Thermosensitive controlled-release hydrogel composite material for osteoarthritis repair and preparation method thereof

Abstract

The present invention relates to the technical field of biomaterials, and specifically to a temperature-sensitive controlled-release hydrogel composite material for osteoarthritis repair and a preparation method thereof. First, the chondroitin sulfate-modified liposomes encapsulating FGF18 are prepared by a chloroform dissolution method to ensure a high encapsulation rate of FGF18; subsequently, 3-allyl-2-hydroxybenzaldehyde is reacted with sodium alginate to introduce an aldehyde group to obtain modified sodium alginate; a hydrogel solution is formed by mixing the modified sodium alginate, hydroxybutyl chitosan and gelatin; the chondroitin sulfate-modified liposomes encapsulating FGF18 are added to the hydrogel mixed solution to obtain an upper-layer hydrogel mixed solution; nano-hydroxyapatite is added to the hydrogel mixed liquid to obtain a lower-layer hydrogel mixed solution; the lower-layer hydrogel mixed solution is injected into a mold, and after 10 minutes, the upper-layer hydrogel mixed solution is added, and the temperature is raised to form a gel, thereby obtaining the temperature-sensitive controlled-release hydrogel composite material.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and specifically to a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair and a preparation method thereof. Background Technique

[0002] Osteoarthritis is a complex disease that affects joints. In damaged joints, macrophages can be activated and polarized into M1 type, and secrete pro-inflammatory factors such as IL-1, IL-6 and TNF-α, resulting in the degradation of the extracellular matrix (ECM); while M2-type macrophages inhibit the inflammatory response by secreting anti-inflammatory factors such as TGF-β and IL-10. Therefore, regulating macrophage polarization is considered an effective strategy for treating osteoarthritis.

[0003] Currently, it is difficult to effectively balance immune regulation and anti-inflammatory effects using a single treatment method, and it is impossible to maintain a long-term immune environment to promote repair. Fibroblast growth factor (FGF18) can promote chondrocyte growth and is related to M2 macrophage polarization; chondroitin sulfate (ChS) is a glucuronic acid polysaccharide, which can relieve joint pain, promote cartilage repair and improve joint function; combining the two and embedding them into a polymer hydrogel to develop a thermosensitive controlled-release hydrogel composite material is of great significance for the treatment of osteoarthritis and the recovery of joint function. Summary of the Invention

[0004] The purpose of the present invention is to provide a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair and a preparation method thereof to solve the problems raised in the above background technique.

[0005] To solve the above technical problems, the present invention provides the following technical solution: A preparation method of a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair, and the preparation method includes the following steps:

[0006] Step 1:

[0007] Take soybean lecithin, cholesterol, and DSPE-PEG-CSA and dissolve them together in chloroform; after evaporating to form a film under reduced pressure, add it to the FGF18 solution for hydration, extrude it after ultrasonic treatment, supplement deionized water and remove the unencapsulated FGF18 by nanofiltration dialysis to obtain chondroitin sulfate-modified liposomes coated with FGF18;

[0008] Step 2:

[0009] S1: Disperse 3 - allyl - 2 - hydroxybenzaldehyde in ultrapure water to obtain a 3 - allyl - 2 - hydroxybenzaldehyde dispersion; mix the 3 - allyl - 2 - hydroxybenzaldehyde dispersion with a sodium alginate solution, add the initiator potassium persulfate, stir for 10 - 15 min, then raise the temperature to 80 - 90 °C and react for 3 - 5 h. After cooling, dialyze to remove impurities, then filter by suction and freeze - dry to obtain modified sodium alginate;

[0010] S2: Mix the modified sodium alginate aqueous solution, hydroxybutyl chitosan aqueous solution, and gelatin aqueous solution to obtain a hydrogel mixed solution;

[0011] Step 3:

[0012] Add the chondroitin sulfate - modified liposome coated with FGF18 to the hydrogel mixed solution to obtain an upper - layer hydrogel mixed solution; add nano - hydroxyapatite to the hydrogel mixed liquid to obtain a lower - layer hydrogel mixed solution; after injecting the lower - layer hydrogel mixed solution into a mold and forming a stable state, add the upper - layer hydrogel mixed solution, raise the temperature to form a gel, and obtain a temperature - sensitive controlled - release hydrogel composite material.

[0013] Furthermore, in step 1, the dialysis pore size is 30 - 50 nm; the encapsulation efficiency of the chondroitin sulfate - modified liposome coated with FGF18 is 55 - 60%.

[0014] Furthermore, in S1, mix the 3 - allyl - 2 - hydroxybenzaldehyde dispersion with the sodium alginate solution according to the mass ratio of 3 - allyl - 2 - hydroxybenzaldehyde to sodium alginate of 1:(8 - 10).

[0015] Furthermore, in S2, the preparation method of hydroxybutyl chitosan is as follows: dissolve chitosan in an aqueous hydrochloric acid solution, filter off the insoluble matter, then add a sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash it with ultrapure water until neutral, add ethanol for desalting, dehydration, drying, and grinding to obtain purified chitosan powder; disperse the purified chitosan powder in an aqueous NaOH solution, stir evenly under nitrogen protection, filter, then add isopropanol and stir evenly, add 1,2 - epoxybutane, react for 96 - 120 h, and precipitate with acetone, then wash the precipitate until neutral.

[0016] Furthermore, in S2, the mass concentration of the modified sodium alginate aqueous solution is 10 - 15%; the mass concentration of the hydroxybutyl chitosan aqueous solution is 5 - 10%; the mass concentration of the gelatin aqueous solution is 8 - 12%.

[0017] Furthermore, in S2, the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:(0.5 - 0.8).

[0018] Further, in step 3, chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution at a mass ratio of (1-2):10.

[0019] Further, in step 3, nano-hydroxyapatite was added to the hydrogel mixed liquid at a mass ratio of (6-10):100.

[0020] Further, in step 3, the gelation temperature was 35-40 °C.

[0021] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention constructs a temperature-sensitive controlled-release hydrogel composite material by preparing chondroitin sulfate-modified liposomes encapsulating FGF18 and combining components such as modified sodium alginate, hydroxybutyl chitosan, and gelatin. First, chondroitin sulfate-modified liposomes encapsulating FGF18 were prepared by chloroform dissolution method to ensure a high encapsulation rate of FGF18; subsequently, 3-allyl-2-hydroxybenzaldehyde was reacted with sodium alginate to introduce aldehyde groups to obtain modified sodium alginate. On the one hand, the aldehyde groups on the modified sodium alginate can undergo Schiff base reactions with the amino groups on the molecular chains of gelatin and hydroxybutyl chitosan, acting as a macromolecular cross-linking agent; on the other hand, the hydrogen bonds and physical entanglement between the macromolecules also further promote the cross-linking of the three. The hydrogel composite material of the present invention includes a two-layer structure: the upper layer structure contains chondroitin sulfate-modified liposomes encapsulating FGF18, and the effect of repairing bone joints is achieved by slowly releasing FGF18 and chondroitin sulfate; nano-hydroxyapatite is added to the lower layer structure. Nano-hydroxyapatite is a natural component of bone tissue, has biocompatibility and biological activity, and can induce the osteogenic differentiation of mesenchymal stem cells.

[0022] In addition, in the present invention, hydroxybutyl chitosan has unique temperature sensitivity and good biological activity, and its aqueous solution can form a gel at about 35 °C; therefore, introducing hydroxybutyl chitosan into the hydrogel system prepared in the present invention can accelerate gelation by raising the temperature; however, it should be added that due to the strong water solubility of hydroxybutyl chitosan, too high a dosage thereof will lead to a decrease in the gelation performance of the hydrogel. Through experimental comparison, it was found that the hydrogel mixed solution obtained by mixing the aqueous solutions of modified sodium alginate, hydroxybutyl chitosan, and gelatin at a mass ratio of 1:1:(0.5-0.8) has better gelation performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention.

[0024] Figure 1 It is an electron microscopy characterization diagram of chondroitin sulfate-modified liposomes. DETAILED DESCRIPTION OF THE INVENTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0026] Example 1: A preparation method of a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair, comprising the following steps:

[0027] Step 1:

[0028] Take soybean lecithin, cholesterol, and DSPE-PEG-CSA and dissolve them together in 2 mL of chloroform; after evaporating to form a film under reduced pressure, add it to 1.5 mL of FGF18 solution for hydration, extrude it after ultrasonic treatment, supplement deionized water to make the volume constant to 2 mL, and remove the unencapsulated FGF18 by nano dialysis, the dialysis pore size is 30 nm; obtain chondroitin sulfate-modified liposomes encapsulating FGF18, and the encapsulation efficiency is 55%;

[0029] Step 2:

[0030] Dissolve chitosan in a 1% hydrochloric acid aqueous solution by volume, filter out the insoluble substances, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash it with ultrapure water until neutral, add ethanol for desalting, dehydration, drying, and grinding to obtain purified chitosan powder; disperse 1 g of purified chitosan powder in 10 mL of a 40% - 60% sodium hydroxide aqueous solution by mass fraction, stir it evenly under nitrogen protection, filter it, add 20 mL of isopropanol and stir it evenly, add 20 mL of 1,2-epoxybutane, react for 96 h, precipitate it with acetone, wash the precipitate until neutral, and dry it to obtain hydroxybutyl chitosan;

[0031] Step 3:

[0032] S1: Disperse 3-allyl-2-hydroxybenzaldehyde in ultrapure water to obtain a 3-allyl-2-hydroxybenzaldehyde dispersion; mix the 3-allyl-2-hydroxybenzaldehyde dispersion and sodium alginate solution according to the mass ratio of 3-allyl-2-hydroxybenzaldehyde to sodium alginate of 1:8, add the initiator potassium persulfate, stir for 10 min, then raise the temperature to 80 °C and react for 3 h, after cooling, dialyze to remove impurities, then filter and freeze-dry to obtain modified sodium alginate;

[0033] S2: Mix the modified sodium alginate aqueous solution, hydroxybutyl chitosan aqueous solution, and gelatin aqueous solution to obtain a hydrogel mixed solution. Among them, the mass concentration of the modified sodium alginate aqueous solution is 10%; the mass concentration of the hydroxybutyl chitosan aqueous solution is 5%; the mass concentration of the gelatin aqueous solution is 8%; the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:0.5;

[0034] Step 4:

[0035] Add the chondroitin sulfate-modified liposome coated with FGF18 to the hydrogel mixed solution according to a mass ratio of 1:10 to obtain an upper-layer hydrogel mixed solution; add nano-hydroxyapatite to the hydrogel mixed liquid according to a mass ratio of 6:100 to obtain a lower-layer hydrogel mixed solution; after injecting the lower-layer hydrogel mixed solution into a mold and forming a stable state, add the upper-layer hydrogel mixed solution, heat to 35 °C to form a gel, and obtain a temperature-sensitive controlled-release hydrogel composite material.

[0036] Example 2: A preparation method of a temperature-sensitive controlled-release hydrogel composite material for osteoarthritis repair, comprising the following steps:

[0037] Step 1:

[0038] Take soybean lecithin, cholesterol, and DSPE-PEG-CSA and dissolve them together in 2 mL of chloroform; after evaporating to form a film under reduced pressure, add it to 1.5 mL of FGF18 solution for hydration, extrude after ultrasonic treatment, supplement deionized water to a constant volume of 2 mL, and remove unencapsulated FGF18 by nano-dialysis, the dialysis pore size is 30 nm; obtain the chondroitin sulfate-modified liposome coated with FGF18, and the encapsulation efficiency is 55%;

[0039] Step 2:

[0040] Dissolve chitosan in a hydrochloric acid aqueous solution with a volume fraction of 1%, filter out the insoluble matter, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash it with ultrapure water to neutrality, add ethanol for desalting, dehydration, drying, and grinding to obtain purified chitosan powder; disperse 1 g of purified chitosan powder in 10 mL of a sodium hydroxide aqueous solution with a mass fraction of 40% - 60%, stir evenly under nitrogen protection, filter, add 20 mL of isopropanol and stir evenly, add 20 mL of 1,2-epoxybutane, react for 108 h, precipitate with acetone, wash the precipitate to neutrality, and dry to obtain hydroxybutyl chitosan;

[0041] Step 3:

[0042] S1: Disperse 3 - allyl - 2 - hydroxybenzaldehyde in ultrapure water to obtain a 3 - allyl - 2 - hydroxybenzaldehyde dispersion; mix the 3 - allyl - 2 - hydroxybenzaldehyde dispersion with a sodium alginate solution according to the mass ratio of 3 - allyl - 2 - hydroxybenzaldehyde to sodium alginate of 1:9, add potassium persulfate as an initiator, stir for 13 min, then raise the temperature to 85 °C and react for 4 h. After cooling, dialyze to remove impurities, then perform suction filtration and freeze - dry to obtain modified sodium alginate;

[0043] S2: Mix the modified sodium alginate aqueous solution, hydroxybutyl chitosan aqueous solution, and gelatin aqueous solution to obtain a hydrogel mixed solution; among them, the mass concentration of the modified sodium alginate aqueous solution is 13%; the mass concentration of the hydroxybutyl chitosan aqueous solution is 8%; the mass concentration of the gelatin aqueous solution is 10%; the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:0.6;

[0044] Step 4:

[0045] Add chondroitin sulfate - modified liposomes coated with FGF18 to the hydrogel mixed solution according to the mass ratio of 1.5:10 to obtain an upper - layer hydrogel mixed solution; add nano - hydroxyapatite to the hydrogel mixed liquid according to the mass ratio of 8:100 to obtain a lower - layer hydrogel mixed solution; inject the lower - layer hydrogel mixed solution into a mold and after forming a stable state, add the upper - layer hydrogel mixed solution, raise the temperature to 35 °C to form a gel, and obtain a temperature - sensitive controlled - release hydrogel composite material.

[0046] Example 3: A preparation method of a temperature - sensitive controlled - release hydrogel composite material for osteoarthritis repair, comprising the following steps:

[0047] Step 1:

[0048] Take soy lecithin, cholesterol, and DSPE - PEG - CSA and dissolve them together in 2 mL of chloroform; after evaporating to form a film under reduced pressure, add it to 1.5 mL of FGF18 solution for hydration, extrude after ultrasonic treatment, supplement deionized water to make up the volume to 2 mL, and perform nano - dialysis to remove unencapsulated FGF18, the dialysis pore size is 30 nm; obtain chondroitin sulfate - modified liposomes coated with FGF18, and the encapsulation efficiency is 55%;

[0049] Step 2:

[0050] Dissolve chitosan in an aqueous hydrochloric acid solution with a volume fraction of 1%. After filtering off the insoluble matter, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate. Wash it with ultrapure water until neutral, add ethanol for desalting, dehydration, drying, and grinding to obtain purified chitosan powder; Disperse 1 g of purified chitosan powder in 10 mL of an aqueous sodium hydroxide solution with a mass fraction of 40% - 60%. Stir it evenly under nitrogen protection, filter, add 20 mL of isopropanol and stir evenly, then add 20 mL of 1,2-epoxybutane and react for 120 h. After precipitation with acetone, wash the precipitate until neutral and dry to obtain hydroxybutyl chitosan;

[0051] Step 3:

[0052] S1: Disperse 3-allyl-2-hydroxybenzaldehyde in ultrapure water to obtain a 3-allyl-2-hydroxybenzaldehyde dispersion; Mix the 3-allyl-2-hydroxybenzaldehyde dispersion and sodium alginate solution according to the mass ratio of 3-allyl-2-hydroxybenzaldehyde to sodium alginate of 1:10. Add the initiator potassium persulfate, stir for 15 min, then raise the temperature to 90 °C and react for 5 h. After cooling, dialyze to remove impurities, then filter and freeze-dry to obtain modified sodium alginate;

[0053] S2: Mix the aqueous solution of modified sodium alginate, the aqueous solution of hydroxybutyl chitosan, and the aqueous solution of gelatin to obtain a hydrogel mixed solution; Among them, the mass concentration of the aqueous solution of modified sodium alginate is 15%; the mass concentration of the aqueous solution of hydroxybutyl chitosan is 10%; the mass concentration of the aqueous solution of gelatin is 12%; the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:0.8;

[0054] Step 4:

[0055] Add chondroitin sulfate-modified liposomes coated with FGF18 to the hydrogel mixed solution according to the mass ratio of 2:10 to obtain an upper-layer hydrogel mixed solution; Add nano-hydroxyapatite to the hydrogel mixed liquid according to the mass ratio of 10:100 to obtain a lower-layer hydrogel mixed solution; After injecting the lower-layer hydrogel mixed solution into a mold and forming a stable state, add the upper-layer hydrogel mixed solution, raise the temperature to 35 °C to form a gel, and obtain a temperature-sensitive controlled-release hydrogel composite material.

[0056] Comparative Example 1: Do not modify sodium alginate, and the other parameters are the same as those in Example 1.

[0057] Step 1:

[0058] Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved together in 2 mL of chloroform; after evaporation to form a film under reduced pressure, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, and deionized water was added to make up the volume to 2 mL, and unencapsulated FGF18 was removed by nano-dialysis with a dialysis pore size of 30 nm; chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained with an encapsulation efficiency of 55%;

[0059] Step 2:

[0060] Chitosan was dissolved in a 1% hydrochloric acid aqueous solution by volume, and after filtering off the insoluble matter, 1 mol / L sodium hydroxide solution was added to the filtrate to obtain a flocculent precipitate, which was washed with ultrapure water until neutral, and then ethanol was added for desalting, dehydration, drying, and grinding to obtain purified chitosan powder; 1 g of purified chitosan powder was dispersed in 10 mL of a 40% - 60% sodium hydroxide aqueous solution by mass fraction, stirred evenly under nitrogen protection, filtered, then 20 mL of isopropanol was added and stirred evenly, 20 mL of 1,2-epoxybutane was added, and the reaction was carried out for 96 h. After precipitation with acetone, the precipitate was washed until neutral and dried to obtain hydroxybutyl chitosan;

[0061] Step 3:

[0062] An aqueous sodium alginate solution, an aqueous hydroxybutyl chitosan solution, and an aqueous gelatin solution were mixed to obtain a hydrogel mixed solution; among them, the mass concentration of the aqueous sodium alginate solution was 10%; the mass concentration of the aqueous hydroxybutyl chitosan solution was 5%; the mass concentration of the aqueous gelatin solution was 8%; the mass ratio of sodium alginate, gelatin, and hydroxybutyl chitosan was 1:1:0.5;

[0063] Step 4:

[0064] The chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution according to a mass ratio of 1:10 to obtain an upper-layer hydrogel mixed solution; nano-hydroxyapatite was added to the hydrogel mixed liquid according to a mass ratio of 6:100 to obtain a lower-layer hydrogel mixed solution; after injecting the lower-layer hydrogel mixed solution into a mold and forming a stable state, the upper-layer hydrogel mixed solution was added, and the temperature was raised to 35 °C to form a gel, obtaining a temperature-sensitive controlled-release hydrogel composite material.

[0065] Comparative Example 2: Nano-hydroxyapatite was not added to the lower-layer hydrogel mixed solution, and the other parameters were the same as in Example 2.

[0066] Step 1:

[0067] Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved together in 2 mL of chloroform; after evaporation to form a film under reduced pressure, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, and deionized water was added to make up the volume to 2 mL, and unencapsulated FGF18 was removed by nano-dialysis with a dialysis pore size of 30 nm; chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained with an encapsulation efficiency of 55%;

[0068] Step 2:

[0069] Chitosan was dissolved in a 1% hydrochloric acid aqueous solution by volume, and after filtering off the insoluble matter, 1 mol / L sodium hydroxide solution was added to the filtrate to obtain a flocculent precipitate, which was washed with ultrapure water until neutral, and then ethanol was added for desalting, dehydration, drying, and grinding to obtain purified chitosan powder; 1 g of purified chitosan powder was dispersed in 10 mL of a 40% - 60% sodium hydroxide aqueous solution by mass, stirred evenly under nitrogen protection, filtered, 20 mL of isopropanol was added and stirred evenly, 20 mL of 1,2-epoxybutane was added, reacted for 108 h, precipitated with acetone, and the precipitate was washed until neutral and dried to obtain hydroxybutyl chitosan;

[0070] Step 3:

[0071] S1: 3-Allyl-2-hydroxybenzaldehyde was dispersed in ultrapure water to obtain a 3-allyl-2-hydroxybenzaldehyde dispersion; according to the mass ratio of 3-allyl-2-hydroxybenzaldehyde to sodium alginate of 1:9, the 3-allyl-2-hydroxybenzaldehyde dispersion was mixed with a sodium alginate solution, potassium persulfate as an initiator was added, after stirring for 13 min, the temperature was raised to 85 °C and reacted for 4 h, after cooling, impurities were removed by dialysis, and then filtered and freeze-dried to obtain modified sodium alginate;

[0072] S2: The modified sodium alginate aqueous solution, hydroxybutyl chitosan aqueous solution, and gelatin aqueous solution were mixed to obtain a hydrogel mixed solution; among them, the mass concentration of the modified sodium alginate aqueous solution was 13%; the mass concentration of the hydroxybutyl chitosan aqueous solution was 8%; the mass concentration of the gelatin aqueous solution was 10%; the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan was 1:1:0.6;

[0073] Step 4:

[0074] The chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution according to a mass ratio of 1.5:10 to obtain an upper-layer hydrogel mixed solution; after the hydrogel mixed solution was injected into a mold and formed a stable state, the upper-layer hydrogel mixed solution was added, and the temperature was raised to 35 °C to form a gel, obtaining a temperature-sensitive controlled-release hydrogel composite material.

[0075] Comparative Example 3: Increase the dosage of hydroxybutyl chitosan in the hydrogel mixed solution, and the other parameters are the same as those in Example 3.

[0076] Step 1:

[0077] Take soy lecithin, cholesterol, and DSPE-PEG-CSA and dissolve them together in 2 mL of chloroform; after evaporating to form a film under reduced pressure, add it to 1.5 mL of FGF18 solution for hydration, extrude it after ultrasonic treatment, supplement deionized water to make the volume up to 2 mL, and remove the unencapsulated FGF18 by nano-dialysis, with the dialysis pore size of 30 nm; obtain chondroitin sulfate-modified liposomes encapsulating FGF18, with an encapsulation efficiency of 55%;

[0078] Step 2:

[0079] Dissolve chitosan in a 1% (v / v) hydrochloric acid aqueous solution, filter off the insoluble substances, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash it with ultrapure water until neutral, add ethanol for desalting, dehydration, drying, and grinding to obtain purified chitosan powder; disperse 1 g of purified chitosan powder in 10 mL of a 40% - 60% (w / w) sodium hydroxide aqueous solution, stir it evenly under nitrogen protection, filter it, add 20 mL of isopropanol and stir it evenly, add 20 mL of 1,2-epoxybutane, react for 120 h, precipitate it with acetone, wash the precipitate until neutral, and dry it to obtain hydroxybutyl chitosan;

[0080] Step 3:

[0081] S1: Disperse 3-allyl-2-hydroxybenzaldehyde in ultrapure water to obtain a 3-allyl-2-hydroxybenzaldehyde dispersion; mix the 3-allyl-2-hydroxybenzaldehyde dispersion with a sodium alginate solution according to the mass ratio of 3-allyl-2-hydroxybenzaldehyde to sodium alginate of 1:10, add the initiator potassium persulfate, stir for 15 min, then raise the temperature to 90 °C and react for 5 h, cool it down, remove impurities by dialysis, filter it by suction, and freeze-dry it to obtain modified sodium alginate;

[0082] S2: Mix the modified sodium alginate aqueous solution, hydroxybutyl chitosan aqueous solution, and gelatin aqueous solution to obtain a hydrogel mixed solution; among them, the mass concentration of the modified sodium alginate aqueous solution is 15%; the mass concentration of the hydroxybutyl chitosan aqueous solution is 10%; the mass concentration of the gelatin aqueous solution is 12%; the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:1;

[0083] Step 4:

[0084] The chondroitin sulfate-modified liposomes coated with FGF18 were added to the hydrogel mixed solution at a mass ratio of 2:10 to obtain the upper-layer hydrogel mixed solution; nano-hydroxyapatite was added to the hydrogel mixed liquid at a mass ratio of 10:100 to obtain the lower-layer hydrogel mixed solution; after injecting the lower-layer hydrogel mixed solution into a mold and forming a stable state, the upper-layer hydrogel mixed solution was added, and the temperature was raised to 35 °C to form a gel, obtaining a temperature-sensitive controlled-release hydrogel composite material.

[0085] Experiment: (1) The hydrogel composite materials prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were tested, and the experimental results are shown in Table 1 below.

[0086] Mechanical properties: A universal material testing machine was used to conduct a compression experiment at a downward pressing speed of 8 mm / min. The hydrogel scaffold was prepared into a cylindrical shape with a diameter of 15 mm and a height of 10 mm to test the maximum stress that the sample could withstand.

[0087] Thermosensitive property: The forming time of the hydrogel composite material at 35 °C was tested.

[0088] Repair effect: An electric bone drill was used to drill a hole in the center of the articular surface of the femoral condyle of the hind limb of a rabbit. The diameter of the hole was 4.5 mm and the depth was 3 mm. Subsequently, the hydrogel was used to fill the defect site. After suturing the wound and disinfecting with iodophor, it was bandaged with a sterile dressing. After raising the rabbits for 12 weeks, the samples were euthanized and the femoral condyle samples were taken out to observe the repair situation.

[0089] (2) The chondroitin sulfate-modified liposomes in Example 1 were tested by electron microscopy, and the experimental results are as Figure 1 shown.

[0090] Table 1 shows the test results of various properties of the hydrogel composite material

[0091] Project Maximum allowable stress / MPa Molding time / s Repair effect Example 1 0.53 183 Good regenerative repair effect on the cartilage defect site Example 2 0.57 192 Good regenerative repair effect on the cartilage defect site Example 3 0.60 204 Good regenerative repair effect on the cartilage defect site Comparative example 1 0.49 207 Poor integration of the repaired tissue with the surrounding cartilage tissue, with obvious defects Comparative example 2 0.56 192 Poor integration of the repaired tissue with the surrounding cartilage tissue, with obvious defects Comparative example 3 0.55 248 Poor integration of the repaired tissue with the surrounding cartilage tissue, with obvious defects

[0092] Conclusion: The data of Examples 1 to 3 show that the hydrogel composite material prepared by the present invention has good properties. The data of Example 1 and Comparative Example 1 show that after modifying sodium alginate, chemical bonds are introduced, which is easier to crosslink, and at the same time, the mechanical strength of the composite material is improved. The data of Example 2 and Comparative Example 2 show that after adding nano-hydroxyapatite to the lower-layer hydrogel mixed solution, the repair effect is better. The data of Example 3 and Comparative Example 3 show that too high a dosage of hydroxybutyl chitosan in the hydrogel mixed solution is not conducive to forming and the effect is poor.

[0093] The phase transition temperature at which the hydrogel changes from a liquid state to a gel state is 35 degrees Celsius.

[0094] It should be noted that in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0095] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair, characterized in that: The following steps are involved: Step 1: Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved in chloroform; After the film is formed by evaporation under reduced pressure, it is added to the FGF18 solution for hydration, extruded after ultrasonic treatment, supplemented with deionized water and nanodialysis to remove the unencapsulated FGF18, thereby obtaining the chondroitin sulfate-modified liposomes encapsulating FGF18; Step 2: S1: Dispersing 3-allyl-2-hydroxybenzaldehyde in ultrapure water to obtain a 3-allyl-2-hydroxybenzaldehyde dispersion; mixing the 3-allyl-2-hydroxybenzaldehyde dispersion with a sodium alginate solution, adding an initiator potassium persulfate, stirring for 10-15 minutes, heating to 80-90°C for reaction for 3-5 hours, cooling, dialyzing to remove impurities, filtering, and freeze-drying to obtain modified sodium alginate; S2: mixing the modified sodium alginate aqueous solution, the hydroxybutyl chitosan aqueous solution and the gelatin aqueous solution to obtain a hydrogel mixed solution; Step 3: Adding chondroitin sulfate modified liposomes coated with FGF18 into the hydrogel mixed solution to obtain an upper layer of hydrogel mixed solution; adding nano-hydroxyapatite into the hydrogel mixed liquid to obtain a lower layer of hydrogel mixed solution; After the lower layer of hydrogel mixed solution is injected into the mold and forms a stable state, the upper layer of hydrogel mixed solution is added, and the temperature is increased to form a gel to obtain a temperature-sensitive controlled-release hydrogel composite material; Among them, in S2, the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:(0.5~0.8).

2. The method for preparing a thermosensitive controlled-release hydrogel composite material for repairing osteoarthritis according to claim 1, characterized in that: In step 1, the dialysis pore size is 30-50 nm; the encapsulation efficiency of the chondroitin sulfate-modified liposomes encapsulating FGF18 is 55-60%.

3. The method for preparing a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair according to claim 1, characterized in that: In S1, 3-allyl-2-hydroxybenzaldehyde dispersion and sodium alginate solution are mixed at a mass ratio of 3-allyl-2-hydroxybenzaldehyde to sodium alginate of 1:(8-10).

4. The method for preparing a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair according to claim 1, characterized in that: In S2, the preparation method of hydroxybutyl chitosan is as follows: dissolving chitosan in a hydrochloric acid aqueous solution, filtering out insoluble matter, adding a sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, washing with ultrapure water until neutral, adding ethanol for desalting, dehydrating, drying, and grinding to obtain a purified chitosan powder; dispersing the purified chitosan powder in a NaOH aqueous solution, stirring until uniform under nitrogen protection, filtering, adding isopropanol, stirring until uniform, adding 1,2-butylene oxide, reacting for 96 to 120 hours, precipitating with acetone, and washing the precipitate to neutrality.

5. The method for preparing a thermosensitive controlled-release hydrogel composite material for repairing osteoarthritis according to claim 1, characterized in that: In S2, the mass concentration of the modified sodium alginate aqueous solution is 10-15%; the mass concentration of the hydroxybutyl chitosan aqueous solution is 5-10%; and the mass concentration of the gelatin aqueous solution is 8-12%.

6. The method for preparing a thermosensitive controlled-release hydrogel composite material for repairing osteoarthritis according to claim 1, characterized in that: In step 3, the chondroitin sulfate-modified liposomes encapsulating FGF18 are added to the hydrogel mixed solution at a mass ratio of (1-2):

10.

7. The method for preparing a thermosensitive controlled-release hydrogel composite material for repairing osteoarthritis according to claim 1, characterized in that: In step 3, nano-hydroxyapatite is added to the hydrogel mixed liquid at a mass ratio of (6-10):

100.

8. The method for preparing a thermosensitive controlled-release hydrogel composite material for repairing osteoarthritis according to claim 1, characterized in that: In step 3, the gelling temperature is 35-40°C.

9. A thermosensitive controlled-release hydrogel composite material for osteoarthritis repair, characterized in that: The thermosensitive controlled-release hydrogel composite material for osteoarthritis repair is prepared according to the preparation method of any one of claims 1 to 8.

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