Temperature-sensitive controlled-release hydrogel composite material for osteoarthritis repair and preparation method of temperature-sensitive controlled-release hydrogel composite material

Abstract

The invention relates to the technical field of biological materials, in particular to a temperature-sensitive controlled-release hydrogel composite material for osteoarthritis repair and a preparation method. The preparation method comprises the following steps: firstly, preparing chondroitin sulfate modified lipidosome coated with FGF18 through a chloroform dissolution method, and ensuring high encapsulation efficiency of the FGF18; then, 3-allyl-2-hydroxybenzaldehyde and sodium alginate are subjected to a reaction, an aldehyde group is introduced, and modified sodium alginate is obtained; the preparation method comprises the following steps: mixing modified sodium alginate, hydroxybutyl chitosan and gelatin to form a hydrogel solution; adding chondroitin sulfate modified lipidosome coated with FGF18 into the hydrogel mixed solution to obtain an upper layer hydrogel mixed solution; adding nano-hydroxyapatite into the hydrogel mixed solution to obtain a lower-layer hydrogel mixed solution; injecting the lower-layer hydrogel mixed solution into a mold, adding the upper-layer hydrogel mixed solution after 10 minutes, and heating to form gel, so as to obtain the temperature-sensitive release-control hydrogel composite material.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, in particular to a temperature-sensitive controlled-release hydrogel composite material for repairing osteoarthritis and a preparation method thereof. Background Art

[0002] Osteoarthritis is a complex disease that affects joints. In damaged joints, macrophages can be activated and polarized to the M1 type, and secrete pro-inflammatory factors such as IL-1, IL-6, and TNF-α, leading to extracellular matrix (ECM) degradation; while M2 macrophages suppress 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] At present, it is difficult to effectively balance immune regulation and anti-inflammatory effects with 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 associated with M2 macrophage polarization; chondroitin sulfate (ChS) is a glucuronic acid that can relieve joint pain, promote cartilage repair, and improve joint function; combining the two and embedding them into polymer hydrogels 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 temperature-sensitive controlled-release hydrogel composite material for osteoarthritis repair and a preparation method thereof, so as to solve the problems raised in the above-mentioned background technology.

[0005] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing a thermosensitive controlled-release hydrogel composite material for repairing osteoarthritis, the preparation method comprising the following steps: Step 1: Soybean lecithin, cholesterol and DSPE-PEG-CSA are dissolved in chloroform; after evaporation under reduced pressure to form a film, 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 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: The chondroitin sulfate modified liposomes coated with FGF18 are added to the hydrogel mixed solution to obtain an upper hydrogel mixed solution; nanohydroxyapatite is added to the hydrogel mixed liquid to obtain a lower hydrogel mixed solution; after the lower hydrogel mixed solution is injected into a mold and forms a stable state, the upper hydrogel mixed solution is added, the temperature is increased to form a gel, and a temperature-sensitive controlled-release hydrogel composite material is obtained.

[0006] Furthermore, in step 1, the dialysis pore size is 30-50 nm; and the encapsulation efficiency of the chondroitin sulfate-modified liposomes encapsulating FGF18 is 55-60%.

[0007] Further, in S1, the 3-allyl-2-hydroxybenzaldehyde dispersion is mixed with the sodium alginate solution at a mass ratio of 3-allyl-2-hydroxybenzaldehyde to sodium alginate of 1:(8-10).

[0008] Furthermore, in S2, the preparation method of hydroxybutyl chitosan is: dissolving chitosan in a hydrochloric acid aqueous solution, filtering out insoluble matter, adding sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, washing with ultrapure water to neutrality, adding ethanol for desalting, dehydrating, drying, and grinding to obtain purified chitosan powder; dispersing the purified chitosan powder in a NaOH aqueous solution, stirring until uniform under nitrogen protection, filtering, adding isopropanol and stirring until uniform, adding 1,2-butylene oxide, reacting for 96 to 120 hours, precipitating with acetone, and washing the precipitate to neutrality.

[0009] 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%; and the mass concentration of the gelatin aqueous solution is 8-12%.

[0010] Furthermore, in S2, the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:(0.5~0.8).

[0011] Furthermore, 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.

[0012] Furthermore, in step 3, nano-hydroxyapatite is added to the hydrogel mixed liquid at a mass ratio of (6-10):100.

[0013] Furthermore, in step 3, the gelling temperature is 35-40°C.

[0014] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: the present invention constructs a thermosensitive 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 are prepared by chloroform dissolution method to ensure a high encapsulation rate of FGF18; then, 3-allyl-2-hydroxybenzaldehyde is used to react 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 react with the amino groups on the molecular chains of gelatin and hydroxybutyl chitosan to react with Schiff bases, acting as macromolecular crosslinkers; on the other hand, hydrogen bonds and physical entanglement between macromolecules further promote the crosslinking of the three. The hydrogel composite material of the present invention comprises a two-layer structure: the upper layer structure contains chondroitin sulfate-modified liposomes encapsulating FGF18, and achieves the effect of repairing bone joints by sustained-release of FGF18 and chondroitin sulfate; the lower layer structure is added with nano-hydroxyapatite, which is a natural component of bone tissue, has biocompatibility and bioactivity, and can induce osteogenic differentiation of mesenchymal stem cells.

[0015] In addition, in the present invention, hydroxybutyl chitosan has unique temperature sensitivity and good biological activity, and its aqueous solution can gel at about 35°C; therefore, hydroxybutyl chitosan is introduced into the hydrogel system prepared by the present invention, and the gelation can be accelerated by heating; however, it should be supplemented that due to the strong water solubility of hydroxybutyl chitosan, excessive use of hydroxybutyl chitosan 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 modified sodium alginate aqueous solution, the hydroxybutyl chitosan aqueous solution, and the gelatin aqueous solution in a mass ratio of 1:1:(0.5~0.8) has better gelation performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0017] Figure 1 This is an electron microscopy characterization of chondroitin sulfate modified liposomes. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1: A method for preparing a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair, comprising the following steps: Step 1: Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved in 2 mL of chloroform; after evaporation under reduced pressure to form a film, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, supplemented with deionized water to make the volume 2 mL, and nanodialysis was performed to remove the unencapsulated FGF18, wherein the dialysis pore size was 30 nm; and chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained, and the encapsulation efficiency was 55%; Step 2: The chitosan was dissolved in a 1% volume fraction hydrochloric acid aqueous solution, and after filtering out the insoluble matter, a 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 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% to 60% mass fraction NaOH aqueous solution, and stirred to be uniform under nitrogen protection, and after filtering, 20 mL of isopropanol was added and stirred to be uniform, and 20 mL of 1,2-butylene oxide was added, and the reaction was carried out for 96 hours, and the precipitate was washed to be neutral after precipitation with acetone, and dried to obtain hydroxybutyl chitosan; Step 3: 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 at a mass ratio of 1:8, adding an initiator potassium persulfate, stirring for 10 minutes, heating to 80°C for reaction for 3 hours, cooling, dialyzing to remove impurities, filtering, and freeze-drying to obtain modified sodium alginate; S2: Mixing a modified sodium alginate aqueous solution, a hydroxybutyl chitosan aqueous solution and a gelatin aqueous solution to obtain a hydrogel mixed solution; wherein 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%; and the mass ratio of the modified sodium alginate, gelatin and hydroxybutyl chitosan is 1:1:0.5; Step 4: The chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution at a mass ratio of 1:10 to obtain an upper hydrogel mixed solution; nanohydroxyapatite was added to the hydrogel mixed liquid at a mass ratio of 6:100 to obtain a lower hydrogel mixed solution; after the lower hydrogel mixed solution was injected into the mold and a stable state was formed, the upper hydrogel mixed solution was added, and the temperature was raised to 35°C to form a gel, thereby obtaining a thermosensitive controlled-release hydrogel composite material.

[0020] Example 2: A method for preparing a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair, comprising the following steps: Step 1: Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved in 2 mL of chloroform; after evaporation under reduced pressure to form a film, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, supplemented with deionized water to make the volume 2 mL, and nanodialysis was performed to remove the unencapsulated FGF18, wherein the dialysis pore size was 30 nm; and chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained, and the encapsulation efficiency was 55%; Step 2: Dissolve chitosan in a 1% volume fraction hydrochloric acid aqueous solution, filter out insoluble matter, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash 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% to 60% mass fraction NaOH aqueous solution, stir until uniform under nitrogen protection, filter, add 20 mL of isopropanol, stir until uniform, add 20 mL of 1,2-butylene oxide, react for 108 h, precipitate with acetone, wash the precipitate until neutral, and dry to obtain hydroxybutyl chitosan; Step 3: 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 at a mass ratio of 1:9, adding an initiator potassium persulfate, stirring for 13 minutes, heating to 85°C for reaction for 4 hours, cooling, dialyzing to remove impurities, filtering, and freeze-drying to obtain modified sodium alginate; S2: Mixing a modified sodium alginate aqueous solution, a hydroxybutyl chitosan aqueous solution and a gelatin aqueous solution to obtain a hydrogel mixed solution; wherein 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%; and the mass ratio of the modified sodium alginate, gelatin and hydroxybutyl chitosan is 1:1:0.6; Step 4: The chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution at a mass ratio of 1.5:10 to obtain an upper hydrogel mixed solution; nanohydroxyapatite was added to the hydrogel mixed liquid at a mass ratio of 8:100 to obtain a lower hydrogel mixed solution; after the lower hydrogel mixed solution was injected into a mold and a stable state was formed, the upper hydrogel mixed solution was added, and the temperature was raised to 35°C to form a gel, thereby obtaining a thermosensitive controlled-release hydrogel composite material.

[0021] Example 3: A method for preparing a thermosensitive controlled-release hydrogel composite material for osteoarthritis repair, comprising the following steps: Step 1: Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved in 2 mL of chloroform; after evaporation under reduced pressure to form a film, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, supplemented with deionized water to make the volume 2 mL, and nanodialysis was performed to remove the unencapsulated FGF18, wherein the dialysis pore size was 30 nm; and chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained, and the encapsulation efficiency was 55%; Step 2: Dissolve chitosan in a 1% volume fraction hydrochloric acid aqueous solution, filter out insoluble matter, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash 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% to 60% mass fraction NaOH aqueous solution, stir until uniform under nitrogen protection, filter, add 20 mL of isopropanol, stir until uniform, add 20 mL of 1,2-butylene oxide, react for 120 h, precipitate with acetone, wash the precipitate until neutral, and dry to obtain hydroxybutyl chitosan; Step 3: 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 at a mass ratio of 1:10, adding an initiator potassium persulfate, stirring for 15 minutes, heating to 90°C for reaction for 5 hours, cooling, dialyzing to remove impurities, filtering, and freeze-drying to obtain modified sodium alginate; S2: Mixing a modified sodium alginate aqueous solution, a hydroxybutyl chitosan aqueous solution and a gelatin aqueous solution to obtain a hydrogel mixed solution; wherein 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%; and the mass ratio of the modified sodium alginate, gelatin and hydroxybutyl chitosan is 1:1:0.8; Step 4: The chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution at a mass ratio of 2:10 to obtain an upper hydrogel mixed solution; nanohydroxyapatite was added to the hydrogel mixed liquid at a mass ratio of 10:100 to obtain a lower hydrogel mixed solution; after the lower hydrogel mixed solution was injected into a mold and a stable state was formed, the upper hydrogel mixed solution was added, and the temperature was raised to 35°C to form a gel, thereby obtaining a temperature-sensitive controlled-release hydrogel composite material.

[0022] Comparative Example 1: Sodium alginate was not modified, and other parameters were the same as those in Example 1.

[0023] Step 1: Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved in 2 mL of chloroform; after evaporation under reduced pressure to form a film, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, supplemented with deionized water to make the volume 2 mL, and nanodialysis was performed to remove the unencapsulated FGF18, wherein the dialysis pore size was 30 nm; and chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained, and the encapsulation efficiency was 55%; Step 2: The chitosan was dissolved in a 1% volume fraction hydrochloric acid aqueous solution, and after filtering out the insoluble matter, a 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 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% to 60% mass fraction NaOH aqueous solution, and stirred to be uniform under nitrogen protection, and after filtering, 20 mL of isopropanol was added and stirred to be uniform, and 20 mL of 1,2-butylene oxide was added, and the reaction was carried out for 96 hours, and the precipitate was washed to be neutral after precipitation with acetone, and dried to obtain hydroxybutyl chitosan; Step 3: Mixing a sodium alginate aqueous solution, a hydroxybutyl chitosan aqueous solution and a gelatin aqueous solution to obtain a hydrogel mixed solution; wherein the mass concentration of the 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%; and the mass ratio of sodium alginate, gelatin and hydroxybutyl chitosan is 1:1:0.5; Step 4: The chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution at a mass ratio of 1:10 to obtain an upper hydrogel mixed solution; nanohydroxyapatite was added to the hydrogel mixed liquid at a mass ratio of 6:100 to obtain a lower hydrogel mixed solution; after the lower hydrogel mixed solution was injected into the mold and a stable state was formed, the upper hydrogel mixed solution was added, and the temperature was raised to 35°C to form a gel, thereby obtaining a thermosensitive controlled-release hydrogel composite material.

[0024] Comparative Example 2: No nano-hydroxyapatite was added to the lower layer of the hydrogel mixed solution, and the other parameters were the same as those in Example 2.

[0025] Step 1: Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved in 2 mL of chloroform; after evaporation under reduced pressure to form a film, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, supplemented with deionized water to make the volume 2 mL, and nanodialysis was performed to remove the unencapsulated FGF18, wherein the dialysis pore size was 30 nm; and chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained, and the encapsulation efficiency was 55%; Step 2: Dissolve chitosan in a 1% volume fraction hydrochloric acid aqueous solution, filter out insoluble matter, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash 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% to 60% mass fraction NaOH aqueous solution, stir until uniform under nitrogen protection, filter, add 20 mL of isopropanol, stir until uniform, add 20 mL of 1,2-butylene oxide, react for 108 h, precipitate with acetone, wash the precipitate until neutral, and dry to obtain hydroxybutyl chitosan; Step 3: 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 at a mass ratio of 1:9, adding an initiator potassium persulfate, stirring for 13 minutes, heating to 85°C for reaction for 4 hours, cooling, dialyzing to remove impurities, filtering, and freeze-drying to obtain modified sodium alginate; S2: Mixing a modified sodium alginate aqueous solution, a hydroxybutyl chitosan aqueous solution and a gelatin aqueous solution to obtain a hydrogel mixed solution; wherein 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%; and the mass ratio of the modified sodium alginate, gelatin and hydroxybutyl chitosan is 1:1:0.6; Step 4: Chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution at a mass ratio of 1.5:10 to obtain an upper hydrogel mixed solution; after the hydrogel mixed solution was injected into a mold and a stable state was formed, the upper hydrogel mixed solution was added, and the temperature was raised to 35°C to form a gel to obtain a thermosensitive controlled-release hydrogel composite material.

[0026] Comparative Example 3: The amount of hydroxybutyl chitosan in the hydrogel mixed solution was increased, and the other parameters were the same as those in Example 3.

[0027] Step 1: Soybean lecithin, cholesterol, and DSPE-PEG-CSA were dissolved in 2 mL of chloroform; after evaporation under reduced pressure to form a film, it was added to 1.5 mL of FGF18 solution for hydration, extruded after ultrasonic treatment, supplemented with deionized water to make the volume 2 mL, and nanodialysis was performed to remove the unencapsulated FGF18, wherein the dialysis pore size was 30 nm; and chondroitin sulfate-modified liposomes encapsulating FGF18 were obtained, and the encapsulation efficiency was 55%; Step 2: Dissolve chitosan in a 1% volume fraction hydrochloric acid aqueous solution, filter out insoluble matter, add 1 mol / L sodium hydroxide solution to the filtrate to obtain a flocculent precipitate, wash 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% to 60% mass fraction NaOH aqueous solution, stir until uniform under nitrogen protection, filter, add 20 mL of isopropanol, stir until uniform, add 20 mL of 1,2-butylene oxide, react for 120 h, precipitate with acetone, wash the precipitate until neutral, and dry to obtain hydroxybutyl chitosan; Step 3: 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 at a mass ratio of 1:10, adding an initiator potassium persulfate, stirring for 15 minutes, heating to 90°C for reaction for 5 hours, cooling, dialyzing to remove impurities, filtering, and freeze-drying to obtain modified sodium alginate; S2: Mixing a modified sodium alginate aqueous solution, a hydroxybutyl chitosan aqueous solution and a gelatin aqueous solution to obtain a hydrogel mixed solution; wherein 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%; and the mass ratio of the modified sodium alginate, gelatin and hydroxybutyl chitosan is 1:1:1; Step 4: The chondroitin sulfate-modified liposomes encapsulating FGF18 were added to the hydrogel mixed solution at a mass ratio of 2:10 to obtain an upper hydrogel mixed solution; nanohydroxyapatite was added to the hydrogel mixed liquid at a mass ratio of 10:100 to obtain a lower hydrogel mixed solution; after the lower hydrogel mixed solution was injected into a mold and a stable state was formed, the upper hydrogel mixed solution was added, and the temperature was raised to 35°C to form a gel, thereby obtaining a temperature-sensitive controlled-release hydrogel composite material.

[0028] Experiment: (1) The hydrogel composite materials prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were tested. The experimental results are shown in Table 1 below.

[0029] Mechanical properties: A compression test was carried out using a universal material testing machine at a downward pressure 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 tolerable stress of the sample.

[0030] Thermosensitive performance: Testing the molding time of hydrogel composites at 35°C.

[0031] Repair effect: An electric bone drill was used to drill a hole in the middle of the femoral condyle articular surface of the rabbit's hind limb, with a hole diameter of 4.5 mm and a depth of 3 mm. The defect was then filled with hydrogel, and the incision was sutured and disinfected with iodine, then bandaged with sterile dressings. After 12 weeks of feeding, the sample was euthanized and the femoral condyle sample was taken out to observe the repair condition.

[0032] (2) The chondroitin sulfate modified liposomes in Example 1 were tested by electron microscopy. The experimental results are as follows: Figure 1 shown.

[0033] Table 1 shows the test results of various properties of hydrogel composite materials project Maximum stress that can be tolerated / MPa Molding time / s Repair effect Example 1 0.53 183 Good regeneration and repair effect of cartilage defect Example 2 0.57 192 Good regeneration and repair effect of cartilage defect Example 3 0.60 204 Good regeneration and repair effect of cartilage defect Comparative Example 1 0.49 207 The repair tissue is poorly integrated with the surrounding cartilage tissue and has obvious defects Comparative Example 2 0.56 192 The repair tissue is poorly integrated with the surrounding cartilage tissue and has obvious defects Comparative Example 3 0.55 248 The repair tissue is poorly integrated with the surrounding cartilage tissue and has obvious defects , Conclusion: The data of Examples 1 to 3 show that the hydrogel composite material prepared by the present invention has good performance. The data of Example 1 and Comparative Example 1 show that after the sodium alginate is modified, chemical bonds are introduced, which makes it easier to cross-link, 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 hydrogel mixed solution, the repair effect is better. The data of Example 3 and Comparative Example 3 show that the use of too high an amount of hydroxybutyl chitosan in the hydrogel mixed solution is not conducive to molding and the effect is not good.

[0034] The phase transition temperature of the hydrogel from liquid to gel is 35 degrees Celsius.

[0035] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0036] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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.

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 S2, the mass ratio of modified sodium alginate, gelatin, and hydroxybutyl chitosan is 1:1:(0.5~0.8).

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, the chondroitin sulfate-modified liposomes encapsulating FGF18 are added to the hydrogel mixed solution at a mass ratio of (1-2):

10.

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, nano-hydroxyapatite is added to the hydrogel mixed liquid at a mass ratio of (6-10):

100.

9. 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.

10. 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 9.

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