Preparation method of 3D printed hydrogel scaffold for cartilage repair

Abstract

This invention discloses a method for preparing a 3D-printed hydrogel scaffold for cartilage repair, comprising: providing methacrylamide chitosan; providing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres; obtaining a 3D-printed bio-ink for cartilage repair using methacrylamide chitosan, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres, recombinant collagen CF-1552, and lithium magnesium silicate as main raw materials; and obtaining a 3D-printed bio-ink for cartilage repair using the 3D-printed bio-ink for cartilage repair as a raw material to obtain a 3D-printed hydrogel scaffold for cartilage repair. This 3D-printed hydrogel scaffold has advantages such as antibacterial properties and the ability to promote the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into chondrocytes, and can be used for cartilage repair, potentially bringing new curative opportunities for cartilage injury diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing a 3D-printed hydrogel scaffold for cartilage repair. Background Technology

[0002] The main chemical components of cartilage are water, collagen, and polysaccharides. Articular cartilage plays a vital role in load-bearing, resisting mechanical stress, and shock absorption. Once cartilage is damaged, it lacks sufficient blood supply to provide adequate nutrition, making it difficult to repair itself. Trauma, disease, and cartilage degeneration often lead to cartilage defects. If these defects are not adequately treated, the joint will undergo irreversible degeneration, potentially resulting in disability.

[0003] Tissue engineering is an emerging technology for cartilage repair and regeneration, and a key objective of this technology is to construct biomimetic extracellular matrix (ECM) biomaterials. As a biomacromial complex secreted by cells and immobilized around them, the extracellular matrix plays a crucial role in providing physical support to cells and maintaining cellular evolution and physiological homeostasis. Constructing ECM biomaterials requires that the materials themselves exhibit promoting effects in various aspects of regulating cell behavior and functional expression. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for preparing a 3D-printed hydrogel scaffold for cartilage repair. This method is inspired by recombinant collagen, which is widely used in biomaterials such as soft tissue fillers, vascular scaffolds, and wound dressings. It creatively combines 3D printing technology to obtain a 3D-printed hydrogel scaffold. This hydrogel scaffold has advantages such as antibacterial properties and the ability to promote the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into chondrocytes. It can be used for cartilage repair and is expected to bring new curative opportunities for cartilage injury diseases.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a 3D-printed hydrogel scaffold for cartilage repair, characterized in that it includes:

[0006] Provides methacrylamide chitosan;

[0007] We provide 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres;

[0008] Provides 3D printing bio-inks for cartilage repair;

[0009] Using 3D printing technology, a 3D-printed hydrogel scaffold for cartilage repair was obtained using the aforementioned 3D-printed bio-ink for cartilage repair as a raw material.

[0010] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized in that providing methacrylamide chitosan specifically includes: adding methacrylic anhydride dropwise to a chitosan acetic acid solution, stirring and reacting in a water bath at 60°C for 3-6 hours, dialyzing, and freeze-drying to obtain methacrylamide chitosan.

[0011] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized in that the chitosan acetate solution is a chitosan acetate solution obtained by dissolving chitosan in an acetic acid solution, wherein the volume percentage concentration of the acetic acid solution is 1% to 2%; the relative molecular mass of the chitosan is 1200 kDa to 2500 kDa; and the amount of methacrylic anhydride is 1 to 4 times the amount of chitosan.

[0012] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized by providing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres, specifically comprising:

[0013] Low-concentration polyvinyl alcohol solution, polylactic acid-glycolic acid copolymer solution, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution and high-concentration polyvinyl alcohol solution are provided respectively;

[0014] A mixture of polylactic acid-hydroxyacetic acid copolymer solution and 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution was added dropwise to the high-concentration polyvinyl alcohol solution to obtain a mixed system;

[0015] The mixture was sonicated in an ice bath for 4 to 8 minutes to obtain a primary emulsion.

[0016] Under stirring conditions, the primary emulsion was added dropwise to a low-concentration polyvinyl alcohol solution, and stirring was continued for 4 to 16 hours. After centrifugation, washing, and freeze-drying, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres were obtained.

[0017] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized in that,

[0018] The low-concentration polyvinyl alcohol solution is a deionized aqueous solution of polyvinyl alcohol, wherein the concentration of polyvinyl alcohol in the low-concentration polyvinyl alcohol solution is 0.08% w / v to 0.12% w / v; the method for providing the low-concentration polyvinyl alcohol solution includes: placing polyvinyl alcohol in deionized water, stirring at room temperature until uniformly dispersed, then heating to 80°C and continuing to stir until completely dissolved to obtain the low-concentration polyvinyl alcohol solution; the molecular weight of the polyvinyl alcohol is 13000 to 23000;

[0019] The polylactic acid-glycolic acid copolymer solution is a polylactic acid-glycolic acid copolymer solution obtained by dissolving polylactic acid-glycolic acid copolymer in dichloromethane, wherein the concentration of polylactic acid-glycolic acid copolymer in the polylactic acid-glycolic acid copolymer solution is 20 mg / ml to 50 mg / ml;

[0020] The 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution is a 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution obtained by dissolving 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid in dimethyl sulfoxide, wherein the concentration of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid in the 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution is 10 mg / ml to 20 mg / ml;

[0021] The high-concentration polyvinyl alcohol solution is a deionized aqueous solution of polyvinyl alcohol, wherein the concentration of polyvinyl alcohol in the high-concentration polyvinyl alcohol solution is 0.8% w / v to 1.2% w / v; and the molecular weight of the polyvinyl alcohol is 13,000 to 23,000.

[0022] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized by providing a 3D-printed bio-ink for cartilage repair, specifically comprising:

[0023] Solution A containing methacrylamide chitosan and a photoinitiator, solution B containing recombinant collagen, a microsphere suspension containing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres, and a colloidal dispersion containing lithium magnesium silicate are provided respectively.

[0024] The microsphere suspension was added to the system after mixing liquid A and liquid B to obtain mixed system D;

[0025] Under stirring conditions, mixing system D is added to the colloidal dispersion, and stirring is continued to obtain a gel solution; in the gel solution, the mass percentage of recombinant collagen is 5% to 10%, the mass percentage of methacrylamide chitosan is 0.5% to 1%, the mass percentage of photoinitiator is 0.1% to 0.5%, the mass percentage of lithium magnesium silicate is 2.5% to 3.5%, and the content of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres is 1 μg / ml to 3 μg / ml;

[0026] The gel solution was left to stand in a refrigerator at 4°C for 8 to 36 hours to obtain bio-ink.

[0027] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized in that,

[0028] The solution A containing methacrylamide chitosan and a photoinitiator is obtained by dissolving methacrylamide chitosan and a photoinitiator in deionized water under light-protected conditions; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate.

[0029] The B solution containing recombinant collagen is obtained by dissolving recombinant collagen in deionized water under water bath conditions; the recombinant collagen is recombinant collagen CF-1552.

[0030] The microsphere suspension containing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres is a microsphere suspension obtained by ultrasonically dispersing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres in deionized water.

[0031] The colloidal dispersion containing lithium magnesium silicate is obtained by stirring and dispersing lithium magnesium silicate in deionized water.

[0032] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized by using 3D printing technology and the aforementioned 3D-printed bio-ink for cartilage repair as raw material to obtain a 3D-printed hydrogel scaffold for cartilage repair, specifically including:

[0033] The bio-ink is loaded into a 3D printer, and the extrudate is irradiated with 365nm ultraviolet light while being extruded to obtain a photocured cross-linked product. The photocured cross-linked product is then sterilized by Co60 irradiation to obtain a 3D printed hydrogel scaffold for cartilage repair.

[0034] The above-mentioned method for preparing a 3D-printed hydrogel scaffold for cartilage repair is characterized in that the cross-linking time is 5 min to 15 min; the nozzle specification of the 3D printer is 23G to 26G; the printing speed is 20 mm / s to 50 mm / s; the printing temperature is 30℃; and the extrusion amount is 4% to 8%.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] 1. The present invention relates to a method for preparing a 3D-printed hydrogel scaffold for cartilage repair. Inspired by recombinant collagen, which is widely used in biomaterials such as soft tissue fillers, vascular scaffolds, and wound dressings, this invention creatively combines 3D printing technology to obtain a 3D-printed hydrogel scaffold. This hydrogel scaffold has advantages such as antibacterial properties and the ability to promote the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into chondrocytes. It can be used for cartilage repair and is expected to bring new curative opportunities for cartilage injury diseases.

[0037] 2. The present invention provides a method for preparing a 3D-printed hydrogel scaffold for cartilage repair. The raw material includes chitosan modified with methacrylic anhydride, whose photosensitizing properties can effectively promote the ultraviolet curing process of bio-ink to obtain the target 3D-printed hydrogel scaffold.

[0038] 3. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to the present invention includes lithium magnesium silicate as a raw material. As a synthetic hydrophilic layered silicate, it can give full play to its excellent rheological properties in the preparation method of the present invention, so that the bio-ink has fluidity and mechanical stability, which facilitates the extrusion of bio-ink and the molding of extrudate.

[0039] 4. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to the present invention includes first obtaining 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres by an oil-in-water emulsion solvent evaporation method, and then incorporating the microspheres into the scaffold material. By utilizing the characteristic of continuous release of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres from the hydrogel scaffold, the 3D-printed hydrogel scaffold can induce bone marrow mesenchymal stem cells to differentiate into chondrocytes, thereby promoting the cartilage repair effect of the hydrogel scaffold.

[0040] 5. The preparation method of the present invention is reliable in principle and is conducive to its widespread application.

[0041] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0042] Instruction manual illustrations

[0043] Figure 1 a is a scanning electron microscope image of the KGN@PLGA microspheres in Example 1;

[0044] Figure 1 b represents Image-J quantization. Figure 1 The particle size distribution map obtained after the SEM image of a;

[0045] Figure 2 a is a schematic diagram showing the test results of the extrudability performance of the supports in Example 1 and Comparative Example 2;

[0046] Figure 2 b is a schematic diagram of the stability test results of the stents in Example 1 and Comparative Example 2;

[0047] Figure 3 a is a scanning electron microscope image of bracket A in Example 1 at a scale of 100 μm;

[0048] Figure 3 b is a scanning electron microscope image of bracket A in Example 1 at a scale of 2μm;

[0049] Figure 3 c Figure 3 A magnified view of part b;

[0050] Figure 4 This is a topographic view of the stent A in Example 1;

[0051] Figure 5 This is a schematic diagram of the cytotoxicity test results of scaffold A in Example 1;

[0052] Figure 6 a is a graph showing the number of bacteria cultured during the antibacterial performance test of scaffold A in Example 1;

[0053] Figure 6 b is a graph showing the Escherichia coli count results after culture in the antibacterial performance test of scaffold A in Example 1;

[0054] Figure 6 c is a graph showing the Staphylococcus aureus count results after culture in the antibacterial performance test of scaffold A in Example 1;

[0055] Figure 7 a represents the toluidine blue staining results of scaffold-induced differentiation of human bone marrow mesenchymal stem cells into chondrocytes in Example 1 and Comparative Example 2;

[0056] Figure 7 b represents the GAG ​​quantification results of scaffold-induced differentiation of human bone marrow mesenchymal stem cells into chondrocytes in Example 1 and Comparative Example 2;

[0057] Figure 7 c represents the ColII quantification results of scaffold-induced differentiation of human bone marrow mesenchymal stem cells into chondrocytes in Example 1 and Comparative Example 2. Detailed Implementation

[0058] This invention provides a method for preparing a 3D-printed hydrogel scaffold for cartilage repair, comprising mixing methacrylamide chitosan, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres, recombinant collagen and lithium magnesium silicate as main raw materials to obtain a gel solution, allowing the gel solution to stand at 4°C to obtain a bio-ink, and loading the bio-ink into a 3D printer for printing to obtain a 3D-printed hydrogel scaffold for cartilage repair.

[0059] The following description, in conjunction with specific embodiments, illustrates the content of the present invention. However, the following description is not intended to limit the scope of the present invention.

[0060] Example 1

[0061] This embodiment provides a method for preparing a 3D-printed hydrogel scaffold for cartilage repair, including:

[0062] Step 1: Provide methacrylamide chitosan, specifically including:

[0063] Step 101: Dissolve 1g of chitosan in 50ml of 2% (v / v) acetic acid solution to obtain chitosan acetic acid solution; the relative molecular mass of the chitosan is 1200 kDa to 2500 kDa.

[0064] Step 102: Add 1.96 ml of methacrylic anhydride dropwise to the chitosan acetate solution, stir in a water bath at 60°C for 3 h, and collect the product;

[0065] Step 103: Dialyze the product continuously in deionized water for 7 days, then freeze-dry to obtain methacrylamide chitosan; the cutoff molecular weight of the dialysis bag is 3500 Da;

[0066] Step 2: Provide 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres (KGN@PLGA microspheres), specifically including:

[0067] Step 201: Place 60 mg of polyvinyl alcohol (PVA) in 60 ml of deionized water, stir at room temperature until evenly dispersed, heat to 80 °C and continue stirring until completely dissolved to obtain polyvinyl alcohol solution A; the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 0.1% w / v; the stirring time until evenly dispersed is 10 min; the stirring time until completely dissolved is 30 min; the molecular weight of the polyvinyl alcohol is 13000-23000;

[0068] Step 202: Place 30 mg of polylactic acid-glycolic acid copolymer (PLGA) in 1 ml of dichloromethane and sonicate until dissolved to obtain a polylactic acid-glycolic acid copolymer solution; the monomer ratio of polylactic acid and glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50, the relative molecular mass of the polylactic acid-glycolic acid copolymer is 38,000 to 54,000, and it was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number P832792;

[0069] Step 203: Place 10 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (KGN) in 1 ml of dimethyl sulfoxide and sonicate until dissolved to obtain a 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution;

[0070] Step 204: Place 30 mg of polyvinyl alcohol in 3 ml of deionized water and sonicate until dissolved to obtain polyvinyl alcohol solution B; the concentration of polyvinyl alcohol in polyvinyl alcohol solution B is 1% w / v; the molecular weight of the polyvinyl alcohol is 13000-23000.

[0071] Step 205: Mix the polylactic acid-glycolic acid copolymer solution described in step 202 and the 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution described in step 203 evenly to obtain an oil phase;

[0072] Step 206: Add the oil phase dropwise to the polyvinyl alcohol solution B obtained in step 204 to obtain a mixed system;

[0073] Step 207: Under ice bath conditions, the mixture is sonicated for 6 minutes to obtain a primary emulsion; the frequency of the sonication is 40W.

[0074] Step 208: Under stirring conditions at room temperature and 300 rpm, the primary emulsion is added dropwise to the polyvinyl alcohol solution A obtained in step 201, and stirring is continued for 12 hours. After centrifugation, washing, and freeze-drying, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres (KGN@PLGA microspheres) are obtained. The washing is performed by washing with deionized water 5 times.

[0075] Step 3: Provide 3D printing bio-inks for cartilage repair, specifically including:

[0076] Step 301: Place 60 mg of methacrylamide chitosan and 20 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2.5 ml of deionized water, seal with aluminum foil to protect from light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature can be 12 h.

[0077] Step 302: Place 500 mg of recombinant collagen CF-1552 in 2.5 ml of deionized water and stir in a water bath at 37°C until completely dissolved to obtain solution B; the stirring time in the water bath can be 2 hours; the recombinant collagen CF-1552 is derived from human collagen mRNA reverse transcription to generate cDNA, which is then expressed in Escherichia coli BL21. Its structure and acquisition method can be found in, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method";

[0078] Step 303: Place 1.4 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres in 1 ml of deionized water and sonicate for 1 h until uniformly dispersed to obtain a microsphere suspension;

[0079] Step 304: Place 300 mg of lithium magnesium silicate in 5 ml of deionized water and immediately stir at 500 rpm for 5 min to obtain a colloidal dispersion; the lithium magnesium silicate is lithium magnesium silicate Laponite-XLG([Mg 5.34 Li 0.66 Si8O20 [OH]Na 0.66 Purchased from BYK, Germany;

[0080] Step 305: Mix liquid A described in step 301 and liquid B described in step 302 to obtain mixed system C;

[0081] Step 306: Place 10 μl of the microsphere suspension described in step 303 into the mixing system C described in step 305 to obtain the mixing system D;

[0082] Step 307: While stirring at 500 rpm, add the mixture D described in step 306 to the colloidal dispersion described in step 304, and continue stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0083] Step 308: Let the gel solution stand in a refrigerator at 4°C for 12 hours to obtain bio-ink; the purpose of placing the gel solution at 4°C for about 12 hours is to allow the gel solution to fully cross-link and defoam, thereby obtaining bio-ink.

[0084] Step 4: Using the 3D printing bio-ink for cartilage repair as a raw material, a 3D printed hydrogel scaffold for cartilage repair is obtained, specifically including:

[0085] The bio-ink was loaded into the barrel of a 3D printer, and the product generation platform was irradiated with 365nm ultraviolet light. The 3D printer was started, and the extrudate was irradiated simultaneously with the extrusion. After extrusion, irradiation continued for 10 minutes to allow cross-linking. The cross-linked product was then sterilized by irradiation with Co60 to obtain a 3D-printed hydrogel scaffold for cartilage repair, denoted as scaffold A. The diameter of scaffold A is 15mm and the height is 1mm. The 3D printer model is FOODBOT-S2.

[0086] The nozzle specification of the 3D printer is 25G;

[0087] The printing speed is 30mm / s;

[0088] The printing temperature is 30℃;

[0089] The extrusion rate is 5%.

[0090] Comparative Example 1

[0091] This comparative study investigated the influencing factors on the bio-inkability of gel solutions, and the preparation methods included:

[0092] Step 1: Place 30 mg of methacrylamide chitosan described in Example 1 and 10 mg of photoinitiator LAP in 2.5 ml of deionized water, seal with aluminum foil to protect from light, and stir at room temperature until completely dissolved to obtain solution A;

[0093] Step 2: Place 250 mg of the recombinant collagen CF-1552 described in Example 1 into 2.5 ml of deionized water and stir in a 37°C water bath until completely dissolved to obtain solution B;

[0094] Step 3: Mix liquid A and liquid B thoroughly to obtain mixed system C;

[0095] Step 4: Place the mixture C in an ultraviolet curing chamber and irradiate it with 365nm light for 10 minutes, then sterilize it by Co60 irradiation to obtain a hydrogel.

[0096] The hydrogel in this comparative example is not printable, possibly because the mixture C has excessive fluidity and lacks shear-thinning properties.

[0097] Comparative Example 2

[0098] This comparative study investigated the effect of KGN@PLGA microspheres on the performance of 3D-printed hydrogel scaffolds. The resulting 3D-printed hydrogel scaffold was designated scaffold B. Its preparation method was the same as in Example 1, except that step two was not included, and step three was as follows:

[0099] Step 301: Place 60 mg of methacrylamide chitosan and 20 mg of photoinitiator LAP in 2.5 ml of deionized water, seal with aluminum foil to protect from light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature can be 12 h.

[0100] Step 302: Place 500 mg of recombinant collagen CF-1552 in 2.5 ml of deionized water and stir in a water bath at 37°C until completely dissolved to obtain solution B; the stirring time in the water bath can be 2 hours.

[0101] Step 303: Place 300 mg of Laponite XLG in 5 ml of deionized water and immediately stir at 500 rpm for 5 min to obtain a colloidal dispersion;

[0102] Step 304: Mix liquid A described in step 301 and liquid B described in step 302 to obtain mixed system C;

[0103] Step 305: While stirring at 500 rpm, add the mixture C described in step 304 to the colloidal dispersion described in step 303, and continue stirring for 1 hour to obtain a gel solution;

[0104] Step 306: Let the gel solution stand in a refrigerator at 4°C for 12 hours to obtain bio-ink.

[0105] Example 2

[0106] This embodiment provides a method for preparing a 3D-printed hydrogel scaffold for cartilage repair, including:

[0107] Step 1: Provide methacrylamide chitosan, specifically including:

[0108] Step 101: Dissolve 1g of chitosan in 50ml of 1% acetic acid solution to obtain chitosan acetic acid solution; the relative molecular mass of the chitosan is 1200Kda~2500Kda;

[0109] Step 102: Add 0.98 ml of methacrylic anhydride dropwise to the chitosan acetate solution, stir in a water bath at 60°C for 6 h, and collect the product;

[0110] Step 103: Dialyze the product continuously in deionized water for 7 days, then freeze-dry to obtain methacrylamide chitosan; the cutoff molecular weight of the dialysis bag is 3500 Da;

[0111] Step 2: Provide 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres (KGN@PLGA microspheres), specifically including:

[0112] Step 201: Place 48 mg of polyvinyl alcohol (PVA) in 60 ml of deionized water, stir at room temperature until evenly dispersed, heat to 80°C and continue stirring until completely dissolved to obtain polyvinyl alcohol solution A; the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 0.08% w / v; the stirring time until evenly dispersed is 20 min; the stirring time until completely dissolved is 60 min; the molecular weight of the polyvinyl alcohol is 13000-23000;

[0113] Step 202: Place 20 mg of polylactic acid-glycolic acid copolymer (PLGA) in 1 ml of dichloromethane and sonicate until dissolved to obtain a polylactic acid-glycolic acid copolymer solution; the monomer ratio of polylactic acid and glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50, the relative molecular mass of the polylactic acid-glycolic acid copolymer is 38,000 to 54,000, and it was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number P832792;

[0114] Step 203: Place 15 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (KGN) in 1 ml of dimethyl sulfoxide and sonicate until dissolved to obtain a 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution;

[0115] Step 204: Place 24 mg of polyvinyl alcohol in 3 ml of deionized water and sonicate until dissolved to obtain polyvinyl alcohol solution B; the concentration of polyvinyl alcohol in polyvinyl alcohol solution B is 0.8% w / v; the molecular weight of the polyvinyl alcohol is 13000-23000.

[0116] Step 205: Mix the polylactic acid-glycolic acid copolymer solution described in step 202 and the 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution described in step 203 evenly to obtain an oil phase;

[0117] Step 206: Add the oil phase dropwise to the polyvinyl alcohol solution B obtained in step 204 to obtain a mixed system;

[0118] Step 207: Under ice bath conditions, the mixture is sonicated for 4 minutes to obtain a primary emulsion; the frequency of the sonication is 40W.

[0119] Step 208: Under stirring conditions at room temperature and 300 rpm, the primary emulsion is added dropwise to the polyvinyl alcohol solution A obtained in step 201, and stirring is continued for 4 hours. After centrifugation, washing, and freeze-drying, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres (KGN@PLGA microspheres) are obtained; the washing is performed by washing with deionized water 5 times.

[0120] Step 3: Provide 3D printing bio-inks for cartilage repair, specifically including:

[0121] Step 301: Place 50 mg of methacrylamide chitosan and 10 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2.5 ml of deionized water, seal with aluminum foil to protect from light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature can be 12 h.

[0122] Step 302: Place 800 mg of recombinant collagen CF-1552 in 2.5 ml of deionized water and stir in a water bath at 37°C until completely dissolved to obtain solution B; the stirring time in the water bath can be 2 hours; the recombinant collagen CF-1552 is derived from human collagen mRNA reverse transcription to generate cDNA, which is then expressed in Escherichia coli BL21. Its structure and acquisition method can be found in, for example, the patent application document with patent number ZL01106757.8 and patent title "A human-like collagen and its production method";

[0123] Step 303: Place 1.0 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres in 1 ml of deionized water and sonicate for 1 h until uniformly dispersed to obtain a microsphere suspension;

[0124] Step 304: Place 250 mg of lithium magnesium silicate in 5 ml of deionized water and immediately stir at 500 rpm for 5 min to obtain a colloidal dispersion; the lithium magnesium silicate is lithium magnesium silicate Laponite-XLG([Mg 5.34 Li0.66 Si8O 20 [OH]Na 0.66 Purchased from BYK, Germany;

[0125] Step 305: Mix liquid A described in step 301 and liquid B described in step 302 to obtain mixed system C;

[0126] Step 306: Place 10 μl of the microsphere suspension described in step 303 into the mixing system C described in step 305 to obtain the mixing system D;

[0127] Step 307: While stirring at 500 rpm, add the mixture D described in step 306 to the colloidal dispersion described in step 304, and continue stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0128] Step 308: Let the gel solution stand in a refrigerator at 4°C for 8 hours to obtain bio-ink;

[0129] Step 4: Using the 3D printing bio-ink for cartilage repair as a raw material, a 3D printed hydrogel scaffold for cartilage repair is obtained, specifically including:

[0130] The bio-ink is loaded into the barrel of a 3D printer, and the product generation platform is irradiated with 365nm ultraviolet light. The 3D printer is started, and the extrudate is irradiated simultaneously. After extrusion, irradiation continues for 10 minutes to allow cross-linking. The cross-linked product is then sterilized by irradiation with Co60 to obtain a 3D printed hydrogel scaffold for cartilage repair. The 3D printer model is FOODBOT-S2.

[0131] The nozzle specification of the 3D printer is 25G;

[0132] The printing speed is 30mm / s;

[0133] The printing temperature is 30℃;

[0134] The extrusion rate is 5%.

[0135] The performance of the 3D-printed hydrogel scaffold for cartilage repair in this embodiment is basically the same as that in Embodiment 1.

[0136] Example 3

[0137] This embodiment provides a method for preparing a 3D-printed hydrogel scaffold for cartilage repair, including:

[0138] Step 1: Provide methacrylamide chitosan, specifically including:

[0139] Step 101: Dissolve 1g of chitosan in 50ml of 2% (v / v) acetic acid solution to obtain chitosan acetic acid solution; the relative molecular mass of the chitosan is 1200 kDa to 2500 kDa.

[0140] Step 102: Add 3.92 ml of methacrylic anhydride dropwise to the chitosan acetate solution, stir in a water bath at 60°C for 5 h, and collect the product;

[0141] Step 103: Dialyze the product continuously in deionized water for 7 days, then freeze-dry to obtain methacrylamide chitosan; the cutoff molecular weight of the dialysis bag is 3500 Da;

[0142] Step 2: Provide 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres (KGN@PLGA microspheres), specifically including:

[0143] Step 201: Place 72 mg of polyvinyl alcohol (PVA) in 60 ml of deionized water, stir at room temperature until evenly dispersed, heat to 80 °C and continue stirring until completely dissolved to obtain polyvinyl alcohol solution A; the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 0.12% w / v; the stirring time until evenly dispersed is 15 min; the stirring time until completely dissolved is 40 min; the molecular weight of the polyvinyl alcohol is 13000-23000;

[0144] Step 202: Place 50 mg of polylactic acid-glycolic acid copolymer (PLGA) in 1 ml of dichloromethane and sonicate until dissolved to obtain a polylactic acid-glycolic acid copolymer solution; the monomer ratio of polylactic acid and glycolic acid in the polylactic acid-glycolic acid copolymer is 50:50, the relative molecular mass of the polylactic acid-glycolic acid copolymer is 38,000 to 54,000, and it was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., product number P832792;

[0145] Step 203: Place 20 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid (KGN) in 1 ml of dimethyl sulfoxide and sonicate until dissolved to obtain a 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution;

[0146] Step 204: Place 36 mg of polyvinyl alcohol in 3 ml of deionized water and sonicate until dissolved to obtain polyvinyl alcohol solution B; the concentration of polyvinyl alcohol in polyvinyl alcohol solution B is 1.2% w / v; the molecular weight of the polyvinyl alcohol is 13000-23000.

[0147] Step 205: Mix the polylactic acid-glycolic acid copolymer solution described in step 202 and the 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution described in step 203 evenly to obtain an oil phase;

[0148] Step 206: Add the oil phase dropwise to the polyvinyl alcohol solution B obtained in step 204 to obtain a mixed system;

[0149] Step 207: Under ice bath conditions, the mixture is sonicated for 8 minutes to obtain a primary emulsion; the frequency of the sonication is 40W.

[0150] Step 208: Under stirring conditions at room temperature and 300 rpm, the primary emulsion is added dropwise to the polyvinyl alcohol solution A obtained in step 201, and stirring is continued for 16 hours. After centrifugation, washing, and freeze-drying, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres (KGN@PLGA microspheres) are obtained. The washing is performed by washing with deionized water 5 times.

[0151] Step 3: Provide 3D printing bio-inks for cartilage repair, specifically including:

[0152] Step 301: Place 100 mg of methacrylamide chitosan and 50 mg of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) in 2.5 ml of deionized water, seal with aluminum foil to protect from light, and stir at room temperature until completely dissolved to obtain solution A; the stirring time at room temperature can be 12 h.

[0153] Step 302: Place 1000 mg of recombinant collagen CF-1552 in 2.5 ml of deionized water and stir in a water bath at 37°C until completely dissolved to obtain solution B; the stirring time in the water bath can be 2 hours; the recombinant collagen CF-1552 is derived from human collagen mRNA reverse transcription to generate cDNA, which is then expressed in Escherichia coli BL21. Its structure and acquisition method can be found in, for example, the patent application document with patent number ZL01106757.8 and patent name "A human-like collagen and its production method";

[0154] Step 303: Place 3 mg of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres in 1 ml of deionized water and sonicate for 1 h until uniformly dispersed to obtain a microsphere suspension;

[0155] Step 304: Place 350 mg of lithium magnesium silicate in 5 ml of deionized water and immediately stir at 500 rpm for 5 min to obtain a colloidal dispersion; the lithium magnesium silicate is lithium magnesium silicate Laponite-XLG([Mg 5.34 Li 0.66 Si8O20 [OH]Na 0.66 Purchased from BYK, Germany;

[0156] Step 305: Mix liquid A described in step 301 and liquid B described in step 302 to obtain mixed system C;

[0157] Step 306: Place 10 μl of the microsphere suspension described in step 303 into the mixing system C described in step 305 to obtain the mixing system D;

[0158] Step 307: While stirring at 500 rpm, add the mixture D described in step 306 to the colloidal dispersion described in step 304, and continue stirring for 1 hour to obtain a gel solution; the gel solution has no obvious fluidity and is milky white;

[0159] Step 308: Let the gel solution stand in a refrigerator at 4°C for 36 hours to obtain bio-ink;

[0160] Step 4: Using the 3D printing bio-ink for cartilage repair as a raw material, a 3D printed hydrogel scaffold for cartilage repair is obtained, specifically including:

[0161] The bio-ink is loaded into the barrel of a 3D printer, and the product generation platform is irradiated with 365nm ultraviolet light. The 3D printer is started, and the extrudate is irradiated simultaneously. After extrusion, irradiation continues for 10 minutes to allow cross-linking. The cross-linked product is then sterilized by irradiation with Co60 to obtain a 3D printed hydrogel scaffold for cartilage repair. The 3D printer model is FOODBOT-S2.

[0162] The nozzle specification of the 3D printer is 25G;

[0163] The printing speed is 30mm / s;

[0164] The printing temperature is 30℃;

[0165] The extrusion rate is 5%.

[0166] The performance of the 3D-printed hydrogel scaffold for cartilage repair in this embodiment is basically the same as that in Embodiment 1.

[0167] Example 4

[0168] This embodiment is the same as Embodiment 1, except that in step four, the nozzle specification of the 3D printer is 23G, the printing speed is 20mm / s, the extrusion amount is 4%, and the crosslinking time is 5min.

[0169] Example 5

[0170] This embodiment is the same as Embodiment 1, except that in step four, the nozzle specification of the 3D printer is 26G, the printing speed is 50mm / s, the extrusion amount is 8%, and the crosslinking time is 15min.

[0171] Both Examples 4 and 5 yielded 3D-printed hydrogel scaffolds. From the perspective of scaffold structure, the hydrogel scaffold of Example 1 has a more consistent and symmetrical scaffold structure, indicating that the size of the bio-ink extrusion filament, the extrusion amount per unit time, and the printing speed of the present invention affect the morphology of the 3D-printed hydrogel scaffold.

[0172] Performance Evaluation

[0173] Figure 1 The images show scanning electron microscope (SEM) images and particle size distribution diagrams of the KGN@PLGA microspheres in Example 1. Figure 1 a is a scanning electron microscope image of the KGN@PLGA microspheres from Example 1, based on Figure 1 As shown in Figure a, the KGN@PLGA microspheres are uniformly spherical with smooth surfaces. Figure 1 b represents Image-J quantization. Figure 1 The particle size distribution map obtained after the SEM image of a is based on Figure 1 As can be seen from b, the average particle size of KGN@PLGA microspheres is 185.05±32.86nm.

[0174] Figure 2 This is a schematic diagram of the rheological properties of the scaffolds in Example 1 and Comparative Example 2. The testing method includes: placing the bio-ink described in step 308 of Example 1 and the bio-ink described in step 306 of Comparative Example 2 on the rotary rheometer stage, and performing a steady-state rate scan (0.1–1000 s). -1 The extrudability of the gel was tested; the stability of the hydrogel was tested by scanning the oscillation frequency (0.1-100Hz, strain 1%) of the scaffold A described in Example 1 and the scaffold B described in Comparative Example 2. Figure 2 a is a schematic diagram showing the extrudability test results of the bio-inks of Example 1 and Comparative Example 2. Figure 2 As shown in Figure a, the viscosity of both samples decreases with increasing shear rate, indicating that the samples have shear-thinning properties and are printable.

[0175] Figure 2 b is a schematic diagram showing the stability test results of the stents in Example 1 and Comparative Example 2. According to... Figure 2 As shown in b, the storage modulus (G') and loss modulus (G”) of the sample remain essentially unchanged with the change of oscillation frequency, indicating that both scaffolds have a solid-like elasticity, which confirms the stability of the hydrogel scaffold of the present invention.

[0176] Figure 3 This is a scanning electron microscope image of the stent A in Example 1. Figure 3 a is a scanning electron microscope image of support A at a scale of 100 μm, based on... Figure 3 As can be seen, scaffold A has a three-dimensional porous structure, which can provide a microenvironment for chondrocyte growth; Figure 3 b is a scanning electron microscope image of support A at a scale of 2 μm. Figure 3 c is Figure 3 A magnified view of b, based on Figure 3 As shown in b and 3c, there are spherical and disc-shaped structures on the wall of the A hole of the support, indicating the successful loading of KGN@PLGA microspheres and lithium magnesium silicate Laponite XLG-XR.

[0177] Figure 4 The image shows the morphology of scaffold A in Example 1, demonstrating that the method of the present invention can successfully print hydrogels containing recombinant collagen CF-1552, lithium magnesium silicate Laponite XLG-XR, methacrylamide chitosan, and KGN@PLGA microspheres.

[0178] Figure 5 This is a schematic diagram of the cytotoxicity test results for scaffold A in Example 1, and the test method includes:

[0179] The preparation of DMEM complete culture medium specifically includes: adding fetal bovine serum and antibiotics to DMEM basal culture medium to obtain DMEM complete culture medium; wherein the mass percentage of fetal bovine serum in the DMEM complete culture medium is 10%, the mass percentage of antibiotics is 1%, and the antibiotics are streptomycin and penicillin;

[0180] The preparation of MTT solution specifically includes: dissolving MTT in sterile PBS, filtering through a 0.22 μm filter membrane to obtain the MTT solution, and storing it protected from light; the concentration of MTT in the MTT solution is 5 mg / ml;

[0181] The preparation of the stent extract includes: placing the stent A from Example 1 into a 50mL sterile centrifuge tube, adding 1mL of DMEM complete culture medium for every 0.1g of stent A, and incubating the centrifuge tube in an incubator at 37℃ and 5% CO2 for 72h to obtain the stent extract;

[0182] hBMSCs were 1×10 4Cells were seeded at a density of 1:1 in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The old culture medium was removed, and scaffold extraction medium was added for further incubation. DMEM complete culture medium was used as a control group. After 24 h and 48 h of incubation, MTT solution was added to each well at a rate of 50 μL / well. Cells were incubated for another 2–4 h, and the supernatant was removed. DMSO was added to each well at a rate of 150 μL / well. The OD value of each well was measured at 490 nm using a microplate reader. Cell viability was calculated based on the OD values. The cell viability calculation results are shown below. Figure 5 As shown, according to Figure 5 It is evident that the 3D scaffold material prepared by this invention has no obvious cytotoxicity and can be used in organisms.

[0183] Figure 6 This is a schematic diagram of the antibacterial performance results of stent A in Example 1. The test method includes:

[0184] Prepare solid and liquid culture media. The solid culture media are prepared by adding 10g tryptone, 5g yeast extract, 10g sodium chloride, and 15g agar to a 1L volumetric flask, adding deionized water to bring the volume to 1L, dissolving by sonication, dispensing into Erlenmeyer flasks, sealing, and sterilizing at 121℃ for 30min. The liquid culture media are prepared by adding 10g tryptone, 5g yeast extract, and 10g sodium chloride to a 1L volumetric flask, adding deionized water to bring the volume to 1L, dissolving by sonication, dispensing into Erlenmeyer flasks, sealing, and sterilizing at 121℃ for 30min.

[0185] Staphylococcus aureus and Escherichia coli were activated separately using liquid culture medium to obtain bacterial suspensions.

[0186] According to the concentration of scaffold A of 0.1 g / mL, scaffold A from Example 1 was added to the bacterial suspension and incubated for 24 h. After incubation, 100 μL was plated on a solid culture medium plate. The bacterial suspension without scaffold was used as a control group. After incubation at 37°C for 24 h, the plates were photographed and colony counts were performed. The results are as follows: Figure 6 As shown, where Figure 6 a is a graph showing the number of bacteria cultured during the antibacterial performance test of scaffold A in Example 1; Figure 6 b is a graph showing the Escherichia coli count results after culture in the antibacterial performance test of scaffold A in Example 1; Figure 6 c is a graph showing the Staphylococcus aureus count results after culture in the antibacterial performance test of scaffold A in Example 1. According to... Figure 6 As can be seen, the survival rate of Escherichia coli in scaffold A of Example 1 was 5.43%, and the survival rate of Staphylococcus aureus was 1.92%. The scaffold A of the present invention has anti-Escherichia coli and anti-Staphylococcus aureus effects.

[0187] Figure 7 This is a schematic diagram illustrating the experimental results of scaffold-induced differentiation of hBMSCs into chondrocytes in Example 1 and Comparative Example 2. The testing method included: placing scaffold A from Example 1 and scaffold B from Comparative Example 2 into 24-well cell culture plates, adding 1 ml of chondrocyte induction medium to each, incubating for 24 h, aspirating the original medium, and then adding 5 × 10⁻⁶ ml of medium to each plate. 5 hBMSCs were seeded at a specific cell / scaffold density and cultured at 37°C with 5% CO2 for 24 hours. The medium was changed with fresh chondrogenic induction medium, and then every two days thereafter. hBMSCs induced by chondrogenic induction medium without scaffold addition served as a control group. After 21 days of culture, differentiated cells were stained with toluidine blue. Based on the fact that Col II and GAG are specific matrices for chondrogenesis, the levels of these two substances secreted during cell culture can be used as evaluation criteria for assessing the scaffold's chondrogenic effect. In this invention, the Col II ELISA kit and the 1,9-dimethylmethylene blue assay were used to determine the Col II and GAG levels during culture for cells co-cultured with the scaffold for 7, 14, and 21 days, as well as for control cells. The results are as follows: Figure 7 As shown. Figure 7 a represents the toluidine blue staining results. The staining depth of the cells co-cultured with the scaffold is similar to that of the cells in the positive control group (induced by the induction medium), indicating that the scaffold group was successfully induced. The staining depth and density of scaffold A in Example 1 are greater than those of scaffold B in Comparative Example 2, indicating that the 3D printed hydrogel scaffold containing KGN@PLGA microspheres of the present invention has a better induction effect.

[0188] Figure 7 b represents the GAG ​​content detection result. Figure 7 c represents the result of the Col II content detection. According to... Figure 7 As can be seen from b and 7c, the Col II and GAG contents produced by scaffold A in Example 1 are higher than those of the control group and scaffold B in Comparative Example 2, indicating that the 3D printed hydrogel scaffold containing KGN@PLGA microspheres of the present invention has a better induction effect, and the scaffold prepared by the present invention is expected to be applied to the repair of cartilage defects.

[0189] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a 3D-printed hydrogel scaffold for cartilage repair, characterized in that, include: Provides methacrylamide chitosan; We provide 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres; Provides 3D printing bio-inks for cartilage repair; specifically including: Solution A containing methacrylamide chitosan and a photoinitiator, solution B containing recombinant collagen, a microsphere suspension containing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres, and a colloidal dispersion containing lithium magnesium silicate are provided respectively. The microsphere suspension was added to the system after mixing liquid A and liquid B to obtain mixed system D; Under stirring conditions, mixing system D is added to the colloidal dispersion, and stirring is continued to obtain a gel solution; in the gel solution, the mass percentage of recombinant collagen is 5% to 10%, the mass percentage of methacrylamide chitosan is 0.5% to 1%, the mass percentage of photoinitiator is 0.1% to 0.5%, the mass percentage of lithium magnesium silicate is 2.5% to 3.5%, and the content of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres is 1 μg / ml to 3 μg / ml; The gel solution was left to stand in a refrigerator at 4°C for 8 to 36 hours to obtain bio-ink; Using 3D printing technology, a 3D-printed hydrogel scaffold for cartilage repair was obtained using the aforementioned 3D-printed bio-ink for cartilage repair as a raw material.

2. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to claim 1, characterized in that, The specific steps for providing methacrylated chitosan include: adding methacrylic anhydride dropwise to a chitosan acetic acid solution, stirring the mixture in a water bath at 60°C for 3-6 hours, dialyzing, and freeze-drying to obtain methacrylated chitosan.

3. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to claim 2, characterized in that, The chitosan acetate solution is obtained by dissolving chitosan in an acetic acid solution, wherein the volume percentage concentration of the acetic acid solution is 1% to 2%; the relative molecular mass of the chitosan is 1200 kDa to 2500 kDa; and the amount of methacrylic anhydride is 1 to 4 times the amount of chitosan.

4. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to claim 1, characterized in that, We provide 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-glycolic acid copolymer microspheres, specifically comprising: Low-concentration polyvinyl alcohol solution, polylactic acid-glycolic acid copolymer solution, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution and high-concentration polyvinyl alcohol solution are provided respectively; A mixture of polylactic acid-hydroxyacetic acid copolymer solution and 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution was added dropwise to the high-concentration polyvinyl alcohol solution to obtain a mixed system; The mixture was sonicated in an ice bath for 4 to 8 minutes to obtain a primary emulsion. Under stirring conditions, the primary emulsion was added dropwise to a low-concentration polyvinyl alcohol solution, and stirring was continued for 4 to 16 hours. After centrifugation, washing, and freeze-drying, 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres were obtained.

5. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to claim 4, characterized in that, The low-concentration polyvinyl alcohol solution is a deionized aqueous solution of polyvinyl alcohol, wherein the concentration of polyvinyl alcohol in the low-concentration polyvinyl alcohol solution is 0.08% w / v to 0.12% w / v; the method for providing the low-concentration polyvinyl alcohol solution includes: placing polyvinyl alcohol in deionized water, stirring at room temperature until uniformly dispersed, then heating to 80°C and continuing to stir until completely dissolved to obtain the low-concentration polyvinyl alcohol solution; the molecular weight of the polyvinyl alcohol is 13000 to 23000; The polylactic acid-glycolic acid copolymer solution is a polylactic acid-glycolic acid copolymer solution obtained by dissolving polylactic acid-glycolic acid copolymer in dichloromethane, wherein the concentration of polylactic acid-glycolic acid copolymer in the polylactic acid-glycolic acid copolymer solution is 20 mg / ml to 50 mg / ml; The 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution is a 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution obtained by dissolving 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid in dimethyl sulfoxide, wherein the concentration of 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid in the 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid solution is 10 mg / ml to 20 mg / ml; The high-concentration polyvinyl alcohol solution is a deionized aqueous solution of polyvinyl alcohol, wherein the concentration of polyvinyl alcohol in the high-concentration polyvinyl alcohol solution is 0.8% w / v to 1.2% w / v; and the molecular weight of the polyvinyl alcohol is 13,000 to 23,000.

6. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to claim 1, characterized in that, The solution A containing methacrylamide chitosan and a photoinitiator is obtained by dissolving methacrylamide chitosan and a photoinitiator in deionized water under light-protected conditions; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate. The B solution containing recombinant collagen is obtained by dissolving recombinant collagen in deionized water under water bath conditions; the recombinant collagen is recombinant collagen CF-1552. The microsphere suspension containing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres is a microsphere suspension obtained by ultrasonically dispersing 2-([1,1-biphenyl]-4-ylcarbamoyl)benzoic acid@polylactic acid-hydroxyacetic acid copolymer microspheres in deionized water. The colloidal dispersion containing lithium magnesium silicate is obtained by stirring and dispersing lithium magnesium silicate in deionized water.

7. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to claim 1, characterized in that, Using 3D printing technology and the aforementioned 3D printing bio-ink for cartilage repair as raw material, a 3D printed hydrogel scaffold for cartilage repair is obtained, specifically comprising: The bio-ink is loaded into a 3D printer, and the extrudate is irradiated with 365nm ultraviolet light while being extruded to obtain a photocured cross-linked product. The photocured cross-linked product is then sterilized by Co60 irradiation to obtain a 3D printed hydrogel scaffold for cartilage repair.

8. The method for preparing a 3D-printed hydrogel scaffold for cartilage repair according to claim 7, characterized in that, The crosslinking time is 5 min to 15 min; the nozzle specification of the 3D printer is 23G to 26G, the printing speed is 20 mm / s to 50 mm / s, the printing temperature is 30℃, and the extrusion amount is 4% to 8%.

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