Articular cartilage repair material and preparation method thereof

Abstract

The present invention discloses an articular cartilage repair material and a preparation method thereof, and relates to the technical field of medical materials; the articular cartilage repair material is composed of graphene oxide-based composite microspheres, a stabilizer, a lithium strontium cross-linked gel carrier and icariin; the lithium strontium cross-linked gel carrier is composed of a hydrogel matrix, lithium chloride and strontium chloride; the graphene oxide-based composite microspheres are combined and doped with the lithium strontium cross-linked gel carrier, and under the bidirectional cross-linking action of the stabilizer, a porous, multi-layered, multi-cross-linked adhesive network structure is formed, which improves the mechanical properties and antibacterial properties of the material, is better suitable for support and attachment of articular cartilage, and can reduce the risk of infection, and then carries icariin, cooperates with multiple metal elements and bioactive substances to promote cartilage repair and regeneration, and significantly improves the repair effect of articular cartilage.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and specifically relates to an articular cartilage repair material and a preparation method thereof. Background Art

[0002] Degenerative changes or defects in articular cartilage caused by advanced age, unreasonable exercise, mechanical injury or certain infectious diseases are common in clinical practice, often leading to joint pain and limited mobility, seriously affecting people's health. Articular cartilage is composed of chondrocytes and extracellular matrix, lacks the distribution of blood vessels, nerves, and lymphatic vessels, and nutrient intake mainly depends on the circulation of synovial fluid, which limits the activity of chondrocytes. At the same time, there are fewer chondrocytes and their repair ability is limited. These reasons lead to the self-healing process of articular cartilage being hindered once it is damaged. Although existing treatments such as autologous cartilage transplantation and stem cell therapy have improved the treatment effect of cartilage damage to a certain extent, due to the difficulty of cartilage repair, the efficacy of existing treatments is still limited.

[0003] With the continuous development of the field of tissue engineering, the research and preparation of tissue engineering scaffold materials with excellent repair properties have opened up new research avenues for the treatment of cartilage injuries. Among them, hydrogels have good biocompatibility, low immunogenicity and biodegradability, and are ideal materials for bone tissue engineering scaffolds. These scaffold materials interact with surrounding cells, guide the formation of extracellular matrix, and provide the necessary structural support for the newly formed tissue.

[0004] The existing technology currently has the following problems:

[0005] The mechanical properties of hydrogel scaffold materials are poor and cannot withstand the repeated stress of joint movement, which affects the stability of the scaffold structure. In addition, the antibacterial properties of hydrogels are insufficient, which easily leads to infection risks, thus limiting the repair effect on articular cartilage. Summary of the Invention

[0006] In view of the above situation, in order to overcome the defects of the prior art, the present invention proposes an articular cartilage repair material, comprising the following components in parts by weight: 50-60 parts of graphene oxide-based composite microspheres, 10-20 parts of a stabilizer, 20-30 parts of a lithium strontium cross-linked gel carrier, and 30-40 parts of icariin.

[0007] The graphene oxide-based composite microspheres include the following components in parts by weight: 10-20 parts of graphene oxide, 10-30 parts of lysine, and 20-30 parts of poly(glycidyl methacrylate) microspheres.

[0008] The lithium strontium cross-linked gel carrier comprises the following components in parts by weight: 30-50 parts of hydrogel matrix, 5-10 parts of lithium chloride, and 5-10 parts of strontium chloride.

[0009] The preparation method of the graphene oxide-based composite microspheres specifically comprises the following steps:

[0010] (1) 81 mL of anhydrous ethanol, 9 mL of water, and 3.0 g of polyvinyl pyrrolidone were added to a round-bottom flask in sequence, and then 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile were added and stirred evenly. The solution system was ultrasonicated and nitrogen was passed through to remove oxygen. Then, the temperature was controlled at 60-70 ° C in a nitrogen atmosphere and the reaction was stirred at 100-150 rpm for 24 hours. The product was centrifuged, washed, and freeze-dried. The poly(glycidyl methacrylate) microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for the attachment and proliferation of chondrocytes. The glycidyl groups on the surface are easily coupled with bioactive molecules, further promoting cell adhesion and differentiation, thereby obtaining poly(glycidyl methacrylate) microspheres.

[0011] (2) The poly(glycidyl methacrylate) microspheres described in step (1) are uniformly mixed with ethylenediamine and 50 mL of deionized water, stirred at 70-80° C. for 10-12 h, and the product is centrifuged and washed multiple times until the pH of the solution is 7.0, and freeze-dried. Ethylenediamine introduces amino groups into the molecular chain of poly(glycidyl methacrylate), thereby improving the positive charge and hydrophilicity of the microsphere surface. It is not only easy to adsorb extracellular matrix proteins such as collagen and promote the adhesion of chondrocytes, but also enhances the penetration and killing of bacteria to exert an antibacterial effect, thereby obtaining ethylenediamine-functionalized microspheres;

[0012] (3) Disperse 100-200 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 10-20 min, and then stir at 100-200 rpm to form a graphene oxide suspension. Graphene oxide has good electrical conductivity, high specific surface area and excellent mechanical properties, and can be used as a cartilage repair material. In addition, graphene oxide can upregulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycans and collagen, and can regulate macrophage polarization and reduce joint inflammation, thereby exerting the biological activity of cartilage repair. Add 0.25 g of 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide, and finally adding lysine powder, reacting at room temperature for 24 hours, collecting the precipitate, washing with deionized water 3-5 times, and freeze-drying. The surface of graphene oxide is grafted with lysine, thereby improving the dispersibility of graphene oxide and the safety of graphene oxide in the body. It also has good hydrophilicity and excellent protein adsorption capacity, which is conducive to the growth and adhesion of cells and proteins on the surface of the material. The amino group of lysine can destroy the cell membrane of bacteria, thereby enhancing the physical antibacterial effect of graphene oxide in a chemical antibacterial manner, thereby obtaining lysine graphene oxide nanoparticles;

[0013] (4) The lysine graphene oxide nanoparticles described in step (3) are added to 100 mL of deionized water, followed by adding 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinamide, stirring for 20-30 min, and then adding the ethylenediamine functionalized microspheres described in step (2), reacting at 50-60 ° C for 1-2 h, centrifuging, and freeze-drying the precipitate. By coating the surface of the ethylenediamine functionalized microspheres with lysine graphene oxide nanoparticles, a core-shell composite material with internal rigidity and outer flexibility is formed, which is more suitable for supporting articular cartilage, improves the mechanical properties of the material, and makes it more pressure-resistant and deformation-resistant. The ethylenediamine functionalized microspheres serve as the core structure, further avoiding the stacking of graphene oxide, exposing more active groups, enhancing the stimulating effect on cartilage tissue, and is conducive to improving the repair effect. The presence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine functionalized microspheres in the body, improve the stability of the structure, and obtain graphene oxide-based composite microspheres;

[0014] Preferably, in step (2), the amount of ethylenediamine added is 55-75 mL. Ethylenediamine can serve as a chemical cross-linking point to enhance the interaction between cells and materials, which is beneficial to the stability of the material.

[0015] Preferably, in step (3), the amount of lysine added is 0.1-0.3 g. Lysine, as an important amino acid component of collagen, can not only activate the signal pathway of growth factors, promote chondrocyte proliferation and secretion of matrices such as proteoglycans and collagen, thereby accelerating the repair of cartilage defects, but also form hydroxylysine through hydroxylation, participate in the cross-linking of collagen fibers, thereby enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cellular energy metabolism, providing chondrocytes with the nutritional support required for repair.

[0016] The preparation method of the lithium strontium cross-linked gel carrier specifically comprises the following steps:

[0017] a. Add 1.0-2.0 g of chitosan to 50 mL of 0.6% acetic acid solution for standby use, weigh 60-80 mg of sodium alginate and 20.0-25.0 g of sodium glycerophosphate and add them to 100 mL and 50 mL of deionized water respectively, then add the sodium glycerophosphate solution dropwise to the chitosan solution at 4 ° C. Mix evenly, and then add the sodium alginate solution dropwise under magnetic stirring until fully fused, and adjust the pH to neutral. A thermosensitive double network gel is formed by this process, which can be injected into the defect site of articular cartilage to play a filling and repair role, guide cells to arrange in order, stimulate the synthesis of type II collagen and glycosaminoglycans in the cartilage matrix, and accelerate the regeneration of functional cartilage tissue. Chitosan provides biological activity, sodium glycerophosphate imparts thermosensitivity, and sodium alginate enhances mechanical properties. The three complement each other to optimize performance to obtain a hydrogel matrix;

[0018] b. Lithium chloride and strontium chloride are added to 50 mL of sterile double-distilled water, stirred evenly, and then added to the hydrogel matrix described in step a, stirred at a speed of 200-500 rpm for 20-30 min, poured into a mold and transferred to a 37°C incubator. Lithium chloride is combined with chitosan through electrostatic interaction, and strontium chloride is cross-linked with the carboxyl group of sodium alginate, both of which enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy collapse of structure, poor cell adhesion and bone inductivity of calcium ion cross-linked gel, and give the gel the function of promoting bone regeneration. The combined use of lithium and strontium effectively inhibits the biofilm of various bacteria and has excellent antibacterial properties, thereby obtaining a lithium strontium cross-linked gel carrier;

[0019] Preferably, in step b, the amount of lithium chloride added is 0.5-1.0 g, and the amount of strontium chloride added is 0.5-1.0 g. Lithium chloride has certain antibacterial properties, reducing the risk of infection after implantation, and lithium and strontium elements can stimulate chondrocyte proliferation and differentiation by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway.

[0020] The present invention also provides a method for preparing an articular cartilage repair material, which specifically comprises the following steps:

[0021] S1. Dissolve 3.0-4.0g of tannic acid in 100mL of ultrapure water under magnetic stirring, then add 0.3-0.4g of ferric chloride and 0.5g of copper sulfate, and stir evenly. Tannic acid and metallic iron ions and copper ions form a coordination network, which enhances the mechanical strength of the material and simulates the mechanical properties of natural cartilage. The release of metal ions then promotes cartilage regeneration. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol group imparts strong adhesion ability, allowing it to closely adhere to the cartilage defect site and reduce the risk of shedding. The release of metal ions also further enhances the antibacterial properties of polyphenols, thereby obtaining a stabilizer.

[0022] S2. Add the stabilizer described in step S1 to 50 mL of MOPS buffer with a mass fraction of 0.1-0.5% under ice bath conditions, quickly add the graphene oxide-based composite microspheres, ultrasonically treat for 1-5 minutes, then add the lithium strontium cross-linked gel carrier, ultrasonically treat for 5-10 minutes, and finally add icariin, ultrasonically treat for 5-10 minutes, and freeze-dry. The addition of the stabilizer enhances the binding stability and complexity of the graphene oxide-based composite microspheres and the lithium strontium cross-linked gel carrier, forming a porous, multi-layered, multi-cross-linked network structure with excellent mechanical properties and antibacterial properties, better simulating the supporting role of natural cartilage in the joints, and then through multiple metal elements, icariin Chinese medicinal ingredients and other active substances, a multi-pathway and multi-target cartilage repair effect is exerted, effectively promoting cartilage regeneration to obtain an articular cartilage repair material;

[0023] Preferably, in step S2, the amount of icariin added is 0.3-0.5 g. Icariin can enhance cell activity and improve the microenvironment around the joint by promoting chondrocyte proliferation and differentiation, inhibiting excessive apoptosis, reducing the release of inflammatory factors, and promoting the synthesis of cartilage extracellular matrix, thereby promoting the repair of cartilage damage.

[0024] The beneficial effects achieved by the present invention are as follows:

[0025] The present invention combines and dopes graphene oxide-based composite microspheres with lithium strontium cross-linked gel carriers, and forms a porous, multi-layer, multi-cross-linked adhesive network structure under the bidirectional cross-linking action of a stabilizer, thereby improving the mechanical properties and antibacterial properties of the material, making it better suitable for supporting and attaching to articular cartilage, and reducing the risk of infection. Then, icariin is loaded, and a variety of metal elements and bioactive substances are coordinated to promote cartilage repair and regeneration, significantly improving the repair effect of articular cartilage. In the graphene oxide-based composite microspheres, lysine The graphene oxide is grafted with acid to improve the dispersibility of graphene oxide and enhance the antibacterial property of graphene oxide. Then the ethylenediamine functionalized microspheres are coated to form core-shell microspheres with a combination of rigidity and flexibility, which improves the mechanical properties of the material and makes it more resistant to pressure and deformation. The ethylenediamine functionalized microspheres serve as the core structure to avoid the stacking of graphene oxide, expose more active groups, and act more fully on the cartilage tissue to play a stimulating and promoting role. At the same time, the presence of the coating layer reduces the degradation of the ethylenediamine functionalized microspheres in the body, thereby improving the material's mechanical properties. The stability of the microspheres is enhanced, and the killing effect of the microspheres on bacteria is enhanced; in the lithium strontium cross-linked gel carrier, lithium chloride and strontium chloride replace calcium ions and are cross-linked with the hydrogel matrix formed by chitosan, sodium glycerophosphate and sodium alginate to obtain a gel carrier with uniform cross-linking, stable structure, cell adhesion and bone induction, and can also effectively inhibit the biofilm of various bacteria and has excellent antibacterial properties; the polyphenol-metal network structure of the stabilizer serves as a dynamic cross-linking point, which can be combined with the lithium strontium cross-linked gel carrier and cross-linked with the graphene oxide-based composite microspheres. The present invention prepares an articular cartilage repair material using graphene oxide-based composite microspheres, stabilizers, lithium strontium cross-linked gel carriers and icariin, which effectively enhances the mechanical properties and antibacterial properties of the material and significantly strengthens the repair effect on articular cartilage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a scanning electron micrograph of the articular cartilage repair material prepared in Example 1 of the present invention;

[0027] Figure 2 The mechanical strength results of Examples 1-4 and Comparative Examples 1-3 of the present invention are shown in FIG.

[0028] Figure 3 The diagram shows the diameters of the inhibition zones of Examples 1-4 and Comparative Examples 1-3 of the present invention;

[0029] Figure 4 Graphs showing the results of articular cartilage repair in Examples 1-4 of the present invention and Comparative Examples 1-3. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0033] Example 1

[0034] This embodiment provides an articular cartilage repair material, comprising the following components in parts by weight: 60 parts of graphene oxide-based composite microspheres, 20 parts of a stabilizer, 30 parts of a lithium strontium cross-linked gel carrier, and 40 parts of icariin.

[0035] The graphene oxide-based composite microspheres include the following components in parts by weight: 20 parts of graphene oxide, 30 parts of lysine, and 30 parts of poly(glycidyl methacrylate) microspheres.

[0036] The lithium strontium cross-linked gel carrier comprises the following components in parts by weight: 50 parts of hydrogel matrix, 10 parts of lithium chloride and 10 parts of strontium chloride.

[0037] The preparation method of graphene oxide-based composite microspheres specifically comprises the following steps:

[0038] (1) 81 mL of anhydrous ethanol, 9 mL of water, and 3.0 g of polyvinyl pyrrolidone were added to a round-bottom flask in sequence, and then 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile were added and stirred evenly. The solution system was ultrasonicated and nitrogen was passed through to remove oxygen. Then, the temperature was controlled at 70 ° C in a nitrogen atmosphere and the reaction was stirred at 150 rpm for 24 h. The product was centrifuged, washed, and freeze-dried. The poly(glycidyl methacrylate) microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for the attachment and proliferation of chondrocytes. The glycidyl groups on the surface are easily coupled with bioactive molecules, further promoting cell adhesion and differentiation, thereby obtaining poly(glycidyl methacrylate) microspheres.

[0039] (2) The poly(methyl methacrylate) glycidyl ester microspheres described in step (1) were mixed with ethylenediamine and 50 mL of deionized water, and stirred at 80°C for 12 h. The amount of ethylenediamine added was 75 mL. Ethylenediamine can serve as a chemical cross-linking point to enhance the interaction between cells and materials, which is beneficial to the stability of the material. The product was centrifuged and washed several times until the pH of the solution was 7.0, and freeze-dried. Ethylenediamine introduced amino groups into the poly(methyl methacrylate) glycidyl ester molecular chain, which improved the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbed extracellular matrix proteins such as collagen and promoted the adhesion of chondrocytes, but also enhanced the penetration and killing of bacteria to exert an antibacterial effect, thereby obtaining ethylenediamine-functionalized microspheres.

[0040] (3) Disperse 200 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 20 minutes, and then stir at 200 rpm to form a graphene oxide suspension. Graphene oxide can be used as a cartilage repair material due to its good electrical conductivity, high specific surface area and excellent mechanical properties. In addition, graphene oxide can upregulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycans and collagen, and can regulate macrophage polarization and reduce joint inflammation, thereby exerting the biological activity of cartilage repair. Under continuous stirring, 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added, and finally lysine powder was added. The amount of lysine added was 0.3 g. Lysine, as an important component of collagen, can not only activate the signal pathway of growth factors, but also promote chondrocyte Proliferation and secretion of matrices such as proteoglycans and collagen, thereby accelerating the repair of cartilage defects, and forming hydroxylysine through hydroxylation, participating in the cross-linking of collagen fibers, thereby enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cellular energy metabolism, providing chondrocytes with the nutritional support required for repair. The reaction was carried out at room temperature for 24 hours, and the precipitate was collected, washed with deionized water 5 times, and freeze-dried. The surface of graphene oxide was grafted with lysine, thereby improving the dispersibility of graphene oxide and the safety of graphene oxide in the body. It also has good hydrophilicity and excellent protein adsorption capacity, which is conducive to the growth and adhesion of cells and proteins on the surface of the material. The amino group of lysine can destroy the cell membrane of bacteria, and enhance the physical antibacterial effect of graphene oxide in a chemical antibacterial manner to obtain lysine graphene oxide nanoparticles;

[0041] (4) The lysine graphene oxide nanoparticles described in step (3) are added to 100 mL of deionized water, followed by adding 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinamide, stirring for 30 min, and then adding the ethylenediamine functionalized microspheres described in step (2), reacting at 60 ° C for 2 h, centrifuging, and freeze-drying the precipitate. By coating the surface of the ethylenediamine functionalized microspheres with lysine graphene oxide nanoparticles, a core-shell composite material with internal rigidity and outer flexibility is formed, which is more suitable for supporting articular cartilage, improves the mechanical properties of the material, and makes it more pressure-resistant and deformation-resistant. Among them, the ethylenediamine functionalized microspheres as the core structure further avoid the stacking of graphene oxide, expose more active groups, enhance the stimulation and promotion effect on cartilage tissue, and are beneficial to improving the repair effect. The presence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine functionalized microspheres in the body, improve the stability of the structure, and obtain graphene oxide-based composite microspheres.

[0042] The preparation method of the lithium strontium cross-linked gel carrier specifically comprises the following steps:

[0043] a. Add 2.0 g of chitosan to 50 mL of 0.6% acetic acid solution for standby use. Weigh 80 mg of sodium alginate and 25.0 g of sodium glycerophosphate and add them to 100 mL and 50 mL of deionized water, respectively. Then, add the sodium glycerophosphate solution dropwise to the chitosan solution at 4 ° C. Mix evenly, and then add the sodium alginate solution dropwise under magnetic stirring until fully fused. Adjust the pH to neutral. Through this process, a thermosensitive double-network gel is formed, which can be injected into the defect site of articular cartilage to play a filling and repair role, guide the orderly arrangement of cells, stimulate the synthesis of type II collagen and glycosaminoglycans in the cartilage matrix, and accelerate the regeneration of functional cartilage tissue. Chitosan provides biological activity, sodium glycerophosphate imparts thermosensitivity, and sodium alginate enhances mechanical properties. The three complement each other to optimize performance to obtain a hydrogel matrix;

[0044] b. Lithium chloride and strontium chloride are added to 50 mL of sterile double-distilled water, the amount of lithium chloride added is 1.0 g, and the amount of strontium chloride added is 1.0 g. Lithium chloride has certain antibacterial properties, reducing the risk of infection after implantation, and lithium and strontium elements can stimulate chondrocyte proliferation and differentiation by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, the mixture is added to the hydrogel matrix described in step a, stirred at 500 rpm for 30 minutes, poured into a mold and transferred to a 37°C incubator. Lithium chloride binds to chitosan through electrostatic action, and strontium chloride cross-links with the carboxyl groups of sodium alginate. Both enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy structural collapse, poor cell adhesion and bone induction of calcium ion cross-linked gel, and give the gel the function of promoting bone regeneration. In addition, the combined use of lithium and strontium effectively inhibits the biofilm of various bacteria and has excellent antibacterial properties, thereby obtaining a lithium-strontium cross-linked gel carrier.

[0045] This embodiment provides a method for preparing an articular cartilage repair material, which specifically includes the following steps:

[0046] S1. Dissolve 4.0 g of tannic acid in 100 mL of ultrapure water under magnetic stirring, then add 0.4 g of ferric chloride and 0.5 g of copper sulfate and stir evenly. Tannic acid and metallic iron ions and copper ions form a coordination network, enhancing the mechanical strength of the material and simulating the mechanical properties of natural cartilage. The release of metal ions then promotes cartilage regeneration. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol group imparts strong adhesion, allowing it to closely adhere to the cartilage defect site and reduce the risk of shedding. The release of metal ions also further enhances the antibacterial properties of polyphenols, thereby obtaining a stabilizer.

[0047] S2. Add the stabilizer described in step S1 to 50 mL of 0.5% MOPS buffer under ice bath conditions, then quickly add graphene oxide-based composite microspheres, ultrasonically treat for 5 minutes, then add lithium strontium cross-linked gel carrier, ultrasonically treat for 10 minutes, and finally add icariin, the amount of icariin added is 0.5 g. Icariin can enhance cell activity and improve periarticular microstructure by promoting chondrocyte proliferation and differentiation, inhibiting excessive apoptosis, reducing the release of inflammatory factors, and promoting the synthesis of cartilage extracellular matrix. environment, thereby promoting the repair of cartilage damage, ultrasonic treatment for 10 minutes, freeze drying, and the addition of stabilizers enhance the binding stability and complexity of graphene oxide-based composite microspheres and lithium strontium cross-linked gel carriers, forming a porous, multi-layered, multi-cross-linked network structure with excellent mechanical and antibacterial properties, better simulating the supporting role of natural cartilage in the joints, and then through a variety of metal elements, icariin Chinese medicine ingredients and other active substances, multi-pathway and multi-target cartilage repair effects are exerted, effectively promoting cartilage regeneration and obtaining articular cartilage repair materials.

[0048] In this example, the prepared articular cartilage repair material was subjected to scanning electron microscopy to observe its microscopic morphology. Figure 1 This is a 100-fold magnified SEM image of the articular cartilage repair material prepared in Example 1. Figure 1 The articular cartilage repair material prepared in this embodiment presents a porous, multi-layered, and multi-cross-linked network structure.

[0049] Example 2

[0050] This embodiment provides an articular cartilage repair material, comprising the following components in parts by weight: 50 parts of graphene oxide-based composite microspheres, 10 parts of a stabilizer, 20 parts of a lithium strontium cross-linked gel carrier, and 30 parts of icariin.

[0051] The graphene oxide-based composite microspheres include the following components in parts by weight: 10 parts of graphene oxide, 10 parts of lysine, and 20 parts of poly(glycidyl methacrylate) microspheres.

[0052] The lithium strontium cross-linked gel carrier comprises the following components in parts by weight: 30 parts of hydrogel matrix, 5 parts of lithium chloride and 5 parts of strontium chloride.

[0053] The preparation method of graphene oxide-based composite microspheres specifically comprises the following steps:

[0054] (1) 81 mL of anhydrous ethanol, 9 mL of water, and 3.0 g of polyvinylpyrrolidone were added to a round-bottom flask in sequence, and then 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile were added and stirred evenly. The solution system was ultrasonicated and nitrogen was passed through to remove oxygen. Then, the temperature was controlled at 60 ° C in a nitrogen atmosphere and the reaction was stirred at 100 rpm for 24 h. The product was centrifuged, washed, and freeze-dried. The poly(glycidyl methacrylate) microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for the attachment and proliferation of chondrocytes. The glycidyl groups on the surface are easily coupled with bioactive molecules, further promoting cell adhesion and differentiation, thereby obtaining poly(glycidyl methacrylate) microspheres.

[0055] (2) The poly(methyl methacrylate) glycidyl ester microspheres described in step (1) were mixed with ethylenediamine and 50 mL of deionized water, and stirred at 70°C for 10 h. The amount of ethylenediamine added was 55 mL. Ethylenediamine can serve as a chemical cross-linking point to enhance the interaction between cells and materials, which is beneficial to the stability of the material. The product was centrifuged and washed several times until the pH of the solution was 7.0, and freeze-dried. Ethylenediamine introduced amino groups into the poly(methyl methacrylate) glycidyl ester molecular chain, thereby improving the positive charge and hydrophilicity of the microsphere surface. It is not only easy to adsorb extracellular matrix proteins such as collagen and promote the adhesion of chondrocytes, but also enhances the penetration and killing of bacteria to exert an antibacterial effect, thereby obtaining ethylenediamine-functionalized microspheres.

[0056] (3) Disperse 100 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 10 minutes, and then stir at 100 rpm to form a graphene oxide suspension. Graphene oxide can be used as a cartilage repair material due to its good electrical conductivity, high specific surface area and excellent mechanical properties. In addition, graphene oxide can upregulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycans and collagen, and can regulate macrophage polarization and reduce joint inflammation, thereby exerting the biological activity of cartilage repair. Under continuous stirring conditions, 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added, and finally lysine powder was added. The amount of lysine added was 0.1 g. Lysine, as an important component of collagen, can not only activate the signal pathway of growth factors, but also promote chondrocyte Proliferation and secretion of matrices such as proteoglycans and collagen, thereby accelerating the repair of cartilage defects. It can also form hydroxylysine through hydroxylation and participate in the cross-linking of collagen fibers, thereby enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cellular energy metabolism and provides chondrocytes with the nutritional support required for repair. The reaction is carried out at room temperature for 24 hours, and the precipitate is collected, washed three times with deionized water, and freeze-dried. The surface of graphene oxide is grafted with lysine, thereby improving the dispersibility of graphene oxide and the safety of graphene oxide in the body. It also has good hydrophilicity and excellent protein adsorption capacity, which is conducive to the growth and adhesion of cells and proteins on the surface of the material. The amino group of lysine can destroy the cell membrane of bacteria, and enhance the physical antibacterial effect of graphene oxide in a chemical antibacterial manner to obtain lysine graphene oxide nanoparticles;

[0057] (4) The lysine graphene oxide nanoparticles described in step (3) are added to 100 mL of deionized water, followed by adding 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinamide, stirring for 20 min, and then adding the ethylenediamine functionalized microspheres described in step (2), reacting at 50 ° C for 1 h, centrifuging, and freeze-drying the precipitate. By coating the surface of the ethylenediamine functionalized microspheres with lysine graphene oxide nanoparticles, a core-shell composite material with internal rigidity and outer flexibility is formed, which is more suitable for supporting articular cartilage, improves the mechanical properties of the material, and makes it more pressure-resistant and deformation-resistant. Among them, the ethylenediamine functionalized microspheres as the core structure further avoid the stacking of graphene oxide, expose more active groups, enhance the stimulation and promotion effect on cartilage tissue, and are beneficial to improving the repair effect. The presence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine functionalized microspheres in the body, improve the stability of the structure, and obtain graphene oxide-based composite microspheres.

[0058] The preparation method of the lithium strontium cross-linked gel carrier specifically comprises the following steps:

[0059] a. Add 1.0 g of chitosan to 50 mL of 0.6% acetic acid solution for standby use. Weigh 60 mg of sodium alginate and 20.0 g of sodium glycerophosphate and add them to 100 mL and 50 mL of deionized water, respectively. Then, add the sodium glycerophosphate solution dropwise to the chitosan solution at 4 ° C and mix evenly. Then, add the sodium alginate solution dropwise under magnetic stirring until fully fused. Adjust the pH to neutral. Through this process, a thermosensitive double-network gel is formed, which can be injected into the defect site of articular cartilage to play a filling and repair role, guide the orderly arrangement of cells, stimulate the synthesis of type II collagen and glycosaminoglycans in the cartilage matrix, and accelerate the regeneration of functional cartilage tissue. Among them, chitosan provides biological activity, sodium glycerophosphate imparts thermosensitivity, and sodium alginate enhances mechanical properties. The three complement each other to optimize performance to obtain a hydrogel matrix;

[0060] b. Lithium chloride and strontium chloride are added to 50 mL of sterile double-distilled water, the amount of lithium chloride added is 0.5 g, and the amount of strontium chloride added is 0.5 g. Lithium chloride has certain antibacterial properties, reducing the risk of infection after implantation, and lithium and strontium elements can stimulate chondrocyte proliferation and differentiation by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, the mixture is added to the hydrogel matrix described in step a, stirred at 200 rpm for 20 minutes, poured into a mold and transferred to a 37°C incubator. Lithium chloride binds to chitosan through electrostatic action, and strontium chloride cross-links with the carboxyl groups of sodium alginate. Both enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy structural collapse, poor cell adhesion and bone induction of calcium ion cross-linked gel, and give the gel the function of promoting bone regeneration. In addition, the combined use of lithium and strontium effectively inhibits the biofilm of various bacteria and has excellent antibacterial properties, thereby obtaining a lithium-strontium cross-linked gel carrier.

[0061] This embodiment provides a method for preparing an articular cartilage repair material, which specifically includes the following steps:

[0062] S1. Dissolve 3.0g of tannic acid in 100mL of ultrapure water under magnetic stirring, then add 0.3g of ferric chloride and 0.5g of copper sulfate and stir evenly. Tannic acid and metallic iron ions and copper ions form a coordination network, enhancing the mechanical strength of the material and simulating the mechanical properties of natural cartilage. The release of metal ions then promotes cartilage regeneration. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol group imparts strong adhesion, allowing it to closely adhere to the cartilage defect site and reduce the risk of shedding. The release of metal ions also further enhances the antibacterial properties of polyphenols, thereby obtaining a stabilizer.

[0063] S2. The stabilizer described in step S1 was added to 50 mL of 0.1% MOPS buffer under ice bath conditions, and then the graphene oxide-based composite microspheres were quickly added and ultrasonically treated for 1 minute. Then, the lithium strontium cross-linked gel carrier was added and ultrasonically treated for 5 minutes. Finally, icariin was added in an amount of 0.3 g. Icariin can enhance cell activity and improve periarticular microstructure by promoting chondrocyte proliferation and differentiation, inhibiting excessive apoptosis, reducing the release of inflammatory factors, and promoting the synthesis of cartilage extracellular matrix. environment, thereby promoting the repair of cartilage damage, ultrasonic treatment for 5 minutes, freeze drying, and the addition of stabilizers enhance the binding stability and complexity of graphene oxide-based composite microspheres and lithium strontium cross-linked gel carriers, forming a porous, multi-layered, multi-cross-linked network structure with excellent mechanical and antibacterial properties, better simulating the supporting role of natural cartilage in the joints, and then through a variety of metal elements, icariin Chinese medicinal ingredients and other active substances, multi-pathway and multi-target cartilage repair effects are exerted, effectively promoting cartilage regeneration and obtaining articular cartilage repair materials.

[0064] Example 3

[0065] This embodiment provides an articular cartilage repair material, comprising the following components in parts by weight: 55 parts of graphene oxide-based composite microspheres, 15 parts of a stabilizer, 25 parts of a lithium strontium cross-linked gel carrier, and 35 parts of icariin.

[0066] The graphene oxide-based composite microspheres include the following components in parts by weight: 15 parts of graphene oxide, 20 parts of lysine, and 25 parts of poly(glycidyl methacrylate) microspheres.

[0067] The lithium strontium cross-linked gel carrier comprises the following components in parts by weight: 40 parts of hydrogel matrix, 7.5 parts of lithium chloride and 7.5 parts of strontium chloride.

[0068] The preparation method of graphene oxide-based composite microspheres specifically comprises the following steps:

[0069] (1) 81 mL of anhydrous ethanol, 9 mL of water, and 3.0 g of polyvinyl pyrrolidone were added to a round-bottom flask in sequence, and then 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile were added and stirred evenly. The solution system was ultrasonicated and nitrogen was passed through to remove oxygen. Then, the temperature was controlled at 65 ° C in a nitrogen atmosphere and the reaction was stirred at 125 rpm for 24 h. The product was centrifuged, washed, and freeze-dried. The poly(glycidyl methacrylate) microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for the attachment and proliferation of chondrocytes. The glycidyl groups on the surface are easily coupled with bioactive molecules, further promoting cell adhesion and differentiation, thereby obtaining poly(glycidyl methacrylate) microspheres.

[0070] (2) The poly(methyl methacrylate) glycidyl ester microspheres described in step (1) were mixed with ethylenediamine and 50 mL of deionized water, and stirred at 75°C for 11 h. The amount of ethylenediamine added was 65 mL. Ethylenediamine can serve as a chemical cross-linking point to enhance the interaction between cells and materials, which is beneficial to the stability of the material. The product was centrifuged and washed several times until the pH of the solution was 7.0, and freeze-dried. Ethylenediamine introduced amino groups into the poly(methyl methacrylate) glycidyl ester molecular chain, which improved the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbed extracellular matrix proteins such as collagen and promoted the adhesion of chondrocytes, but also enhanced the penetration and killing of bacteria to exert an antibacterial effect, thereby obtaining ethylenediamine-functionalized microspheres.

[0071] (3) Disperse 150 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 15 minutes, and then stir at 150 rpm to form a graphene oxide suspension. Graphene oxide can be used as a cartilage repair material due to its good electrical conductivity, high specific surface area and excellent mechanical properties. In addition, graphene oxide can upregulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycans and collagen, and can regulate macrophage polarization and reduce joint inflammation, thereby exerting the biological activity of cartilage repair. Under continuous stirring, 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added, and finally lysine powder was added. The amount of lysine added was 0.2 g. Lysine, as an important component of collagen, can not only activate the signal pathway of growth factors, but also promote chondrocyte Proliferation and secretion of matrices such as proteoglycans and collagen, thereby accelerating the repair of cartilage defects. It can also form hydroxylysine through hydroxylation and participate in the cross-linking of collagen fibers, thereby enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cellular energy metabolism and provides chondrocytes with the nutritional support required for repair. The reaction is carried out at room temperature for 24 hours, and the precipitate is collected, washed 4 times with deionized water, and freeze-dried. The surface of graphene oxide is grafted with lysine, thereby improving the dispersibility of graphene oxide and the safety of graphene oxide in the body. It also has good hydrophilicity and excellent protein adsorption capacity, which is conducive to the growth and adhesion of cells and proteins on the surface of the material. The amino group of lysine can destroy the cell membrane of bacteria, and enhance the physical antibacterial effect of graphene oxide in a chemical antibacterial manner to obtain lysine graphene oxide nanoparticles.

[0072] (4) The lysine graphene oxide nanoparticles described in step (3) are added to 100 mL of deionized water, followed by the addition of 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinamide, stirred for 25 min, and then the ethylenediamine functionalized microspheres described in step (2) are added, reacted at 55 ° C for 1.5 h, centrifuged, and the precipitate is freeze-dried. By coating the surface of the ethylenediamine functionalized microspheres with lysine graphene oxide nanoparticles, a core-shell composite material with internal rigidity and outer flexibility is formed, which is more suitable for supporting articular cartilage, improves the mechanical properties of the material, and makes it more pressure-resistant and deformation-resistant. Among them, the ethylenediamine functionalized microspheres as the core structure further avoid the stacking of graphene oxide, expose more active groups, enhance the stimulation and promotion effect on cartilage tissue, and are beneficial to improving the repair effect. The presence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine functionalized microspheres in the body, improve the stability of the structure, and obtain graphene oxide-based composite microspheres.

[0073] The preparation method of the lithium strontium cross-linked gel carrier specifically comprises the following steps:

[0074] a. Add 1.5 g of chitosan to 50 mL of 0.6% acetic acid solution for standby use. Weigh 70 mg of sodium alginate and 22.5 g of sodium glycerophosphate and add them to 100 mL and 50 mL of deionized water, respectively. Then, add the sodium glycerophosphate solution dropwise to the chitosan solution at 4 ° C and mix evenly. Then, add the sodium alginate solution dropwise under magnetic stirring until fully fused. Adjust the pH to neutral. Through this process, a thermosensitive double-network gel is formed, which can be injected into the defect site of articular cartilage to play a filling and repair role, guide cells to arrange in order, stimulate the synthesis of type II collagen and glycosaminoglycans in the cartilage matrix, and accelerate the regeneration of functional cartilage tissue. Chitosan provides biological activity, sodium glycerophosphate imparts thermosensitivity, and sodium alginate enhances mechanical properties. The three complement each other to optimize performance to obtain a hydrogel matrix.

[0075] b. Lithium chloride and strontium chloride are added to 50 mL of sterile double-distilled water, the amount of lithium chloride added is 0.75 g, and the amount of strontium chloride added is 0.75 g. Lithium chloride has certain antibacterial properties, reducing the risk of infection after implantation, and lithium and strontium elements can stimulate chondrocyte proliferation and differentiation by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, the mixture is added to the hydrogel matrix described in step a, stirred at 350 rpm for 25 minutes, poured into a mold and transferred to a 37°C incubator. Lithium chloride binds to chitosan through electrostatic action, and strontium chloride cross-links with the carboxyl groups of sodium alginate. Both enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy structural collapse, poor cell adhesion and bone induction of calcium ion cross-linked gel, and give the gel the function of promoting bone regeneration. In addition, the combined use of lithium and strontium effectively inhibits the biofilm of various bacteria and has excellent antibacterial properties, thereby obtaining a lithium-strontium cross-linked gel carrier.

[0076] This embodiment provides a method for preparing an articular cartilage repair material, which specifically includes the following steps:

[0077] S1. Dissolve 3.5g of tannic acid in 100mL of ultrapure water under magnetic stirring, then add 0.35g of ferric chloride and 0.5g of copper sulfate and stir evenly. Tannic acid and metallic iron ions and copper ions form a coordination network, enhancing the mechanical strength of the material and simulating the mechanical properties of natural cartilage. The release of metal ions then promotes cartilage regeneration. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol group imparts strong adhesion, allowing it to closely adhere to the cartilage defect site and reduce the risk of shedding. The release of metal ions also further enhances the antibacterial properties of polyphenols, thereby obtaining a stabilizer.

[0078] S2. The stabilizer described in step S1 was added to 50 mL of 0.3% MOPS buffer under ice bath conditions, and then the graphene oxide-based composite microspheres were quickly added and ultrasonically treated for 3 minutes. Then, the lithium strontium cross-linked gel carrier was added and ultrasonically treated for 7.5 minutes. Finally, icariin was added in an amount of 0.4 g. Icariin can enhance cell activity and improve periarticular microstructure by promoting chondrocyte proliferation and differentiation, inhibiting excessive apoptosis, reducing the release of inflammatory factors, and promoting the synthesis of cartilage extracellular matrix. environment, thereby promoting the repair of cartilage damage, ultrasonic treatment for 7.5 minutes, freeze drying, and the addition of stabilizers enhance the binding stability and complexity of graphene oxide-based composite microspheres and lithium strontium cross-linked gel carriers, forming a porous, multi-layered, multi-cross-linked network structure with excellent mechanical and antibacterial properties, better simulating the supporting role of natural cartilage in the joints, and then through a variety of metal elements, icariin Chinese medicine ingredients and other active substances, multi-pathway and multi-target cartilage repair effects are exerted, effectively promoting cartilage regeneration and obtaining articular cartilage repair materials.

[0079] Example 4

[0080] This embodiment provides an articular cartilage repair material, comprising the following components in parts by weight: 50 parts of graphene oxide-based composite microspheres, 10 parts of a stabilizer, 30 parts of a lithium strontium cross-linked gel carrier, and 40 parts of icariin.

[0081] The graphene oxide-based composite microspheres include the following components in parts by weight: 20 parts of graphene oxide, 10 parts of lysine, and 30 parts of poly(glycidyl methacrylate) microspheres.

[0082] The lithium strontium cross-linked gel carrier comprises the following components in parts by weight: 50 parts of hydrogel matrix, 5 parts of lithium chloride and 10 parts of strontium chloride.

[0083] The preparation method of graphene oxide-based composite microspheres specifically comprises the following steps:

[0084] (1) 81 mL of anhydrous ethanol, 9 mL of water, and 3.0 g of polyvinyl pyrrolidone were added to a round-bottom flask in sequence, and then 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile were added and stirred evenly. The solution system was ultrasonicated and nitrogen was passed through to remove oxygen. Then, the temperature was controlled at 70 ° C in a nitrogen atmosphere and the reaction was stirred at 150 rpm for 24 h. The product was centrifuged, washed, and freeze-dried. The poly(glycidyl methacrylate) microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for the attachment and proliferation of chondrocytes. The glycidyl groups on the surface are easily coupled with bioactive molecules, further promoting cell adhesion and differentiation, thereby obtaining poly(glycidyl methacrylate) microspheres.

[0085] (2) The poly(methyl methacrylate) glycidyl ester microspheres described in step (1) were mixed with ethylenediamine and 50 mL of deionized water, and stirred at 80°C for 10 h. The amount of ethylenediamine added was 75 mL. Ethylenediamine can serve as a chemical cross-linking point to enhance the interaction between cells and materials, which is beneficial to the stability of the material. The product was centrifuged and washed several times until the pH of the solution was 7.0, and freeze-dried. Ethylenediamine introduced amino groups into the poly(methyl methacrylate) glycidyl ester molecular chain, thereby improving the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbed extracellular matrix proteins such as collagen and promoted the adhesion of chondrocytes, but also enhanced the penetration and killing of bacteria to exert an antibacterial effect, thereby obtaining ethylenediamine-functionalized microspheres.

[0086] (3) Disperse 200 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 10 minutes, and then stir at 200 rpm to form a graphene oxide suspension. Graphene oxide can be used as a cartilage repair material due to its good electrical conductivity, high specific surface area and excellent mechanical properties. In addition, graphene oxide can upregulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycans and collagen, and can regulate macrophage polarization and reduce joint inflammation, thereby exerting the biological activity of cartilage repair. Under continuous stirring, 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added, and finally lysine powder was added. The amount of lysine added was 0.1 g. Lysine, as an important component of collagen, can not only activate the signal pathway of growth factors, but also promote the growth of chondrocytes. Proliferation and secretion of matrices such as proteoglycans and collagen, thereby accelerating the repair of cartilage defects, and forming hydroxylysine through hydroxylation, participating in the cross-linking of collagen fibers, thereby enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cellular energy metabolism, providing chondrocytes with the nutritional support required for repair. The reaction was carried out at room temperature for 24 hours, and the precipitate was collected, washed with deionized water 5 times, and freeze-dried. The surface of graphene oxide was grafted with lysine, thereby improving the dispersibility of graphene oxide and the safety of graphene oxide in the body. It also has good hydrophilicity and excellent protein adsorption capacity, which is conducive to the growth and adhesion of cells and proteins on the surface of the material. The amino group of lysine can destroy the cell membrane of bacteria, and enhance the physical antibacterial effect of graphene oxide in a chemical antibacterial manner to obtain lysine graphene oxide nanoparticles;

[0087] (4) The lysine graphene oxide nanoparticles described in step (3) are added to 100 mL of deionized water, followed by the addition of 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinamide, stirred for 20 min, and then the ethylenediamine functionalized microspheres described in step (2) are added, reacted at 60 ° C for 1 h, centrifuged, and the precipitate is freeze-dried. By coating the surface of the ethylenediamine functionalized microspheres with lysine graphene oxide nanoparticles, a core-shell composite material with internal rigidity and outer flexibility is formed, which is more suitable for supporting articular cartilage, improves the mechanical properties of the material, and makes it more pressure-resistant and deformation-resistant. The ethylenediamine functionalized microspheres serve as the core structure, further avoiding the stacking of graphene oxide, exposing more active groups, enhancing the stimulating effect on cartilage tissue, and is beneficial to improving the repair effect. The presence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine functionalized microspheres in the body, improve the stability of the structure, and obtain graphene oxide-based composite microspheres.

[0088] The preparation method of the lithium strontium cross-linked gel carrier specifically comprises the following steps:

[0089] a. Add 2.0 g of chitosan to 50 mL of 0.6% acetic acid solution for standby use. Weigh 80 mg of sodium alginate and 25.0 g of sodium glycerophosphate and add them to 100 mL and 50 mL of deionized water, respectively. Then, add the sodium glycerophosphate solution dropwise to the chitosan solution at 4 ° C. Mix evenly, and then add the sodium alginate solution dropwise under magnetic stirring until fully fused. Adjust the pH to neutral. Through this process, a thermosensitive double-network gel is formed, which can be injected into the defect site of articular cartilage to play a filling and repair role, guide the orderly arrangement of cells, stimulate the synthesis of type II collagen and glycosaminoglycans in the cartilage matrix, and accelerate the regeneration of functional cartilage tissue. Chitosan provides biological activity, sodium glycerophosphate imparts thermosensitivity, and sodium alginate enhances mechanical properties. The three complement each other to optimize performance to obtain a hydrogel matrix;

[0090] b. Lithium chloride and strontium chloride are added to 50 mL of sterile double-distilled water, the amount of lithium chloride added is 0.5 g, and the amount of strontium chloride added is 1.0 g. Lithium chloride has certain antibacterial properties, reducing the risk of infection after implantation, and lithium and strontium elements can stimulate chondrocyte proliferation and differentiation by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, the mixture is added to the hydrogel matrix described in step a, stirred at 500 rpm for 20 minutes, poured into a mold and transferred to a 37°C incubator. Lithium chloride binds to chitosan through electrostatic action, and strontium chloride cross-links with the carboxyl groups of sodium alginate. Both enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy structural collapse, poor cell adhesion and bone induction of calcium ion cross-linked gel, and give the gel the function of promoting bone regeneration. In addition, the combined use of lithium and strontium effectively inhibits the biofilm of various bacteria and has excellent antibacterial properties, thereby obtaining a lithium-strontium cross-linked gel carrier.

[0091] This embodiment provides a method for preparing an articular cartilage repair material, which specifically includes the following steps:

[0092] S1. Dissolve 4.0 g of tannic acid in 100 mL of ultrapure water under magnetic stirring, then add 0.3 g of ferric chloride and 0.5 g of copper sulfate and stir evenly. Tannic acid and metallic iron ions and copper ions form a coordination network, enhancing the mechanical strength of the material and simulating the mechanical properties of natural cartilage. The release of metal ions then promotes cartilage regeneration. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol group imparts strong adhesion, allowing it to fit closely to the cartilage defect site and reduce the risk of falling off. The release of metal ions also further enhances the antibacterial properties of polyphenols, thereby obtaining a stabilizer.

[0093] S2. The stabilizer described in step S1 was added to 50 mL of 0.5% MOPS buffer under ice bath conditions, and then the graphene oxide-based composite microspheres were quickly added and ultrasonically treated for 1 min. Then, the lithium strontium cross-linked gel carrier was added and ultrasonically treated for 5 min. Finally, icariin was added in an amount of 0.5 g. Icariin can enhance cell activity and improve periarticular microstructure by promoting chondrocyte proliferation and differentiation, inhibiting excessive apoptosis, reducing the release of inflammatory factors, and promoting the synthesis of cartilage extracellular matrix. environment, thereby promoting the repair of cartilage damage, ultrasonic treatment for 5 minutes, freeze drying, and the addition of stabilizers enhance the binding stability and complexity of graphene oxide-based composite microspheres and lithium strontium cross-linked gel carriers, forming a porous, multi-layered, multi-cross-linked network structure with excellent mechanical and antibacterial properties, better simulating the supporting role of natural cartilage in the joints, and then through a variety of metal elements, icariin Chinese medicinal ingredients and other active substances, multi-pathway and multi-target cartilage repair effects are exerted, effectively promoting cartilage regeneration and obtaining articular cartilage repair materials.

[0094] Comparative Example 1

[0095] This comparative example provides an articular cartilage repair material, which differs from Example 1 in that the graphene oxide-based composite microspheres do not contain graphene oxide and lysine; the preparation method of the graphene oxide-based composite microspheres does not include step (3); the preparation method of the lithium strontium cross-linked gel carrier is the same as that of Example 1; and the preparation method of the articular cartilage repair material is the same as that of Example 1.

[0096] Comparative Example 2

[0097] This comparative example provides an articular cartilage repair material, which differs from Example 1 in that the lithium strontium cross-linked gel carrier does not contain lithium chloride and strontium chloride; the preparation method of graphene oxide-based composite microspheres is the same as that in Example 1; sterile double distilled water containing lithium chloride and strontium chloride is not added in step b of the preparation method of the lithium strontium cross-linked gel carrier; the preparation method of the articular cartilage repair material is the same as that in Example 1.

[0098] Comparative Example 3

[0099] This comparative example provides an articular cartilage repair material, which differs from Example 1 in that the articular cartilage repair material does not contain a stabilizer; the preparation method of graphene oxide-based composite microspheres is the same as that of Example 1; the preparation method of the lithium strontium cross-linked gel carrier is the same as that of Example 1; and the preparation method of the articular cartilage repair material does not include step S1.

[0100] Experimental Example 1

[0101] Mechanical properties test

[0102] Test samples: articular cartilage repair materials prepared in Examples 1-4 and Comparative Examples 1-3.

[0103] Test method: The test samples were subjected to compression tests using an electronic universal material testing machine. A 100N sensor was used for the compression test. The compression rate was 5mm / min and the samples were compressed to a strain of 50%. During the compression test, 10 repeated loading and unloading cycles were performed. The compressive strain was kept constant at 40%. Three parallel tests were performed for each group of samples and the average value was taken.

[0104] Figure 2 It is a graph of the mechanical strength results of Examples 1-4 and Comparative Examples 1-3; as shown in the figure, the mechanical strength of Examples 1-4 is 10.5-12.6 MPa, indicating that the mechanical properties are better; the mechanical strength of Comparative Examples 1-3 is 5.1-7.8 MPa, indicating that the mechanical properties are poor; the graphene oxide-based composite microspheres of Comparative Example 1 do not contain graphene oxide and lysine, and cannot form a coating on the ethylenediamine functionalized microspheres, lacking the resistance of the flexible shell, resulting in poor mechanical properties; the lithium strontium cross-linked gel carrier of Comparative Example 2 does not contain lithium chloride and strontium chloride, and cannot form a gel structure with uniform cross-linking and not easy to collapse, resulting in poor mechanical properties; the articular cartilage repair material of Comparative Example 3 does not contain a stabilizer, and cannot achieve multi-component cross-linking of the lithium strontium cross-linked gel carrier and the graphene oxide-based composite microspheres, which reduces the stability of the porous multi-layer network structure and results in poor mechanical properties.

[0105] Experimental Example 2

[0106] Antibacterial performance test

[0107] Test samples: articular cartilage repair materials prepared in Examples 1-4 and Comparative Examples 1-3.

[0108] Test method: There are many different types of bacteria that can cause infection in implants. In this experiment, we selected common Staphylococcus aureus and Escherichia coli as experimental bacteria for antibacterial experiments. The test sample was designed into a flat cylinder with a diameter of 10 mm and a thickness of 2 mm. After UV sterilization, it was placed on a plate coated with 10 6 cfu / mL of bacterial solution on agar medium, turn the culture dish upside down and place it in a constant temperature incubator at 37℃ for 24 hours, then take it out to observe the growth of the inhibition zone. The larger the diameter of the inhibition zone (mm), the stronger the inhibitory effect on the experimental bacteria.

[0109] Figure 3The results of the inhibition zone diameters of Examples 1-4 and Comparative Examples 1-3 are shown in FIG. As shown in the figure, the diameters of the inhibition zones of Staphylococcus aureus and Escherichia coli in Examples 1-4 are 40-48 mm and 38-45 mm, respectively, both of which are >35 mm, indicating that the antibacterial activity is strong and the risk of infection is effectively reduced; the diameters of the inhibition zones of Staphylococcus aureus and Escherichia coli in Comparative Examples 1-3 are 9-19 mm and 7-16 mm, respectively, both of which are <20 mm, indicating that the antibacterial activity is general and the risk of infection cannot be effectively reduced; the graphene oxide-based composite microspheres in Comparative Example 1 do not contain graphene oxide and lysine, and cannot exert the dual killing effect of physical antibacterial and chemical sterilization, nor can they be sterilized by The antibacterial properties of the ethylenediamine functionalized microspheres are improved by coating, resulting in general antibacterial properties, which cannot effectively reduce the risk of infection; the lithium strontium cross-linked gel carrier of comparative example 2 does not contain lithium chloride and strontium chloride, and cannot exert the inhibitory effect of lithium and strontium on the biofilms of various bacteria, and is not conducive to the uniformity and stability of the gel carrier, and thus cannot better prevent the invasion and migration of bacteria, resulting in general antibacterial properties, and cannot effectively reduce the risk of infection; the articular cartilage repair material of comparative example 3 does not contain a stabilizer, which is not conducive to the formation of a porous, multi-layer, multi-cross-linked network structure, weakens the barrier and adhesion to bacteria, resulting in general antibacterial properties, and cannot effectively reduce the risk of infection.

[0110] Experimental Example 3

[0111] Repair effect experiment

[0112] Test samples: articular cartilage repair materials prepared in Examples 1-4 and Comparative Examples 1-3.

[0113] Test method: 70 New Zealand white rabbits, 3 months old and weighing 1.8-2.5 kg, were randomly divided into 7 groups, with 70 rabbits in each group. They were anesthetized with 2.5% sodium pentobarbital injected intravenously at the ear margin. After proper anesthesia, their limbs were properly fixed, and the lateral edge of the patellar ligament was cut in sequence to fully expose the weight-bearing area of ​​the lateral condyle of the lower end of the femur. A cylindrical defect with a diameter of 4 mm and a depth of 3 mm was created thereon. The subchondral bone was drilled through, and after removing the blood clot in the defect, the test samples were placed and filled. The wounds were sutured layer by layer. After the operation, the wounds were disinfected with iodine and bandaged with gauze without external fixation. Penicillin-streptomycin was injected intramuscularly to prevent infection. The rabbits were housed in single cages after the operation. CT scans were performed 16 weeks later to observe the repair of the cartilage. The International Cartilage Repair Society (ICRS) scoring criteria were used to score and calculate the average value. The specific scoring criteria for the degree of defect repair and overall repair are as follows:

[0114] The scoring criteria for the degree of defect repair are as follows:

[0115] the same as the surrounding normal cartilage, 4 points;

[0116] 75% of the defect depth was repaired, 3 points;

[0117] 2 points for repairing 50% of the defect depth;

[0118] 1 point for repairing 25% of the defect depth;

[0119] No repair, 0 points;

[0120] The scoring criteria for boundary integration are as follows:

[0121] No obvious border, completely integrated with the surrounding cartilage, 4 points;

[0122] margin <1 mm, 3 points;

[0123] 75% integration, 25% significant gap >1 mm, 2 points;

[0124] 50% integration, 50% significant gaps >1 mm, 1 point;

[0125] less than 25% integration, 0 points;

[0126] The scoring criteria for general appearance observation are as follows:

[0127] Smooth and intact surface, 4 points;

[0128] Surface is fibrous, 3 points;

[0129] Small cracks or scars can be seen on the surface, 2 points;

[0130] A small number of large cracks can be seen on the surface, 1 point;

[0131] No repair or complete degeneration, 0 points;

[0132] The scoring criteria for overall restoration are as follows:

[0133] Grade I, completely normal, 12 points;

[0134] Grade II, good repair, 8-11 points;

[0135] Grade III: repaired but with poor results, 4-7 points;

[0136] Grade IV, no repair effect, 0-3 points.

[0137] Figure 4The results of articular cartilage repair of Examples 1-4 and Comparative Examples 1-3 are shown in FIG. As shown in the figure, the scores of defect repair degree and overall repair of Examples 1-4 are 3.5-3.9 and 10.2-11.9, respectively, indicating that the repair effect is better; the scores of defect repair degree and overall repair of Comparative Examples 1-3 are 1.8-2.8 and 5.3-7.2, respectively, indicating that the repair effect is average; the graphene oxide-based composite microspheres of Comparative Example 1 do not contain graphene oxide and lysine, and cannot be coated with ethylenediamine functionalized microspheres to form a flexible shell, which neither can play the repair role of graphene oxide and lysine, nor is it conducive to the repair of cells and proteins. The growth and adhesion of white on the surface of the microspheres cannot ensure the stable repair of the microspheres in the body, resulting in a mediocre repair effect; the lithium strontium cross-linked gel carrier of Comparative Example 2 does not contain lithium chloride and strontium chloride, and cannot give the gel carrier cell adhesion and bone induction, resulting in a mediocre repair effect; the articular cartilage repair material of Comparative Example 3 does not contain a stabilizer, and cannot form a porous, multi-layer, multi-cross-linked adhesive network structure, which is not conducive to the support and adhesion of the articular cartilage, reduces the load of icariin, and is not conducive to the effective coordination of multiple active substances to stably play a repair role on the cartilage, resulting in a mediocre repair effect.

[0138] The above experimental results show that the mechanical properties, antibacterial properties and articular cartilage repair of Examples 1-4 of the present invention are significantly better than those of the samples of Comparative Examples 1-3. Among them, Example 1 using graphene oxide-based composite microspheres, lithium strontium cross-linked gel carriers and stabilizers has better mechanical properties, stronger antibacterial properties and better repair effect. The graphene oxide-based composite microspheres are combined and doped with the lithium strontium cross-linked gel carrier. Under the bidirectional cross-linking action of the stabilizer, a porous, multi-layer, multi-cross-linked adhesive network structure is formed, which improves the mechanical properties and antibacterial properties of the material and is better suitable for support and attachment of articular cartilage. Then, it is loaded with icariin and cooperates with various metal elements and bioactive substances to promote cartilage repair and regeneration, significantly improving the repair effect of articular cartilage. In the lithium strontium cross-linked gel carrier, lithium chloride and strontium chloride replace calcium ions and are cross-linked with the hydrogel matrix formed by chitosan, sodium glycerophosphate and sodium alginate to obtain a gel carrier with uniform cross-linking, stable structure, cell adhesion and bone induction.

[0139] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions, and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

[0140] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. An articular cartilage repair material, characterized by: The articular cartilage repair material comprises the following components in parts by weight: 50-60 parts of graphene oxide-based composite microspheres, 10-20 parts of a stabilizer, 20-30 parts of a lithium strontium cross-linked gel carrier, and 30-40 parts of icariin; the graphene oxide-based composite microspheres comprise the following components in parts by weight: 10-20 parts of graphene oxide, 10-30 parts of lysine, and 20-30 parts of poly(glycidyl methacrylate) microspheres; the lithium strontium cross-linked gel carrier comprises the following components in parts by weight: 30-50 parts of a hydrogel matrix, 5-10 parts of lithium chloride, and 5-10 parts of strontium chloride; The method for preparing the graphene oxide-based composite microspheres specifically comprises the following steps: (1) 81 mL of anhydrous ethanol, 9 mL of water, and 3.0 g of polyvinyl pyrrolidone were added to a round-bottom flask in sequence, and then 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile were added and stirred evenly. The solution system was ultrasonicated and nitrogen was passed through to remove oxygen. The temperature was controlled at 60-70 ° C in a nitrogen atmosphere and the reaction was stirred at 100-150 rpm for 24 h. The product was centrifuged, washed, and freeze-dried to obtain poly(glycidyl methacrylate) microspheres. (2) The poly(glycidyl methacrylate) microspheres described in step (1) were mixed with ethylenediamine and 50 mL of deionized water, stirred at 70-80° C. for 10-12 h, and the product was centrifuged and washed several times until the pH of the solution was 7.0, and freeze-dried to obtain ethylenediamine-functionalized microspheres; (3) Disperse 100-200 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 10-20 min, then stir at 100-200 rpm to form a graphene oxide suspension, add 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide under continuous stirring, and finally add lysine powder. React at room temperature for 24 h, collect the precipitate, wash it with deionized water 3-5 times, and freeze-dry to obtain lysine graphene oxide nanoparticles; (4) The lysine graphene oxide nanoparticles described in step (3) were added to 100 mL of deionized water, followed by the addition of 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinamide, stirred for 20-30 min, and then the ethylenediamine functionalized microspheres described in step (2) were added, reacted at 50-60° C. for 1-2 h, centrifuged, and the precipitate was freeze-dried to obtain graphene oxide-based composite microspheres; The preparation method of the lithium strontium cross-linked gel carrier specifically comprises the following steps: a. Add 1.0-2.0 g of chitosan to 50 mL of 0.6% acetic acid solution and set aside. Weigh 60-80 mg of sodium alginate and 20.0-25.0 g of sodium glycerophosphate and add them to 100 mL and 50 mL of deionized water, respectively. Then, add the sodium glycerophosphate solution dropwise to the chitosan solution at 4°C and mix well. Then, add the sodium alginate solution dropwise under magnetic stirring until fully fused. Adjust the pH to neutral to obtain a hydrogel matrix. b. Add lithium chloride and strontium chloride to 50 mL of sterile double-distilled water, stir evenly, and then add to the hydrogel matrix described in step a. Stir at 200-500 rpm for 20-30 minutes. Pour into a mold and transfer to a 37°C incubator to obtain a lithium-strontium cross-linked gel carrier. The preparation method of the articular cartilage repair material specifically comprises the following steps: S1. Dissolve 3.0-4.0 g of tannic acid in 100 mL of ultrapure water under magnetic stirring, then add 0.3-0.4 g of ferric chloride and 0.5 g of copper sulfate, and stir well to obtain a stabilizer; S2. Add the stabilizer described in step S1 to 50 mL of MOPS buffer with a mass fraction of 0.1-0.5% under ice bath conditions, then quickly add the graphene oxide-based composite microspheres, ultrasonically treat for 1-5 minutes, then add the lithium strontium cross-linked gel carrier, ultrasonically treat for 5-10 minutes, and finally add icariin, ultrasonically treat for 5-10 minutes, and freeze-dry to obtain the articular cartilage repair material.

2. A method for preparing the articular cartilage repair material according to claim 1, characterized in that: The specific steps include: S1. Dissolve 3.0-4.0 g of tannic acid in 100 mL of ultrapure water under magnetic stirring, then add 0.3-0.4 g of ferric chloride and 0.5 g of copper sulfate, and stir well to obtain a stabilizer; S2. Add the stabilizer described in step S1 to 50 mL of 0.1-0.5% MOPS buffer under ice bath conditions, then quickly add the graphene oxide-based composite microspheres, sonicate for 1-5 minutes, then add the lithium strontium cross-linked gel carrier, sonicate for 5-10 minutes, and finally add icariin, sonicate for 5-10 minutes, and freeze-dry to obtain the articular cartilage repair material; The method for preparing the graphene oxide-based composite microspheres specifically comprises the following steps: (1) 81 mL of anhydrous ethanol, 9 mL of water, and 3.0 g of polyvinyl pyrrolidone were added to a round-bottom flask in sequence, and then 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile were added and stirred evenly. The solution system was ultrasonicated and nitrogen was passed through to remove oxygen. The temperature was controlled at 60-70 ° C in a nitrogen atmosphere and the reaction was stirred at 100-150 rpm for 24 h. The product was centrifuged, washed, and freeze-dried to obtain poly(glycidyl methacrylate) microspheres. (2) The poly(glycidyl methacrylate) microspheres described in step (1) were mixed with ethylenediamine and 50 mL of deionized water, stirred at 70-80° C. for 10-12 h, and the product was centrifuged and washed several times until the pH of the solution was 7.0, and freeze-dried to obtain ethylenediamine-functionalized microspheres; (3) Disperse 100-200 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 10-20 min, then stir at 100-200 rpm to form a graphene oxide suspension, add 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide under continuous stirring, and finally add lysine powder. React at room temperature for 24 h, collect the precipitate, wash it with deionized water 3-5 times, and freeze-dry to obtain lysine graphene oxide nanoparticles; (4) The lysine graphene oxide nanoparticles described in step (3) were added to 100 mL of deionized water, followed by the addition of 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinamide, stirred for 20-30 min, and then the ethylenediamine functionalized microspheres described in step (2) were added, reacted at 50-60° C. for 1-2 h, centrifuged, and the precipitate was freeze-dried to obtain graphene oxide-based composite microspheres; The preparation method of the lithium strontium cross-linked gel carrier specifically comprises the following steps: a. Add 1.0-2.0 g of chitosan to 50 mL of 0.6% acetic acid solution and set aside. Weigh 60-80 mg of sodium alginate and 20.0-25.0 g of sodium glycerophosphate and add them to 100 mL and 50 mL of deionized water, respectively. Then, add the sodium glycerophosphate solution dropwise to the chitosan solution at 4°C and mix well. Then, add the sodium alginate solution dropwise under magnetic stirring until fully fused. Adjust the pH to neutral to obtain a hydrogel matrix. b. Add lithium chloride and strontium chloride to 50 mL of sterile double-distilled water, stir evenly, and then add to the hydrogel matrix described in step a. Stir at 200-500 rpm for 20-30 minutes. Pour into a mold and transfer to a 37°C incubator to obtain a lithium-strontium cross-linked gel carrier.

3. The method for preparing the articular cartilage repair material according to claim 2, wherein: In step S2, the amount of icariin added is 0.3-0.5 g.

4. The method for preparing the articular cartilage repair material according to claim 3, wherein: In step (2), the amount of ethylenediamine added is 55-75 mL.

5. The method for preparing the articular cartilage repair material according to claim 4, characterized in that: In step (3), the amount of lysine added is 0.1-0.3 g.

6. The method for preparing the articular cartilage repair material according to claim 5, characterized in that: In step b, the amount of lithium chloride added is 0.5-1.0 g, and the amount of strontium chloride added is 0.5-1.0 g.

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