Articular cartilage repair material and preparation method thereof

Abstract

The 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 prepared from 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 and the lithium-strontium cross-linked gel carrier are combined and doped, and a porous, multilayer and multi-cross-linked adhesive network structure is formed under the bidirectional cross-linking action of the stabilizer, so that the mechanical property and antibacterial property of the material are improved, the material is better suitable for supporting and adhesion at articular cartilage, the risk of infection can be reduced, and the application prospect is wide. Then icariin is loaded and cooperates with various metal elements and bioactive substances to jointly promote cartilage repair and regeneration, and the repair effect of articular cartilage is remarkably improved.

Description

Technical Field

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

[0002] Degenerative changes or defects of articular cartilage caused by advanced age, unreasonable exercise, mechanical injury or certain infectious diseases are relatively common clinically, often leading to joint pain and limited mobility, seriously affecting people's health level. Articular cartilage is composed of chondrocytes and extracellular matrix, lacking the distribution of blood vessels, nerves and lymphatic vessels. The nutrition uptake mainly depends on the circulation of synovial fluid, which limits the activity of chondrocytes. At the same time, the number of chondrocytes is small and the repair ability is limited. These reasons lead to the obstruction of the self-healing process once the articular cartilage is damaged. Although existing treatment methods such as autologous cartilage transplantation and stem cell therapy have improved the treatment effect of cartilage injury to a certain extent, due to the great difficulty of cartilage repair, the curative effect of existing therapies 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 performance have opened up new research avenues for the treatment of cartilage injury. 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 to guide the formation of extracellular matrix and provide necessary structural support for the newly formed tissue.

[0004] Currently, the following main problems exist in the prior art:

[0005] The mechanical properties of hydrogel scaffold materials are poor, unable to withstand the repeated stress of joint activities, affecting the stability of the scaffold structure, and the antibacterial properties of hydrogels are insufficient, prone to infection risks, thus limiting the repair effect on articular cartilage. Summary of the Invention

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

[0007] 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.

[0008] The lithium-strontium crosslinked 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 includes the following steps:

[0010] (1) Add 81 mL of absolute ethanol, 9 mL of water, and 3.0 g of polyvinylpyrrolidone into a round-bottom flask in sequence, then add 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile, stir evenly. After ultrasonic treatment of the solution system and purging with nitrogen to remove oxygen, under a nitrogen atmosphere, control the temperature at 60 - 70 °C, stir and react at a speed of 100 - 150 rpm for 24 h. Centrifuge, wash, and freeze-dry the product. The polyglycidyl methacrylate microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for the adhesion and proliferation of chondrocytes. The glycidyl groups on its surface are easy to couple with bioactive molecules, further promoting cell adhesion and differentiation, and obtain polyglycidyl methacrylate microspheres;

[0011] (2) Mix the polyglycidyl methacrylate microspheres described in step (1) with ethylenediamine and 50 mL of deionized water evenly, stir at 70 - 80 °C for 10 - 12 h. Centrifuge and wash the product multiple times until the solution pH is 7.0, then freeze-dry. Ethylenediamine introduces amino groups on the polyglycidyl methacrylate molecular chain, improving the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbs extracellular matrix proteins such as collagen, promotes the adhesion of chondrocytes, but also enhances the penetration and killing of bacteria to play an antibacterial role, and obtains ethylenediamine-functionalized microspheres;

[0012] (3) Disperse 100 - 200 mg of graphene oxide in 50 mL of deionized water, first perform ultrasonic treatment for 10 - 20 min, and then stir at a speed of 100 - 200 rpm to form a graphene oxide suspension. Graphene oxide can be used as a cartilage repair material with good electrical conductivity, high specific surface area, and excellent mechanical properties. Moreover, graphene oxide can up-regulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycans and collagen, and can alleviate joint inflammation by regulating macrophage polarization, thereby playing a biological active role in cartilage repair. Under continuous stirring conditions, add 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and finally add lysine powder, react at room temperature for 24 h, collect the precipitate, wash it 3 - 5 times with deionized water, and freeze-dry. Graft modification of the graphene oxide surface is carried out through lysine, thereby improving the dispersibility of graphene oxide, enhancing the safety of graphene oxide in vivo, and also having good hydrophilicity and excellent protein adsorption ability, which is beneficial to the growth and adhesion of cells and proteins on the material surface. The amino group of lysine can damage the cell membrane of bacteria and enhance the physical antibacterial property of graphene oxide in a chemical antibacterial manner, and obtain lysine-functionalized graphene oxide nanoparticles;

[0013] (4) Add the lysine-functionalized graphene oxide nanoparticles described in step (3) to 100 mL of deionized water. Then add 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinimide, and stir for 20 - 30 min. Next, add the ethylenediamine-functionalized microspheres described in step (2), and react at 50 - 60 °C for 1 - 2 h. Centrifuge, and freeze-dry the precipitate. By coating the surface of the ethylenediamine-functionalized microspheres with lysine-functionalized graphene oxide nanoparticles, a core-shell composite material with a rigid interior and a flexible outer layer is formed, which is more suitable for supporting the articular cartilage, improving the mechanical properties of the material, making it more pressure-resistant and resistant to deformation. Among them, the ethylenediamine-functionalized microspheres serve as the core structure, further avoiding the stacking of graphene oxide, exposing more active groups, enhancing the stimulatory and promoting effect on cartilage tissue, and being beneficial to improving the repair effect. The existence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine-functionalized microspheres in vivo, improving the stability of the structure, and obtaining graphene oxide-based composite microspheres;

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

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

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

[0017] a. Add 1.0 - 2.0 g of chitosan to 50 mL of acetic acid solution with a mass fraction of 0.6% for later 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 gradually add the sodium glycerophosphate solution to the chitosan solution dropwise at 4 °C, mix evenly, and then gradually 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 articular cartilage defect site to play a filling and repair role, guiding the orderly arrangement of cells, stimulating the synthesis of type II collagen and glycosaminoglycans in the cartilage matrix, and accelerating 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 the performance, and a hydrogel matrix is obtained;

[0018] b. Add lithium chloride and strontium chloride into 50 mL of sterile double-distilled water. After stirring evenly, add them into the hydrogel matrix described in step a, stir at a speed of 200 - 500 rpm for 20 - 30 min, pour into a mold and transfer to an incubator at 37°C. Lithium chloride binds to chitosan through electrostatic interaction, and strontium chloride crosslinks and binds with the carboxyl groups of sodium alginate. The two enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy structural collapse, poor cell adhesion and osteoinductivity existing in the calcium ion-crosslinked gel, endow the gel with the function of promoting bone regeneration, and the combined use of lithium element and strontium element effectively inhibits the biofilms of various bacteria and has excellent antibacterial properties, thus obtaining a lithium-strontium crosslinked gel carrier;

[0019] Preferably, in step b, the addition amount of lithium chloride is 0.5 - 1.0 g, and the addition amount of strontium chloride is 0.5 - 1.0 g. Lithium chloride has certain antibacterial properties, reducing the infection risk after implantation, and lithium element and strontium element 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 preparation method of an articular cartilage repair material, which specifically includes the following steps:

[0021] 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 evenly. Tannic acid forms a coordination network with metal iron ions and copper ions, enhancing the mechanical strength of the material, simulating the mechanical properties of natural cartilage, and playing a role in promoting cartilage regeneration through the release of metal ions. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol groups therein endow strong adhesion ability, can closely fit the cartilage defect site, reduce the risk of shedding, and the release of metal ions further enhances the antibacterial properties of polyphenols, thus obtaining a stabilizer;

[0022] S2. Add the stabilizer described in step S1 into 50 mL of MOPS buffer solution with a mass fraction of 0.1 - 0.5% under ice bath conditions, then quickly add graphene oxide-based composite microspheres, ultrasonically treat for 1 - 5 min, then add the lithium-strontium crosslinked gel carrier, ultrasonically treat for 5 - 10 min, and finally add icariin, ultrasonically treat for 5 - 10 min, and freeze-dry. The addition of the stabilizer enhances the binding stability and complexity between the graphene oxide-based composite microspheres and the lithium-strontium crosslinked gel carrier, forms a porous and multi-layered multi-crosslinked network structure, has excellent mechanical properties and antibacterial properties, better simulates the supporting role of natural cartilage at the joint, and then exerts a multi-path and multi-target cartilage repair effect through various metal elements, the traditional Chinese medicine component icariin and other active substances, effectively promoting cartilage regeneration, thus obtaining an articular cartilage repair material;

[0023] Preferably, in step S2, the addition amount of icariin 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, promoting the synthesis of chondrocyte extracellular matrix and other ways, so as to promote cartilage injury repair.

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

[0025] In the present invention, by combining and doping graphene oxide-based composite microspheres with a lithium-strontium crosslinked gel carrier, under the bidirectional crosslinking action of a stabilizer, a porous, multi-layered, multi-crosslinked adhesive network structure is formed, which improves the mechanical properties and antibacterial properties of the material, is more suitable for the support and adhesion at the articular cartilage, and can reduce the risk of infection. Then, it is loaded with icariin, and together with a variety of metal elements and bioactive substances, it jointly promotes cartilage repair and regeneration, significantly improving the repair effect of articular cartilage; in the graphene oxide-based composite microspheres, lysine graft-modifies graphene oxide, improves the dispersibility of graphene oxide, enhances the antibacterial property of graphene oxide, and then coats the ethylenediamine-functionalized microspheres to form a core-shell microsphere combining rigidity and flexibility, improving the mechanical properties of the material, being more resistant to pressure and deformation. And the ethylenediamine-functionalized microspheres as the core structure 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 existence of the coating layer reduces the degradation of the ethylenediamine-functionalized microspheres in the body, improves the stability of the material, and also enhances the bactericidal effect of the microspheres on bacteria; in the lithium-strontium crosslinked gel carrier, lithium chloride and strontium chloride replace calcium ions and are crosslinked and combined with the hydrogel matrix formed by chitosan, sodium glycerophosphate and sodium alginate to obtain a gel carrier with uniform crosslinking, stable structure and cell adhesion and osteoinductivity, and can also effectively inhibit the biofilms of a variety of bacteria, having excellent antibacterial properties; the polyphenol-metal network structure of the stabilizer as a dynamic crosslinking point can not only combine with the lithium-strontium crosslinked gel carrier, but also crosslink with the graphene oxide-based composite microspheres to form a multi-crosslinked network structure, which is more conducive to the support and adhesion at the articular cartilage. Among them, the synergistic effect of a variety of metal elements and active substances not only improves the antibacterial property of the material, but also significantly promotes cartilage repair and regeneration in a multi-pathway manner, achieving an excellent repair effect of articular cartilage; the present invention uses graphene oxide-based composite microspheres, a stabilizer, a lithium-strontium crosslinked gel carrier and icariin to prepare an articular cartilage repair material, effectively enhancing the mechanical properties and antibacterial properties of the material and significantly strengthening the repair effect on articular cartilage. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 2 It is the graph of the mechanical strength results of Examples 1-4 and Comparative Examples 1-3 of the present invention;

[0028] Figure 3 It is the graph of the inhibition zone diameter results of Examples 1-4 and Comparative Examples 1-3 of the present invention;

[0029] Figure 4 It is the graph of the articular cartilage repair results of Examples 1-4 and Comparative Examples 1-3 of the present invention. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for illustrative purposes and do not limit the content of this application.

[0032] The experimental methods in the following embodiments are all conventional methods unless otherwise specified; the test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0033] Example 1

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

[0035] The graphene oxide-based composite microspheres comprise 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 crosslinked 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 the graphene oxide-based composite microspheres specifically comprises the following steps:

[0038] (1) Add 81 mL of absolute ethanol, 9 mL of water, and 3.0 g of polyvinylpyrrolidone into a round-bottom flask in sequence. Then add 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile, and stir evenly. After ultrasonic treatment and purging with nitrogen to remove oxygen from the solution system, under a nitrogen atmosphere, control the temperature at 70 °C and stir the reaction at a speed of 150 rpm for 24 h. Centrifuge, wash, and freeze-dry the product. The glycidyl methacrylate microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for the adhesion and proliferation of chondrocytes. The glycidyl groups on its surface are easy to couple with bioactive molecules, further promoting cell adhesion and differentiation, and glycidyl methacrylate microspheres are obtained;

[0039] (2) Mix the glycidyl methacrylate microspheres described in step (1) with ethylenediamine and 50 mL of deionized water evenly, and stir at 80 °C for 12 h. The addition amount of ethylenediamine is 75 mL. Ethylenediamine can serve as a chemical cross-linking point to enhance the interaction between cells and materials and is beneficial to the stability of the material. The product is centrifuged and washed repeatedly until the solution pH is 7.0, and then freeze-dried. Ethylenediamine introduces amino groups on the molecular chain of glycidyl methacrylate, improving the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbs extracellular matrix proteins such as collagen, promotes the adhesion of chondrocytes, but also enhances the penetration and killing of bacteria to play an antibacterial role, and ethylenediamine-functionalized microspheres are obtained;

[0040] (3) Disperse 200 mg of graphene oxide in 50 mL of deionized water. First, ultrasonically treat it for 20 min, and then stir it at a speed of 200 rpm to form a graphene oxide suspension. Graphene oxide, with good electrical conductivity, high specific surface area, and excellent mechanical properties, can be used as a cartilage repair material. Moreover, graphene oxide can upregulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycan and collagen, and can reduce joint inflammation by regulating macrophage polarization, thus playing a bioactive role in cartilage repair. Under continuous stirring conditions, add 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and finally add lysine powder. The addition amount of lysine is 0.3 g. As an important component amino acid of collagen, lysine can not only activate the signal pathway of growth factors, promote the proliferation of chondrocytes and the secretion of matrix such as proteoglycan and collagen, thereby accelerating the repair of cartilage defects, but also participate in the cross-linking of collagen fibers by forming hydroxylysine through hydroxylation, thus enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cell energy metabolism and provides the nutritional support required for cartilage cell repair. React at room temperature for 24 h, collect the precipitate, wash it 5 times with deionized water, and freeze-dry it. Graft modification of the graphene oxide surface is carried out through lysine, thereby improving the dispersibility of graphene oxide, enhancing the safety of graphene oxide in vivo, and also having good hydrophilicity and excellent protein adsorption ability, which is beneficial to the growth and adhesion of cells and proteins on the material surface. The amino group of lysine can damage the cell membrane of bacteria and enhance the physical antibacterial property of graphene oxide in a chemical antibacterial manner to obtain lysine-functionalized graphene oxide nanoparticles;

[0041] (4) Add the lysine-functionalized graphene oxide nanoparticles described in step (3) to 100 mL of deionized water, then add 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinimide, stir for 30 min, and then add the ethylenediamine-functionalized microspheres described in step (2). React at 60 °C for 2 h, centrifuge, and freeze-dry the precipitate. Through the coating of the ethylenediamine-functionalized microspheres with lysine-functionalized graphene oxide nanoparticles, a core-shell composite material with a combination of internal rigidity and outer flexibility is formed, which is more suitable for the support at the articular cartilage, improves the mechanical properties of the material, makes it more pressure-resistant and resistant to deformation. Among them, the ethylenediamine-functionalized microspheres serve as the core structure, further avoiding the stacking of graphene oxide, exposing more active groups, enhancing the stimulatory promotion effect on cartilage tissue, and being beneficial to improving the repair effect. The existence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine-functionalized microspheres in vivo and improve the structural stability to obtain graphene oxide-based composite microspheres.

[0042] A preparation method of a lithium-strontium cross-linked gel carrier specifically includes the following steps:

[0043] a. Add 2.0 g of chitosan to 50 mL of acetic acid solution with a mass fraction of 0.6% for later 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, slowly add the sodium glycerophosphate solution to the chitosan solution drop by drop at 4 °C and mix evenly. Next, slowly add the sodium alginate solution drop by drop under magnetic stirring until fully integrated. Adjust the pH to neutral. Through this process, a thermosensitive double-network gel is formed, which can be injected into the articular cartilage defect site to play a filling and repairing 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 the performance and obtain a hydrogel matrix;

[0044] b. Add lithium chloride and strontium chloride to 50 mL of sterile double-distilled water. The addition amount of lithium chloride is 1.0 g, and the addition amount of strontium chloride is 1.0 g. Lithium chloride has certain antibacterial properties and reduces the risk of infection after implantation. And lithium and strontium elements can stimulate the proliferation and differentiation of chondrocytes by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, add it to the hydrogel matrix described in step a, stir at a speed of 500 rpm for 30 min, pour it into a mold and transfer it to a 37 °C incubator. Lithium chloride binds to chitosan through electrostatic interaction, and strontium chloride crosslinks and binds with the carboxyl group of sodium alginate. The two enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy collapse of the structure, poor cell adhesion and osteoinductivity existing in the calcium ion crosslinked gel, endow the gel with the function of promoting bone regeneration, and the combined use of lithium and strontium elements effectively inhibits the biofilms of various bacteria and has excellent antibacterial properties, obtaining a lithium-strontium crosslinked gel carrier.

[0045] This example provides a preparation method of 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, and then add 0.4 g of ferric chloride and 0.5 g of copper sulfate and stir evenly. Tannic acid forms a coordination network with metal iron ions and copper ions, enhancing the mechanical strength of the material, simulating the mechanical properties of natural cartilage, and playing a role in promoting cartilage regeneration by releasing metal ions. Iron ions participate in collagen synthesis, and copper ions support the vascularization of subchondral bone, providing nutritional support for cartilage repair. The catechol groups therein endow strong adhesion ability, can closely adhere to the articular cartilage defect site, reduce the risk of shedding, and the release of metal ions further enhances the antibacterial properties of polyphenols, 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 the graphene oxide-based composite microspheres, sonicate for 5 min, then add the lithium-strontium crosslinked gel carrier, sonicate for 10 min, and finally add icariin. The addition amount of icariin is 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 chondrocyte extracellular matrix, etc., so as to promote cartilage injury repair. Sonicate for 10 min and then freeze-dry. The addition of the stabilizer enhances the binding stability and complexity between the graphene oxide-based composite microspheres and the lithium-strontium crosslinked gel carrier, forming a porous and multi-layered multi-crosslinked network structure with excellent mechanical properties and antibacterial properties, better simulating the supporting role of natural cartilage at the joint. Then, through various metal elements, the traditional Chinese medicine component icariin, and other active substances, it plays a multi-path and multi-target cartilage repair role, effectively promoting cartilage regeneration, and obtaining a joint cartilage repair material.

[0048] In this example, the prepared joint cartilage repair material was observed by scanning electron microscopy to observe its microscopic morphology. Figure 1 The SEM image of the joint cartilage repair material prepared in Example 1 magnified 100 times is as Figure 1 , and the joint cartilage repair material prepared in this example presents a porous, multi-layered and multi-crosslinked network structure.

[0049] Example 2

[0050] This example presents a joint cartilage repair material, which includes the following components in parts by weight: 50 parts of graphene oxide-based composite microspheres, 10 parts of stabilizer, 20 parts of lithium-strontium crosslinked 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 crosslinked gel carrier includes 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 the graphene oxide-based composite microspheres specifically includes the following steps:

[0054] (1) Add 81 mL of absolute ethanol, 9 mL of water, and 3.0 g of polyvinylpyrrolidone into a round-bottom flask in sequence. Then add 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile, and stir evenly. After ultrasonic treatment and purging with nitrogen to remove oxygen from the solution system, under a nitrogen atmosphere, control the temperature at 60 °C and stir the reaction at a speed of 100 rpm for 24 h. Centrifuge the product, wash it, and freeze-dry it. The glycidyl methacrylate microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for chondrocyte attachment and proliferation. The glycidyl groups on its surface are easy to couple with bioactive molecules, further promoting cell adhesion and differentiation, and glycidyl methacrylate microspheres are obtained;

[0055] (2) Mix the glycidyl methacrylate microspheres described in step (1) with ethylenediamine and 50 mL of deionized water evenly, and stir at 70 °C for 10 h. The addition amount of ethylenediamine is 55 mL. Ethylenediamine can serve as a chemical cross-linking point to enhance the interaction between cells and materials and is beneficial to the stability of the materials. Centrifuge and wash the product multiple times until the solution pH is 7.0, and then freeze-dry it. Ethylenediamine introduces amino groups on the molecular chain of glycidyl methacrylate, improving the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbs extracellular matrix proteins such as collagen to promote chondrocyte adhesion but also enhances the penetration and killing of bacteria to play an antibacterial role, and ethylenediamine-functionalized microspheres are obtained;

[0056] (3) Disperse 100 mg of graphene oxide in 50 mL of deionized water. First, ultrasonically treat it for 10 min, and then stir it at a speed of 100 rpm to form a graphene oxide suspension. Graphene oxide, with good electrical conductivity, a high specific surface area, and excellent mechanical properties, can be used as a cartilage repair material. Moreover, graphene oxide can up-regulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycan and collagen, and can reduce joint inflammation by regulating macrophage polarization, thereby playing a bioactive role in cartilage repair. Under continuous stirring conditions, add 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and finally add lysine powder. The addition amount of lysine is 0.1 g. As an important component amino acid of collagen, lysine can not only activate the signal pathway of growth factors, promote the proliferation of chondrocytes and the secretion of matrix such as proteoglycan and collagen, thereby accelerating the repair of cartilage defects, but 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 cell energy metabolism and provides the nutritional support required for cartilage cell repair. React at room temperature for 24 h, collect the precipitate, wash it 3 times with deionized water, and freeze-dry it. Graft modification of the graphene oxide surface is carried out through lysine, thereby improving the dispersibility of graphene oxide, enhancing the safety of graphene oxide in vivo, and also having good hydrophilicity and excellent protein adsorption ability, which is beneficial to the growth and adhesion of cells and proteins on the material surface. The amino group of lysine can destroy the cell membrane of bacteria and enhance the physical antibacterial property of graphene oxide in a chemical antibacterial manner to obtain lysine-functionalized graphene oxide nanoparticles;

[0057] (4) Add the lysine-functionalized graphene oxide nanoparticles described in step (3) to 100 mL of deionized water, then add 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinimide, stir for 20 min, and then add the ethylenediamine-functionalized microspheres described in step (2). React at 50 °C for 1 h, centrifuge, and freeze-dry the precipitate. Through the coating of the ethylenediamine-functionalized microspheres with lysine-functionalized graphene oxide nanoparticles, a core-shell composite material with a rigid interior and a flexible outer layer is formed, which is more suitable for the support at the joint cartilage, improves the mechanical properties of the material, makes it more resistant to pressure and deformation. Among them, the ethylenediamine-functionalized microspheres serve as the core structure, further avoiding the stacking of graphene oxide, exposing more active groups, enhancing the stimulatory promotion effect on cartilage tissue, and being beneficial to improving the repair effect. The existence of the coating layer can reduce the enzymatic hydrolysis or hydrolysis of the ethylenediamine-functionalized microspheres in vivo and improve the structural stability to obtain graphene oxide-based composite microspheres.

[0058] A preparation method of a lithium-strontium cross-linked gel carrier specifically includes the following steps:

[0059] a. Add 1.0 g of chitosan to 50 mL of acetic acid solution with a mass fraction of 0.6% for later 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, gradually add the sodium glycerophosphate solution dropwise to the chitosan solution at 4 °C, mix evenly, and then gradually 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 articular cartilage defect site to play a filling and repairing role, guide the orderly arrangement of cells, stimulate the synthesis of type II collagen and glycosaminoglycan 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 the performance and obtain a hydrogel matrix;

[0060] b. Add lithium chloride and strontium chloride to 50 mL of sterile double-distilled water. The addition amount of lithium chloride is 0.5 g, and the addition amount of strontium chloride is 0.5 g. Lithium chloride has certain antibacterial properties and reduces the risk of infection after implantation. And lithium and strontium elements can stimulate the proliferation and differentiation of chondrocytes by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, add it to the hydrogel matrix described in step a, stir at a speed of 200 rpm for 20 min, pour it into a mold and transfer it to a 37 °C incubator. Lithium chloride binds to chitosan through electrostatic interaction, and strontium chloride crosslinks and binds with the carboxyl group of sodium alginate. The two enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy collapse of the structure, poor cell adhesion and osteoinductivity existing in the calcium ion crosslinked gel, endow the gel with the function of promoting bone regeneration, and the combined use of lithium and strontium elements effectively inhibits the biofilms of various bacteria and has excellent antibacterial properties, obtaining a lithium-strontium crosslinked gel carrier.

[0061] This example provides a preparation method of an articular cartilage repair material, which specifically includes the following steps:

[0062] S1. Dissolve 3.0 g of tannic acid in 100 mL of ultrapure water under magnetic stirring, and then add 0.3 g of ferric chloride and 0.5 g of copper sulfate, and stir evenly. Tannic acid forms a coordination network with metal iron ions and copper ions, enhancing the mechanical strength of the material, simulating the mechanical properties of natural cartilage, and playing a role in promoting cartilage regeneration by releasing metal ions. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol groups therein endow strong adhesion ability, can closely fit the articular cartilage defect site, reduce the risk of shedding, and the release of metal ions further enhances the antibacterial properties of polyphenols, obtaining a stabilizer;

[0063] S2. Add the stabilizer described in step S1 to 50 mL of MOPS buffer with a mass fraction of 0.1% under ice bath conditions, then quickly add the graphene oxide-based composite microspheres, ultrasonically treat for 1 min, then add the lithium-strontium cross-linked gel carrier, ultrasonically treat for 5 min, and finally add icariin. The addition amount of icariin is 0.3 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, promoting the synthesis of chondrocyte extracellular matrix and other multiple ways, so as to promote cartilage injury repair. Ultrasonically treat for 5 min and then freeze-dry. The addition of the stabilizer enhances the binding stability and complexity between the graphene oxide-based composite microspheres and the lithium-strontium cross-linked gel carrier, forming a porous and multi-layered multi-crosslinked network structure with excellent mechanical properties and antibacterial properties, better simulating the supporting role of natural cartilage at the joint. Then, through multiple metal elements, the traditional Chinese medicine component icariin and other active substances, it plays a multi-path and multi-target cartilage repair role, effectively promoting cartilage regeneration, and obtaining a joint cartilage repair material.

[0064] Example 3

[0065] This example presents a joint cartilage repair material, which includes the following components in parts by weight: 55 parts of graphene oxide-based composite microspheres, 15 parts of stabilizer, 25 parts of 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 includes 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 the graphene oxide-based composite microspheres specifically includes the following steps:

[0069] (1) Add 81 mL of absolute ethanol, 9 mL of water and 3.0 g of polyvinylpyrrolidone to a round-bottom flask in sequence, then add 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile, stir evenly. After ultrasonically treating the solution system and purging with nitrogen to remove oxygen, under a nitrogen atmosphere, control the temperature at 65 °C and stir and react at a speed of 125 rpm for 24 h. Centrifuge the product, wash, and freeze-dry. The poly(glycidyl methacrylate) microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, provide a microenvironment for chondrocyte attachment and proliferation, and the glycidyl groups on its surface are easy to couple with bioactive molecules, further promoting cell adhesion and differentiation, obtaining poly(glycidyl methacrylate) microspheres;

[0070] (2) Mix the glycidyl methacrylate microspheres described in step (1) evenly with ethylenediamine and 50 mL of deionized water, stir at 75 °C for 11 h, and the addition amount of ethylenediamine is 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 is centrifuged and washed repeatedly until the solution pH is 7.0, and then freeze-dried. Ethylenediamine introduces amino groups on the molecular chain of glycidyl methacrylate, improving the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbs extracellular matrix proteins such as collagen, promotes the adhesion of chondrocytes, but also enhances the penetration and killing of bacteria to play an antibacterial role, obtaining ethylenediamine-functionalized microspheres;

[0071] (3) Disperse 150 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 15 min, and then stir at a speed of 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. Moreover, graphene oxide can up-regulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycans and collagen, and can reduce joint inflammation by regulating macrophage polarization, thereby playing a biological activity role in cartilage repair. Under continuous stirring conditions, add 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and finally add lysine powder. The addition amount of lysine is 0.2 g. As an important constituent amino acid of collagen, lysine can not only activate the signal pathway of growth factors, promote the proliferation of chondrocytes and the secretion of matrix such as proteoglycans and collagen, thus accelerating the repair of cartilage defects, but also participate in the cross-linking of collagen fibers by forming hydroxylysine through hydroxylation, thereby enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cell energy metabolism, providing the nutritional support required for cartilage cell repair. React at room temperature for 24 h, collect the precipitate, wash it 4 times with deionized water, and then freeze-dry. Graft modification of the graphene oxide surface is carried out through lysine, which improves the dispersibility of graphene oxide, enhances the safety of graphene oxide in vivo, and also has good hydrophilicity and excellent protein adsorption ability, which is beneficial to the growth and adhesion of cells and proteins on the material surface. The amino group of lysine can destroy the cell membrane of bacteria, enhancing the physical antibacterial property of graphene oxide in a chemical antibacterial manner, obtaining lysine-functionalized graphene oxide nanoparticles;

[0072] (4) Add the lysine-functionalized graphene oxide nanoparticles described in step (3) to 100 mL of deionized water, then add 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinimide, stir for 25 min, and then add the ethylenediamine-functionalized microspheres described in step (2). React at 55 °C for 1.5 h, centrifuge, and freeze-dry the precipitate. By coating the surface of the ethylenediamine-functionalized microspheres with lysine-functionalized graphene oxide nanoparticles, a core-shell composite material with a rigid interior and a flexible outer layer is formed, which is more suitable for supporting at the articular cartilage, improving the mechanical properties of the material, making it more pressure-resistant and resistant to deformation. Among them, the ethylenediamine-functionalized microspheres serve as the core structure, further avoiding the stacking of graphene oxide, exposing more active groups, enhancing the stimulatory promotion effect on cartilage tissue, and being beneficial to improving the repair effect. The existence of the coating layer can reduce the enzymatic or hydrolytic degradation of the ethylenediamine-functionalized microspheres in vivo, improve the stability of the structure, and obtain graphene oxide-based composite microspheres.

[0073] A preparation method of a lithium-strontium cross-linked gel carrier specifically includes the following steps:

[0074] a. Add 1.5 g of chitosan to 50 mL of an acetic acid solution with a mass fraction of 0.6% and set aside. 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, slowly add the sodium glycerophosphate solution to the chitosan solution drop by drop at 4 °C, mix evenly, and then slowly add the sodium alginate solution drop by drop under magnetic stirring until fully integrated. Adjust the pH to neutral. Through this process, a thermosensitive double-network gel is formed, which can be injected into the articular cartilage defect site to play a filling and repair role, guide the orderly arrangement of cells, stimulate the synthesis of type II collagen and glycosaminoglycan 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 the performance, and a hydrogel matrix is obtained;

[0075] b. Add lithium chloride and strontium chloride to 50 mL of sterile double-distilled water. The addition amount of lithium chloride is 0.75 g, and the addition amount of strontium chloride is 0.75 g. Lithium chloride has certain antibacterial properties, reducing the risk of infection after implantation. Moreover, lithium and strontium elements can stimulate the proliferation and differentiation of chondrocytes by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, add it to the hydrogel matrix described in step a, stir at a speed of 350 rpm for 25 min, pour it into a mold and transfer it to a 37°C incubator. Lithium chloride binds to chitosan through electrostatic interaction, and strontium chloride crosslinks and binds to the carboxyl group of sodium alginate. The two enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy structural collapse, poor cell adhesion and osteoinductivity existing in the calcium ion crosslinked gel, endow the gel with the function of promoting bone regeneration, and the combined use of lithium and strontium elements effectively inhibits the biofilms of various bacteria and has excellent antibacterial properties, obtaining a lithium-strontium crosslinked gel carrier.

[0076] This example provides a preparation method of an articular cartilage repair material, which specifically includes the following steps:

[0077] S1. Dissolve 3.5 g of tannic acid in 100 mL of ultrapure water under magnetic stirring, and then add 0.35 g of ferric chloride and 0.5 g of copper sulfate, and stir evenly. Tannic acid and metal iron ions and copper ions form a coordination network to enhance the mechanical strength of the material, simulate the mechanical properties of natural cartilage, and play a role in promoting cartilage regeneration by releasing metal ions. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol groups therein endow strong adhesion ability, can closely adhere to the cartilage defect site, reduce the risk of shedding, and the release of metal ions further enhances the antibacterial properties of polyphenols, obtaining a stabilizer.

[0078] S2. Add the stabilizer described in step S1 to 50 mL of MOPS buffer with a mass fraction of 0.3% under ice bath conditions, then quickly add the graphene oxide-based composite microspheres, ultrasonically treat for 3 min, then add the lithium-strontium cross-linked gel carrier, ultrasonically treat for 7.5 min, and finally add icariin. The addition amount of icariin is 0.4 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, promoting the synthesis of chondrocyte extracellular matrix and other multiple pathways, so as to promote cartilage injury repair. Ultrasonically treat for 7.5 min, and then freeze-dry. The addition of the stabilizer enhances the binding stability and complexity between the graphene oxide-based composite microspheres and the lithium-strontium cross-linked gel carrier, forming a porous and multi-layered multi-crosslinked network structure with excellent mechanical properties and antibacterial properties, better simulating the supporting role of natural cartilage at the joint. Then, through multiple metal elements, the traditional Chinese medicine component icariin, and other active substances, it plays a multi-path and multi-target cartilage repair role, effectively promoting cartilage regeneration, and obtaining a joint cartilage repair material.

[0079] Example 4

[0080] This example presents a joint cartilage repair material, which includes the following components in parts by weight: 50 parts of graphene oxide-based composite microspheres, 10 parts of stabilizer, 30 parts of 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 includes 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 the graphene oxide-based composite microspheres specifically includes the following steps:

[0084] (1) Add 81 mL of absolute ethanol, 9 mL of water, and 3.0 g of polyvinylpyrrolidone to a round-bottom flask in sequence, then add 10.0 g of glycidyl methacrylate and 0.2 g of azobisisobutyronitrile, stir evenly. After ultrasonically treating the solution system and purging with nitrogen to remove oxygen, under a nitrogen atmosphere, control the temperature at 70 °C and stir and react at a speed of 150 rpm for 24 h. Centrifuge the product, wash, and freeze-dry. The poly(glycidyl methacrylate) microspheres can form a porous three-dimensional structure, simulate the extracellular matrix, and provide a microenvironment for chondrocyte attachment and proliferation. The glycidyl groups on its surface are easy to couple with bioactive molecules, further promoting cell adhesion and differentiation, and obtaining poly(glycidyl methacrylate) microspheres;

[0085] (2) Mix the glycidyl methacrylate microspheres described in step (1) evenly with ethylenediamine and 50 mL of deionized water, stir at 80 °C for 10 h, and the addition amount of ethylenediamine is 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 is centrifuged and washed repeatedly until the solution pH is 7.0, and then freeze-dried. Ethylenediamine introduces amino groups on the molecular chain of glycidyl methacrylate, improving the positive charge and hydrophilicity of the microsphere surface. It not only easily adsorbs extracellular matrix proteins such as collagen, promotes the adhesion of chondrocytes, but also enhances the penetration and killing of bacteria to play an antibacterial role, obtaining ethylenediamine-functionalized microspheres;

[0086] (3) Disperse 200 mg of graphene oxide in 50 mL of deionized water, first ultrasonically treat for 10 min, and then stir at a speed of 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. Moreover, graphene oxide can up-regulate the expression of cartilage-specific genes, promote the synthesis of glycosaminoglycan and collagen, and can reduce joint inflammation by regulating macrophage polarization, thereby playing a biological activity role in cartilage repair. Under continuous stirring conditions, add 0.25 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and finally add lysine powder. The addition amount of lysine is 0.1 g. As an important component amino acid of collagen, lysine can not only activate the signal pathway of growth factors, promote the proliferation of chondrocytes and the secretion of matrix such as proteoglycan and collagen, thereby accelerating the repair of cartilage defects, but also participate in the cross-linking of collagen fibers by forming hydroxylysine through hydroxylation, thereby enhancing the mechanical strength and stability of cartilage. At the same time, lysine participates in cell energy metabolism, providing the nutritional support required for cartilage cell repair. React at room temperature for 24 h, collect the precipitate, wash it 5 times with deionized water, and then freeze-dry. Graft modification of the graphene oxide surface is carried out through lysine, which improves the dispersibility of graphene oxide, enhances the safety of graphene oxide in vivo, and also has good hydrophilicity and excellent protein adsorption ability, which is beneficial to the growth and adhesion of cells and proteins on the material surface. The amino group of lysine can destroy the cell membrane of bacteria, enhancing the physical antibacterial property of graphene oxide in a chemical antibacterial manner, obtaining lysine-functionalized graphene oxide nanoparticles;

[0087] (4) Add the lysine-functionalized graphene oxide nanoparticles described in step (3) to 100 mL of deionized water, then add 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinimide, stir for 20 min, and then add the ethylenediamine-functionalized microspheres described in step (2). React at 60 °C for 1 h, centrifuge, and freeze-dry the precipitate. By coating the surface of the ethylenediamine-functionalized microspheres with lysine-functionalized graphene oxide nanoparticles, a core-shell composite material with a rigid interior and a flexible outer layer is formed, which is more suitable for supporting the articular cartilage, improving the mechanical properties of the material, making it more pressure-resistant and resistant to deformation. Among them, the ethylenediamine-functionalized microspheres serve as the core structure, further avoiding the stacking of graphene oxide, exposing more active groups, enhancing the stimulatory and promoting effect on cartilage tissue, and being beneficial to improving the repair effect. The presence of the coating layer can reduce the enzymatic or hydrolytic degradation of the ethylenediamine-functionalized microspheres in vivo, improving the structural stability, and obtaining graphene oxide-based composite microspheres.

[0088] Preparation method of lithium-strontium cross-linked gel carrier, specifically including the following steps:

[0089] a. Add 2.0 g of chitosan to 50 mL of acetic acid solution with a mass fraction of 0.6% and set aside. 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, slowly add the sodium glycerophosphate solution to the chitosan solution drop by drop at 4 °C, mix evenly, and then slowly add the sodium alginate solution drop by drop 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 articular cartilage defect site to play a filling and repair role, guide the orderly arrangement of cells, stimulate the synthesis of type II collagen and glycosaminoglycan 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 the mechanical properties. The three complement each other to optimize the performance, and a hydrogel matrix is obtained;

[0090] b. Add lithium chloride and strontium chloride to 50 mL of sterile double-distilled water. The addition amount of lithium chloride is 0.5 g, and the addition amount of strontium chloride is 1.0 g. Lithium chloride has certain antibacterial properties, reducing the risk of infection after implantation. Moreover, lithium and strontium elements can stimulate the proliferation and differentiation of chondrocytes by activating the Wnt signaling pathway and the mitogen-activated protein kinase signaling pathway. After stirring evenly, add it to the hydrogel matrix described in step a, stir at a speed of 500 rpm for 20 min, pour it into a mold and transfer it to a 37°C incubator. Lithium chloride binds to chitosan through electrostatic interaction, and strontium chloride crosslinks and binds to the carboxyl groups of sodium alginate. The two enhance the crosslinking and stability of the gel, improve the problems of uneven crosslinking, easy collapse of the structure, poor cell adhesion and osteoinductivity of the calcium ion crosslinked gel, endow the gel with the function of promoting bone regeneration, and the combined use of lithium and strontium elements effectively inhibits the biofilms of various bacteria and has excellent antibacterial properties, obtaining a lithium-strontium crosslinked gel carrier.

[0091] This example provides a preparation method of 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 forms a coordination network with metal iron ions and copper ions, enhancing the mechanical strength of the material, simulating the mechanical properties of natural cartilage, and playing a role in promoting cartilage regeneration through the release of metal ions. Iron ions participate in collagen synthesis, and copper ions support subchondral bone vascularization, providing nutritional support for cartilage repair. The catechol groups therein endow strong adhesion ability, can closely fit the cartilage defect site, reduce the risk of shedding, and the release of metal ions further enhances the antibacterial properties of polyphenols, obtaining a stabilizer.

[0093] S2. Add the stabilizer described in step S1 to 50 mL of MOPS buffer with a mass fraction of 0.5% under ice bath conditions, then quickly add the graphene oxide-based composite microspheres, ultrasonically treat for 1 min, then add the lithium-strontium cross-linked gel carrier, ultrasonically treat for 5 min, and finally add icariin. The addition amount of icariin is 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, promoting the synthesis of chondrocyte extracellular matrix and other multiple pathways, so as to promote cartilage injury repair. Ultrasonically treat for 5 min and then freeze-dry. The addition of the stabilizer enhances the binding stability and complexity between the graphene oxide-based composite microspheres and the lithium-strontium cross-linked gel carrier, forming a porous and multi-layered multi-crosslinked network structure with excellent mechanical properties and antibacterial properties, better simulating the supporting role of natural cartilage at the joint. Then, through multiple metal elements, the traditional Chinese medicine component icariin and other active substances, it plays a cartilage repair role through multiple pathways and multiple targets, effectively promoting cartilage regeneration, and obtaining a joint cartilage repair material.

[0094] Comparative Example 1

[0095] This comparative example provides a joint cartilage repair material, which is different 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 in Example 1; the preparation method of the joint cartilage repair material is the same as that in Example 1.

[0096] Comparative Example 2

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

[0098] Comparative Example 3

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

[0100] Experimental Example 1

[0101] Mechanical Property Experiment

[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, and the compression rate was 5 mm / min. The samples were compressed to a strain of 50%. During the compression test, 10 repeated loadings and unloadings were performed, and the compression strain was kept constant at 40%. Three parallel tests were set for each group of samples, and the average value was taken.

[0104] Figure 2 Figure showing 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 was 10.5-12.6 MPa, indicating good mechanical properties; the mechanical strength of Comparative Examples 1-3 was 5.1-7.8 MPa, indicating poor mechanical properties; the graphene oxide-based composite microspheres in Comparative Example 1 did not contain graphene oxide and lysine, could not form a coating on the ethylenediamine-functionalized microspheres, and lacked the resistance of the flexible shell, resulting in poor mechanical properties; the lithium-strontium crosslinked gel carrier in Comparative Example 2 did not contain lithium chloride and strontium chloride, could not form a crosslinked and homogeneous gel structure that was not easily collapsed, resulting in poor mechanical properties; the articular cartilage repair material in Comparative Example 3 did not contain a stabilizer, could not achieve the multi-crosslinking of the lithium-strontium crosslinked gel carrier and the graphene oxide-based composite microspheres, reduced the stability of the porous multi-layer network structure, and resulted in poor mechanical properties.

[0105] Experimental Example 2

[0106] Antibacterial property experiment

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

[0108] Test method: There are various types of bacteria that can cause infections by implants. In this experiment, common Staphylococcus aureus and Escherichia coli were selected as the experimental bacteria for the antibacterial experiment; the test samples were designed as flat cylinders with a diameter of 10 mm and a thickness of 2 mm. After ultraviolet sterilization, they were placed on an agar medium coated with a bacterial solution of 10 6 cfu / mL. The culture dish was inverted and placed in an incubator at 37°C for 24 h. Then, it was taken out to observe the growth of the inhibition zone. The larger the diameter (mm) of the inhibition zone, the stronger the inhibitory effect on the experimental bacteria.

[0109] Figure 3The figure shows the results of the diameters of the antibacterial zones for Examples 1-4 and Comparative Examples 1-3. As shown in the figure, the diameters of the antibacterial zones against Staphylococcus aureus and Escherichia coli in Examples 1-4 are 40-48 mm and 38-45 mm respectively, both > 35 mm, indicating strong antibacterial properties and effectively reducing the risk of infection. The diameters of the antibacterial zones against Staphylococcus aureus and Escherichia coli in Comparative Examples 1-3 are 9-19 mm and 7-16 mm respectively, both < 20 mm, indicating general antibacterial properties and being unable to effectively reduce the risk of infection. In Comparative Example 1, the graphene oxide-based composite microspheres do not contain graphene oxide and lysine, and thus cannot exert the dual killing effects of physical antibacterial and chemical sterilization, nor can they improve the antibacterial properties of ethylenediamine-functionalized microspheres through coating, resulting in general antibacterial properties and being unable to effectively reduce the risk of infection. In Comparative Example 2, the lithium-strontium crosslinked gel carrier does not contain lithium chloride and strontium chloride, and thus cannot exert the inhibitory effects of lithium and strontium elements on the biofilms of various bacteria, nor is it conducive to the uniformity and stability of the gel carrier, and further cannot better prevent the invasion and migration of bacteria, resulting in general antibacterial properties and being unable to effectively reduce the risk of infection. In Comparative Example 3, the articular cartilage repair material does not contain a stabilizer, which is not conducive to the formation of a porous multi-layer and multi-element crosslinked network structure, weakening the barrier and adhesion to bacteria, resulting in general antibacterial properties and being unable to effectively reduce the risk of infection.

[0110] Experimental Example 3

[0111] Repair effect experiment

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

[0113] Test method: Seventy New Zealand white rabbits, 3 months old and weighing 1.8-2.5 kg, were randomly divided into 7 groups with 10 rabbits in each group. They were anesthetized by intravenous injection of 2.5% sodium pentobarbital through the ear margin. After proper anesthesia, their limbs were fixed properly. The lateral edge of the patellar ligament was incised successively 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 made on it, drilling through the subchondral bone. After removing the blood clots in the defect, the test samples were filled in respectively, and the wound was sutured layer by layer. After the operation, the wound was disinfected with iodophor, bandaged with gauze without external fixation, intramuscularly injected with penicillin and streptomycin double antibiotics to prevent infection, and fed individually in a cage after the operation. After 16 weeks, CT examinations were performed respectively to observe the repair of the cartilage, and the International Cartilage Repair Society (ICRS) scoring standard was used for scoring and calculating the average value. The specific scoring criteria for the defect repair degree and overall repair are as follows:

[0114] Scoring criteria for the defect repair degree, specifically:

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

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

[0117] Repair 50% of the defect depth, 2 points;

[0118] Repair 25% of the defect depth, 1 point;

[0119] No repair, 0 points;

[0120] Scoring criteria for boundary integration are as follows:

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

[0122] Boundary < 1mm, 3 points;

[0123] 75% integrated, 25% with a significant gap > 1mm, 2 points;

[0124] 50% integrated, 50% with a significant gap > 1mm, 1 point;

[0125] Less than 25% integration degree, 0 points;

[0126] 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 fissures can be seen on the surface, 1 point;

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

[0132] Scoring criteria for overall repair are as follows:

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

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

[0135] Grade III, there is repair, but the effect is not good, 4 - 7 points;

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

[0137] Figure 4Graphs showing the results of articular cartilage repair in Examples 1-4 and Comparative Examples 1-3; as shown in the figure, the defect repair degrees and overall repair scores of Examples 1-4 were 3.5-3.9 and 10.2-11.9, indicating good repair effects; the defect repair degrees and overall repair scores of Comparative Examples 1-3 were 1.8-2.8 and 5.3-7.2, indicating average repair effects; the graphene oxide-based composite microspheres in Comparative Example 1 did not contain graphene oxide and lysine, and could not coat the ethylenediamine-functionalized microspheres to form a flexible shell, so it could neither exert the repair effects of graphene oxide and lysine, nor was it conducive to the growth and adhesion of cells and proteins on the surface of the microspheres, nor could it ensure the stable repair of the microspheres in vivo, resulting in average repair effects; the lithium-strontium crosslinked gel carrier in Comparative Example 2 did not contain lithium chloride and strontium chloride, and could not endow the gel carrier with cell adhesion and osteoinductivity, resulting in average repair effects; the articular cartilage repair material in Comparative Example 3 did not contain a stabilizer, and could not form a porous, multi-layered, multi-crosslinked adhesive network structure, which was not conducive to the support and adhesion at the articular cartilage, reducing both the loading of icariin and the effective synergistic effect of multiple active substances to stably exert the repair effect on cartilage, resulting in average repair effects.

[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 in Comparative Examples 1-3. Among them, Example 1 using graphene oxide-based composite microspheres, lithium-strontium crosslinked gel carrier, and stabilizer has better mechanical properties, stronger antibacterial properties, and better repair effects. By combining and doping the graphene oxide-based composite microspheres with the lithium-strontium crosslinked gel carrier, under the bidirectional crosslinking effect of the stabilizer, a porous, multi-layered, multi-crosslinked adhesive network structure is formed, improving the mechanical properties and antibacterial properties of the material, and being more suitable for the support and attachment at the articular cartilage. Then, it is loaded with icariin, and together with multiple metal elements and bioactive substances, it jointly promotes cartilage repair and regeneration, significantly improving the repair effect of articular cartilage. Among the lithium-strontium crosslinked gel carriers, lithium chloride and strontium chloride replace calcium ions and are crosslinked and combined with the hydrogel matrix formed by chitosan, sodium glycerophosphate, and sodium alginate to obtain a gel carrier with uniform crosslinking, stable structure, and cell adhesion and osteoinductivity.

[0139] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention.

[0140] The above describes the present invention and its implementation manners. Such description is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual application is not limited thereto. In general, if those of ordinary skill in the art are inspired by it and, without departing from the gist of the present invention, design similar manners and embodiments to this technical solution without creative efforts, they shall fall within the protection scope of the present invention.

Claims

1. A kind of articular cartilage repair material, characterized in that: 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.

2. A preparation method of 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, stir evenly, and 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.

3. The preparation method of the articular cartilage repair material according to claim 2, characterized in that: In step S2, the amount of icariin added is 0.3-0.5 g.

4. The preparation method of the articular cartilage repair material according to claim 3, wherein: 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 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 h. The product was centrifuged, washed, and freeze-dried to obtain polyglycidyl methacrylate microspheres; (2) The poly(glycidyl methacrylate) microspheres described in step (1) are mixed with ethylenediamine and 50 mL of deionized water, stirred at 70-80° C. for 10-12 h, the product is centrifuged and washed several times until the pH of the solution is 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 it to obtain lysine graphene oxide nanoparticles; (4) Add the lysine-functionalized graphene oxide nanoparticles described in step (3) to 100 mL of deionized water, then add 0.25 g of carbodiimide and 0.15 g of N-hydroxysuccinimide, stir for 20 - 30 min, and then add the ethylenediamine-functionalized microspheres described in step (2). React at 50 - 60 °C for 1 - 2 h, centrifuge, and freeze-dry the precipitate to obtain graphene oxide-based composite microspheres.

5. The preparation method of the articular cartilage repair material according to claim 4, characterized in that: In step (2), the addition amount of ethylenediamine is 55 - 75 mL.

6. The preparation method of the articular cartilage repair material according to claim 5, wherein: In step (3), the addition amount of lysine is 0.1 - 0.3 g.

7. The preparation method of the articular cartilage repair material according to claim 6, wherein: The preparation method of the lithium-strontium cross-linked gel carrier specifically includes the following steps: a. Add 1.0 - 2.0 g of chitosan to 50 mL of an acetic acid solution with a mass fraction of 0.6% 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, slowly add the sodium glycerophosphate solution to the chitosan solution drop by drop at 4 °C, mix evenly, and then slowly add the sodium alginate solution drop by drop under magnetic stirring until fully integrated. 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 it to the hydrogel matrix described in step a. Stir at a speed of 200 - 500 rpm for 20 - 30 min, pour it into a mold, and transfer it to an incubator at 37 °C to obtain a lithium-strontium cross-linked gel carrier.

8. The preparation method of the articular cartilage repair material according to claim 7, wherein: In step b, the addition amount of lithium chloride is 0.5 - 1.0 g, and the addition amount of strontium chloride is 0.5 - 1.0 g.

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