Chitosan / poly-p-dioxanone composite hydrogel and preparation method and application thereof

Abstract

The present invention discloses a chitosan / polydioxanone composite hydrogel and its preparation method and application. The hydrogel is composed of chitosan with a deacetylation degree of ≥95% and a polydioxanone with a viscosity average molecular weight of 1×10 4 ~1×10 6 The invention discloses a chitosan / polydioxanone composite hydrogel, which is a cross-linked polymer swelling liquid formed by polydioxanone and prepared by repeated freeze-thaw treatment; wherein the amount of polydioxanone used is 1wt% to 18wt% of the weight of chitosan; the chitosan / polydioxanone composite hydrogel introduces polydioxanone molecular chains with excellent flexibility and mechanical properties into a chitosan hydrogel with a network structure to form a hydrogel with a network interpenetrating structure, thereby greatly improving the mechanical strength of the chitosan hydrogel; at the same time, the hydrogel has good degradability and biocompatibility, and its good biocompatibility enables it to be used in tissue engineering; in addition, the preparation method of the hydrogel is simple and the conditions are easy to control, and it has broad application prospects in the biomedical field.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and in particular to a chitosan / polydioxanone composite hydrogel and a preparation method and application thereof. Background Art

[0002] Materials used as tissue engineering scaffolds must have interconnected and porous pore structures to facilitate cell attachment, proliferation, and differentiation, so as to facilitate tissue growth and functional recovery, and provide channels for nutrient transport and the discharge of metabolic waste. Tissue engineering scaffolds should have sufficient mechanical strength to provide support for tissue regeneration at the implant site; tissue engineering scaffolds must also have good biocompatibility, suitable biodegradability, non-toxicity, and characteristics that promote good integration with tissues around the implant site. Studies have shown that chitosan is one of the ideal materials in the field of tissue engineering scaffolds. Compared with traditional surgical methods, chitosan gel treats or repairs defective tissues in a minimally invasive manner, and can also control tissue induction factors through chemical modification; in addition, chitosan hydrogels also have structural characteristics similar to the extracellular matrix of the human body, which can transport and store cells.

[0003] Based on this, the use of chitosan to form porous gel scaffolds is a research and development direction in the field of tissue engineering scaffolds. However, porous gel scaffolds formed by chitosan have some shortcomings, such as: weak mechanical strength of chitosan scaffolds, lack of cell-specific binding sites, susceptibility of scaffold structure to destruction in physiological environments, and low in vivo degradation of chitosan hydrogels, which seriously limit their application in tissue engineering. Therefore, researchers have combined chitosan with synthetic polymers or natural biopolymers to improve the mechanical strength and bioactivity of gel scaffold materials in a complementary manner, providing the necessary mechanical support for tissue regeneration.

[0004] Currently, there are a variety of chitosan bioengineering scaffolds on the market. Published patent CN105944150A discloses a chitosan / collagen hydrogel that, by adding collagen to chitosan, can achieve a biomimetic structure similar to natural bone, increase the production of calcium and sulfate glycosaminoglycans, and have the potential for osteogenesis and chondrogenesis, thereby improving the mechanical properties of the chitosan / collagen gel material; however, the presence of collagen in the body increases the risk of immune rejection in the human body. Published patent CN113929819A discloses a chitosan / polyacrylamide hydrogel that uses chitosan as the main body and polyacrylamide hydrogel as the porous skeleton to prepare a porous composite hydrogel with good mechanical properties, that is, the overall mechanical strength is provided by the polyacrylamide porous skeleton; however, acrylamide has certain neurotoxicity and has shown carcinogenic potential in animal studies. Long-term presence increases health risks, so this hydrogel material is generally used as an adsorption material. In addition, there is a commonly used polyvinyl alcohol-based composite hydrogel. Although this hydrogel has many advantages in performance, polyvinyl alcohol may cause skin irritation and stinging when used on the human body for a long time, and may even cause allergic reactions in some people.

[0005] Therefore, in order to solve the above problems, it is necessary to further develop and modify the porous gel formed by chitosan, so as to ensure that the mechanical strength and flexibility of the hydrogel are improved while making it have good biocompatibility and complete degradability, and effectively avoid health risks such as antigen rejection. Summary of the Invention

[0006] The purpose of the present invention is to provide a chitosan / polydioxanone hydrogel having both good mechanical strength and good biological properties.

[0007] Another object of the present invention is to provide a method for preparing the chitosan / polydioxanone hydrogel.

[0008] Another object of the present invention is to provide an application of the chitosan / polydioxanone hydrogel.

[0009] To this end, the technical solution of the present invention is as follows:

[0010] A chitosan / polydioxanone composite hydrogel is composed of chitosan with a deacetylation degree of ≥95% and a polydioxanone with a viscosity-average molecular weight of 1×10 4 ~1×10 6 The cross-linked polymer swelling liquid formed by polydioxanone is prepared by repeated freeze-thaw treatment; wherein the amount of polydioxanone is 1wt% to 18wt% of the weight of chitosan, and more preferably 3.5wt% to 12wt%.

[0011] The chitosan / polydioxanone composite hydrogel uses chitosan as the main material and polydioxanone as the reinforcing material. By dispersing polydioxanone in chitosan for cross-linking reaction and adopting a physical freeze-thaw method, polydioxanone molecular chains with excellent flexibility and mechanical properties are introduced into the chitosan hydrogel with a network structure, forming a hydrogel with a network interpenetrating structure, thereby modifying the chitosan hydrogel, in order to obtain a kind of chitosan hydrogel.

[0012] It should be noted that in the process of selecting the main material, in addition to chitosan, the applicant also tried to use sodium alginate and gelatin, which are also biocompatible and degradable. However, in the process of preparing hydrogels, although sodium alginate also has good biocompatibility, water absorption and moisture retention, is widely available, safe and non-toxic, and degradable; its gel controllability is poor and its mechanical strength is low, which poses great difficulties in the preparation process. Although gelatin also has good bioaffinity and degradability, the uncross-linked gelatin film has disadvantages such as being easily soluble in water, hard and brittle, and having poor mechanical properties. Therefore, chitosan was finally used as the material for preparing the gel main body.

[0013] A method for preparing the chitosan / polydioxanone composite hydrogel comprises the following steps:

[0014] S1, dissolving chitosan in an acidic aqueous solution to prepare a chitosan solution;

[0015] S2, dissolving the cross-linking agent in the chitosan solution to prepare a chitosan solution containing the cross-linking agent;

[0016] S3, dissolving polydioxanone in an organic solvent to prepare a PPDO solution;

[0017] S4. Slowly add the PPDO solution to the chitosan solution containing the crosslinker, and keep it at a constant temperature of 55°C to 60°C and stir for 1 hour to mix the two evenly; continue stirring for 3 hours to 6 hours to allow the chitosan to fully hydrate and swell; then cool to room temperature to obtain a CS / PPDO swelling solution;

[0018] S5. Repeatedly freeze-thaw the CS / PPDO swelling solution to obtain a freeze-thawed CS / PPDO hydrogel;

[0019] S6. The freeze-thawed CS / PPDO hydrogel is freeze-dried and then placed in deionized water to fully absorb water and swell, thereby obtaining a CS / PPDO composite hydrogel that is in swelling equilibrium and does not contain an organic solvent.

[0020] In step S1 , the acidic aqueous solution is an acetic acid aqueous solution, a formic acid aqueous solution, an oxalic acid aqueous solution or a carbonic acid aqueous solution with a pH of 3-6, preferably an acetic acid aqueous solution with a pH of 3-6.

[0021] In step S1, the amount of the acidic aqueous solution is preferably 20 to 25 times the weight of the chitosan.

[0022] In step S1, the preparation temperature of the chitosan solution is 45°C to 60°C, preferably 55°C.

[0023] In step S2, the cross-linking agent is sodium citrate, calcium chloride or glutaraldehyde, preferably sodium citrate; the amount of the cross-linking agent is preferably 0.25 to 0.3 times the weight of the chitosan.

[0024] In step S3, the organic solvent is dimethyl sulfoxide, N-dimethylformamide or chloroform, preferably dimethyl sulfoxide. The amount of the organic solvent used is determined by the amount of the organic solvent that can dissolve the polydioxanone.

[0025] In step S3, the preparation temperature of the PPDO solution is preferably 60°C to 80°C, preferably 75°C.

[0026] Preferably, before performing step S5, the CS / PPDO swelling solution is defoamed at room temperature to obtain a uniform CS / PPDO swelling solution.

[0027] Defoaming treatment methods include, but are not limited to, ultrasonic defoaming, vacuum defoaming, or room temperature quiescence. Ultrasonic defoaming involves placing the CS / PPDO swelling solution in an ultrasonic cleaner for 10 to 30 minutes to remove bubbles. Vacuum defoaming involves placing the CS / PPDO swelling solution in a vacuum oven at a pressure of 0 to -0.1 MPa for 10 to 30 minutes. Room temperature quiescence involves allowing the solution to stand at room temperature for 2 to 4 hours.

[0028] Preferably, in step S5, the method of repeated freeze-thaw treatment is: freezing the CS / PPDO swelling solution at -48°C to -12°C for 8h to 12h, and then thawing it at room temperature for 3h to 6h; repeating the above freeze-thaw operation 4 to 6 times to obtain a frozen-thawed CS / PPDO hydrogel.

[0029] Preferably, in step S6, the temperature of the freeze-drying treatment is -60°C to -40°C, the pressure is 0.4 mbar to 0.01 mbar, and the freeze-drying time is 24 hours to 48 hours.

[0030] Preferably, in step S6, the water absorption and swelling method is: placing the freeze-dried CS / PPDO hydrogel in deionized water to absorb and swell for 12 hours to 24 hours, so that the hydrogel fully absorbs water.

[0031] An application of the chitosan / polydioxanone composite hydrogel as claimed in claim 1 for skin tissue engineering, soft tissue engineering and bone tissue engineering.

[0032] Compared with the existing technology, the chitosan / polydioxanone composite hydrogel is effective in that: on the one hand, the chitosan / polydioxanone hydrogel uses chitosan as the main material and polydioxanone as the reinforcing material. By dispersing polydioxanone in the chitosan hydrogel containing a cross-linking agent and adopting a physical freeze-thaw method, the polydioxanone molecular chain with excellent flexibility and mechanical properties is introduced into the chitosan hydrogel with a network structure, forming a hydrogel with a network interpenetrating structure; the hydrogel structure greatly improves the mechanical strength of the chitosan hydrogel, and its mechanical properties are greatly improved, including tensile and compressive properties. On the other hand, both chitosan and polydioxanone have good degradability and biocompatibility, and their decomposition products are harmless to the human body, greatly reducing the damage and side effects to human organs, showing good biocompatibility. Moreover, due to the network interpenetrating structure characteristics of the hydrogel, it has a porous structure. The biological properties of the two can also make it more conducive to the adhesion and growth of cells when applied to tissue engineering, giving it broad application prospects in the biomedical field. In addition, the preparation method of the chitosan / polydioxanone hydrogel is simple and the conditions are easy to control, so it has broad application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1( a ) is a photograph of a columnar sample of chitosan / polydioxanone hydrogel prepared in Examples 1, 2, and 3 of the present invention placed in a sample bottle;

[0034] FIG1( b ) is a photograph of a sheet sample of the chitosan / polydioxanone hydrogel prepared in Example 1 of the present invention;

[0035] Figure 2 This is a comparison of infrared spectra of the CS / PPDO hydrogel prepared in Example 3 of the present invention and the chitosan hydrogel prepared in Comparative Example 1;

[0036] Figure 3 This is a scanning electron micrograph of the brittle fracture surface of the chitosan / polydioxanone hydrogel prepared in Example 3 of the present invention;

[0037] Figure 4 A comparison graph of mechanical tensile test result curves of the chitosan / polydioxanone hydrogels prepared in Examples 1, 3, 6, and 7 of the present invention and the chitosan hydrogel prepared in Comparative Example 1;

[0038] Figure 5 This is a bar chart comparing the water absorption and swelling test results of the chitosan / polydioxanone hydrogels prepared in Examples 3, 6 and 7 of the present invention and the chitosan hydrogel of Comparative Example 1 in deionized water;

[0039] Figure 6 This is a line graph comparing the mass loss of the chitosan / polydioxanone hydrogel prepared in Example 3 of the present invention and the chitosan hydrogel prepared in Comparative Example 1 after seven weeks of simulated degradation experiments in phosphate buffer. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the following embodiments are by no means intended to limit the present invention in any way.

[0041] Example 1

[0042] A chitosan / polydioxanone composite hydrogel is prepared by the following method:

[0043] S1. Add 0.85 g of chitosan (CS) to 20 mL of a mixed solution prepared by acetic acid and water in a weight ratio of 1:1, and stir and dissolve in a constant temperature oil bath at 55° C. to obtain a chitosan solution; wherein the chitosan is produced by Shanghai Aladdin Biochemical Technology Co., Ltd., and has a deacetylation degree of ≥95% and a viscosity of 100 mPa.s to 200 mPa.s. The same applies to the following Examples 2 to 7;

[0044] S2. Add 0.25 g of sodium citrate to the chitosan solution, and continue to stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution containing sodium citrate;

[0045] S3, 0.01g viscosity average molecular weight 5×10 5 Add polydioxanone (PPDO) into 20 mL of dimethyl sulfoxide aqueous solution and stir to dissolve at 75°C to obtain PPDO solution;

[0046] S4. Slowly add the PPDO solution to the chitosan solution containing sodium citrate, and continue stirring in a 55°C constant temperature oil bath for 1 hour to mix evenly. Stir for another 6 hours to allow the chitosan to fully hydrate and swell, and then cool to room temperature to obtain a CS / PPDO swelling solution.

[0047] S5. At room temperature, the CS / PPDO swelling solution was placed in an ultrasonic cleaner and ultrasonicated for 30 minutes to remove bubbles to obtain a uniform CS / PPDO swelling solution. The uniform CS / PPDO swelling solution was then frozen at -24°C for 12 hours and returned to room temperature for 6 hours to thaw. The freeze-thaw operation was repeated 5 times to obtain a CS / PPDO hydrogel.

[0048] S6. Place the hydrogel after freeze-thawing in step S5 into a freeze dryer and freeze-dry it at -40°C and 0.1 mbar for 24 hours to completely dry the hydrogel, thereby removing organic solvents such as acetic acid and dimethyl sulfoxide from the hydrogel and further enhancing the mechanical strength of the hydrogel by freeze-thawing. Remove the gel and place it in deionized water to fully absorb water for 12 hours to achieve swelling equilibrium and produce a CS / PPDO composite hydrogel free of organic solvents.

[0049] Example 2

[0050] A chitosan / polydioxanone composite hydrogel is prepared by the following method:

[0051] S1. Add 0.85 g of chitosan (CS) to 20 mL of a mixed solution of acetic acid and water in a weight ratio of 1:1, and stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution.

[0052] S2. Add 0.255 g of calcium chloride to the chitosan solution, and continue to stir and dissolve in a constant temperature oil bath at 60°C to obtain a chitosan solution containing calcium chloride;

[0053] S3, 0.03g viscosity average molecular weight 1.0×10 4 Add polydioxanone (PPDO) into 20 mL of dimethyl sulfoxide solution and stir to dissolve at 75°C to obtain PPDO solution;

[0054] S4. Slowly add the PPDO solution to the chitosan solution containing calcium chloride, place in a 60°C constant temperature oil bath and continue stirring for 1 hour to mix evenly, then stir for another 5 hours to allow the chitosan to fully hydrate and swell, and then cool to room temperature to obtain a CS / PPDO swelling solution;

[0055] S5. At room temperature, the CS / PPDO swelling solution was placed in an ultrasonic cleaner and ultrasonicated for 30 minutes to remove bubbles to obtain a uniform CS / PPDO swelling solution. The uniform CS / PPDO swelling solution was then frozen at -48°C for 8 hours and then thawed at room temperature for 3 hours. The freeze-thaw operation was repeated four times to obtain a CS / PPDO hydrogel.

[0056] S6. Place the hydrogel after freeze-thawing in step S5 into a freeze dryer and freeze-dry it at -60°C and 0.4 mbar for 24 hours to completely dry the hydrogel, thereby removing organic solvents such as acetic acid and dimethyl sulfoxide from the hydrogel and further enhancing the mechanical strength of the hydrogel by freeze-thawing. Remove the gel and place it in deionized water to fully absorb water for 12 hours to achieve swelling equilibrium and produce a CS / PPDO composite hydrogel free of organic solvents.

[0057] Example 3

[0058] A chitosan / polydioxanone composite hydrogel is prepared by the following method:

[0059] S1. Add 0.85 g of chitosan (CS) to 20 mL of a mixed solution of acetic acid and water in a weight ratio of 1:1, and stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution.

[0060] S2. Add 0.25 g of sodium citrate to the chitosan solution, and continue to stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution containing sodium citrate as a crosslinker;

[0061] S3, 0.05g viscosity average molecular weight of 5.0×10 5 Add polydioxanone (PPDO) into 20 mL of dimethyl sulfoxide solution and stir to dissolve at 75°C to obtain PPDO solution;

[0062] S4. Slowly add the PPDO solution to the chitosan solution containing sodium citrate, and continue stirring in a 55°C constant temperature oil bath for 1 hour to mix evenly. Stir for another 6 hours to allow the chitosan to fully hydrate and swell, and then cool to room temperature to obtain a CS / PPDO swelling solution.

[0063] S5. At room temperature, the CS / PPDO swelling solution was placed in an ultrasonic cleaner and ultrasonicated for 30 minutes to remove bubbles to obtain a uniform CS / PPDO swelling solution. The uniform CS / PPDO swelling solution was then frozen at -24°C for 12 hours and then thawed at room temperature for 6 hours. The freeze-thaw operation was repeated 5 times to obtain a CS / PPDO hydrogel.

[0064] S6. Place the hydrogel after freeze-thawing in step S5 into a freeze dryer and freeze-dry it at -40°C and 0.1 mbar for 24 hours to completely dry the hydrogel, thereby removing organic solvents such as acetic acid and dimethyl sulfoxide from the hydrogel and further enhancing the mechanical strength of the hydrogel by freeze-thawing. Remove the gel and place it in deionized water to fully absorb water for 12 hours to achieve swelling equilibrium and produce a CS / PPDO composite hydrogel free of organic solvents.

[0065] Example 4

[0066] A chitosan / polydioxanone composite hydrogel is prepared by the following method:

[0067] S1. Add 0.85 g of chitosan (CS) to 20 mL of a mixed solution of acetic acid and water in a weight ratio of 1:1, and stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution.

[0068] S2. Add 0.213 g of sodium citrate to the chitosan solution, and continue to stir and dissolve in a constant temperature oil bath at 45°C to obtain a chitosan solution containing sodium citrate as a crosslinker;

[0069] S3, 0.07g viscosity average molecular weight 1.0×10 6 Add polydioxanone (PPDO) into 20 mL of dimethyl sulfoxide solution and stir to dissolve at 80°C to obtain PPDO solution;

[0070] S4. Slowly add the PPDO solution to the chitosan solution containing sodium citrate, and continue stirring in a 60°C constant temperature oil bath for 2 h to mix evenly. Stir for another 6 h to allow the chitosan to fully hydrate and swell, and then cool to room temperature to obtain a CS / PPDO swelling solution.

[0071] S5. At room temperature, the CS / PPDO swelling solution was placed in an ultrasonic cleaner and ultrasonicated for 30 minutes to remove bubbles to obtain a uniform CS / PPDO swelling solution. The uniform CS / PPDO swelling solution was then frozen at -36°C for 10 hours and then thawed at room temperature for 5 hours. The freeze-thaw operation was repeated four times to obtain a CS / PPDO hydrogel.

[0072] S6. Place the hydrogel after freeze-thawing in step S5 into a freeze dryer and freeze-dry it at -45°C and 0.05 mbar for 48 hours to completely dry the hydrogel, thereby removing organic solvents such as acetic acid and dimethyl sulfoxide from the hydrogel and further enhancing the mechanical strength of the hydrogel by freeze-thawing. Remove the gel and place it in deionized water to fully absorb water for 24 hours to achieve swelling equilibrium and produce a CS / PPDO composite hydrogel free of organic solvents.

[0073] Example 5

[0074] A chitosan / polydioxanone composite hydrogel is prepared by the following method:

[0075] S1. Add 0.85 g of chitosan (CS) to 20 mL of a mixed solution of acetic acid and water in a weight ratio of 1:1, and stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution.

[0076] S2. Add 0.25 g of sodium citrate to the chitosan solution, and continue to stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution containing sodium citrate as a crosslinker;

[0077] S3, 0.09g viscosity average molecular weight of 5.0×10 5 Add polydioxanone (PPDO) into 20 mL of dimethyl sulfoxide solution and stir to dissolve at 75°C to obtain PPDO solution;

[0078] S4, slowly add the PPDO solution to the chitosan solution containing sodium citrate, and place it in a constant temperature oil bath at 55°C and continue to stir for 1 h to mix evenly, then stir for 6 h to allow the chitosan to fully hydrate and swell, and then cool to room temperature to obtain a CS / PPDO swelling solution;

[0079] S5, place the CS / PPDO swelling solution at room temperature in an ultrasonic cleaner and ultrasonically treat it for 30 min to remove bubbles, to obtain a uniform CS / PPDO swelling solution; then place the uniform CS / PPDO swelling solution in a freezer at -24°C for 12 h, and then take it out and thaw it at room temperature for 6 h; repeat the freezing-thawing operation 5 times to obtain a CS / PPDO hydrogel;

[0080] S6, place the hydrogel after the freezing-thawing operation in step S5 in a freeze dryer, and freeze dry it at -40°C and 0.1 mbar for 24 h to completely dry the hydrogel, so as to remove organic solvents such as acetic acid and dimethyl sulfoxide in the hydrogel, and also to further strengthen the mechanical strength of the hydrogel by freezing and thawing; take out the gel and place it in deionized water to fully absorb water for 12 h, so as to obtain a CS / PPDO composite hydrogel that has reached swelling equilibrium and does not contain organic solvents.

[0081] Example 6

[0082] A chitosan / poly-p-dioxanone composite hydrogel is prepared by the following method:

[0083] S1, add 0.85 g of chitosan (CS) to 20 mL of a mixed solution prepared by mixing acetic acid and water at a weight ratio of 1:1, and place it in a constant temperature oil bath at 55°C and stir to dissolve, to obtain a chitosan solution;

[0084] S2, add 0.25 g of sodium citrate to the chitosan solution, and continue to place it in a constant temperature oil bath at 55°C and stir to dissolve, to obtain a chitosan solution to which a sodium citrate crosslinking agent has been added;

[0085] S3, add 0.10 g of poly-p-dioxanone (PPDO) with a viscosity average molecular weight of 5.0 x 10 5 to 20 mL of dimethyl sulfoxide solution, and stir to dissolve at 75°C, to obtain a PPDO solution;

[0086] S4, slowly add the PPDO solution to the chitosan solution containing sodium citrate, and place it in a constant temperature oil bath at 55°C and continue to stir for 1 h to mix evenly, then stir for 6 h to allow the chitosan to fully hydrate and swell, and then cool to room temperature to obtain a CS / PPDO swelling solution;

[0087] S5. At room temperature, the CS / PPDO swelling solution was placed in an ultrasonic cleaner and ultrasonicated for 30 minutes to remove bubbles to obtain a uniform CS / PPDO swelling solution. The uniform CS / PPDO swelling solution was then frozen at -24°C for 12 hours and then thawed at room temperature for 6 hours. The freeze-thaw operation was repeated 5 times to obtain a CS / PPDO hydrogel.

[0088] S6. Place the hydrogel after freeze-thawing in step S5 into a freeze dryer and freeze-dry it at -40°C and 0.1 mbar for 24 hours to completely dry the hydrogel, thereby removing organic solvents such as acetic acid and dimethyl sulfoxide from the hydrogel and further enhancing the mechanical strength of the hydrogel by freeze-thawing. Remove the gel and place it in deionized water to fully absorb water for 12 hours to achieve swelling equilibrium and produce a CS / PPDO composite hydrogel free of organic solvents.

[0089] Example 7

[0090] A chitosan / polydioxanone composite hydrogel is prepared by the following method:

[0091] S1. Add 0.85 g of chitosan (CS) to 20 mL of a mixed solution of acetic acid and water in a weight ratio of 1:1, and stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution.

[0092] S2. Add 0.25 g of sodium citrate to the chitosan solution, and continue to stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution containing sodium citrate as a crosslinker;

[0093] S3, 0.15g viscosity average molecular weight of 5.0×10 5 Add polydioxanone (PPDO) into 20 mL of dimethyl sulfoxide solution and stir to dissolve at 75°C to obtain PPDO solution;

[0094] S4. Slowly add the PPDO solution to the chitosan solution containing sodium citrate, and continue stirring in a 55°C constant temperature oil bath for 1 hour to mix evenly. Stir for another 6 hours to allow the chitosan to fully hydrate and swell, and then cool to room temperature to obtain a CS / PPDO swelling solution.

[0095] S5. At room temperature, the CS / PPDO swelling solution was placed in an ultrasonic cleaner and ultrasonicated for 30 minutes to remove bubbles to obtain a uniform CS / PPDO swelling solution. The uniform CS / PPDO swelling solution was then frozen at -24°C for 12 hours and then thawed at room temperature for 6 hours. The freeze-thaw operation was repeated 5 times to obtain a CS / PPDO hydrogel.

[0096] S6. Place the hydrogel after freeze-thawing in step S5 into a freeze dryer and freeze-dry it at -40°C and 0.1 mbar for 24 hours to completely dry the hydrogel, thereby removing organic solvents such as acetic acid and dimethyl sulfoxide from the hydrogel and further enhancing the mechanical strength of the hydrogel by freeze-thawing. Remove the gel and place it in deionized water to fully absorb water for 12 hours to achieve swelling equilibrium and produce a CS / PPDO composite hydrogel free of organic solvents.

[0097] Comparative Example 1

[0098] A chitosan hydrogel is prepared by the following method:

[0099] S1. Add 0.85 g of chitosan (CS) to 20 mL of a mixed solution of acetic acid and water in a weight ratio of 1:1, and stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution.

[0100] S2. Add 0.25 g of sodium citrate to the chitosan solution, and continue to stir and dissolve in a constant temperature oil bath at 55°C to obtain a chitosan solution containing sodium citrate as a crosslinker;

[0101] S3. Add 20 mL of dimethyl sulfoxide aqueous solution to the chitosan solution and stir at 55°C for 6 h to allow the chitosan to fully hydrate and swell.

[0102] S4. At room temperature, the chitosan solution was placed in an ultrasonic cleaning machine and ultrasonicated for 30 minutes to remove bubbles to obtain a uniform chitosan swelling solution; the uniform chitosan swelling solution was frozen at -24°C for 12 hours, and then returned to room temperature for 6 hours to thaw; the freeze-thaw operation was repeated 5 times to obtain a chitosan hydrogel;

[0103] S5. The hydrogel after freeze-thawing in step S5 is placed in a freeze dryer and freeze-dried at -40°C and 0.1 mbar for 24 hours. The hydrogel is then placed in deionized water and fully absorbed for 12 hours to achieve swelling equilibrium, thereby obtaining a CS hydrogel free of organic solvent.

[0104] Comparative Example 2

[0105] A chitosan / polydioxanone composite hydrogel is prepared by a method substantially the same as that in Example 1, except that the chitosan in step S1 is chitosan with a deacetylation degree of ≈65%.

[0106] Performance testing:

[0107] (I) Macro- and microstructural characterization of chitosan / polydioxanone hydrogels:

[0108] 1. Macrostructure characterization:

[0109] Figure 1(a) shows a photograph of columnar chitosan / polydioxanone hydrogels prepared in Examples 1, 2, and 3 placed in sample vials. As can be seen, the hydrogels prepared in Examples 1-3 are slightly yellowish and transparent. They were placed inverted in the three sample vials and did not exhibit fluidity, demonstrating successful hydrogel preparation. Similarly, the chitosan / polydioxanone hydrogels prepared in Examples 4-7 exhibited similar performance.

[0110] As shown in Figure 1(b), the chitosan / polydioxanone hydrogel swelling solution prepared in Example 1 is placed in a sample plate slot, and then a sheet sample is obtained after repeated freeze-thaw, freeze-drying, and swelling. As can be seen from the figure, when the sheet sample is held at both ends with both hands and stretched, the sheet sample is obviously not easily broken and has certain tensile properties.

[0111] 2. Microstructure characterization:

[0112] The chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 were subjected to infrared testing to verify whether the PPDO long chains were successfully introduced into the CS / PPDO hydrogel structure. Figure 2 The infrared spectra of the CS / PPDO hydrogel prepared in Example 3 and the chitosan hydrogel prepared in Comparative Example 1 are shown. -1 The left and right sides are carbonyl C=O stretching vibration absorption peaks; 3251cm in the structure of chitosan -1 The broad peaks on the left and right are the overlapping peaks of NH and OH; at 2922cm -1 The peaks near it can represent OH functional groups; and the wave number 1715cm -1 It is usually related to the stretching vibration of carbonyl (C=O), and the composite hydrogel has a wavelength of 1715 cm -1 The peak at 1715 cm is obviously higher, and PPDO mainly contains carbonyl (C=O), which indicates that PPDO is successfully added into CS / PPDO hydrogel as a reinforcing material. -1 The left and right are carbonyl C=O stretching vibration absorption peaks. Similarly, the infrared spectra of other examples also have the same characterization results as above.

[0113] The brittle fracture surfaces of the chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 were scanned by electron microscopy to determine their microstructural characteristics. Figure 3Shown is a scanning electron micrograph of a brittle fracture surface of the CS / PPDO hydrogel prepared in Example 3. The image demonstrates that the microstructure of the CS / PPDO hydrogel prepared in Example 3 exhibits a continuous interpenetrating network, with uniformly distributed micron-sized pores ranging from 1 μm to 8 μm visible on the fracture surface. This porous structure not only facilitates cell growth and adhesion but also facilitates the hydrogel's absorption of tissue fluid, providing an excellent environment for cell growth. Similarly, scanning electron micrographs of brittle fracture surfaces of the CS / PPDO hydrogels prepared in other examples exhibit similar microstructural features.

[0114] (2) Mechanical strength test of chitosan / polydioxanone hydrogel;

[0115] The chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 and the CS hydrogel prepared in Comparative Example 1 were subjected to tensile testing. The specific testing method was to affix a 35 mm × 20 mm × 2 mm (length × width × height) strip of gel to both ends of an instrument. The tensile strain and tensile strength of the hydrogels were then calculated at a tensile speed of 200 mm / min. The specific test results are shown in Table 1 below.

[0116] Table 1:

[0117]

[0118]

[0119] From the test results in Table 1, it can be seen that compared with the pure CS hydrogel prepared in Comparative Example 1, the tensile strain at break and the tensile strength at break of the chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 are significantly improved. Specifically, the tensile strain at break is increased from 7% to 57%, and the tensile strength at break is increased from 170 kPa to 563 kPa.

[0120] In Examples 1, 3, 5, 6 and 7, while keeping other conditions unchanged, as the amount of polydioxanone added to chitosan gradually increased, the tensile strain at break and the tensile strength at break of the chitosan / polydioxanone hydrogel showed a trend of first increasing and then decreasing; therefore, in the modification of the chitosan hydrogel network structure using polydioxanone, the amount of polydioxanone added is also an important modification factor.

[0121] In the present application, the suitable range of the addition amount of polydioxanone relative to chitosan is 1wt% to 18wt%. When the addition amount of polydioxanone relative to chitosan is 1wt%, it can be seen from Table 1 that the tensile strain at break and the tensile strength at break decrease more significantly than when the addition amount is 3.5%. According to actual experimental conditions, when the addition amount of polydioxanone relative to chitosan is less than 1wt%, the tensile strain at break and the tensile strength at break of the hydrogel are not significantly improved. When the addition amount of polydioxanone relative to chitosan exceeds 18wt%, it is difficult for polydioxanone to be dispersed in the chitosan solution in the form of a solution, which also results in no significant improvement in the tensile strain at break and the tensile strength at break of the hydrogel.

[0122] Among them, when the addition amount of polydioxanone relative to chitosan is in the range of 3.5wt% to 12%, the tensile strength at break of the chitosan / polydioxanone hydrogel is greater than 400KPa, and the tensile strain at break is greater than 400KPa, which is consistent with the tensile strength at break requirement of hydrogels used as tissue engineering scaffolds; and when the addition amount of polydioxanone relative to chitosan is less than 3.5wt% or greater than 12%, at least one of the tensile strain at break and the tensile strength at break will significantly decrease; therefore, the suitable range of the addition amount of polydioxanone relative to chitosan is 1wt% to 18wt%, more preferably 3.5wt% to 12wt%.

[0123] like Figure 4 Graphs showing the mechanical tensile properties of the CS / PPDO hydrogels prepared in Examples 1, 3, 6, and 7, as well as the CS hydrogel prepared in Comparative Example 1, show that the mechanical properties of the hydrogels prepared after the addition of PPDO are superior to those of pure chitosan hydrogels. The data and graphs also indicate that the mechanical properties of the composite hydrogels gradually increase with the addition of polydioxanone, reaching peak performance at 5 wt%, before gradually declining. This is likely due to the formation of an interpenetrating network in the hydrogels due to the addition of the macromolecular polydioxanone, which enhances their mechanical properties. However, an excess of macromolecules can disrupt the hydrogel's internal three-dimensional structure, leading to a decrease in mechanical properties. In summary, the hydrogels exhibited optimal mechanical properties when the polydioxanone addition reached 5 wt%.

[0124] The tensile strain at break and tensile strength at break of the chitosan / polydioxanone hydrogel prepared in Comparative Example 2 were too low because the deacetylation degree of chitosan was too low, which resulted in reduced solubility of chitosan and corresponding reduced crystallinity and moldability, resulting in too low mechanical properties.

[0125] The PPDO / CS hydrogels prepared in Examples 1 to 7 and the chitosan hydrogel in Comparative Example 1 were subjected to compression tests. The specific test method is as follows: a 22 mm × 17 mm (diameter × height) cylindrical hydrogel is placed in a testing instrument. The gel column sample is compressed at a compression rate of 1 mm / s at room temperature, and the compression deformation is set to 30%. The specific test results are shown in Table 2 below.

[0126] Table 2:

[0127]

[0128]

[0129] As can be seen from the test results in Table 2, compared with the pure CS hydrogel prepared in Comparative Example 1, the compressive strength and compressive yield strain of the chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 were significantly improved. Specifically, the compressive strength increased from 70 kPa to 110 kPa, and the compressive yield strain increased from 9.25% to 25.51%.

[0130] In Examples 1, 3, 5, 6 and 7, while keeping other conditions unchanged, as the amount of polydioxanone added to chitosan gradually increased, the tensile strain at break and the tensile strength at break of the chitosan / polydioxanone hydrogel showed a trend of first increasing and then decreasing; therefore, in the modification of the chitosan hydrogel network structure using polydioxanone, the amount of polydioxanone added is also an important modification factor.

[0131] However, the low-deacetylation chitosan hydrogel prepared in Comparative Example 2 has too low mechanical strength and poor processing performance after modification with polydioxanone. Its mechanical properties can no longer meet the material requirements. Therefore, a series of subsequent tests such as compression were not performed, and only other properties of chitosan with a deacetylation degree ≥95% were explored.

[0132] As the amount of polydioxanone added increases, the compressive strength of chitosan / polydioxanone hydrogel shows a trend of first increasing and then decreasing. The reason is that an appropriate amount of polydioxanone and chitosan form a cross-linked network, which improves the compressive strength of the hydrogel. The presence of excessive polydioxanone destroys the cross-linked network structure and the compressive performance decreases. However, the increase in compressive strength is beneficial to its application in tissue engineering.

[0133] The above two mechanical property test results prove that: in this application, polydioxanone is used as a reinforcing material to structurally modify chitosan. By dispersing polydioxanone in a chitosan hydrogel containing a cross-linker and adopting a physical freeze-thaw method, a network interpenetrating structure is formed between the polydioxanone molecular chain and the chitosan hydrogel with a network structure, thereby achieving the purpose of significantly improving both the tensile and compressive properties of the hydrogel.

[0134] (3) Swelling rate test of chitosan / polydioxanone hydrogel;

[0135] The chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 and the CS hydrogel prepared in Comparative Example 1 were tested for swelling properties. The specific testing method was to measure the dry mass m1 of the hydrogel, then place it in deionized water until it fully swelled, recording its mass m2. The swelling ratio R = (m2 - m1) / m1. The specific test results are shown in Table 3 below.

[0136] Table 3:

[0137] name Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Swelling rate 629％ 561％ 552％ 540.2％ 532％ 523％ 505％ 705％

[0138] As can be seen from the test results in Table 3, although the swelling ratios of the chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 decreased compared with the pure CS hydrogel prepared in Comparative Example 1, the swelling ratios of the chitosan / polydioxanone hydrogels were still stably maintained at above 500%, indicating a good swelling state. Furthermore, the swelling ratios of the chitosan / polydioxanone hydrogels hardly changed with the addition amount of polydioxanone.

[0139] like Figure 5 Shown are the swelling rate curves in water for the chitosan / polydioxanone hydrogels prepared in Examples 3, 6, and 7, and the chitosan hydrogel prepared in Comparative Example 1. The figure shows that, while the water absorption rates of the three chitosan / polydioxanone hydrogels are lower than those of the pure CS hydrogel, they remain stable at over 550%, demonstrating good swelling properties.

[0140] (IV) Degradation performance test:

[0141] The chitosan / polydioxanone hydrogels prepared in Examples 1 to 7 and the CS hydrogel prepared in Comparative Example 1 were subjected to a seven-week degradation experiment. Specifically, the degradation was simulated in vitro in a phosphate buffer solution with an initial pH of 7.4 in a 37°C constant temperature water bath. The results are shown in Table 4 below.

[0142] Table 4:

[0143] name Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Comparative Example 1 Degradation amount 20.47％ 21.36％ 22.5％ 23.1％ 23.4％ 24.1％ 24.3％ 19.54％

[0144] As can be seen from the test results in Table 4, the chitosan hydrogel of Comparative Example 1 is well known. Although it has the advantages of good biocompatibility and in vivo degradation, the strong hydrogen bonds in the chitosan molecules make it only soluble in acidic media and its solubility in water is also low. The degradation performance of the chitosan / polydioxanone hydrogels prepared according to Examples 1 to 7, on the other hand, ranged from 20.47% to 24.3% after seven weeks of degradation, which is significantly improved compared to the chitosan hydrogel of Comparative Example 1. In other words, after structural modification, the chitosan / polydioxanone hydrogel of the present application has a shortened degradation cycle, making it more suitable for human application requirements.

[0145] like Figure 6 A graph showing the mass loss changes during the degradation experiments for Example 3 and Comparative Example 1 is shown. The graph shows that the degradation rate of Example 3 was significantly faster than that of the comparative example during the first two weeks of the experiment. This is because the degradation cycle of polydioxanone is shorter than that of chitosan, resulting in the majority of polydioxanone beginning to degrade within the first two weeks. Chitosan, on the other hand, degrades slowly throughout the entire process due to its longer degradation cycle. This also results in the overall degradation rate of the composite hydrogel being greater than that of the chitosan hydrogel in the comparative example.

[0146] The degradation experiment proves that the chitosan / polydioxanone hydrogel of the present application optimizes the degradation rate of the chitosan hydrogel by structurally modifying the chitosan hydrogel, making it more suitable for use as a tissue engineering scaffold.

Claims

1. A chitosan / polydioxanone composite hydrogel, characterized in that: It is composed of chitosan with a deacetylation degree of ≥95% and a viscosity-average molecular weight of 1×10 4 ~1×10 6 The cross-linked polymer swelling liquid formed by polydioxanone is prepared by repeated freeze-thaw treatment; wherein the amount of polydioxanone is 1wt%~18wt% of the weight of chitosan.

2. A method for preparing the chitosan / polydioxanone composite hydrogel according to claim 1, characterized in that: Here are the steps: S1, dissolving chitosan in an acidic aqueous solution to prepare a chitosan solution; S2, dissolving the cross-linking agent in the chitosan solution to prepare a chitosan solution containing the cross-linking agent; S3, dissolving polydioxanone in an organic solvent to prepare a PPDO solution; S4. Slowly add the PPDO solution to the chitosan solution containing the crosslinker, and keep it at a constant temperature of 55°C to 60°C and stir for 1 hour to mix the two evenly; continue stirring for 3 hours to 6 hours to allow the chitosan to fully hydrate and swell; then cool to room temperature to obtain a CS / PPDO swelling solution; S5. Repeatedly freeze-thaw the CS / PPDO swelling solution to obtain a freeze-thawed CS / PPDO hydrogel; S6. The freeze-thawed CS / PPDO hydrogel is freeze-dried and then placed in deionized water to fully absorb water and swell, thereby obtaining a CS / PPDO composite hydrogel that is in swelling equilibrium and does not contain an organic solvent.

3. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 2, characterized in that: In step S1 , the acidic aqueous solution is an acetic acid aqueous solution, a formic acid aqueous solution, an oxalic acid aqueous solution, or a carbonic acid aqueous solution with a pH of 3 to 6.

4. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 2, wherein: In step S2, the cross-linking agent is sodium citrate, calcium chloride or glutaraldehyde; the amount of the cross-linking agent is 0.25 to 0.3 times the weight of the chitosan.

5. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 4, characterized in that: The cross-linking agent is sodium citrate.

6. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 2, wherein: In step S3, the organic solvent is dimethyl sulfoxide, N,N-dimethylformamide or chloroform.

7. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 2, wherein: Before step S5, the CS / PPDO swelling solution is defoamed at room temperature to obtain a uniform CS / PPDO swelling solution.

8. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 2, wherein: In step S5, the freeze-thaw treatment is repeated multiple times as follows: the CS / PPDO swelling solution is frozen at -48°C to -12°C for 8 h to 12 h, and then returned to room temperature for 3 h to 6 h to thaw; the freeze-thaw operation is repeated 4 to 6 times to obtain a frozen-thawed CS / PPDO hydrogel.

9. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 2, wherein: In step S6, the freeze-drying treatment temperature is -60°C to -40°C, the pressure is 0.4 mbar to 0.01 mbar, and the freeze-drying time is 24 hours to 48 hours.

10. The method for preparing the chitosan / polydioxanone composite hydrogel according to claim 2, characterized in that: In step S6, the water absorption and swelling method is: placing the freeze-dried CS / PPDO hydrogel in deionized water to absorb and swell for 12 hours to 24 hours, so that the hydrogel can fully absorb water.

11. Use of the chitosan / polydioxanone composite hydrogel according to claim 1 in preparing products for skin tissue engineering, soft tissue engineering or bone tissue engineering.

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