and the product PVA-BA was obtained after freeze-drying.

[0052] (4) Preparation of hydrogels

[0053] CECT-ADH, CES-DOD, and PVA-BA were respectively dissolved in PBS buffer solution with pH = 7.4. As Figures 2 - 4 shown, CECT-ADH, CES-DOD, and PVA-BA can be dissolved in PBS at room temperature to obtain a clear aqueous solution. With a constant solid content of 5.0 wt%, PVA-BA and the composite solution containing CECT-ADH and CES-DOD were respectively formulated into 0, 0.2, 0.5, 0.8, and 1.0 at different molar ratios (R = M-NH2:M-CHO). CECT-ADH and CEC-DOD were first mixed evenly, then the PVA-BA solution was added, and then vortexed and mixed evenly quickly and transferred to a polytetrafluoroethylene mold. After curing at 37 °C for 60 min, the CECT-ADH / PVA-BA / CES-DOD hydrogel was obtained. The composition of the CPD0-4 gel is shown in Table 1.

[0054] Table 1. Composition of each component of the hydrogel.

[0055]

[0056] Test Example 1 Rheological Analysis

[0057] The mechanical properties of the hydrogel were investigated by dynamic frequency sweep rheological tests.

[0058] Specifically, a Discovery HR-2 rheometer (TA Instruments, USA) was used to measure the gel time and mechanical properties of the specimens. A 40-mm diameter flat plate was used as the fixture, and the plate gap was set to 1 mm. Before the time and frequency sweeps, a strain amplitude sweep was first performed to determine the linear viscoelastic region of the specimens. During the test, silicone oil was coated on the edge of the specimens to prevent water evaporation. With a fixed frequency of 1 Hz and a strain of 1.0%, a time sweep was carried out to evaluate the gel time of the hydrogel. The three precursor solutions were quickly mixed and transferred to the fixture, and then the curves of the storage modulus (G′) and loss modulus (G″) versus time at 37 °C were recorded to investigate the mechanical properties of the hydrogel.

[0059] As Figure 5 shown, when the molar ratio of [M-NH2:M-CHO] increased from 0 to 1.0, the G' of the CPD hydrogel decreased significantly, from 15.18 ± 0.52 kPa to 2.11 ± 0.77 kPa. At this time, the concentration of CES-DOD and the corresponding Schiff base bonds increased, while the concentration of CECT-ADH and the corresponding acylhydrazone bonds decreased. The increase in the concentration of Schiff base bonds would lead to a poorer network stability and lower strength of the hydrogel.

[0060] Test Example 2 Self - Healing and Injectability Performance Evaluation

[0061] The self-healing ability of the hydrogel CPD2 was evaluated by macroscopic visual method. First, the heart-shaped hydrogels stained with rhodamine B and methyl blue respectively were each cut in half. Then the two heart-shaped hydrogel pieces of different colors were put together along the cut surface, placed in the original mold, and healed at 37 °C in a dryer with high humidity. The self-healing process of the hydrogel was recorded with a digital camera. In addition, the hydrogel CPD2 was prepared in situ in a 21G syringe, and the word "WHU" was written in a petri dish with the syringe.

[0062] As Figure 6 shown, the two hydrogel pieces stained with rhodamine B and methyl blue respectively were each cut in half; then the two heart-shaped hydrogel pieces of different colors were put together. After healing at 37 °C for 0.5 h, the hydrogel began to fuse; after healing for 2 h, the two heart-shaped hydrogel pieces healed into a complete hydrogel piece, and the cutting marks completely disappeared after healing for 7 h. It can be seen that during the repair process, the two dye molecules continuously diffused towards the cut surface and finally fused into purple at the cut surface. The above results indicate that the hydrogel can effectively achieve self-healing.

[0063] As Figure 7As shown, the methyl blue-stained CECT-ADH solution, CES-DOD solution, and PVA-BA solution were quickly mixed in a centrifuge tube and loaded into a syringe barrel, and then aged at 37 °C for 0.5 h. At room temperature, after pressing the syringe plunger, the gel in the syringe was easily extruded through a fine needle (21G), and finally a complete hydrogel was rapidly formed in situ, confirming the excellent injectability of the chitin / chitosan composite hydrogel.

[0064] Test Example 3 Adhesion Experiment

[0065] The adhesion strength of the hydrogel to the surfaces of various materials was evaluated using an adhesion experiment. The steps were as follows: The component solutions of the hydrogel were quickly mixed in proportion and dropped onto the surface of porcine skin to form a hydrogel in situ. After standing at 37 °C for 30 min, the hydrogel was stretched, twisted, bent, and quickly rinsed with water to test the adhesion of the hydrogel to fresh porcine skin. In addition, the above operations were repeated to determine the adhesion strength of the hydrogel to materials such as the heart, kidney, lung, spleen, liver, steel plate, wooden board, rubber, glass, and plastic.

[0066] As Figure 8 shown, in order to vividly demonstrate the adhesion performance of CPD2, images of CPD2 adhering to various surfaces such as steel plates, wooden boards, rubber, glass, and plastic solid materials, as well as biological tissues such as skin, heart, liver, spleen, lung, and kidney were obtained. As can be seen from the figure, after the porcine skin adhered with CPD2 was stretched, twisted, bent, and washed with water, the hydrogel still firmly adhered to the surface of the porcine skin, indicating that the hydrogel can be applied to adhesion in vivo in a moist and dynamic environment. The adhesion mechanism of the chitin / chitosan composite hydrogel is attributed to the amino and dodecyl groups on the natural polysaccharide, which have been proven to be able to insert into the cell membrane bilayer to play a fixing role.

[0067] Test Example 4 In Vivo Degradation Performance

[0068] Eighteen BALB / C mice were used to evaluate the in vivo degradation performance of the hydrogel. First, the mice were anesthetized with sodium pentobarbital, and then 400 μL of the in situ formed hydrogel was injected subcutaneously into the back of the mice using a syringe. Three mice were sacrificed by overdose anesthesia at 5 minutes, 2 weeks, 4 weeks, 6 weeks, and 8 weeks after injection. The back skin of the mice was cut open with ophthalmic scissors to expose the hydrogel, and a camera was used to take pictures to evaluate the degradation of the hydrogel in vivo.

[0069] As Figure 9As shown, the hydrogel was injected subcutaneously into mice, and the intact hydrogel was observed immediately after injection. CPD0 and CPD4 basically disappeared at the 4th week and the 6th week after injection, respectively, while CPD2 still remained at about 24.4% at the 8th week after injection. Therefore, the moderate degradation rate provides sufficient time for the newly formed bone to gradually replace the degraded hydrogel scaffold. Moreover, there was no obvious redness or swelling in the skin tissue around the injection site, which is related to the good biocompatibility of the hydrogel. Therefore, the chitin / chitosan composite hydrogel has ideal in vivo degradation properties and can be used in bone defect repair experiments.

[0070] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A preparation method of an injectable and self-healing hydrogel for bone repair, comprising the following steps: (1) Synthesis of carboxyethyl chitin grafted adipic dihydrazide CECT-ADH; Dissolve carboxyethyl chitin in deionized water, add adipic dihydrazide, morpholineethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide into the above solution, stir and react in the dark, and obtain the product CECT-ADH after dialysis and freeze-drying; (2) Synthesis of carboxyethyl chitosan grafted dodecanal CES-DOD; Introduce morpholineethanesulfonic acid and sodium cyanoborohydride into the carboxyethyl chitosan solution. Subsequently, dissolve dodecanal in ethanol and drop it into the above solution under dark conditions. React the obtained mixture, and obtain the product CES-DOD after dialysis and freeze-drying; (3) Synthesis of polyvinyl alcohol grafted benzaldehyde PVA-BA; Under a nitrogen atmosphere, add 4-hydroxybenzaldehyde, 4-formylbenzoic acid and a catalytic amount of anhydrous pyridine to the polyvinyl alcohol dimethyl sulfoxide solution. After heating and stirring the reaction, add succinic anhydride, continue the reaction, then cool to room temperature, pour it into ice ether for reprecipitation, and obtain the product PVA-BA after redissolution, dialysis and freeze-drying; (4) Preparation of hydrogel; Dissolve CECT-ADH, CES-DOD and PVA-BA in PBS buffer respectively. Then, mix the CECT-ADH solution and the CEC-DOD solution evenly first, and then add the PVA-BA solution. Transfer the obtained mixed solution to a polytetrafluoroethylene mold after vortexing and mixing quickly, and cure to obtain the CECT-ADH / PVA-BA / CES-DOD hydrogel.

2. The preparation method according to claim 1, wherein In step (1), the molar ratio of the carboxyethyl chitin, adipic dihydrazide, morpholineethanesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.8 - 2.2:4.5 - 5.5:0.2 - 0.3:0.2 - 0.

3.

3. The preparation method according to claim 1, wherein In step (1), the temperature of the stirring reaction in the dark is 20 - 30 °C, and the time is 6 - 36 h.

4. The preparation method according to claim 1, wherein In step (2), the molar ratio of dodecanal, morpholineethanesulfonic acid, sodium cyanoborohydride, and carboxyethyl chitosan is 1:40 - 45:8 - 12:8 - 12.

5. The preparation method according to claim 1, wherein In step (2), the dropping rate of the dodecanal ethanol solution is 1 mL / 30 min - 1 mL / min, the temperature of the reaction is 20 - 30 °C, and the time is 12 - 48 h.

6. The preparation method according to claim 1, wherein In step (3), the molar ratio of 4-hydroxybenzaldehyde, 4-formylbenzoic acid, polyvinyl alcohol and succinic anhydride is 1:6 - 10:4 - 6:4 - 6.

7. The preparation method according to claim 1, wherein In step (3), the temperature of the heating reaction is 75 - 85 °C, and the time is 2 - 4 h; The time for continuing the reaction after adding succinic anhydride is 2 - 10 h.

8. The preparation method according to claim 1, wherein In step (4), the pH value of the PBS buffer is 7.2 - 7.6; The mixing of the PVA-BA solution with the CECT-ADH solution and the CES-DOD solution makes the amino / aldehyde ratio in the mixture 1.0 - 3.0; The mixing of the PVA-BA solution with the CECT-ADH solution and the CES-DOD solution makes the solid content of the mixture 1.0 - 5.0 wt%; The concentration of the PVA-BA solution is 1.0 - 5.0 wt%, the concentration of the CECT-ADH solution is 1.0 - 5.0 wt%, and the concentration of the CES-DOD solution is 1.0 - 5.0 wt%. The aging temperature is 37 °C and the time is 10 min - 120 min.

9. A hydrogel prepared by the preparation method according to any one of claims 1-8.

10. Use of the hydrogel according to claim 9 in the preparation of a bone repair material.

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