Design and preparation method of high-strength cartilage repair material

Abstract

The invention relates to the field of biomedical engineering, and provides a design and a preparation method of a high-strength cartilage repair material. The cartilage repair material disclosed by the invention is high-strength cartilage repair hydrogel formed by carrying out free radical polymerization reaction and crosslinking on a mixed solution of a glycidyl methacrylate modified natural material derivative, sulfobetaine methacrylate and a photoinitiator under the irradiation of ultraviolet light. The cartilage repair material prepared by the method can be injected and implanted by virtue of a minimally invasive arthroscope device, can be quickly formed in situ under ultraviolet irradiation, does not release heat obviously during curing, and can effectively avoid burning surrounding tissues. In addition, the rapid photo-crosslinking reaction endows the high-strength injectable hydrogel with excellent compression strength, overcomes the defects that the traditional natural polymer hydrogel is unstable in mechanical property and too high in degradation rate, and provides a feasible scheme for preparation of the high-performance injectable cartilage repair material.

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 common defects of existing injectable cartilage repair materials, namely, insufficient mechanical properties and rapid degradation. 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 and aging usually cannot recover to the level of hyaline cartilage before damage. In recent years, the incidence of cartilage damage has been increasing year by year, bringing huge physical, psychological pressure and economic burden to patients. At present, the treatment methods for cartilage defects in clinical practice 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, making it difficult to achieve satisfactory cartilage repair effect. Therefore, it is urgent 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 hot topic in the field of cartilage repair. Hydrogel is a scaffold material with high water content and three-dimensional porous network structure, which 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 with their shape plasticity and tissue adhesion. It is worth noting that injectable hydrogels are expected to fill irregular defect areas and firmly adhere to the surface of defective tissue to form in situ, while combining with the matrix proteins of the surrounding cartilage to promote the migration of cells of the surrounding tissue into the hydrogel matrix and achieve cartilage regeneration at the defect. Therefore, the development of injectable hydrogel materials is of great significance to the realization of high-quality in situ cartilage repair.

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

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

[0006] 2. Degradation rate is too fast: The degradation performance of tissue repair materials is an important factor affecting tissue repair. The degradation rate of ideal tissue repair materials should match the rate of tissue regeneration. However, the degradation rate of single cross-linked 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 the common natural hydrogels, which have insufficient mechanical properties and rapid degradation. The hydrogel can be minimally invasively implanted into irregular cartilage defects to provide mechanical support and a repair microenvironment for the defect site, and is an injectable cartilage repair material with great application potential.

[0009] In a first aspect of the present invention, a hydrogel is provided, wherein the hydrogel is prepared by the following method, the method comprising: in the presence of a photoinitiator and a first solvent, component A and component B are cross-linked and cured under ultraviolet light irradiation conditions to obtain the hydrogel;

[0010] Wherein, the component A is a natural material derivative modified by glycidyl methacrylate (GMA); and the component B is sulfobetaine methacrylate (SBMA).

[0011] In another preferred embodiment, the raw materials for synthesizing the hydrogel do not include other raw materials except component A and component B.

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

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

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

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

[0016] In another preferred embodiment, 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 embodiment, in the component A, glycidyl methacrylate is modified with 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, for example 1:10.

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

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

[0020] In another preferred embodiment, 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 embodiment, the hydrogel has a multi-level pore structure, the pore size of the primary pore is between 1-10 μm, and the pore size of the secondary pore is between 10-50 μm.

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

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

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

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

[0026] In another preferred embodiment, 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 the component A in the first solvent is 5-200 g / L, preferably 10-100 g / L, more preferably 20-80 g / L.

[0028] In another preferred embodiment, 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 embodiment, 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 embodiment, the hydrogel precursor solution is an injectable solution and can be injected and implanted via a syringe.

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

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

[0062] a first kit comprising a solution of component B in a solvent;

[0063] A second kit, the second kit comprising component A;

[0064] A third kit comprises a photoinitiator.

[0065] In another preferred embodiment, 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 embodiment, the kit further includes an instruction manual, which states: the first medicine box, the second medicine box, and the third medicine box are mixed and cured under ultraviolet light to obtain the hydrogel as described in the first aspect of the present invention.

[0067] In another preferred embodiment, the kit further includes an instruction manual, which states: adding the first medicine box to the second medicine box, and then adding the third medicine box, and then curing under ultraviolet light to obtain the hydrogel as described in the first aspect of the present invention.

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

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

[0070] A pharmaceutically acceptable carrier.

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

[0072] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for repairing cartilage.

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

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

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

[0076] Figure 1 This is a graph showing the injectability results of the cartilage repair material precursor solution.

[0077] Figure 2 This is the result of light-responsive curing of cartilage repair material.

[0078] Figure 3 This is a graph showing the compressive strength test results of cartilage repair materials.

[0079] Figure 4 The microscopic morphology observation results of cartilage repair materials. (A) HAMA 1000×; (B) HAMA-SBMA 1000×; (C) HAMA 200×; (D) HAMA-SBMA 200×.

[0080] Figure 5 This is a graph showing the in vitro degradation performance test results of cartilage repair materials.

[0081] Figure 6 This is a diagram showing the cell compatibility test results of cartilage repair materials.

[0082] Figure 7 Schematic diagram of the role of cartilage repair materials in repairing articular cartilage defects in rats.

[0083] Figure 8 The results of evaluating the repair effect of cartilage repair materials on rat articular cartilage. (A) HE staining; (B) Safranin O / Fast Green staining; (C) Toluidine blue staining; (D) Immunohistochemical staining of COL II. (The black arrow points to the boundary between the new cartilage and the natural cartilage). DETAILED DESCRIPTION

[0084] The inventors of the present application have developed a high-strength cartilage repair material after extensive and in-depth research. The cartilage repair material of the present invention is a high-strength cartilage repair hydrogel formed by 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 through free radical polymerization. The hydrogel precursor solution prepared by the present invention is injectable and photoresponsive, can be directly injected into the area in need of treatment, and quickly polymerizes in situ to form a gel in a short time. The formed hydrogel has the advantages of high strength, degradation performance and excellent biocompatibility, and is a high-quality cartilage repair material for minimally invasive treatment with great application potential.

[0085] Therefore, the high-strength injectable hydrogel prepared by 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" are used interchangeably, and refer to a high-strength injectable hydrogel formed by cross-linking of a mixed solution of a natural material derivative modified with glycidyl methacrylate (GMA), sulfobetaine methacrylate (SBMA) and a photoinitiator under ultraviolet light irradiation through free radical polymerization reaction.

[0088] In another preferred embodiment, 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 embodiment, in the cartilage repair material, the content of the natural material derivative is 1-10% (w / v), and the content of SBMA is 0-70wt%.

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

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

[0092] In another preferred embodiment, the cartilage repair material is biodegradable within 7 to 45 days.

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

[0094] In another preferred embodiment, 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 one of the important components of natural articular cartilage. It plays a lubricating role in cartilage and provides a microenvironment for chondrocyte adhesion, proliferation and differentiation.

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

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

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

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

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

[0102] Sulfobetaine methacrylate (SBMA) is a biocompatible zwitterionic monomer rich in double bond structure. It plays an important role in enhancing the strength and lubricity of hydrogels. It is a highly potential cartilage repair material and has good application prospects in the field of biomedical materials.

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

[0104] Preparation method

[0105] The present invention provides a method for preparing the high-strength cartilage repair material, comprising the following steps:

[0106] The injectable cartilage repair hydrogel is obtained by using GMA-modified natural material derivatives and SBMA as reaction raw materials, through the action of photoinitiator and ultraviolet light irradiation, 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 embodiment, the precursor solution can be rapidly photocured.

[0121] Compared with the prior art, the present invention has the following advantages:

[0122] 1. Injectable: The hydrogel precursor prepared by the present invention has fluidity and can be minimally invasively injected into irregular defects of articular cartilage for filling, and then rapidly polymerizes in situ to form a gel in a short time. As a convenient minimally invasive treatment method, the injectable hydrogel is expected to achieve the repair of irregular cartilage defects.

[0123] 2. Rapid curing: The injectable cartilage repair hydrogel prepared by the present invention is photoresponsive and can be injected and implanted with the aid of a minimally invasive arthroscopic device. It can be rapidly cured in situ under ultraviolet light irradiation. There is no obvious heat release during curing, which can effectively avoid burns to 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 monomer, which significantly improves the mechanical properties of the hydrogel. In addition, by changing the concentration of SBMA in the hydrogel precursor solution, its compressive strength can be controlled and adjusted (0.07-3.26MPa) 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, and has the biodegradability required for tissue repair materials. The degradation rate of the hydrogel can be regulated by controlling the concentration of SBMA, which effectively solves the problem of too fast degradation rate of natural hydrogels, makes the degradation rate of the hydrogel adapt to the tissue regeneration rate, and 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, has good biocompatibility, provides an excellent microenvironment for cell growth, proliferation and differentiation, and has application potential in cartilage tissue engineering.

[0127] 6. Potential for cartilage repair: The present invention uses widely available natural material derivatives 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 examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0129] Example 1

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

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

[0132] Example 2

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

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

[0135] Example 3

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

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

[0138] Example 4

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

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

[0141] Example 5

[0142] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

[0143] Weigh 2.333 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare SBMA solution. Weigh 20 mg of HAMA sample and place it in a 1.5 mL centrifuge tube, add 1 mL of SBMA aqueous solution and fully dissolve it. Add 0.1 mg of I2959 initiator, mix well, and inject it into the mold. Irradiate it under ultraviolet light for 10 minutes to solidify and obtain HAMA-SBMA hydrogel.

[0144] Example 6

[0145] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

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

[0147] Example 7

[0148] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

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

[0150] Example 8

[0151] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

[0152] Weigh 0.111 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare SBMA solution. Weigh 70 mg of HAMA sample and place it in a 1.5 mL centrifuge tube, add 1 mL of SBMA aqueous solution and fully dissolve it. Add 1.5 mg of LAP initiator, mix well, and inject it into the mold. Irradiate under ultraviolet light for 2 minutes to solidify to obtain HAMA-SBMA hydrogel.

[0153] Example 9

[0154] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

[0155] Weigh 0.111 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare SBMA solution. Weigh 10 mg of HAMA sample and place it in a 1.5 mL centrifuge tube, add 1 mL of SBMA aqueous solution to fully dissolve it. Add 0.1 mg of LAP initiator, mix well, and inject into the mold. Irradiate under ultraviolet light for 10 minutes to solidify to obtain HAMA-SBMA hydrogel.

[0156] Example 10

[0157] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

[0158] Weigh 0.429 g of SBMA powder and dissolve it in 1 mL of ultrapure water to prepare SBMA solution. Weigh 20 mg of HAMA sample and place it in a 1.5 mL centrifuge tube, add 1 mL of SBMA aqueous solution and fully dissolve it. Add 0.5 mg of LAP initiator, mix well, and inject it into the mold. Irradiate under ultraviolet light for 5 minutes to solidify to obtain HAMA-SBMA hydrogel.

[0159] Embodiment 11

[0160] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

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

[0162] Example 12

[0163] This embodiment provides a method for preparing a high-strength injectable cartilage repair hydrogel material, comprising the following steps:

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

[0165] Embodiment 13

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

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

[0168] Weigh 70 mg of HAMA into a 1.5 mL centrifuge tube, add 1 mL of ultrapure water to fully dissolve it, and obtain a HAMA aqueous solution. Add 1.5 mg of LAP initiator, mix well, and inject into a mold. Irradiate under ultraviolet light for 2 minutes to solidify and obtain a HAMA hydrogel.

[0169] Embodiment 14

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

[0171] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 as an example, the hydrogel precursor was placed in a syringe. After the syringe was pushed, the hydrogel precursor was smoothly squeezed out of the syringe through the syringe needle without clogging, indicating that the prepared cartilage repair hydrogel precursor was injectable. Figure 1 shown.

[0172] Embodiment 15

[0173] This embodiment provides a method for evaluating the photoresponsiveness of a prepared high-strength injectable cartilage repair hydrogel.

[0174] Taking the high-strength injectable cartilage repair hydrogel prepared in Example 12 as an example, the hydrogel precursor solution was placed in a vial and tilted at 45°. It can be seen that the transparent precursor solution has flowability. After ultraviolet light irradiation, the precursor solution quickly gelled to obtain a non-flowable transparent solid hydrogel, indicating that the prepared cartilage repair hydrogel has light responsiveness. The results are shown in Figure 12. Figure 2 shown.

[0175] Example 16

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

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

[0178] Embodiment 17

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

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

[0181] Embodiment 18

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

[0183] The high-strength injectable cartilage repair hydrogel prepared in Example 12 and the HAMA hydrogel prepared in Example 13 were used as examples to evaluate the degradation performance. The freeze-dried hydrogel was weighed to its dry weight m 0 Then soak in a hyaluronidase solution with a concentration of 100 U / mL. Take out the freeze-dried tissue every 7 days and weigh it, and record the mass as m t , and according to the formula Remaining Mass / % = m t / m 0 ×100% to calculate its degradation rate. The experimental results show that the prepared cartilage repair hydrogel can be biodegraded within 7 to 45 days, and about 50% of its mass is retained after 7 days, while the HAMA hydrogel has been completely degraded within 7 days. The specific results are as follows Figure 5 shown.

[0184] Embodiment 19

[0185] This embodiment provides a method for evaluating the cell compatibility of a 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 culture medium were discarded, the CCK8 working solution was added and incubated at 37°C for 2 hours, the absorbance at 450nm and 650nm was detected, and the cell activity was calculated. The experimental results show that the prepared cartilage repair hydrogel has no obvious cytotoxicity and has excellent cell compatibility. The specific results are as follows. Figure 6 shown.

[0187] Embodiment 20

[0188] This embodiment 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 of rat articular cartilage was evaluated. The evaluation method includes:

[0190] A cartilage defect model (2 mm in diameter and 1 mm in depth) was established at the trochlea of ​​the femoral joint of rats. Specifically, 6 SD rats (male, weighing 200-250 g) were randomly divided into 2 groups, 3 rats in each group. The rats were anesthetized by intraperitoneal injection of sodium pentobarbital and fixed on the operating table. The rat hair in the surgical area was shaved and the skin was prepared, and disinfected with alcohol. The skin and fascia were cut in sequence on the outside of the knee joint, the surrounding muscles were separated to the distal end of the femur, the joint capsule was cut, the femoral condyle was exposed, the patella was dislocated to the outside, and a cartilage defect (2.0 mm in diameter and 1.0 mm in depth) was constructed in the trochlea groove of the left femur of the rat. After modeling, the hydrogel precursor solution was implanted into the defect area via a syringe and cured under ultraviolet light for 2 minutes (the control group only modeled; the HAMA-SBMA hydrogel group was implanted with the HAMA-SBMA hydrogel precursor solution and cured in situ for 2 minutes). The cartilage debris formed by modeling in the joint cavity was thoroughly rinsed with physiological saline, and the wound was closed layer by layer with sutures. The rats were killed 4 weeks later, and the repaired articular cartilage specimens were collected for gross observation to evaluate the in vivo repair effect of HAMA-SBMA hydrogel. Gross observation showed that the defect area of ​​the HAMA-SBMA hydrogel group was partially filled with new tissue, and the morphology of the new tissue was similar to that of the surrounding tissue, showing a relatively ideal repair effect. 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 staining with hematoxylin-eosin (HE), Safranin O / Fast Green, Toluidine Blue, and COLⅡ 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 the defect was deeper, and some of the generated cartilage tissue was mostly infiltrated with fibrous cartilage tissue. In contrast, the HAMA-SBMA hydrogel group successfully formed a certain amount of hyaline cartilage tissue in the cartilage defect area. The specific results are as follows. Figure 8 As shown. Figure 8 It can be seen that the HAMA-SBMA hydrogel prepared in 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 claims attached to this application.

Claims

1. A hydrogel, 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 a natural material derivative modified by glycidyl methacrylate (GMA); and the component B is sulfobetaine methacrylate (SBMA).

2. The hydrogel according to claim 1, characterized in that The natural material derivative is one or more of hyaluronic acid, gelatin, chondroitin sulfate, and γ-polyglutamic acid, preferably hyaluronic acid.

3. The hydrogel according to claim 1, characterized in that 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.

4. The hydrogel according to claim 1, characterized in that The photoinitiator is selected from the following group: phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), or a combination thereof, preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

5. The hydrogel according to claim 1, characterized in that In the method, the mass volume fraction of component A in the solvent is 5-200 g / L, preferably 10-100 g / L, more preferably 20-80 g / L; In the method, the mass volume fraction of component B in the solvent is 0.05-5 g / mL, preferably 0.1-5 g / mL, more preferably 1-5 g / mL; In the method, the mass volume fraction of the photoinitiator in the solvent is 0.05-5 g / L, preferably 0.08-3 g / L, and more preferably 0.1-2 g / L.

6. The hydrogel according to claim 1, wherein The curing time is 1-10 min, preferably 2-5 min.

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. A hydrogel precursor solution, characterized in that: The hydrogel precursor solution comprises: component A, component B, a photoinitiator and a solvent.

9. The hydrogel precursor solution according to claim 8, characterized in that The hydrogel precursor solution is an injectable solution and can be injected and implanted via a syringe.

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

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