Hyaluronic acid-silk fibroin hydrogel for intra-articular injection and preparation method therefor

Abstract

Provided are a hyaluronic acid-silk fibroin hydrogel for intra-articular injection and a preparation method therefor. The hydrogel comprises gel particles and an aqueous medium. The gel particles comprise hyaluronic acid or a salt thereof, silk fibroin, and a crosslinking agent. The crosslinking agent is BDDE, and the molecular weight of silk fibroin is 25-100 kDa. The preparation method comprises: a. dissolving a certain amount of hyaluronic acid or a salt thereof and silk fibroin powder in an alkaline solution, stirring uniformly, and performing defoaming treatment; b. adding an appropriate amount of a crosslinking agent BDDE to the defoamed solution in step a to form a mixed solution, performing crosslinking, and then dialyzing; c. granulating the dialyzed gel in step b through a 40-100 mesh sieve to obtain gel particles; and d. adding an appropriate amount of an aqueous medium to the gel particles in step c for resuspension to obtain the hyaluronic acid-silk fibroin hydrogel.

Description

A hyaluronic acid-silk fibroin hydrogel for intra-articular injection of bone joint and a preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a hyaluronic acid-silk fibroin hydrogel for intra-articular injection of bone joint and a preparation method thereof. BACKGROUND

[0002] Osteoarthritis (OA) is a debilitating joint disease that causes damage to the articular cartilage and underlying bone. Cartilage is composed of chondrocytes and extracellular matrix (ECM), which provides a living environment and nutrient components for cells, and the network structure in the ECM provides the elastic shaping ability of cartilage. Apoptosis of chondrocytes and abnormal degradation of ECM are one of the signs of pathological changes of OA.

[0003] There is an urgent need for a drug for treating osteoarthritis and other bone-related diseases that is safe, has a simple preparation method and is suitable for industrial production. SUMMARY

[0004] The purpose of the present application is to provide a hyaluronic acid-silk fibroin hydrogel that can be used for intra-articular injection of bone joint, has good mechanical ability and viscoelasticity, and has a rich porous structure, and a preparation method thereof.

[0005] The technical scheme of the present application is as follows:

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

[0007] Preferably, the molecular weight of the hyaluronic acid is 40-2.5 million Daltons, and the molecular weight of the silk fibroin is 0.8-0.5 million Daltons.

[0008] In a preferred embodiment, the mass ratio of hyaluronic acid or a salt thereof: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 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.

[0010] In a preferred embodiment, the molecular weight of the hyaluronic acid is 80-2.5 million Daltons, preferably 150-2.5 million Daltons.

[0011] In a preferred embodiment, the elastic modulus of the gel particles is 1 x 10 2 Pa - 1 x 10 4 Pa, preferably 1 x 10 3 Pa - 5 x 10 3 Pa, more preferably 1500 Pa - 3000 Pa.

[0012] In a preferred embodiment, 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.

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

[0014] In a preferred embodiment, the cross-linking 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.

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

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

[0017] In a second aspect, the present application provides a method for preparing the aforementioned composition, characterized in that it comprises the following steps:

[0018] a. dissolving the hyaluronic acid or its salt and the silk fibroin powder in an alkaline solution, stirring until uniform, and defoaming;

[0019] b. adding the cross-linking agent to the defoamed solution of step a to form a mixed solution, cross-linking, and then dialyzing;

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

[0021] d. resuspending the gel particles of step c in an aqueous medium.

[0022] In a preferred embodiment, the mass percentage of 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 silk fibroin is 1%-10%, preferably 2%-10%, more preferably 5%-8%; and the mass percentage of crosslinking agent is 1%-5%, preferably 2%.

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

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

[0025] In a preferred embodiment, the dialysis uses a phosphate buffer as the dialysate, with a pH of 7.0-7.2, and the dialysis time is 24 hours or more, preferably 24 hours.

[0026] In a preferred embodiment, the aqueous medium in step d 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 application provides a kit, characterized in that it comprises gel particles and an aqueous medium, wherein the gel particles comprise hyaluronic acid or its salt, silk fibroin, and a crosslinking agent.

[0028] Preferably, the molecular weight of the hyaluronic acid is 4-2.5 million Daltons, and the molecular weight of the silk fibroin is 0.8-0.5 million 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 molecular weight of the silk fibroin is 2.5-20 million Daltons, preferably 2.5-10 million Daltons.

[0031] In a preferred embodiment, the hyaluronic acid has a molecular weight of 80-2.5 million Dalton, preferably 150-2.5 million Dalton.

[0032] In a preferred embodiment, the gel particles have an elastic modulus of 1 x 10 2 Pa - 1 x 10 4 Pa, preferably 1 x 10 3 Pa - 5 x 10 3 Pa, more preferably 1500 Pa - 3000 Pa.

[0033] In a preferred embodiment, 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.

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

[0035] In a preferred embodiment, the cross-linking 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)ethane (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.

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

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

[0038] In a fourth aspect, the present application provides the use of the aforementioned composition or the composition obtained according to the aforementioned preparation method or the aforementioned kit for the preparation of a medicament for the treatment or prevention of bone joint related diseases.

[0039] In a preferred embodiment, the bone joint related disease is selected from one or more of postoperative fracture, osteoarthritis, rheumatoid arthritis, degenerative arthritis, bursitis, synovitis, cervical spondylosis, lumbar spondylosis, periarthritis of shoulder, osteoporosis, femoral head necrosis and other various joint dysfunction diseases. Advantages:

[0040] The preparation process of the present application is simple, avoids the use of organic solvent ethanol, is safer and has more industrial production prospects.

[0041] The hyaluronic acid-silk fibroin hydrogel of the present application has good injectability and can be used for intra-articular injection, and its mechanical properties, viscoelasticity and rich porous structure are more suitable for the treatment of bone joint related diseases.

[0042] Compared with HA hydrogel, the HA / SF hydrogel of the present application has good mechanical properties and cell compatibility, and has the ability to stimulate cell growth with time; in vivo and in vitro anti-inflammatory tests show that the HA / SF hydrogel of the present application has a significant anti-inflammatory effect and significantly improves the pain threshold of osteoarthritis rats. The HA / SF hydrogel of the present application is conducive to cartilage formation and inhibition of degradation of cartilage extracellular matrix, and can regulate the gene expression levels of pro-apoptotic gene Bcl-2 and anti-apoptotic gene Caspase3 in mitochondria of chondrocytes. The present application is conducive to the treatment of bone joint related diseases.

[0043] Brief Description of the Drawings

[0044] Figure 1 shows the elastic modulus of HA / SF hydrogel with different HA molecular weights, wherein HA-SF-S represents HA / SF hydrogel prepared using S-HA, HA-SF-M represents HA / SF hydrogel prepared using M-HA, and HA-SF-L represents HA / SF hydrogel prepared using L-HA.

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

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

[0047] Figure 4 shows the appearance transparency of HA hydrogel and HA / SF hydrogel.

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

[0049] Figure 6 shows the swelling properties of HA hydrogel and HA / SF hydrogel.

[0050] Figure 7 shows the rheological properties of HA hydrogel and HA / SF hydrogel.

[0051] Figure 8 shows the elastic modulus of HA / SF hydrogel prepared using different contents of gel particles.

[0052] Figure 9 is a live-dead staining image after treating C28 / I2 cells with the leaching solution of HA hydrogel and HA / SF hydrogel of Example 1, wherein the green part represents live cells and the red part represents dead cells.

[0053] Figure 10 shows the cell proliferation activity of the leaching solution of HA hydrogel and HA / SF hydrogel.

[0054] Figure 11 shows the hemolysis rate of HA hydrogel and HA / SF hydrogel.

[0055] Figure 12 shows the in vitro anti-inflammatory activity of HA hydrogel and HA / SF hydrogel. * / ** / *** / **** means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with PC group; # / ## / ### / #### means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with HA 10% group. PC: control group, C28 / I2 cells were induced to be inflammatory cells and were given normal cell culture solution.

[0056] Figure 13 shows the effect of HA hydrogel and HA / SF hydrogel on the gene expression level of in vitro chondrocyte-induced inflammatory cells. * / ** / *** / **** means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with PC group; # / ## / ### / #### means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with HA group; @ / @@ / @@@ / @@@@ means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with NC group. PC: control group, C28 / I2 cells were induced to be inflammatory cells and were given normal cell culture solution; NC: C28 / I2 cells were not induced to be inflammatory and were given normal cell culture solution.

[0057] Figure 14 shows the effect of HA hydrogel and HA / SF hydrogel on the level of mitochondrial gene expression of in vitro chondrocyte-induced inflammatory cells. * / ** / *** / **** means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with PC group; # / ## / ### / #### means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with HA group; @ / @@ / @@@ / @@@@ means P<0.05 / P<0.01 / P<0.001 / P<0.0001 compared with NC group. PC: control group, C28 / I2 cells were induced to be inflammatory cells, and normal cell culture solution was given; NC: C28 / I2 cells were not induced to be inflammatory, and normal cell culture solution was given.

[0058] Figure 15 shows the results of the cold-hot plate test of the osteoarthritis model rats after administration for 4 weeks. * means P<0.05 compared with PBS group; ## means P<0.01 compared with HA group.

[0059] Figure 16 is the SDS gel electrophoresis result of three molecular weight ranges of silk fibroin lyophilized blocks.

[0060] DETAILED DESCRIPTION

[0061] DEFINITIONS

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

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

[0064] As used herein, the term "crosslinking" refers to intermolecular bonds that link individual polymer molecules, macromolecules, and / or monomer chains into more stable structures such as gels. The term "crosslinking agent" refers to a substance that causes intermolecular bonds that link individual polymer molecules, macromolecules, and / or monomer chains. Representative crosslinking agents include 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)ethane (EGDGE), 1,2,7,8-diepoxyoctane (DEO), bis carbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic hydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylene diamine (HMDA), 1-(2,3- epoxypropyl)-2,3-epoxycyclohexane, carbodiimides, and the like.

[0065] As used herein, the term "gel" refers to a thick liquid or semisolid preparation of solution, suspension, or emulsion type. Gel bases are single-phase dispersion systems, which are divided into aqueous and oleaginous. Aqueous gel bases are generally composed of water, glycerin or propylene glycol, and cellulose derivatives, carbomer and alginic acid salts, tragacanth, gelatin, starch, and the like; oleaginous gel bases are composed of liquid paraffin and polyethylene or fatty oils and colloidal silicon or aluminum soaps, zinc soaps, and the like.

[0066] As used herein, the term "salt" includes, for example, salts of inorganic acids and salts of organic acids. Examples of salts can include hydrochloride, phosphate, pyrophosphate, hydrobromide, sulfate, sulfinate, nitrate, malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and alkanoate (e.g., acetate, HOOC-(CH2) n -COOH, where n is 0-4). Furthermore, where the compounds herein are obtained as acid addition salts, the free base can be obtained by basifying a solution of the acid salt. Conversely, where the compounds herein are a free base, an 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 an acid, in accordance with conventional procedures for making acid addition salts from base compounds. Those of skill in the art will appreciate that a wide variety of synthetic procedures are available for preparing nontoxic pharmaceutically acceptable addition salts.

[0067] As used herein, the term "treatment" and related terms, such as treating, refer to therapeutic treatment. When used in reference to a specific condition, treatment indicates: (1) improvement of the condition or one or more of the biological manifestations of the condition, (2) interference with (a) one or more points in a biological cascade that leads to or causes the condition, or (b) one or more of the biological manifestations of the condition, (3) reduction of one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing of the progression of the condition, or slowing of one or more of the biological manifestations of the condition.

[0068] As used herein, "prevention" refers to prophylactic administration to substantially reduce the likelihood or severity of a condition or a biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. Those skilled in the art will appreciate that "prevention" is not an absolute term. For example, prophylactic treatment is appropriate when a subject is deemed to be at high risk of developing a cancer, such as when the subject has a strong family history of cancer or when the subject has been exposed to a carcinogenic substance.

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

[0070] As used herein, the term "kit" can include single or multiple doses of two or more agents, each individually packaged or formulated; or single or multiple doses of two or more agents packaged or formulated in combination. Thus, one or more agents can be present in a first container, and the kit can optionally include one or more agents in a second container. The container(s) is / are placed into a package and the package can optionally include instructions for administration or dosing. The kit can include additional components such as a syringe or other means for administering the agents and diluents or other means for formulation. DETAILED DESCRIPTION

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

[0072] Silk fibroin (SF) is a natural polymer protein, which has good biocompatibility, adjustable mechanical properties and drug loading capacity, and can be applied to bone tissue engineering.

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

[0074] EMBODIMENT

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

[0076] Materials used in the following examples and sources:

[0077] Hyaluronic acid, Shanghai Maikelin Biotechnology Co., Ltd. (molecular weight 40-80 million daltons, batch number C14499337; molecular weight 80-150 million daltons, batch number C14504635; molecular weight 150-250 million daltons, batch number C14554777). Silk fibroin, Shenzhen Huasi Biotechnology Co., Ltd. (batch number SLPSXK1N005). BDDE, Shanghai Maikelin Biotechnology Co., Ltd.

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

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

[0080] (2) Precisely weigh 0.2 g of HA (molecular weight 150-250 million daltons) and 0.1 g of SF (molecular weight 2.5-10 million daltons) in a 10 mL beaker, add 2 mL of 1% sodium hydroxide solution, stir evenly, and defoam at 4°C for 24 h;

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

[0082] (4) Take out the gel and put it into a dialysis bag with a molecular weight cutoff of 8000 for dialysis for 24 hours;

[0083] (5) After dialysis, granulate through a 60-mesh sieve to obtain HA / SF gel particles;

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

[0085] The preparation method of HA hydrogel is as described above, and the preparation process does not add SF.

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

[0087] To further explore which molecular weight of HA is more suitable for joint cavity injection, three molecular weights of HA were investigated, namely 40-80 million Dalton, 80-150 million Dalton and 150-250 million Dalton, denoted as S-HA, M-HA and L-HA. Different molecular weights of HA were crosslinked with SF (molecular weight of 2.5-10 million Dalton) according to the method of Example 1, and the elastic modulus (G') of HA / SF gel particles was detected by a rheometer, and the results are shown in Figure 1.

[0088] The results show that the gel particles prepared by L-HA have a higher elastic modulus, so 150-250 million Dalton of HA is selected for the following experiments.

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

[0090] A three-stage dialysis bag with an outer layer, a middle layer and an inner layer structure was prepared using three different gradient dialysis bags with a molecular weight cut-off of 100 kDa, 25 kDa and 8 kDa. The silk fibroin solution was added to the inner layer dialysis bag and dialysis was started. The outer layer, middle layer and inner layer dialysis bags will finally obtain silk fibroin solutions with different molecular weight ranges. The silk fibroin solutions in the outer layer, middle layer and inner layer dialysis bags were taken out and freeze-dried, respectively, to obtain silk fibroin lyophilized blocks with different molecular weight ranges, i.e. 8 kDa-25 kDa silk fibroin, 25 kDa-100 kDa silk fibroin and >100 kDa silk fibroin. Figure 16 shows the SDS gel electrophoresis results of the three molecular weight ranges of silk fibroin lyophilized blocks.

[0091] The anti-inflammatory effect of the hydrogels prepared from three molecular weights (0.8-2.5, 2.5-10 and above 10 kDa) of SF was compared. The HA / SF hydrogels were prepared according to the method of Example 1 and were denoted as inner (containing 0.8-2.5 kDa SF), middle (containing 2.5-10 kDa SF) and outer (containing above 10 kDa SF), respectively. After freeze-drying, the gels were immersed in the appropriate complete medium to prepare a 8 mg / ml gel extract, and the IL-1β was added to the extract to prepare an IL-1β extract with a final concentration of 50 ng / ml, which 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 determined by using Elisa kits. The results are shown in Figure 2.

[0092] The results show that, compared with the control group (IL-1β induced C28 / I2 cells to be inflammatory cells without drug administration), the HA / SF hydrogel prepared from the middle molecular weight (2.5-10 kDa) SF can significantly increase the expression level of anti-inflammatory factors IL-4 and IL-10 in the cell supernatant of the in vitro induced arthritis model; compared with the inner group and the outer group, the middle group has the effect of inhibiting the expression of pro-inflammatory factor IL-6.

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

[0094] HA and SF with molecular weights of 150-250 kDa and 2.5-10 kDa were used to prepare HA / SF gel particles with different concentrations. According to the method of Example 1, 0.1 g of HA and 0.1 g of SF, 0.16 g of HA and 0.1 g of SF, 0.2 g of HA and 0.1 g of SF, 0.2 g of HA and 0.02 g of SF, 0.2 g of HA and 0.04 g of SF, 0.2 g of HA and 0.16 g of SF, 0.2 g of HA and 0.2 g of SF, and 0.3 g of HA and 0.1 g of SF were precisely weighed into 10 mL beakers, respectively, and 2 mL of 1% sodium hydroxide solution was added. After stirring, HA and SF solutions with different concentrations were obtained. The solutions were prepared into HA / SF gel particles, which were denoted as samples 1-8. The elastic modulus (G’) of the gel particles was determined by using a kinex rheometer. The results are shown in Table 1 and Figure 3.

[0095] The results show that the G’ of the gel particles with different concentrations of HA and SF varies greatly. The G’ of the gel particles with 10%-15% HA and 2%-10% SF is relatively high, but the injectability of the hydrogel prepared from 15% HA and 5% SF is poor and cannot be easily pushed out within 30 seconds. Considering the higher requirements for injectability and elastic modulus for intra-articular injection, the gel particles with 10% HA and 5%-8% SF are better.

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

[0097] Example 5: Characterization of HA / SF hydrogel

[0098] Appearance transparency test

[0099] Take a blank A4 paper, and write HA / SF and HA on it with a black marker, which respectively represent HA / SF hydrogel and HA hydrogel. Then, take appropriate amounts of HA / SF hydrogel and HA hydrogel of Example 1 respectively, and cover them on the font surface to observe the appearance transparency of the hydrogels.

[0100] The results are shown in Figure 4, and the transparency of both hydrogels is good. Among them, the appearance of HA hydrogel is slightly transparent, while the appearance of HA / SF hydrogel is slightly white, which may be related to the change in the appearance of the hydrogel caused by the addition of SF.

[0101] Injectability test

[0102] Label the HA / SF hydrogel and HA hydrogel of Example 1 with citrine and indocyanine green respectively, and observe the injectability of the two hydrogels in a 1 mL syringe.

[0103] The results confirm that both hydrogels can be easily pushed out of a 1 mL syringe within 30 seconds, and the injectability is good.

[0104] Micro-morphology characterization

[0105] Take appropriate amounts of HA hydrogel and HA / SF hydrogel of Example 1, freeze-dry them, and then observe the morphology of the two hydrogels using a scanning electron microscope (SEM), and the results are shown in Figure 5.

[0106] The results show that, compared with HA hydrogel, HA / SF hydrogel has high porosity and more uniform pore distribution, which will be more conducive to cell adhesion and growth.

[0107] Swelling test

[0108] Take appropriate amounts of HA hydrogel and HA / SF hydrogel of Example 1, freeze-dry them, record the initial weight, then immerse them completely in Wahaha water, and take them out to weigh at 15, 30, 45, 60, 75, 90, 105, and 120 minutes respectively. Record the weight of the hydrogels at different time points, and observe their swelling properties, and the results are shown in Figure 6.

[0109] The results show that both hydrogels have good water absorption, and compared with HA hydrogel, the addition of SF can reduce the water absorption and swelling of HA to a certain extent.

[0110] Rheological property test

[0111] 1. Rheological property test of HA hydrogel and HA / SF hydrogel

[0112] An appropriate amount of HA hydrogel and HA / SF hydrogel of Example 1 was taken and freeze-dried, and the freeze-dried powder of HA and HA / SF hydrogel was added to the complete culture medium of human chondrocytes (C28 / I2 cells), which was placed at 37°C for 1, 2 and 3 days to obtain the gel leachate of the two kinds of hydrogel on the 1st, 2nd and 3rd day, respectively. The leachate was co-cultured with C28 / I2 cells, and then CCK-8 (Cell Counting Kit-8) and Calcein-AM / PI kit were used for quantitative and qualitative detection of the proliferation activity of the two kinds of hydrogel on the cells. The results are shown in Figures 9 and 10, respectively. The control group in the figures represents C28 / I2 cells cultured in the complete culture medium without co-culture with the leachate.

[0113] From the results, it can be seen that the addition of SF can significantly improve the elastic modulus of the hydrogel and improve the problem of poor elastic modulus of the hydrogel prepared by using HA alone. The column chart of the elastic modulus of HA / SF hydrogel and HA hydrogel at a shear frequency of 10 Hz shows that the elastic modulus of HA / SF hydrogel is about 900 Pa, and the elastic modulus of HA hydrogel is about 800 Pa.

[0114] 2. Rheological property test of hydrogels with different concentrations

[0115] 0.6 g, 0.8 g and 1.0 g of HA / SF gel particles in Example 1 were weighed, 1 mL of PBS solution was added for resuspension, and the kinex rheometer was used to detect the elastic modulus G' at a shear frequency of 0.1-10 Hz, and the elastic modulus G' of HA / SF hydrogel and HA hydrogel at a shear frequency of 10 Hz.

[0116] The results of Figure 8 show that the hydrogel prepared by resuspending 0.8 g of HA / SF gel particles in 1 mL of PBS solution has good elastic modulus and good injectability.

[0117] Example 6: In vitro test

[0118] Cell compatibility test

[0119] An appropriate amount of HA hydrogel and HA / SF hydrogel of Example 1 was taken and freeze-dried, and the freeze-dried powder of HA and HA / SF hydrogel was added to the complete culture medium of human chondrocytes (C28 / I2 cells), which was placed at 37°C for 1, 2 and 3 days to obtain the gel leachate of the two kinds of hydrogel on the 1st, 2nd and 3rd day, respectively. The leachate was co-cultured with C28 / I2 cells, and then CCK-8 (Cell Counting Kit-8) and Calcein-AM / PI kit were used for quantitative and qualitative detection of the proliferation activity of the two kinds of hydrogel on the cells. The results are shown in Figures 9 and 10, respectively. The control group in the figures represents C28 / I2 cells cultured in the complete culture medium without co-culture with the leachate.

[0120] The results show that the two hydrogels prepared in the application have no cytotoxicity, and the HA / SF hydrogel shows stronger ability to stimulate the proliferation of chondrocytes than the HA hydrogel as time goes on.

[0121] Blood compatibility test

[0122] Fresh blood of rat abdominal aorta was taken, mixed thoroughly, and then transferred to a clean centrifuge tube. After centrifugation at 4°C and 3000 rpm for 15 min, red blood cell precipitate was obtained. The supernatant was discarded and washed with PBS solution for 3 times, and then resuspended with PBS solution (2%, V / V) for standby. 500 μL of HA hydrogel, HA / SF hydrogel in Example 1, and PBS, 1% Triton (n = 4) were mixed thoroughly with 250 μL of 2% red blood cell suspension. They were placed in a 37°C water bath for incubation for 1 h. After incubation, centrifugation was performed at 3000 rpm for 15 min, and hemolysis was observed by naked eye, and the supernatant was taken for OD value detection. The hemolysis rate was calculated according to the OD value, and the formula was as follows:

[0123] Hemolysis rate (%) = [(A-A0) / (A1-A0)]x100.

[0124] A0 is the OD value of the negative control group (PBS), A1 is the OD value of the positive control group (Olay), and A is the OD value of the experimental group. Hemolysis rate less than 5% can be considered as no hemolysis phenomenon. The results are shown in Figure 11, and the hemolysis rates of the two prepared hydrogels, HA hydrogel and HA / SF hydrogel, are less than 5%, indicating that they do not occur hemolysis in vivo.

[0125] In vitro anti-inflammatory test

[0126] The HA hydrogel prepared in Example 1, the HA / SF hydrogel (Example 4 sample 3) and the HA / SF hydrogel prepared in Example 4 sample 6 were freeze-dried, and then added with appropriate complete culture medium to prepare a gel extract solution of 5 mg / ml. Appropriate IL-1β was added to the two extract solutions to prepare an IL-1β extract solution with a final concentration of 50 ng / ml, which was then co-cultured with C28 / I2 cells for 48 hours. The cell supernatant was collected, and the contents of anti-inflammatory factors IL-4, IL-10 and pro-inflammatory factor IL-6 were determined by Elisa kit. The results are shown in Figure 12.

[0127] The results show that compared with the positive control group (C28 / I2 induced inflammatory model, cell culture solution was given, and it is recorded as PC group) and HA hydrogel, the HA / SF hydrogels of samples 3 and 6 can significantly improve 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.

[0128] Regulation of chondrocyte gene level

[0129] The HA hydrogel and HA / SF hydrogel prepared in Example 1 were freeze-dried and added to appropriate complete medium to prepare a gel extract at 5 mg / ml. Appropriate IL-1β was added to the two extracts to prepare an IL-1β extract at a final concentration of 50 ng / ml, which was then co-cultured with C28 / I2 cells for 48 hours, the cells were collected, RNA in the cells was extracted by Trizol method and reverse-transcribed into cDNA, and RT-PCR experiments were further used to explore the gene expression of Sox9, COL II, MMP13, ADAMTS5 and Aggrecan, and the experimental results are shown in Figure 13.

[0130] The results show that, compared with the HA hydrogel, the HA / SF hydrogel can increase the gene expression levels of Sox9, COL II (collagen II) and Aggrecan (proteoglycan) related to cartilage formation, and reduce the gene expression levels of MMP13 (matrix metalloproteinase 13) and ADAMTS5 (platelet reaction protein disintegrin metallopeptidase 5) related to cartilage decomposition metabolism. The HA / SF hydrogel of the application is conducive to the formation of cartilage and the inhibition of the degradation of cartilage extracellular matrix, and is conducive to the treatment of osteoarthritis.

[0131] Effect on mitochondrial gene expression

[0132] The HA hydrogel and HA / SF hydrogel prepared in Example 1 were freeze-dried and added to appropriate complete medium to prepare a gel extract at 5 mg / ml. Appropriate IL-1β was added to the two extracts to prepare an IL-1β extract at a final concentration of 50 ng / ml, which was then co-cultured with C28 / I2 cells for 48 hours, the cells were collected, and RT-PCR experiments were further used to explore the gene expression of pro-apoptotic gene Bcl-2 and anti-apoptotic gene Caspase3 in the mitochondria of the C28 / I2 cells induced to be inflammatory cells, and the results are shown in Figure 14.

[0133] The results show that, compared with the HA hydrogel, the HA / SF hydrogel can significantly increase the gene expression level of anti-apoptotic gene Bcl-2 in the mitochondria, and reduce the gene expression level of pro-apoptotic gene Caspase-3. The HA / SF hydrogel of the application has the function of regulating mitochondria in osteoarthritis chondrocytes.

[0134] Example 7: In vivo efficacy - hot and cold plate test of osteoarthritis model

[0135] SD rats (male, 10 weeks old) were subjected to anterior cruciate ligament transection (ACLT) operation to construct an osteoarthritis model. After the model was successfully constructed, the osteoarthritis rats were randomly divided into 4 groups (Sham group, HA group, HA / SF group and PBS group), 8 rats in each group, and 30 μL of the hydrogel sample prepared in Example 1 was injected into the joint cavity at the 1st and 3rd weeks. The rats in the Sham group only received anesthesia and skin incision, and the rats in the PBS group were treated with PBS as the model group. The hot plate test was performed on all rats at the 4th week to determine the thermal pain threshold, the hot plate temperature was set to 50.0℃, and the time recorded during the animal licking the hind foot was the thermal pain threshold of the animal.

[0136] The results (Figure 15) show that compared with the PBS group and the HA group, the rats in the HA / SF group have a significantly prolonged time of licking the hind foot, which has a statistical difference and is close to the sham group, and even better than the sham group. It is indicated that the HA / SF hydrogel of the application can relieve the pain of the osteoarthritis rats by improving the condition of the osteoarthritis rats.

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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