Design of a High-Strength Cartilage Repair Material and Its Preparation Method

Abstract

The present invention relates to the field of biomedical engineering, and provides a design of a high-strength cartilage repair material and a preparation method thereof. The cartilage repair material of the present invention is a high-strength cartilage repair hydrogel formed by free radical polymerization cross-linking of a mixed solution of a natural material derivative modified with glycidyl methacrylate, sulfobetaine methacrylate, and a photoinitiator under ultraviolet light irradiation. The cartilage repair material prepared by this method can be injected and implanted by means of a minimally invasive arthroscopic device, rapidly form in situ under ultraviolet light irradiation, and there is no obvious heat release during curing, which can effectively avoid burning the surrounding tissues. In addition, the rapid photo-crosslinking reaction endows the high-strength injectable hydrogel with excellent compressive strength, overcomes the disadvantages of traditional natural polymer hydrogels with unstable mechanical properties and too fast degradation rate, and provides a feasible solution for the preparation of high-performance injectable cartilage repair materials.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering. Specifically, it provides a high-strength injectable hydrogel for minimally invasive treatment of cartilage defect repair, which solves the defects of insufficient mechanical properties and too fast degradation commonly existing in existing injectable cartilage repair materials. Background Art

[0002] Due to the structural characteristics of cartilage tissue without blood vessels, lymphatic vessels and nerves, cartilage defects caused by sports trauma, inflammation, aging and other reasons usually cannot spontaneously recover to the level of hyaline cartilage before injury. In recent years, the incidence of cartilage injury has been increasing year by year, bringing huge physical, psychological pressure and economic burden to patients. At present, the clinical treatment methods for cartilage defects mainly include microfracture, arthroscopic debridement, autologous cartilage transplantation, allogeneic cartilage transplantation and artificial joint replacement. These traditional treatment methods have many limitations such as infection risk, weak tissue integration ability and poor repair effect, and it is difficult to achieve satisfactory cartilage repair effect. Therefore, there is an urgent need to explore a more effective strategy to promote cartilage regeneration and integration, and further restore the normal physiological function of articular cartilage.

[0003] With the vigorous development of tissue engineering technology, the development of new cartilage repair materials has become a research hotspot in the field of cartilage repair. Hydrogel is a kind of scaffold material with high water content and three-dimensional porous network structure, and has great application potential in tissue repair. In recent years, minimally invasive surgery has become increasingly common, and injectable hydrogels have shown great advantages in minimally invasive treatment due to their shape plasticity and tissue adhesiveness. It is worth noting that injectable hydrogels are expected to fill irregular defect areas, firmly adhere to the surface of the defect tissue to form in situ, and at the same time bind to the matrix proteins of the surrounding cartilage, promoting the migration of cells in the surrounding tissue into the hydrogel matrix to achieve cartilage regeneration at the defect site. Therefore, the development of injectable hydrogel materials is of great significance for achieving high-quality in situ cartilage repair.

[0004] However, the currently common injectable natural hydrogels generally have the following limitations and cannot meet the needs of cartilage injury repair:

[0005] 1. Poor mechanical properties: Articular cartilage plays a role in load-bearing and lubrication during normal physiological activities, and the compressive strength of natural cartilage can be as high as several megapascals. However, the commonly used injectable natural hydrogels prepared by cross-linking methods such as dynamic bond cross-linking, photo-cross-linking and temperature-responsive cross-linking have too low compressive strength to meet the load-bearing requirements of articular cartilage, which greatly limits their application in cartilage repair.

[0006] 2. Excessive degradation rate: The degradation property of tissue repair materials is an important factor affecting tissue repair. The degradation rate of an ideal tissue repair material should match the rate of tissue regeneration. However, the degradation rate of a single-crosslinked natural hydrogel is too fast, making it difficult to achieve long-term cartilage tissue repair.

[0007] Therefore, there is an urgent need to develop an injectable, biocompatible, biodegradable cartilage repair material with excellent mechanical properties to achieve high-quality cartilage regeneration through minimally invasive surgery. Summary of the Invention

[0008] The purpose of the present invention is to provide a high-strength injectable cartilage repair material to solve the defects of insufficient mechanical properties and excessive degradation of common natural hydrogels at present. This hydrogel can be minimally invasively implanted into irregular cartilage defects, providing mechanical support and a repair microenvironment for the defect site, and is an injectable cartilage repair material with great application potential.

[0009] In the first aspect of the present invention, a hydrogel is provided. The hydrogel is prepared by the following method, which includes: in the presence of a photoinitiator and a first solvent, component A and component B are crosslinked and cured under ultraviolet light irradiation to obtain the hydrogel;

[0010] Among them, component A is a natural material derivative modified with glycidyl methacrylate (GMA); component B is sulfobetaine methacrylate (SBMA).

[0011] In another preferred example, the synthesis raw materials of the hydrogel do not include other raw materials except component A and component B.

[0012] In another preferred example, the natural material derivative is one or more of hyaluronic acid, gelatin, chondroitin sulfate, γ-polyglutamic acid, preferably hyaluronic acid.

[0013] In another preferred example, component A is selected from the group consisting of hyaluronic acid modified with glycidyl methacrylate (HAMA), gelatin modified with glycidyl methacrylate (GelMA), chondroitin sulfate modified with glycidyl methacrylate (CSMA), γ-polyglutamic acid modified with glycidyl methacrylate (m-PGA), or a combination thereof.

[0014] In another preferred example, the molecular weight of the hyaluronic acid is 50 - 500 kDa, preferably 100 - 300 kDa, more preferably 100 - 200 kDa.

[0015] In another preferred example, the molecular weight of the gelatin is 50 - 500 kDa, preferably 50 - 200 kDa, more preferably 50 - 100 kDa.

[0016] In another preferred example, the molecular weight of the γ-polyglutamic acid is 100 to 2000 kDa, preferably 200 to 1000 kDa, and more preferably 300 to 700 kDa.

[0017] In another preferred example, in the component A, glycidyl methacrylate is modified at a molar ratio of carboxyl group to epoxy group of 1:1 to 1:20, preferably 1:2 to 1:15, more preferably 1:3 to 1:15, such as 1:10.

[0018] In another preferred example, the photoinitiator is selected from the group consisting of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), or a combination thereof, preferably lithium phenyl(2,4,6-trimethylbenzoyl)phosphate.

[0019] In another preferred example, the first solvent is water or a PBS solution.

[0020] In another preferred example, the hydrogel has a porous structure with a pore size of 0.1 - 100 μm, preferably 1 - 95 μm, and more preferably 2 - 20 μm.

[0021] In another preferred example, the hydrogel has a hierarchical pore structure, where the pore size of the first - order pores is between 1 - 10 μm and the pore size of the second - order pores is between 10 - 50 μm.

[0022] In another preferred example, the compressive strength of the hydrogel is in the range of 0.05 - 5 MPa (preferably 0.07 - 3.26 MPa).

[0023] In another preferred example, the hydrogel biodegrades within 3 - 50 days (preferably 7 - 45 days).

[0024] In another preferred example, the hydrogel has good biocompatibility.

[0025] In another preferred example, the hydrogel is non - cytotoxic.

[0026] In another preferred example, in the method, the mass ratio of component A to component B is 1:1 - 100, preferably 1:10 - 100, such as 1:20, 1:40, 1:60, 1:80.

[0027] In another preferred example, in the method, the mass - volume fraction of component A in the first solvent is 5 - 200 g / L, preferably 10 - 100 g / L, and more preferably 20 - 80 g / L.

[0028] In another preferred example, in the method, the mass volume fraction of the component B in the first solvent is 0.05-5 g / mL, preferably 0.1-5 g / mL, more preferably 1-5 g / mL.

[0029] In another preferred embodiment, in the method, the mass volume fraction of the photoinitiator in the first solvent is 0.05-5 g / L, preferably 0.08-3 g / L, and more preferably 0.1-2 g / L.

[0030] In another preferred embodiment, the curing is carried out at room temperature, preferably at 10-40°C, more preferably at 15-35°C.

[0031] In another preferred embodiment, the curing time is 1-10 min, preferably 2-5 min.

[0032] In another preferred embodiment, the method is carried out at room temperature, preferably at 10-40°C, more preferably at 15-35°C.

[0033] In another preferred embodiment, the method comprises the following steps:

[0034] Step 101, dissolving component B in a first solvent to obtain a solution of component B;

[0035] Step 102, adding the solution of component B to component A to fully dissolve;

[0036] Step 103, adding a photoinitiator and mixing;

[0037] Step 104: irradiating with ultraviolet light to cause free radical polymerization reaction and cross-linking to obtain the hydrogel.

[0038] In another preferred embodiment, the component A is prepared by the following method, which comprises the following steps:

[0039] Step a: In a second solvent, a natural material derivative is mixed with glycidyl methacrylate, the pH is adjusted, and a reaction is carried out to obtain the component A.

[0040] In another preferred embodiment, the second solvent is water or PBS solution.

[0041] In another preferred embodiment, in step a, the mass volume fraction of the natural material derivative in the second solvent is 5-200 g / L, preferably 10-100 g / L, and more preferably 20-80 g / L.

[0042] In another preferred embodiment, in step a, the volume fraction of the glycidyl methacrylate in the second solvent is 1-100%, preferably 5-80%, more preferably 10-50%.

[0043] In another preferred embodiment, in step a, the materials are added in a molar ratio of the carboxyl group of the natural material derivative to the epoxy group of glycidyl methacrylate of 1:1 to 1:20, preferably 1:2 to 1:15, more preferably 1:3 to 1:15, for example 1:10.

[0044] In another preferred embodiment, in step a, the pH is adjusted to 4-7, for example, 5.

[0045] In another preferred embodiment, in step a, the reaction is carried out at 50-70°C, for example, 60°C.

[0046] In another preferred embodiment, in step a, the reaction time is 6-24 h, preferably 8-12 h.

[0047] In another preferred embodiment, step a further comprises: dialysis and freeze drying to obtain a post-processing step of component A sample.

[0048] In a second aspect of the present invention, a method for preparing the hydrogel according to the first aspect of the present invention is provided, the method comprising: in the presence of a photoinitiator and a solvent, component A and component B are cross-linked and cured under ultraviolet light irradiation to obtain the hydrogel;

[0049] Wherein, the component A is a natural material derivative modified by glycidyl methacrylate; and the component B is methacrylate sulfobetaine.

[0050] In another preferred embodiment, the component A, component B, photoinitiator, solvent and the method are independently as described in the first aspect of the present invention.

[0051] In another preferred embodiment, the method comprises the following steps:

[0052] Step 101, dissolving component B in a solvent to obtain a solution of component B;

[0053] Step 102, adding the solution of component B to component A to fully dissolve;

[0054] Step 103, adding a photoinitiator and mixing;

[0055] Step 104: obtaining the hydrogel by ultraviolet irradiation.

[0056] In a third aspect of the present invention, a hydrogel precursor solution is provided, wherein the hydrogel precursor solution comprises: component A, component B, a photoinitiator and a solvent.

[0057] In another preferred embodiment, the hydrogel precursor solution is a mixed solution of component A, component B and a photoinitiator in a solvent.

[0058] In another preferred example, the component A, component B, photoinitiator, and solvent are each independently as described in the first aspect of the present invention.

[0059] In another preferred example, the hydrogel precursor solution is an injectable solution and can be implanted by injection with a syringe.

[0060] In another preferred example, the hydrogel precursor solution can be rapidly photocured to form the hydrogel described in the first aspect of the present invention.

[0061] In the fourth aspect of the present invention, a kit is provided, which includes:

[0062] A first medicine box containing a solution of component B in a solvent;

[0063] A second medicine box containing component A;

[0064] A third medicine box containing a photoinitiator.

[0065] In another preferred example, the component A, component B, photoinitiator, and solvent are each independently as described in the first aspect of the present invention.

[0066] In another preferred example, the kit further includes an instruction manual, which records: Mix the first medicine box, the second medicine box, and the third medicine box, and cure under ultraviolet light irradiation to obtain the hydrogel described in the first aspect of the present invention.

[0067] In another preferred example, the kit further includes an instruction manual, which records: Add the first medicine box to the second medicine box, then add the third medicine box, and then cure under ultraviolet light irradiation to obtain the hydrogel described in the first aspect of the present invention.

[0068] In the fifth aspect of the present invention, a pharmaceutical composition is provided, which contains:

[0069] The hydrogel described in the first aspect of the present invention, the hydrogel precursor solution described in the third aspect of the present invention, or the kit described in the fourth aspect of the present invention; and

[0070] A pharmaceutically acceptable carrier.

[0071] In another preferred example, the pharmaceutical composition is a gel.

[0072] In another preferred example, the pharmaceutical composition further contains other drugs for cartilage repair.

[0073] In the sixth aspect of the present invention, there is provided the use of a hydrogel as described in the first aspect of the present invention, a hydrogel precursor solution as described in the third aspect of the present invention, a kit as described in the fourth aspect of the present invention, or a pharmaceutical composition as described in the fifth aspect of the present invention, for preparing a pharmaceutical composition for repairing cartilage.

[0074] In the seventh aspect of the present invention, there is provided a method for repairing cartilage, comprising: injecting the hydrogel precursor solution as described in the third aspect of the present invention into a site in need of treatment, thereby repairing cartilage.

[0075] It should be understood that within the scope of the present invention, each of the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is a diagram of the injectability results of the precursor solution for the cartilage repair material.

[0077] Figure 2 It is a diagram of the photo-responsive curing results of the cartilage repair material.

[0078] Figure 3 It is a diagram of the compressive strength test results of the cartilage repair material.

[0079] Figure 4 It is a diagram of the microscopic morphology observation results of the cartilage repair material. (A) HAMA 1000×; (B) HAMA-SBMA 1000×; (C) HAMA 200×; (D) HAMA-SBMA 200×.

[0080] Figure 5 It is a diagram of the in vitro degradation performance test results of the cartilage repair material.

[0081] Figure 6 It is a diagram of the cell compatibility test results of the cartilage repair material.

[0082] Figure 7 It is a schematic diagram of the effect of the cartilage repair material in repairing rat articular cartilage defects.

[0083] Figure 8 It is a diagram of the evaluation results of the repair effect of the cartilage repair material on rat articular cartilage. (A) HE staining; (B) Safranin O / Fast Green staining; (C) Toluidine Blue staining; (D) Immunohistochemical staining of COL II. (Black arrows point to the boundary between the newly formed cartilage and the natural cartilage). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0084] Through extensive and in-depth research, the inventors of the present application have developed a high-strength cartilage repair material. The cartilage repair material of the present invention is a high-strength cartilage repair hydrogel formed by the free radical polymerization reaction cross-linking of a mixed solution of a natural material derivative modified by glycidyl methacrylate (GMA), sulfobetaine methacrylate (SBMA), and a photoinitiator under ultraviolet light irradiation. The hydrogel precursor solution prepared in the present invention has injectability and light responsiveness, can be directly injected into the site to be treated, and rapidly in-situ polymerizes to form a gel in a short time. The formed hydrogel has advantages such as high strength, degradability, and excellent biocompatibility, and is a high-quality minimally invasive treatment cartilage repair material with great application potential.

[0085] Therefore, the high-strength injectable hydrogel prepared in the present invention is expected to achieve the treatment of high-quality cartilage defects through minimally invasive surgery and promote the fusion between the material and natural cartilage. It is a cartilage repair material with broad application prospects.

[0086] Hydrogel

[0087] In the present invention, "hydrogel", "high-strength cartilage repair material", and "cartilage repair material" can be used interchangeably, and refer to a high-strength injectable hydrogel formed by the free radical polymerization reaction cross-linking of a mixed solution of a natural material derivative modified by glycidyl methacrylate (GMA), sulfobetaine methacrylate (SBMA), and a photoinitiator under ultraviolet light irradiation.

[0088] In another preferred example, the natural material derivative is one or more of hyaluronic acid (HAMA), gelatin (GelMA), chondroitin sulfate (CSMA), and γ-polyglutamic acid (m-PGA).

[0089] In another preferred example, in the cartilage repair material, the content of the natural material derivative is 1-10% (w / v), and the content of SBMA is 0-70 wt%.

[0090] In another preferred example, the cartilage repair material has a porous structure with a pore size of 1-95 μm.

[0091] In another preferred example, the compressive strength of the cartilage repair material is 0.07-3.26 MPa.

[0092] In another preferred example, the cartilage repair material can be biodegradable within 7-45 days.

[0093] In another preferred example, the cartilage repair material has good biocompatibility.

[0094] In another preferred example, the cartilage repair material is preferably prepared by the method of Example 12.

[0095] In the cartilage repair material of the present invention, each component has very excellent effects:

[0096] Hyaluronic acid (HA) is an important component of natural articular cartilage, playing a lubricating role in cartilage and providing a microenvironment for chondrocyte adhesion, proliferation, and differentiation simultaneously.

[0097] Chondroitin sulfate (CS) is one of the most abundant glycosaminoglycans in the extracellular matrix of chondrocytes. While lubricating the joint, it can prevent cartilage degeneration and restore the lost extracellular matrix of chondrocytes.

[0098] Gelatin (Gel) is a protein product obtained by partial hydrolysis of collagen, retaining the amino acid composition of collagen, having the advantages of low immunogenicity, cell response, and cell adhesion, and can also be degraded in vivo.

[0099] γ-Polyglutamic acid (γ-PGA) is an anionic polypeptide produced by microbial fermentation in nature, having strong hydrophilicity and water absorption capacity, and can be degraded into non-toxic short peptides and amino acids.

[0100] The above natural polymers are often used to construct injectable cartilage repair hydrogel materials due to their excellent biocompatibility and biodegradability.

[0101] Photo-crosslinking has been widely used to construct injectable hydrogels due to its rapid and mild photo-initiated polymerization reaction. Natural material derivatives modified with methacrylate groups can form a three-dimensional cross-linked hydrogel structure through a rapid photo-initiated free radical polymerization reaction. Its preparation process is relatively simple, and it has adjustable cross-linking degree and physical properties, which are suitable for constructing functional hydrogels. However, its mechanical properties are low and cannot meet the needs of cartilage repair.

[0102] Sulfobetaine methacrylate (SBMA) is an amphoteric ion monomer rich in double bond structures and having biocompatibility, which plays an important role in enhancing the strength and lubricating performance of hydrogels. It is a very promising cartilage repair material and has good application prospects in the field of biomedical materials.

[0103] By combining natural material derivatives with photo-responsiveness and SBMA with mechanical enhancement and lubricating ability, it is expected to construct an injectable hydrogel with excellent mechanical properties and rapid in-situ cross-linking, and it is expected to achieve minimally invasive high-quality cartilage repair.

[0104] Preparation method

[0105] The present invention provides a preparation method of the high-strength cartilage repair material, including the following steps:

[0106] Using GMA-modified natural material derivatives and SBMA as reaction raw materials, photoinitiator action and ultraviolet light irradiation, an injectable cartilage repair hydrogel is obtained, which specifically includes:

[0107] Step 101, dissolving SBMA to obtain a SBMA solution;

[0108] Step 102, adding SBMA solution to the freeze-dried sample of the natural material derivative to fully dissolve it;

[0109] Step 103, adding a photoinitiator and mixing;

[0110] Step 104 , injecting into a cylindrical mold, and irradiating with ultraviolet light to obtain an injectable cartilage repair hydrogel.

[0111] In another preferred embodiment, the natural material derivative is one or more of methacrylated hyaluronic acid (HAMA), gelatin (GelMA), chondroitin sulfate (CSMA), and γ-polyglutamic acid (m-PGA), preferably HAMA.

[0112] In another preferred embodiment, the mass percentage concentration of the SBMA solution in the reaction system in step 101 is 0-70 wt %, preferably 40-50 wt %.

[0113] In another preferred embodiment, the mass fraction of the natural material derivative solution in the reaction system in step 102 is 1-10% (w / v), preferably 4-5%.

[0114] In another preferred embodiment, the initiator in step 103 is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), preferably LAP.

[0115] In another preferred embodiment, the amount of the initiator added to the reaction system in step 103 is 0.01-0.25% (w / v), preferably 0.05-0.1% (w / v).

[0116] In another preferred embodiment, the ultraviolet light irradiation time during the preparation of the injectable cartilage repair hydrogel in step 104 is 0.5 to 10 minutes, preferably 2 to 3 minutes.

[0117] Hydrogel precursor solution

[0118] The invention provides a hydrogel precursor solution, which comprises: a GMA-modified natural material derivative, SBMA, a photoinitiator and a solvent.

[0119] In another preferred embodiment, the precursor solution can be injected and implanted via a syringe.

[0120] In another preferred example, the precursor solution can be rapidly photocured.

[0121] The present invention has the following advantages compared with the prior art:

[0122] 1. Injectable: The hydrogel precursor solution prepared by the present invention has fluidity and can be minimally invasively implanted through injection into the irregular defect sites of articular cartilage for filling, and then rapidly polymerize in situ to form a gel in a short time. The injectable hydrogel, as a convenient minimally invasive treatment means, is expected to achieve the repair of cartilage defects with irregular shapes.

[0123] 2. Rapid curing: The injectable cartilage repair hydrogel prepared by the present invention has light responsiveness, can be injected and implanted with the aid of a minimally invasive arthroscopic device, and rapidly cures in situ under ultraviolet light irradiation. There is no obvious heat release during curing, which can effectively avoid burning the surrounding tissues.

[0124] 3. High strength: The injectable cartilage repair hydrogel prepared by the present invention forms a denser three-dimensional network structure by introducing SBMA monomers, significantly improving the mechanical properties of the hydrogel. In addition, the compressive strength can be controllably adjusted (0.07 - 3.26 MPa) by changing the concentration of SBMA in the hydrogel precursor solution to meet the load-bearing requirements of natural cartilage.

[0125] 4. Degradable: The hydrogel prepared by the present invention has a porous structure and can be gradually degraded under the action of hyaluronidase, possessing the necessary biodegradability of tissue repair materials. By controlling the concentration of SBMA, the degradation rate of the hydrogel can be regulated, effectively solving the problem of too fast degradation rate of natural hydrogels, making the degradation rate of the hydrogel match the tissue regeneration rate, which is more conducive to the continuous repair process of cartilage tissue.

[0126] 5. Biocompatibility: The hydrogel prepared by the present invention has no obvious cytotoxicity and has good biocompatibility, providing an excellent microenvironment for cell growth, proliferation and differentiation, and having the application potential for cartilage tissue engineering.

[0127] 6. Potential for cartilage repair: The present invention uses natural material derivatives with wide sources as raw materials for preparing hydrogels, which can simulate the composition and function of natural cartilage tissue, provide mechanical support and a suitable microenvironment for the defect site, and is a cartilage repair material with great potential.

[0128] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight.

[0129] Example 1

[0130] This example provides a method for preparing a high-strength injectable hydrogel material for cartilage repair, comprising the following steps:

[0131] Weigh 2.333 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 50 mg of HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it thoroughly. Add 0.1 mg of LAP initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 10 min to cure to obtain a HAMA-SBMA hydrogel.

[0132] Example 2

[0133] This example provides a method for preparing a high-strength injectable hydrogel material for cartilage repair, comprising the following steps:

[0134] Weigh 2.333 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 50 mg of GelMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it thoroughly. Add 0.5 mg of LAP initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 5 min to cure to obtain a GelMA-SBMA hydrogel.

[0135] Example 3

[0136] This example provides a method for preparing a high-strength injectable hydrogel material for cartilage repair, comprising the following steps:

[0137] Weigh 2.333 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 50 mg of CSMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it thoroughly. Add 1 mg of LAP initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 3 min to cure to obtain a CSMA-SBMA hydrogel.

[0138] Example 4

[0139] This example provides a method for preparing a high-strength injectable hydrogel material for cartilage repair, comprising the following steps:

[0140] Weigh 2.333 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 50 mg of the m-PGA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it completely. Add 1.5 mg of the LAP initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 2 min to cure and obtain the m-PGA-SBMA hydrogel.

[0141] Example 5

[0142] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0143] Weigh 2.333 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 20 mg of the HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it completely. Add 0.1 mg of the I2959 initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 10 min to cure and obtain the HAMA-SBMA hydrogel.

[0144] Example 6

[0145] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0146] Weigh 1 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 20 mg of the HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it completely. Add 0.5 mg of the I2959 initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 5 min to cure and obtain the HAMA-SBMA hydrogel.

[0147] Example 7

[0148] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0149] Weigh 0.429 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 50 mg of the HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it completely. Add 1 mg of the I2959 initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 3 min to cure and obtain the HAMA-SBMA hydrogel.

[0150] Example 8

[0151] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0152] Weigh 0.111 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 70 mg of the HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it thoroughly. Add 1.5 mg of the LAP initiator, mix well and then inject it into a mold, and irradiate it with ultraviolet light for 2 min to cure to obtain the HAMA-SBMA hydrogel.

[0153] Example 9

[0154] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0155] Weigh 0.111 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 10 mg of the HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it thoroughly. Add 0.1 mg of the LAP initiator, mix well and then inject it into a mold, and irradiate it with ultraviolet light for 10 min to cure to obtain the HAMA-SBMA hydrogel.

[0156] Example 10

[0157] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0158] Weigh 0.429 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Place 20 mg of the HAMA sample weighed in a 1.5 mL centrifuge tube, add 1 mL of the SBMA aqueous solution thereto and dissolve it thoroughly. Add 0.5 mg of the LAP initiator, mix well and then inject it into a mold, and irradiate it with ultraviolet light for 5 min to cure to obtain the HAMA-SBMA hydrogel.

[0159] Example 11

[0160] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0161] Weigh 1 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 50 mg of the HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the SBMA aqueous solution thereto and dissolve it thoroughly. Add 1 mg of the LAP initiator, mix well and then inject it into a mold, and irradiate it with ultraviolet light for 3 min to cure to obtain the HAMA-SBMA hydrogel.

[0162] Example 12

[0163] This example provides a preparation method of a high-strength injectable cartilage repair hydrogel material, including the following steps:

[0164] Weigh 2.333 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare an SBMA solution. Weigh 70 mg of the HAMA sample and place it in a 1.5 mL centrifuge tube. Add 1 mL of the aqueous SBMA solution thereto and dissolve it completely. Add 1.5 mg of the LAP initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 2 min to cure and obtain the HAMA-SBMA hydrogel.

[0165] Example 13

[0166] This example provides a method for preparing a HAMA hydrogel, including the following steps:

[0167] Weigh 2 g of hyaluronic acid (HA) and add it to 100 mL of ultrapure water, stir to dissolve. Add 6.56 mL of GMA, adjust the pH of the solution to 5, and react in a water bath at 60 °C for 8 h. Dialyze for 3 d and freeze-dry to obtain the HAMA sample.

[0168] Weigh 70 mg of HAMA and place it in a 1.5 mL centrifuge tube. Add 1 mL of ultrapure water thereto and dissolve it completely to obtain an aqueous HAMA solution. Add 1.5 mg of the LAP initiator, mix well and inject it into a mold, and irradiate it with ultraviolet light for 2 min to cure and obtain the HAMA hydrogel.

[0169] Example 14

[0170] This example provides a method for evaluating the injectability of the prepared high-strength injectable cartilage repair hydrogel.

[0171] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 as an example, place the hydrogel precursor solution in a syringe. After pushing the syringe, the hydrogel precursor solution smoothly extrudes from the syringe needle without clogging during the process, indicating that the prepared cartilage repair hydrogel precursor solution has injectability. The results are as Figure 1 shown.

[0172] Example 15

[0173] This example provides a method for evaluating the photo-responsive performance of the prepared high-strength injectable cartilage repair hydrogel.

[0174] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 as an example, place the hydrogel precursor solution in a vial and tilt it at 45°. It can be seen that the transparent precursor solution has fluidity. After ultraviolet light irradiation, the precursor solution rapidly gels to obtain a non-flowing transparent solid hydrogel, indicating that the prepared cartilage repair hydrogel has photo-responsiveness. The results are as Figure 2 shown.

[0175] Example 16

[0176] This example provides a method for evaluating the compressive properties of the prepared high-strength injectable cartilage repair hydrogel.

[0177] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 and the HAMA hydrogel prepared in Example 13 as examples, the compressive properties were evaluated. The compressive strength of the hydrogel was characterized by a universal material testing machine. A 5 kN sensor was selected, and the hydrogel was compressed at a rate of 2 mm / min. It can be seen from the experimental results that the compressive strength of the prepared cartilage repair hydrogel can reach 0.07 - 3.26 MPa, while the HAMA hydrogel has basically no compressive strength. The specific results are as Figure 3 shown.

[0178] Example 17

[0179] This example provides a method for characterizing the microscopic morphology of the prepared high-strength injectable cartilage repair hydrogel.

[0180] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 as an example, through scanning electron microscopy observation, it can be found that the prepared cartilage repair hydrogel has a porous structure, and the pore size is about 1 - 95 μm. The specific results are as Figure 4 shown.

[0181] Example 18

[0182] This example provides a method for evaluating the degradation properties of the prepared high-strength injectable cartilage repair hydrogel.

[0183] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 and the HAMA hydrogel prepared in Example 13 as examples, the degradation properties were evaluated. The freeze-dried hydrogel was weighed to obtain its dry weight m 0 , and then immersed in a hyaluronidase solution with a concentration of 100 U / mL. Every 7 days, the freeze-dried hydrogel was taken out and weighed, and the mass was recorded as m t , and the degradation rate was calculated according to the formula Remaining Mass / % = m t / m 0 ×100%. It can be seen from the experimental results that the prepared cartilage repair hydrogel can be biodegradable within 7 - 45 days, and about 50% of the mass remains at 7 days, while the HAMA hydrogel has been degraded within 7 days. The specific results are as Figure 5 shown.

[0184] Example 19

[0185] This example provides a method for evaluating the cell compatibility of the prepared high-strength injectable cartilage repair hydrogel.

[0186] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 as an example, the cartilage repair hydrogel was co-cultured with rat bone marrow mesenchymal stem cells (BMSCs) for 1, 3, and 5 days, and the cell viability was detected by the CCK8 method. Specifically, at each culture time point, the well plate was taken out, the hydrogel and the culture medium were discarded, the CCK8 working solution was added and incubated at 37 °C for 2 h, the absorbance at 450 nm and 650 nm was detected, and the cell activity was calculated. The experimental results showed that the prepared cartilage repair hydrogel had no obvious cytotoxicity and had excellent cell compatibility. The specific results are as Figure 6 shown.

[0187] Example 20

[0188] This example provides a method for evaluating the in vivo cartilage repair effect of the prepared high-strength injectable cartilage repair hydrogel.

[0189] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 as an example, the repair effect evaluation for rat articular cartilage was carried out. The evaluation method includes:

[0190] A cartilage defect model (diameter 2 mm, depth 1 mm) was established at the trochlea of the femoral joint of rats. Specifically, 6 SD rats (male, body weight 200 - 250 g) were randomly divided into 2 groups, with 3 rats in each group. The rats were anesthetized with intraperitoneal injection of sodium pentobarbital and fixed on the operating table. The hair in the surgical area was shaved for skin preparation, and the area was disinfected with alcohol. The skin and fascia were successively incised on the lateral side of the knee joint, the surrounding muscles were bluntly separated to the distal femur, the joint capsule was incised, the femoral condyle was exposed, the patella was dislocated laterally, and a cartilage defect (diameter 2.0 mm, depth 1.0 mm) was constructed in the trochlear groove of the left femur of the rats. After modeling, the hydrogel precursor solution was implanted into the defect area through a syringe and cured under ultraviolet light for 2 min (the control group only modeled; the HAMA-SBMA hydrogel group implanted the HAMA-SBMA hydrogel precursor solution and was cured in situ for 2 min). The joint cavity was thoroughly rinsed with normal saline to remove the cartilage debris formed by modeling, and the wound was closed layer by layer with sutures. After 4 weeks, the rats were sacrificed, and the repaired articular cartilage specimens were collected for gross observation to evaluate the in vivo repair effect of the HAMA-SBMA hydrogel. Gross observation showed that the defect area in the HAMA-SBMA hydrogel group was partially filled with new tissue, and the morphology of the new tissue was relatively close to that of the surrounding tissue, showing an ideal repair effect. The results are as Figure 7 shown.

[0191] Histological staining and immunohistochemical staining were performed to deeply evaluate the quality of the newly formed cartilage tissue. Specifically, the newly formed cartilage tissue was fixed in paraformaldehyde and decalcified, followed by gradient dehydration, infiltration, paraffin embedding, sectioning, dewaxing to water, and stained with hematoxylin-eosin (HE), safranin O / fast green, toluidine blue, and COLⅡ, respectively, to observe the formation of the newly formed tissue. The results of section staining showed that the cartilage defect in the blank control group still existed and was relatively deep, and most of the newly formed cartilage tissue was infiltrated with fibrocartilage tissue. In contrast, a certain amount of hyaline cartilage tissue was successfully regenerated in the cartilage defect area in the HAMA-SBMA hydrogel group, and the specific results are as Figure 8 shown. According to Figure 8 visible, the HAMA-SBMA hydrogel prepared by the present invention can effectively promote the repair of articular cartilage tissue.

[0192] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A hydrogel for repairing cartilage defects, characterized in that: The hydrogel is prepared by the following method, which includes: in the presence of a photoinitiator and a solvent, component A and component B are cross-linked and cured under ultraviolet light irradiation to obtain the hydrogel; Wherein, the component A is hyaluronic acid modified with glycidyl methacrylate; component B is methacrylate sulfobetaine; The component A is prepared by a method comprising the following steps: Step a: mixing hyaluronic acid and glycidyl methacrylate in a solvent, adjusting the pH to 4-5, and reacting to obtain the component A; The materials are added in a molar ratio of 1:3 to 1:15 between the carboxyl group of hyaluronic acid and the epoxy group of glycidyl methacrylate; The mass ratio of component A to component B is 1: 20-100; The photoinitiator is phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate; The compressive strength of the hydrogel is between 0.07 and 5 MPa; The curing time is 1-10 minutes.

2. The hydrogel according to claim 1, characterized in that In step a, the materials are added according to a molar ratio of the carboxyl group of hyaluronic acid to the epoxy group of glycidyl methacrylate of 1:10 to 1:

15.

3. The hydrogel according to claim 1, characterized in that In the method, the mass ratio of component A to component B is 1:40-80.

4. The hydrogel according to claim 1, wherein The molecular weight of the hyaluronic acid is 100-300 kDa.

5. The hydrogel according to claim 1, characterized in that In the method, the mass volume fraction of the component A in the solvent is 10-200 g / L; In the method, the mass volume fraction of the component B in the solvent is 0.05-5 g / mL; In the method, the mass volume fraction of the photoinitiator in the solvent is 0.05-5 g / L.

6. The hydrogel according to claim 1, wherein The curing time is 2-5 minutes.

7. A method for preparing the hydrogel according to any one of claims 1 to 6, the method comprising: In the presence of a photoinitiator and a solvent, component A and component B are cross-linked and cured under ultraviolet light to obtain the hydrogel; Wherein, the component A is a natural material derivative modified by glycidyl methacrylate; and the component B is methacrylate sulfobetaine.

8. An injectable hydrogel precursor solution for cartilage defect repair, characterized in that: The hydrogel precursor solution comprises: component A, component B, a photoinitiator and a solvent; Wherein, the component A is hyaluronic acid modified with glycidyl methacrylate; component B is methacrylate sulfobetaine; The component A is prepared by a method comprising the following steps: Step a: mixing hyaluronic acid and glycidyl methacrylate in a solvent, adjusting the pH to 4-5, and reacting to obtain the component A; The materials are added in a molar ratio of 1:3 to 1:15 between the carboxyl group of hyaluronic acid and the epoxy group of glycidyl methacrylate; The mass ratio of component A to component B is 1: 20-100; The photoinitiator is phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate.

9. The hydrogel precursor solution according to claim 8, characterized in that The hydrogel precursor solution consists of the component A, the component B, a photoinitiator and a solvent.

10. Use of the hydrogel according to any one of claims 1 to 6 or the hydrogel precursor solution according to claim 8 or 9 in preparing a pharmaceutical composition for repairing cartilage defects.

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