Hyaluronic acid-silk fibroin hydrogel injected into bone joint cavity and preparation method of hyaluronic acid-silk fibroin hydrogel

Abstract

The invention relates to the technical field of medicine, and discloses hyaluronic acid-silk fibroin hydrogel injected into a bone joint cavity and a preparation method of the hyaluronic acid-silk fibroin hydrogel. The gel particles contain hyaluronic acid or salt thereof, silk fibroin and a cross-linking agent; the cross-linking agent is BDDE; the molecular weight of the silk fibroin is 2.5-100 thousand Daltons; the preparation method comprises the following steps: a, dissolving a certain amount of hyaluronic acid or salt thereof and silk fibroin powder in an alkaline solution, uniformly stirring, and carrying out defoaming treatment; b, adding a proper amount of cross-linking agent BDDE into the solution defoamed in the step a to form a mixed solution, carrying out cross-linking, and then dialyzing; c, the gel obtained after dialysis in the step b is sieved through a 40-100-mesh sieve to be granulated, and gel particles are obtained; and d, adding a proper amount of aqueous medium into the gel particles in the step c for resuspension to obtain the hyaluronic acid-silk fibroin hydrogel. The hydrogel disclosed by the invention has excellent mechanical properties, viscoelasticity and abundant porous structures, is remarkable in anti-inflammatory effect, can promote the growth of cartilage cells, is used for intraarticular injection and treatment of osteoarticular related diseases, and has a better industrial production prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical technology and relates to a hyaluronic acid-silk fibroin hydrogel for intra-articular injection and its preparation method. Background Technology

[0002] Osteoarthritis (OA) is a weakening joint disease that leads to damage to articular cartilage and underlying bone. Cartilage is composed of chondrocytes and the extracellular matrix (ECM). The ECM provides cells with a suitable environment and nutrients, and its reticular structure provides cartilage with elastic shaping capabilities. Chondrocyte apoptosis and abnormal ECM degradation are hallmarks of the pathological changes in OA.

[0003] There is an urgent need for a drug that is highly safe, has a simple preparation method, and is suitable for industrial production to treat osteoarthritis and other bone-related diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a hyaluronic acid-silk fibroin hydrogel that can be injected into the joint cavity, has good mechanical properties and viscoelasticity, and is rich in porous structure, as well as a method for its preparation.

[0005] The technical solution of this invention is as follows:

[0006] In a first aspect, the present invention provides a composition for intra-articular injection, comprising gel particles and an aqueous medium; said gel particles comprising hyaluronic acid or a salt thereof, silk fibroin and a crosslinking agent;

[0007] Preferably, the hyaluronic acid has a molecular weight of 400,000 to 2,500,000 Daltons, and the silk fibroin has a molecular weight of 8,000 to 500,000 Daltons.

[0008] In a preferred embodiment, the mass ratio of hyaluronic acid or its salt: silk fibroin: crosslinking agent in the gel particles is (2.5-7.5):(0.5-5):1, preferably (4-7.5):(1-5):1, more preferably (4-7.5):(2-4):1, and most preferably 4.5:2.3:1.

[0009] In a preferred embodiment, the silk fibroin has a molecular weight of 25,000-200,000 Daltons, more preferably 25,000-100,000 Daltons, and even more preferably 40,000-50,000 Daltons.

[0010] In a preferred embodiment, the hyaluronic acid has a molecular weight of 800,000 to 2,500,000 Daltons, preferably 1,500,000 to 2,500,000 Daltons.

[0011] In a preferred embodiment, the elastic modulus of the gel particles is 1×10⁻⁶.2 Pa-1×10 4 Pa, preferably 1×10 3 Pa-5×10 3 Pa, more preferably 1500 Pa–3000 Pa.

[0012] In a preferred embodiment, the aqueous medium is selected from one or more of deionized water, physiological saline, phosphate buffer (PBS), Tris buffer, acetate buffer, and HEPES buffer, preferably phosphate buffer.

[0013] In a preferred embodiment, the salt of the hyaluronic acid is selected from sodium hyaluronate, zinc hyaluronate, and potassium hyaluronate, preferably sodium hyaluronate.

[0014] In a preferred embodiment, the crosslinking agent is selected from 1,4-bisglycidoxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipamide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, or any combination thereof, preferably BDDE.

[0015] In a preferred embodiment, the composition is a solution, gel, lyophilized powder, suspension, preferably a hydrogel.

[0016] In a preferred embodiment, the gel particles are prepared by chemical crosslinking of the hyaluronic acid or its salt, the silk fibroin, and the crosslinking agent.

[0017] In a second aspect, the present invention provides a method for preparing the aforementioned composition, characterized by comprising the following steps:

[0018] a. Dissolve hyaluronic acid or its salt and silk fibroin powder in an alkaline solution, stir well, and then perform defoaming treatment;

[0019] b. Add a cross-linking agent to the solution after defoaming in step a to form a mixed solution, perform cross-linking, and then dialyze;

[0020] c. Granulate the gel after dialysis in step b through a 40-100 mesh sieve, preferably through a 60-80 mesh sieve, to obtain gel particles;

[0021] d. Resuspend the gel particles from step c in an aqueous medium.

[0022] In a preferred embodiment, in the mixed solution of step b, based on the total mass of the mixed solution, the mass percentage of hyaluronic acid or its salt is 5%-15%, preferably 10%-15%, more preferably 10%; the mass percentage of silk fibroin is 1%-10%, preferably 2%-10%, more preferably 5%-8%; and the mass percentage of the crosslinking agent is 1%-5%, preferably 2%.

[0023] In a preferred embodiment, in step a, the defoaming treatment is performed at 2-25°C for at least 12 hours; preferably, at 4°C for 24 hours.

[0024] In a preferred embodiment, in step b, the crosslinking conditions are incubation at 37-60°C; preferably, incubation at 37°C for 4 hours.

[0025] In a preferred embodiment, the dialysate used for dialysis is a phosphate buffer solution with a pH of 7.0-7.2, and the dialysis time is more than 24 hours, preferably 24 hours.

[0026] In a preferred embodiment, in step d, the aqueous medium is a phosphate buffer, and the concentration of the hydrogel is 0.6-1.0 g / mL, preferably 0.8 g / mL.

[0027] In a third aspect, the present invention provides a kit characterized in that it comprises gel particles and an aqueous medium, wherein the gel particles comprise hyaluronic acid or a salt thereof, silk fibroin and a crosslinking agent.

[0028] Preferably, the hyaluronic acid has a molecular weight of 400,000 to 2,500,000 Daltons, and the silk fibroin has a molecular weight of 8,000 to 500,000 Daltons.

[0029] In a preferred embodiment, the mass ratio of hyaluronic acid or its salt: silk fibroin: crosslinking agent in the gel particles is (2.5-7.5):(0.5-5):1, preferably (4-7.5):(1-5):1, more preferably (4-7.5):(2-4):1, and most preferably 4.5:2.3:1.

[0030] In a preferred embodiment, the silk fibroin has a molecular weight of 25,000-200,000 Daltons, preferably 25,000-100,000 Daltons.

[0031] In a preferred embodiment, the hyaluronic acid has a molecular weight of 800,000 to 2,500,000 Daltons, preferably 1,500,000 to 2,500,000 Daltons.

[0032] In a preferred embodiment, the elastic modulus of the gel particles is 1×10⁻⁶. 2 Pa-1×10 4 Pa, preferably 1×10 3 Pa-5×10 3 Pa, more preferably 1500 Pa–3000 Pa.

[0033] In a preferred embodiment, the aqueous medium is selected from one or more of deionized water, physiological saline, phosphate buffer (PBS), Tris buffer, acetate buffer, and HEPES buffer, preferably phosphate buffer.

[0034] In a preferred embodiment, the salt of the hyaluronic acid is selected from sodium hyaluronate, zinc hyaluronate, and potassium hyaluronate, preferably sodium hyaluronate.

[0035] In a preferred embodiment, the crosslinking agent is selected from 1,4-bisglycidoxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipamide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, or any combination thereof, preferably BDDE.

[0036] In a preferred embodiment, the gel particles are prepared by chemical crosslinking of the hyaluronic acid or its salt, the silk fibroin, and the crosslinking agent.

[0037] In a preferred embodiment, the gel particles are resuspended in the aqueous medium before use.

[0038] In a fourth aspect, the present invention provides the use of the foregoing composition or the composition obtained according to the foregoing preparation method or the foregoing kit in the preparation of a medicament for the treatment or prevention of osteoarthritis-related diseases.

[0039] In a preferred embodiment, the bone and joint related diseases are selected from one or more of the following: post-fracture surgery, osteoarthritis, rheumatoid arthritis, degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, frozen shoulder, osteoporosis, femoral head necrosis, and other joint dysfunction diseases.

[0040] Beneficial effects:

[0041] The preparation process of this invention is simple, avoids the use of organic solvent ethanol, is safer, and has greater potential for industrial production.

[0042] The hyaluronic acid-silk fibroin hydrogel of this invention has good injectability and can be used for intra-articular injection. Its mechanical properties, viscoelasticity and rich porous structure can better meet the treatment needs of osteoarthritis-related diseases.

[0043] Compared to HA hydrogels, the HA / SF hydrogel of this invention exhibits superior mechanical properties and cell compatibility, and demonstrates the ability to stimulate cell growth over time. In vitro and in vivo anti-inflammatory experiments show that the HA / SF hydrogel of this invention has significant anti-inflammatory effects, significantly increasing the pain threshold in osteoarthritis rats. The HA / SF hydrogel of this invention promotes cartilage formation and inhibits the degradation of the extracellular matrix in chondrocytes, and can regulate the gene expression levels of the pro-apoptotic gene Bcl-2 and the anti-apoptotic gene Caspase3 in chondrocyte mitochondria. This invention is beneficial for the treatment of osteoarthritis-related diseases. Brief description of the attached diagram

[0044] Figure 1 The values ​​represent the elastic modulus of HA / SF hydrogels with different HA molecular weights, where HA-SF-S represents HA / SF hydrogels prepared using S-HA, HA-SF-M represents HA / SF hydrogels prepared using M-HA, and HA-SF-L represents HA / SF hydrogels prepared using L-HA.

[0045] Figure 2 The in vitro anti-inflammatory effects of HA / SF hydrogels with different SF molecular weights are shown. The inner, middle, and outer layers represent HA / SF hydrogels prepared using 8kDa-25kDa silk fibroin, 25kDa-100kDa silk fibroin, and >100kDa silk fibroin, respectively. Control group: C28 / I2 cells were induced into inflammatory cells and given normal cell culture medium. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0046] Figure 3 This represents the elastic modulus of gel particles with different HA and SF concentrations at a shear frequency of 10 Hz.

[0047] Figure 4 This indicates the apparent transparency of HA hydrogels and HA / SF hydrogels.

[0048] Figure 5 SEM images of HA hydrogel and HA / SF hydrogel are shown.

[0049] Figure 6 This indicates the swelling properties of HA hydrogels and HA / SF hydrogels.

[0050] Figure 7 The rheological properties of HA hydrogel and HA / SF hydrogel are represented.

[0051] Figure 8 This indicates the elastic modulus of HA / SF hydrogels made using different amounts of gel particles.

[0052] Figure 9 The images show the live and dead cell staining of C28 / I2 cells after treatment with the HA hydrogel and HA / SF hydrogel extract from Example 1. In the images, the green parts represent live cells and the red parts represent dead cells.

[0053] Figure 10 The cell proliferation activity of HA hydrogel and HA / SF hydrogel extracts was demonstrated.

[0054] Figure 11 The hemolysis rate of HA hydrogel and HA / SF hydrogel is shown.

[0055] Figure 12 The in vitro anti-inflammatory activities of HA hydrogel and HA / SF hydrogel are shown. * / ** / *** / **** indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the PC group; # / ## / ### / #### indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the HA10% group. PC: Control group, C28 / I2 cells induced into inflammatory cells and given normal cell culture medium.

[0056] Figure 13 This diagram illustrates the effects of HA hydrogel and HA / SF hydrogel on gene expression levels in chondrocyte-induced inflammatory cells in vitro. * / ** / *** / **** indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the PC group; # / ## / ### / #### indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the HA group; @ / @@ / @@@ / @@@@ indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the NC group. PC: Control group, C28 / I2 cells induced to become inflammatory cells, given normal cell culture medium; NC: C28 / I2 cells not induced to become inflammatory, given normal cell culture medium.

[0057] Figure 14This diagram illustrates the effects of HA hydrogel and HA / SF hydrogel on mitochondrial gene expression levels in chondrocyte-induced inflammatory cells in vitro. * / ** / *** / **** indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the PC group; # / ## / ### / #### indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the HA group; @ / @@ / @@@ / @@@@ indicates P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared to the NC group. PC: Control group, C28 / I2 cells induced to become inflammatory cells and given normal cell culture medium; NC: C28 / I2 cells not induced to become inflammatory and given normal cell culture medium.

[0058] Figure 15 The results of the hot and cold plate tests in a rat model of osteoarthritis were shown after 4 weeks of drug treatment. * indicates P < 0.05 compared with the PBS group; ## indicates P < 0.01 compared with the HA group.

[0059] Figure 16 SDS gel electrophoresis results for lyophilized blocks of silk fibroin in three molecular weight ranges. Invention Details

[0061] definition

[0062] As used herein, the term "silk fibroin" includes silk fibroin, insect or spider silk fibroin, or recombinant silk fibroin. In one embodiment, the silk fibroin is obtained from domestic silkworms.

[0063] As used herein, the term "hyaluronic acid" is a biodegradable polymeric component composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine. This water-soluble polymer is naturally found in almost all tissues, particularly in the extracellular matrix, synovial fluid of the eye, and joints. HA is commercially available in pure form. Small gel-particle HA fillers can be used to stimulate the production of natural collagen, which is believed to be induced by the mechanical stretching of the dermis and the activation of dermal fibroblasts.

[0064] As used herein, the term "crosslinking" refers to the intermolecular bonds that link individual polymer molecules, macromolecules, and / or monomer chains into a more stable structure such as a gel. The term "crosslinking agent" refers to a substance that links individual polymer molecules, macromolecules, and / or monomer chains into intermolecular bonds. Representative crosslinking agents include 1,4-bisglycidoxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), dicarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic hydrazide (ADH), bis(sulfosuccinimide) octanoate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, etc.

[0065] As used herein, the term "gel" refers to a thick liquid or semi-solid formulation in the form of a solution, suspension, or emulsion. Gel matrices are single-phase dispersion systems and can be aqueous or oil-based. Aqueous gel matrices are generally composed of water, glycerol or propylene glycol with cellulose derivatives, carbomer and alginate, tragacanth gum, gelatin, starch, etc.; oil-based gel matrices are composed of liquid paraffin with polyethylene or fatty oils with colloidal silica or aluminum soaps, zinc soaps, etc.

[0066] As used herein, the term "salt" includes, for example, salts of inorganic acids and salts of organic acids. Examples of salts may include hydrochlorides, phosphates, pyrophosphates, hydrobroms, sulfates, sulfinates, nitrates, malates, maleates, fumarates, tartrates, succinates, citrates, acetates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, and alkylates (e.g., acetates, HOOC-(CH2)). n -COOH, where n is 0-4). Furthermore, if the compound herein is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acid salt. Conversely, if the compound herein is a free base, the addition salt (particularly a pharmaceutically acceptable addition salt) can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with acid, according to conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will understand the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts.

[0067] As used herein, the term “treatment” and its related expressions mean therapeutic treatment. When referring to a specific condition, treatment means: (1) improving the condition or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that cause or contribute to the condition, or (b) one or more biological manifestations of the condition, (3) alleviating one or more symptoms, effects or side effects associated with or related to the condition or its treatment, or (4) slowing the progression of the condition, or slowing one or more biological manifestations of the condition.

[0068] As used herein, “prevention” means prophylactic administration of medication to substantially reduce the likelihood or severity of a disease or its biological manifestations, or to delay the onset of such a disease or its biological manifestations. Those skilled in the art will understand that “prevention” is not an absolute term. For example, prophylactic treatment is appropriate when a subject is considered to be at high risk of developing cancer, such as when the subject has a strong family history of cancer or when the subject has been exposed to a carcinogen.

[0069] As used herein, the term "subject" refers to an animal, such as a mammal (including a human), which has been or will be the subject of treatment, observation, or experimentation. The methods described herein can be used for therapeutic and / or veterinary applications in humans. In some embodiments, the subject is a mammal. In one embodiment, the subject is a human.

[0070] As used herein, the term "kit" may include two or more single-dose or multi-dose pharmaceutical agents, each individually packaged or formulated; or two or more single-dose or multi-dose pharmaceutical agents packaged or formulated in combination. Thus, one or more pharmaceutical agents may be present in a first container, and the kit may optionally include one or more pharmaceutical agents in a second container. One or more containers are housed within a package, and the package may optionally include instructions for administration or dosage. The kit may include additional components, such as syringes or other components for administering the pharmaceutical agents and diluents, or other components for formulation. Detailed Implementation

[0071] Hyaluronic acid (HA), also known as hyaluronic acid, is a natural polysaccharide and an important component of the extracellular matrix in human joint cavities. Studies have shown that the content and viscoelasticity of hyaluronic acid are significantly reduced in patients with osteoarthritis. Injecting hyaluronic acid solution or hydrogel into the joint cavity can replenish the missing components in the synovial fluid. Therefore, hyaluronic acid solution or hydrogel is an excellent viscoelastic supplement.

[0072] Silk fibroin (SF) is a natural macromolecular protein with good biocompatibility, adjustable mechanical properties, and drug loading capacity, and can be used in bone tissue engineering.

[0073] In one embodiment of the present invention, a hyaluronic acid-silk fibroin hydrogel for intra-articular injection is provided. By combining silk fibroin with hyaluronic acid, a gel with good mechanical properties, safety, and efficacy for treating bone-related diseases is obtained.

[0074] Example

[0075] The technical solution of the present invention will be described in detail below with reference to the embodiments. However, those skilled in the art should understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0076] Materials and their sources used in the following embodiments:

[0077] Hyaluronic acid, Shanghai Maclean Biotechnology Co., Ltd. (molecular weight 400,000-800,000 Daltons, batch number C14499337; molecular weight 800,000-1,500,000 Daltons, batch number C14504635; molecular weight 1,500,000-2,500,000 Daltons, batch number C14554777). Silk fibroin, Shenzhen Huasi Biotechnology Co., Ltd. (batch number SLPSXK1N005). BDDE, Shanghai Maclean Biotechnology Co., Ltd.

[0078] Example 1: Preparation of HA / SF hydrogel

[0079] (1) Accurately weigh 0.2g of sodium hydroxide, add 20mL of Wahaha water, stir to dissolve, and prepare a 1% sodium hydroxide solution for later use;

[0080] (2) Accurately weigh 0.2g of HA (molecular weight 1.5-2.5 million Daltons) and 0.1g of SF (molecular weight 2.5-10 million Daltons) into a 10mL beaker, add 2mL of 1% sodium hydroxide solution, stir evenly, and then defoam at 4℃ for 24h.

[0081] (3) After defoaming, add 40 μL of BDDE (0.044 g) to form a mixed solution, and incubate in a water bath at 37°C for 4 h for crosslinking;

[0082] (4) Remove the gel and place it in a dialysis bag with a molecular weight cutoff of 8000 for dialysis for 24 hours;

[0083] (5) After dialysis, the HA / SF gel particles were granulated by passing them through a 60-mesh sieve.

[0084] (6) Weigh 0.8g of HA / SF gel particles, add 1mL of PBS solution, and stir until homogeneous to obtain HA / SF hydrogel.

[0085] The preparation method for HA hydrogel is the same as described above, but SF is not added during the preparation process.

[0086] Example 2: Investigation of HA with different molecular weights

[0087] To further explore which molecular weight of HA is more suitable for intra-articular injection, three molecular weights of HA were investigated: 400,000-800,000 Daltons, 800,000-1,500,000 Daltons, and 1,500,000-2,500,000 Daltons, denoted as S-HA, M-HA, and L-HA. Following the method in Example 1, HA of different molecular weights were crosslinked with SF (molecular weight 25,000-100,000 Daltons). The elastic modulus (G') of the HA / SF gel particles was measured using a rheometer. The results are shown in [Figure 1]. Figure 1 .

[0088] The results showed that the gel particles prepared by L-HA had a high elastic modulus, so HA with a strength of 1.5-2.5 million Daltons was selected for the following experiments.

[0089] Example 3: Investigation of SF with different molecular weights

[0090] A fractional dialysis bag with an outer, middle, and inner layer structure was prepared using dialysis bags with three different molecular weight cutoffs of 100 kDa, 25 kDa, and 8 kDa. Silk fibroin solution was added to the inner dialysis bag, and dialysis was initiated. Ultimately, the outer, middle, and inner dialysis bags yielded silk fibroin solutions with different molecular weight ranges. The silk fibroin solutions from the outer, middle, and inner dialysis bags were then removed and freeze-dried to obtain lyophilized blocks of silk fibroin with different molecular weight ranges: 8 kDa-25 kDa silk fibroin, 25 kDa-100 kDa silk fibroin, and >100 kDa silk fibroin. Figure 16 The results of SDS gel electrophoresis of lyophilized blocks of silk fibroin in three molecular weight ranges are shown.

[0091] The anti-inflammatory effects of hydrogels prepared with three different molecular weights (0.8-2.5 million Daltons, 2.5-10 million Daltons, and over 100,000 Daltons) of SF were compared. HA / SF hydrogels were prepared according to the method in Example 1 and designated as inner (containing 0.8-2.5 million Daltons of SF), middle (containing 2.5-10 million Daltons of SF), and outer (containing over 100,000 Daltons of SF), respectively. After freeze-drying, appropriate complete culture medium was added to prepare an 8 mg / ml gel extract. An appropriate amount of IL-1β was added to the extract to prepare an IL-1β extract with a final concentration of 50 ng / ml. The extract was then co-cultured with C28 / I2 cells for 48 hours. The cell supernatant was collected, and the levels of anti-inflammatory factors IL-4 and IL-10 and pro-inflammatory factor IL-6 were measured using an ELISA kit. The results are shown in [Figure number missing]. Figure 2 .

[0092] The results showed that, compared with the control group (IL-1β-induced C28 / I2 cells were inflammatory cells and no drug was administered), HA / SF hydrogels prepared with medium molecular weight (25,000-100,000 Daltons) SF significantly increased the expression levels of anti-inflammatory factors IL-4 and IL-10 in the cell supernatant of the in vitro induced arthritis model; compared with the internal and external groups, the medium group had the effect of inhibiting the expression of pro-inflammatory factor IL-6.

[0093] Example 4: Investigation of HA and SF concentrations

[0094] Using HA (1.5-2.5 million Daltons) and SF (25,000-100,000 Daltons), following the method in Example 1, accurately weighed 0.1g of HA and 0.1g of SF; 0.16g of HA and 0.1g of SF; 0.2g of HA and 0.1g of SF; 0.2g of HA and 0.02g of SF; 0.2g of HA and 0.04g of SF; 0.2g of HA and 0.16g of SF; 0.2g of HA and 0.2g of SF; and 0.3g of HA and 0.1g of SF, respectively, and added 2mL of 1% sodium hydroxide solution to each 10mL beaker. After stirring evenly, HA and SF solutions of different concentrations were obtained. These solutions were then used to prepare HA / SF gel particles, designated as samples 1-8. Their elastic modulus (G') was measured using a Kinex rheometer. The results are shown in Table 1 and [Table data would be inserted here]. Figure 3 .

[0095] The results showed that the G' of gel particles with different HA and SF concentrations varied greatly. Gel particles with 10%-15% HA and 2%-10% SF had higher G', but the hydrogel prepared with 15% HA and 5% SF had poor injectability and could not be easily injected within 30 seconds. Considering that it is used for intra-articular injection, the requirements for injectability and elastic modulus are higher. Gel particles with 10% HA and 5%-8% SF have better performance.

[0096] Table 1. Elastic modulus (G') of gel particles with different HA and SF concentrations

[0097] sample HA (mass percentage) SF (Percentage by weight) At 10 Hz G'(Pa) 1 5％ 5％ 73.9 2 8％ 5％ 463.9 3 10％ 5％ 2895.3 4 10％ 1％ 589.9 5 10％ 2％ 641.4 6 10％ 8％ 1551.0 7 10％ 10％ 773.5 8 15％ 5％ 3026.7

[0098] Example 5: Characterization of HA / SF hydrogel

[0099] Appearance transparency test

[0100] Take a blank A4 sheet of paper and write HA / SF and HA with a black marker to represent HA / SF hydrogel and HA hydrogel, respectively. Then, take an appropriate amount of HA / SF hydrogel and HA hydrogel from Example 1 and cover the surface of the lettering, and observe the appearance and transparency of the hydrogel.

[0101] The results are as follows Figure 4 As shown, both hydrogels have good transparency. The HA hydrogel appears more transparent, while the HA / SF hydrogel appears more milky white, which may be related to the change in appearance caused by the addition of SF.

[0102] Injectability test

[0103] The HA / SF hydrogel and HA hydrogel in Example 1 were labeled with lemon yellow and indocyanine green, respectively, and the injectability of the two hydrogels in a 1 mL syringe was observed.

[0104] The results confirmed that both hydrogels could be easily dispensed into a 1mL syringe within 30 seconds, demonstrating good injectability.

[0105] Micromorphological characterization

[0106] Appropriate amounts of the HA hydrogel and HA / SF hydrogel from Example 1 were taken, freeze-dried, and their morphology was observed using a scanning electron microscope (SEM). The results are as follows: Figure 5 As shown.

[0107] The results showed that, compared with HA hydrogel, HA / SF hydrogel has higher porosity and more uniform pore distribution, which will be more conducive to cell adhesion and growth.

[0108] Swelling test

[0109] Appropriate amounts of the HA hydrogel and HA / SF hydrogel from Example 1 were taken, freeze-dried, and their initial weights were recorded. They were then completely immersed in Wahaha water and weighed after 15, 30, 45, 60, 75, 90, 105, and 120 minutes, respectively. The weights of the hydrogels at different time points were recorded, and their swelling properties were observed. The results are as follows: Figure 6 As shown.

[0110] The results showed that both hydrogels had good water absorption. Compared with HA hydrogel, the addition of SF could reduce the water absorption and swelling of HA to a certain extent.

[0111] Rheological property testing

[0112] 1. Rheological properties of HA hydrogel and HA / SF hydrogel:

[0113] The elastic modulus G' and viscous modulus G' of HA hydrogel and HA / SF hydrogel from Example 1 were measured using a Kinex rheometer at shear frequencies of 0.1-10 Hz. The elastic modulus of HA / SF hydrogel and HA hydrogel at a shear frequency of 10 Hz is shown in the results. Figure 7 .

[0114] The results show that the addition of SF significantly improves the elastic modulus of the hydrogel, mitigating the poor elastic modulus problem associated with hydrogels prepared using HA alone. The bar chart of elastic modulus for HA / SF hydrogel and HA hydrogel at a shear frequency of 10 Hz shows that the elastic modulus of HA / SF hydrogel is around 900 Pa, while that of HA hydrogel is around 800 Pa.

[0115] 2. Rheological properties of hydrogels at different concentrations:

[0116] Weigh out 0.6g, 0.8g, and 1.0g of the HA / SF gel particles from Example 1, respectively, and resuspend them in 1mL of PBS solution. Use a Kinex rheometer to detect the elastic modulus G' at shear frequencies of 0.1-10Hz, as well as the elastic modulus G' of the HA / SF hydrogel and HA hydrogel at a shear frequency of 10Hz.

[0117] Figure 8 The results showed that the hydrogel prepared by resuspending 0.8g HA / SF gel particles in 1mL PBS solution had good elastic modulus and good injectability.

[0118] Example 6: In vitro test

[0119] Cell compatibility test

[0120] Appropriate amounts of the HA hydrogel and HA / SF hydrogel from Example 1 were taken, freeze-dried, and then the freeze-dried powders of HA and HA / SF hydrogels were added to the complete culture medium of human chondrocytes (C28 / I2 cells). The mixtures were incubated at 37°C for 1, 2, and 3 days to obtain gel extracts of the two hydrogels on days 1, 2, and 3, respectively. The extracts were co-cultured with C28 / I2 cells, and the cell proliferation activity of the two hydrogels was quantitatively and qualitatively detected using the CCK-8 (Cell Counting Kit-8) and Calcein-AM / PI kits, respectively. The results are shown below. Figure 9and 10 As shown, the control group represents C28 / I2 cells cultured only on complete culture medium without co-culturing with the leachate.

[0121] The results showed that neither of the two hydrogels prepared in this invention was cytotoxic, and that the HA / SF hydrogel exhibited a stronger ability to stimulate chondrocyte proliferation than the HA hydrogel over time.

[0122] Blood compatibility test

[0123] Fresh blood was collected from the abdominal aorta of rats, thoroughly mixed, and transferred to a clean centrifuge tube. The tube was centrifuged at 3000 rpm for 15 min at 4°C to obtain erythrocyte pellet. The supernatant was discarded, and the pellet was washed three times with PBS solution. The pellet was then resuspended in PBS solution (2%, V / V) for later use. 500 μL of HA hydrogel and HA / SF hydrogel from Example 1, along with PBS and 1% Triton (n=4), were thoroughly mixed with 250 μL of 2% erythrocyte suspension. The mixture was incubated in a 37°C water bath for 1 h. After incubation, the pellet was centrifuged at 3000 rpm for 15 min, and hemolysis was visually observed. The supernatant was collected for OD value analysis. The hemolysis rate was calculated based on the OD value using the following formula:

[0124] Hemolysis rate (%) = [(A-A0) / (A1-A0)] × 100.

[0125] A0 represents the OD value of the negative control group (PBS), A1 represents the OD value of the positive control group (Wahaha Water), and A represents the OD value of the experimental group. A hemolysis rate of less than 5% can be considered as no hemolysis has occurred. Results are as follows... Figure 11 As shown, the hemolysis rates of the two prepared hydrogels, HA hydrogel and HA / SF hydrogel, are both less than 5%, indicating that they do not undergo hemolysis in vivo.

[0126] In vitro anti-inflammatory test

[0127] The HA hydrogel, HA / SF hydrogel (sample 3 in Example 4), and HA / SF hydrogel prepared in Example 4, sample 6 were freeze-dried and then added to an appropriate complete culture medium to prepare a 5 mg / ml gel extract. An appropriate amount of IL-1β was added to both extracts to prepare an IL-1β extract with a final concentration of 50 ng / ml. These extracts were then co-cultured with C28 / I2 cells for 48 hours. The cell supernatant was collected, and the contents of anti-inflammatory factors IL-4 and IL-10 and pro-inflammatory factor IL-6 were determined using an ELISA kit. The results are as follows: Figure 12 As shown.

[0128] The results showed that, compared with the positive control group (C28 / I2-induced inflammation model, given cell culture medium, denoted as PC group) and HA hydrogel, HA / SF hydrogels of samples 3 and 6 could significantly increase the expression levels of anti-inflammatory factors IL-4 and IL-10 in the cell supernatant of the in vitro induced arthritis model, and inhibit the expression level of pro-inflammatory factor IL-6.

[0129] Regulation of chondrocyte gene levels

[0130] The HA hydrogel and HA / SF hydrogel prepared in Example 1 were freeze-dried and then added to an appropriate complete culture medium to prepare a 5 mg / ml gel extract. An appropriate amount of IL-1β was added to both extracts to prepare an IL-1β extract with a final concentration of 50 ng / ml. These extracts were then co-cultured with C28 / I2 cells for 48 hours. Cells were collected, and RNA was extracted from the cells using the Trizol method and reverse transcribed into cDNA. RT-PCR was used to further explore the gene expression of Sox9, COLII, MMP13, ADAMTS5, and Aggrecan. The experimental results are as follows: Figure 13 As shown.

[0131] The results showed that, compared with HA hydrogel, HA / SF hydrogel could increase the gene expression levels of genes related to cartilage formation, such as Sox9, COLII (collagen II), and Aggrecan (proteoglycan), while decreasing the gene expression levels of genes related to cartilage catabolism, such as MMP13 (matrix metalloproteinase 13) and ADAMTS5 (platelet-reactive protein integrin metallopeptidase 5). The HA / SF hydrogel of this invention promotes cartilage formation and inhibits the degradation of the extracellular matrix of chondrocytes, thus benefiting the treatment of osteoarthritis.

[0132] Effects on mitochondrial gene expression

[0133] The HA hydrogel and HA / SF hydrogel prepared in Example 1 were freeze-dried and then added to an appropriate complete culture medium to prepare a 5 mg / ml gel extract. An appropriate amount of IL-1β was added to both extracts to prepare an IL-1β extract with a final concentration of 50 ng / ml. These extracts were then co-cultured with C28 / I2 cells for 48 hours. Cells were collected, and RT-PCR was used to further investigate the gene expression of the pro-apoptotic gene Bcl-2 and the anti-apoptotic gene Caspase3 in the mitochondria of C28 / I2 chondrocytes induced into inflammatory cells. The results are as follows: Figure 14 As shown.

[0134] The results showed that, compared with HA hydrogel, HA / SF hydrogel significantly increased the gene expression level of the anti-apoptotic gene Bcl-2 and decreased the gene expression level of the pro-apoptotic gene Caspase-3 in mitochondria. The HA / SF hydrogel of this invention has the function of regulating mitochondria in osteoarthritis chondrocytes.

[0135] Example 7: In vivo drug efficacy - hot and cold plate test of osteoarthritis model

[0136] An anterior cruciate ligament (ACLT) transection was performed on male SD rats (10 weeks old) to establish an osteoarthritis model. After successful model establishment, the osteoarthritis rats were randomly divided into four groups (Sham group, HA group, HA / SF group, and PBS group), with eight rats in each group. At weeks 1 and 3, 30 μL of the hydrogel sample prepared in Example 1 was injected into the joint cavity. The Sham group rats received only anesthesia and a skin incision, while the PBS group received PBS treatment. All rats underwent a hot plate test at week 4 to determine their thermal pain threshold. The hot plate temperature was set to 50.0°C, and the timing was stopped when the animal began licking its hind paws; the recorded time was the animal's thermal pain threshold.

[0137] The results show that ( Figure 15 Compared with the PBS and HA groups, the HA / SF group rats exhibited a significantly prolonged hind paw licking time, showing a statistically significant difference, approaching or even surpassing the sham group. This suggests that the HA / SF hydrogel of the present invention can alleviate pain in osteoarthritis rats by improving their condition.

Claims

1. A composition for intra-articular injection of bone joint comprising gel particles and an aqueous medium; the gel particles comprising hyaluronic acid or its salt, silk fibroin and crosslinking agent; Preferably, the molecular weight of the hyaluronic acid is 40-2.5 million Dalton, and the molecular weight of the silk fibroin is 0.8-0.5 million Dalton.

2. The composition of claim 1, wherein, The mass ratio of hyaluronic acid or its salt: silk fibroin: crosslinking agent in the gel particles is (2.5-7.5) :(0.5-5) :1, preferably (4-7.5) :(1-5) :1, more preferably (4-7.5) :(2-4) :1, and most preferably 4.5:2.3:

1.

3. The composition according to claim 1 or 2, characterized in that, The molecular weight of the silk fibroin is 2.5-2 million Dalton, preferably 2.5-1 million Dalton, and more preferably 5-1 million Dalton.

4. The composition according to any one of claims 1 to 3, characterized in that, The molecular weight of the hyaluronic acid is 80-2.5 million Dalton, preferably 150-2.5 million Dalton.

5. The composition according to any one of claims 1-4, characterized in that, The elastic modulus of the gel particles is 1 x 10 2 Pa - 5 x 10 4 Pa, preferably 1 x 10 3 Pa - 5 x 10 3 Pa, more preferably 1500 Pa - 3000 Pa.

6. The composition according to any one of claims 1-5, characterized in that, The aqueous medium is selected from one or several of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, HEPES buffer, preferably phosphate buffer.

7. The composition according to any one of claims 1-6, characterized in that, The salt of the hyaluronic acid is selected from one of sodium hyaluronate, zinc hyaluronate, potassium hyaluronate, preferably sodium hyaluronate.

8. The composition according to any one of claims 1-7, characterized in that, The crosslinking agent is selected from one or any combination of 1, 4-bisglycidyloxybutane, divinyl sulfone (DVS), 1, 4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1, 2-bis (2, 3-epoxypropoxy) ethylene (EGDGE), 1, 2, 7, 8-diepoxyoctane (DEO), bis-carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis (sulfosuccinimidyl) suberate (BS), hexamethylene diamine (HMDA), 1- (2, 3-epoxypropyl) -2, 3-epoxycyclohexane, carbodiimide, preferably BDDE. 9.The composition of any one of claims 1-8, wherein the composition is a solution, a gel, a lyophilized powder, a suspension, preferably a hydrogel. 10.The composition of any one of claims 1-9, wherein the gel particles are prepared by chemical crosslinking of the hyaluronic acid or its salt, the silk fibroin and the crosslinking agent.

11. A process for the preparation of a composition according to any one of claims 1 to 10, characterised in that, comprising the following steps: a. Dissolve the hyaluronic acid or its salt and silk fibroin powder in an alkaline solution, stir uniformly, and defoam; b. Add the crosslinking agent to the defoamed solution of step a to form a mixed solution, crosslink, and then dialyze; c. Granulate the gel after dialysis of step b through a 40-100 mesh sieve, preferably a 60-80 mesh sieve, to obtain gel particles; d. Resuspend the gel particles of step c in the aqueous medium.

12. The method of claim 11, wherein, The mass percentage of the hyaluronic acid or its salt in the mixed solution of step b is 5%-15%, preferably 10%-15%, more preferably 10%, based on the total mass of the mixed solution; the mass percentage of the silk fibroin is 1%-10%, preferably 2%-10%, more preferably 5%-8%; and the mass percentage of the crosslinking agent is 1%-5%, preferably 2%.

13. The production method according to claim 11 or 12, characterized by, In step a, the defoaming treatment is performed at 2-25°C for 12 hours or more; preferably, at 4°C for 24 hours.

14. The production method according to any one of claims 11 to 13, characterized by, In step b, the crosslinking condition is incubation at 37-60°C; preferably, incubation at 37°C for 4 hours.

15. The production method according to any one of claims 11 to 14, characterized by, The dialysis liquid used in the dialysis is phosphate buffer, and the pH value is 7.0-7.

2. The dialysis time is 24 hours or more, preferably 24 hours.

16. The production method according to any one of claims 11 to 15, characterized by, In step d, the aqueous medium is phosphate buffer, and the concentration of the hydrogel is 0.6-1.0 g / mL, preferably 0.8 g / mL.

17. A kit comprising, The gel particles and the aqueous medium, wherein the gel particles comprise hyaluronic acid or its salt, silk fibroin, and a crosslinking agent; Preferably, the molecular weight of the hyaluronic acid is 4-25 million Daltons, and the molecular weight of the silk fibroin is 0.8-5 million Daltons.

18. The kit of claim 17, wherein The mass ratio of hyaluronic acid or its salt:silk fibroin:crosslinking agent in the gel particles is (2.5-7.5):(0.5-5):1, preferably (4-7.5):(1-5):1, more preferably (4-7.5):(2-4):1, and most preferably 4.5:2.3:

1.

19. The kit of claim 17 or 18, wherein, The molecular weight of the silk fibroin is 2.5-20 million Daltons, preferably 2.5-10 million Daltons, and more preferably 4-5 million Daltons.

20. The kit of any one of claims 17-19, wherein, The molecular weight of the hyaluronic acid is 80-250 million Daltons, preferably 150-250 million Daltons.

21. The kit of any one of claims 17-20, wherein, The elastic modulus of the gel particles is 1 x 10 2 Pa - 5 x 10 4 Pa, preferably 1 x 10 3 Pa - 5 x 10 3 Pa, more preferably 1500 Pa - 3000 Pa.

22. The kit of any one of claims 17-21, wherein, The aqueous medium is selected from one or more of deionized water, physiological saline, phosphate (PBS) buffer, Tris buffer, acetate buffer, and HEPES buffer, preferably phosphate buffer.

23. The kit of any one of claims 17-22, wherein, The salt of the hyaluronic acid is selected from one of sodium hyaluronate, zinc hyaluronate, and potassium hyaluronate, preferably sodium hyaluronate.

24. The kit of any one of claims 17-23, wherein, The crosslinking agent is selected from one or any combination of 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis-carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipohydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, and carbodiimide, preferably BDDE.

25. The kit according to any one of claims 17-24, wherein the gel particles are prepared by chemical cross-linking of the hyaluronic acid or salt thereof, the silk fibroin and the cross-linking agent.

26. The kit according to any one of claims 17-25, wherein the gel particles are resuspended in the aqueous medium prior to use.

27. Use of the composition according to any one of claims 1-10 or the composition obtained according to the preparation method of any one of claims 11-16 or the kit according to any one of claims 17-26 for the preparation of a medicament for the treatment or prevention of bone joint related diseases.

28. Use according to claim 27, characterized in that, The bone joint related diseases are selected from one or more of postoperative fracture, osteoarthritis, rheumatoid arthritis, degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, frozen shoulder, osteoporosis, femoral head necrosis and other various joint dysfunction diseases.

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