Composite gel for osteochondral repair as well as preparation method and application of composite gel

Abstract

The invention discloses composite gel for osteochondral repair as well as a preparation method and application of the composite gel. Main raw materials of the composite gel are regenerated silk fibroin (RSF) and recombinant humanized III-type collagen (rhCol-III), and auxiliary raw materials of the composite gel are sodium hyaluronate (HA), fibrinogen and thrombin. The preparation method comprises the following steps: firstly, mixing an RSF solution with an rhCol-III solution, obtaining RSF-rhCol particles through a freeze drying technology and mechanical pulverization, dispersing the particles in an HA solution to obtain composite gel, mixing the gel with a thrombin solution, catalyzing fibrinogen to be split and converted into fibrin by thrombin, and performing freeze drying to obtain the rhCol-RhCol-RhCol-RhCol-RhCol-RhCol-RhCol. And crosslinking the fibrous protein monomer to obtain the injectable composite gel preparation capable of immediately gelling. The prepared RSF-rhCol composite gel can adapt to a defect area, has a certain adhesion effect, has excellent biocompatibility, promotes migration of bone marrow mesenchymal stem cells, induces cell differentiation and promotes cartilage tissue regeneration.

Description

Technical Field

[0001] The invention relates to a composite gel for repairing bone cartilage and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Osteoarthritis is a degenerative disease that seriously affects people's daily lives. It is usually caused by excessive mechanical loads and pathological factors. It is characterized by the degeneration and deformation of articular cartilage, leading to joint pain. Due to the lack of nerves and blood vessels in the articular cartilage, the metabolic activity and migration ability of chondrocytes are low, so the self-healing ability of articular cartilage is limited. At present, the treatment strategies for cartilage repair include microfracture, autologous chondrocyte transplantation and allogeneic / autologous cartilage transplantation, but these repair strategies may cause secondary trauma and immune rejection reactions. Hydrogels have a porous three-dimensional network structure with high water retention, adhesion and good biocompatibility. Hydrogels that introduce specific active substances or load tissue engineering cells can be used in the treatment of cartilage defects.

[0003] Regenerated silk fibroin is a natural polymer protein material extracted from silk. It has good biocompatibility, biodegradability and thermal stability. However, regenerated silk fibroin has low biological activity and low cell adhesion, and needs to be used in combination with other biomaterials. Recombinant humanized collagen is obtained by biofermentation technology using genetic engineering technology to optimize the recombinant expression of the original gene sequence of human skin collagen. Recombinant humanized type III collagen can show extremely similar properties and stability to natural human collagen, with no viral risks, excellent biological compatibility and efficacy, and low immunogenicity. Recombinant humanized type III collagen can promote cell adhesion, proliferation and migration, as well as remodeling of the extracellular matrix, thereby accelerating tissue repair and regeneration, but recombinant humanized type III collagen is metabolized quickly and may not be able to adapt to the recovery cycle of cartilage defects.

[0004] The present invention combines regenerated silk fibroin with recombinant humanized collagen through the cross-linking effect of fibrinogen and thrombin, while giving full play to the specific advantages of the two biomaterials, thereby preparing a composite gel material capable of promoting the repair of osteochondral defects. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a composite gel for osteochondrium repair and a preparation method and application thereof, which can solve the problem of difficulty in cartilage regeneration.

[0006] The present invention provides a composite gel for osteochondrium repair, characterized in that the composite gel is RSF-rhCol gel, and the composite gel comprises RSF microparticles made of regenerated silk fibroin (RSF) and recombinant humanized type III collagen (rhCol-III), sodium hyaluronate, fibrinogen and thrombin.

[0007] In some embodiments, the mass ratio of regenerated silk fibroin (RSF) and recombinant humanized type III collagen (rhCol-III) in the RSF microparticles in the composite gel is: 2-6:1; preferably 2-3:1; most preferably 2:1.

[0008] In some embodiments, the mass ratio of the RSF microparticles to sodium hyaluronate is 5 to 20:1, preferably 10:1.

[0009] In some embodiments, the dosage ratio of the RSF microparticles to thrombin is: 1 mg: 2-10 U, preferably 1 mg: 5 U.

[0010] In some embodiments, the mass ratio of the RSF microparticles to fibrinogen is 5 to 20:1, preferably 10:1.

[0011] The present invention also provides a method for preparing a composite gel for osteochondrium repair. The specific steps of the method for preparing the composite gel for cartilage repair are as follows:

[0012] Step 1: Preparation of composite materials: RSF and rhCol-Ⅲ were dissolved in ultrapure water respectively, the two solutions were mixed, stirred evenly and then freeze-dried;

[0013] Step 2: Preparation of insoluble materials: After freeze-drying, the materials are washed with alcohol and water in sequence, and freeze-dried again;

[0014] Step 3: Preparation of composite particles: mechanically crushing the final material to obtain RSF-rhCol-Ⅲ composite particles;

[0015] Step 4: preparing RSF-rhCol-Ⅲ composite gel: dissolving sodium hyaluronate in PBS solution, and after complete dissolution, uniformly dispersing and mixing the RSF-rhCol-Ⅲ composite microparticles prepared in step 3 in the sodium hyaluronate solution to prepare RSF-rhCol-Ⅲ gel;

[0016] Step 5: Prepare RSF-rhCol-Ⅲ composite gel preparation: dissolve fibrinogen in enzyme-free water by ultrasound, dissolve thrombin in calcium chloride solution, mix the RSF-rhCol-Ⅲ gel prepared in step 4 and the thrombin solution evenly through a two-way connecting tube, and then inject the RSF-rhCol-Ⅲ composite gel preparation with the fibrinogen solution through a double-drug mixing device.

[0017] In some embodiments, the mass ratio of regenerated silk fibroin (RSF) and recombinant humanized type III collagen (rhCol-III) in the RSF microparticles in the composite gel is: 2-6:1; preferably 2-3:1; most preferably 2:1.

[0018] In some embodiments, the mass ratio of the RSF microparticles to sodium hyaluronate is 5 to 20:1, preferably 10:1.

[0019] In some embodiments, the dosage ratio of the RSF microparticles to thrombin is: 1 mg: 2-10 U, preferably 1 mg: 5 U.

[0020] In some embodiments, the mass ratio of the RSF microparticles to fibrinogen is 5 to 20:1, preferably 10:1.

[0021] In some embodiments, in step 1, the concentrations of RSF and rhCol-III are 100-200 mg / ml, preferably 150 mg / ml, respectively.

[0022] In some embodiments, in step 4, the concentration of the sodium hyaluronate solution is 10-50 mg / ml, preferably 20 mg / ml.

[0023] In some embodiments, in step 4, the concentration of the RSF-rhCol composite microparticles is 100-500 mg / ml, preferably 200 mg / ml.

[0024] In some embodiments, in step 5, the thrombin is dissolved in a calcium chloride solution, and the concentration of the thrombin is 2000-7000 U / ml, preferably 5000 U / ml.

[0025] In some embodiments, in step 5, fibrinogen is dissolved in enzyme-free water by ultrasound, and the concentration of fibrinogen is 10-50 mg / ml, preferably 20 mg / ml.

[0026] In some embodiments, in step 5, the volume ratio of the RSF-rhCol-III gel prepared in step 4 to the thrombin solution is 2:1 to 8:1, preferably 4:1.

[0027] In some embodiments, in step 5, the volume ratio of the mixed solution of RSF-rhCol-III gel / thrombin solution to the fibrinogen solution is 1:0.5-3, preferably 1:1.

[0028] The present invention also provides a method for preparing a composite gel for osteochondral repair, the method comprising the following steps:

[0029] Step 1: preparing a composite material: dissolving RSF and rhCol-Ⅲ in ultrapure water to a concentration of 100-200 mg / ml (preferably 150 mg / ml), respectively, mixing the two solutions in a mass ratio of RSF to rhCol-Ⅲ of 2-6:1 (preferably 2-3:1; most preferably 2:1), stirring evenly and then freeze-drying;

[0030] Step 2: Preparation of insoluble materials: After freeze-drying, the freeze-dried RSF-rhCol-Ⅲ block material is washed with alcohol and water in sequence, and freeze-dried again;

[0031] Step 3: Preparation of composite microparticles: mechanically crushing the freeze-dried material to obtain RSF-rhCol composite microparticles;

[0032] Step 4: preparing a composite gel: dissolving sodium hyaluronate (HA) in a PBS solution, wherein the concentration of the sodium hyaluronate solution is 10 to 50 mg / ml, preferably 20 mg / ml after complete dissolution; uniformly dispersing and mixing the prepared RSF-rhCol-III composite microparticles in the sodium hyaluronate solution, wherein the concentration of the RSF-rhCol composite microparticles is 100 to 500 mg / ml, preferably 200 mg / ml; preparing an RSF-rhCol-III composite gel;

[0033] Step 5: Prepare an RSF-rhCol-III composite gel preparation for osteochondrium repair: dissolve thrombin in a calcium chloride solution, wherein the concentration of the thrombin is 2000-7000 U / ml (preferably 5000 U / ml), and dissolve fibrinogen in enzyme-free water by ultrasound, wherein the concentration of the fibrinogen is 10-50 mg / ml, preferably 20 mg / ml; mix the RSF-rhCol-III composite gel prepared in step 4 with the thrombin solution through a two-way connecting tube, and then inject the RSF-rhCol composite gel preparation with the fibrinogen solution through a double drug mixing device.

[0034] The regenerated silk protein of the present invention is a natural high molecular weight fibrous protein with excellent mechanical properties, good biocompatibility, biodegradability and multifunctionality of structural adjustment, and has broad application prospects. Recombinant humanized type III collagen can promote cell adhesion and migration, as well as remodeling of the extracellular matrix, create a good repair environment, and thus accelerate tissue repair and regeneration.

[0035] Beneficial effects of the present invention:

[0036] 1. The present invention utilizes the catalytic cleavage reaction of thrombin on fibrinogen to convert fibrinogen into fibrin. The fibrin monomers are cross-linked to form a stable gel network structure. The composite gel can be directly injected into the defect site through parallel injection of a dual-drug mixing syringe.

[0037] 2. RSF-rhCol material was washed with anhydrous ethanol to induce the protein material to transform into a β-folded conformation, forming an insoluble and more stable composite material.

[0038] 3. The three-dimensional network structure of the hydrogel provides a physiological simulation environment for cell metabolism, is conducive to the transportation of nutrients, and provides a good environment for cartilage repair. Both regenerated silk protein and recombinant humanized type III collagen have good biocompatibility and can accelerate tissue regeneration and repair.

[0039] 4. In vitro and in vivo studies have shown that the RSF-rhCol composite gel preparation prepared by the present invention can promote cell migration, inhibit inflammatory response, promote cartilage tissue regeneration, and has a significant therapeutic effect on cartilage defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic flow chart of the preparation method of RSF-rhCol composite gel.

[0041] Figure 2 Schematic structural flow chart of the preparation method of RSF-rhCol composite gel.

[0042] Figure 3 This is the distribution diagram of rhCol-Ⅲ in 2RSF-rhCol.

[0043] Figure 4 This is a scanning electron micrograph of RSF-rhCol composite particles.

[0044] Figure 5 Figure 2 is the particle size diagram of 2RSF-rhCol composite particles.

[0045] Figure 6 This is the appearance of 2RSF-rhCol composite gel.

[0046] Figure 7 This is a scanning electron micrograph of the RSF-rhCol composite gel.

[0047] Figure 8 This is the rheological diagram of RSF-rhCol composite gel.

[0048] Fig. 9 The figure shows the cytotoxicity data of RSF-rhCol composite gel on bone marrow mesenchymal stem cells.

[0049] Fig.10 This is a statistical diagram of the effect of RSF-rhCol composite gel on the migration of bone marrow mesenchymal stem cells.

[0050] Fig.11This is a graph showing the effect of RSF-rhCol composite gel on the expression of cartilage-related genes in cells.

[0051] Fig.12 Photo of the rat cartilage defect treated with RSF-rhCol composite gel.

[0052] Fig.13 This is a micro-CT scan of the RSF-rhCol composite gel in treating cartilage defects in rats. DETAILED DESCRIPTION

[0053] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0055] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0056] Regenerated silk fibroin, abbreviated as RSF, was purchased from Nanjing Siyuan Medical Technology Co., Ltd.; recombinant humanized type III collagen, abbreviated as rhCol-Ⅲ, was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.; sodium hyaluronate, abbreviated as HA, was purchased from Huaxi Biotechnology Co., Ltd.; fibrinogen was purchased from Beijing Solebao Technology Co., Ltd.; and thrombin was purchased from Hunan Yige Pharmaceutical Co., Ltd.

[0057] like Figure 1 and Figure 2 As shown, the preparation method of the composite gel of the present invention comprises the following steps:

[0058] Step 1: Dissolve RSF and rhCol-Ⅲ, mix the two solutions in a certain mass ratio, and freeze-dry.

[0059] Step 2: The freeze-dried material was washed twice with alcohol, three times with water, and freeze-dried again;

[0060] Step 3: Mechanically crush to obtain RSF-rhCol composite particles;

[0061] Step 4: dissolving HA, dispersing and mixing the RSF-rhCol composite particles in HA to prepare RSF-rhCol gel;

[0062] Step 5: Dissolve fibrinogen and thrombin separately, mix the RSF-rhCol gel and thrombin, and then inject the RSF-rhCol composite gel preparation with the fibrinogen solution through a double-drug mixing device.

[0063] In the present invention, in order to obtain sterile RSF-rhCol composite gel for use in in vitro and in vivo experiments, the prepared RSF-rhCol gel is first sterilized by wet heat at 121°C for 12 min; fibrinogen and thrombin are dissolved using sterile reagents, and the gel is formed in a sterile environment.

[0064] The following is a detailed description with specific embodiments:

[0065] Example 1

[0066] A RSF composite particle is prepared by the following steps:

[0067] Step S1: 630 mg of RSF was dissolved in 4.2 ml of ultrapure water and stirred at room temperature for 2 h to obtain a 150 mg / ml RSF solution. The RSF solution was divided into six-well cell culture plates and placed in a freeze dryer for freeze drying.

[0068] Step S2: After the freeze-drying process is completed, the freeze-dried RSF block material is taken out, immersed in anhydrous ethanol solution for stirring and washing for 1 hour, immersed in ethanol solution for stirring and washing for 1 hour, and finally immersed in ultrapure water for stirring and washing 3 times, each time for 1 hour. After the soaking and washing, the RSF block material is repacked in a six-well cell culture plate and placed in a freeze dryer for freeze drying.

[0069] Step S3: After freeze-drying, the RSF material is mechanically crushed using an electric grinder, and the crushed particles are RSF particles.

[0070] Example 2

[0071] A 6RSF-rhCol composite particle is prepared by the following steps:

[0072] Step S1: Dissolve 540 mg of RSF in 3.6 ml of ultrapure water, stir at room temperature for 2 hours, and obtain a RSF solution with a concentration of 150 mg / ml after the dissolution is completed; dissolve 90 mg of rhCol-Ⅲ in 0.6 ml of ultrapure water, and ultrasonically dissolve to obtain a rhCol-Ⅲ solution with a concentration of 150 mg / ml. Use a dropper to draw the rhCol-Ⅲ solution and add it dropwise to the RSF solution. Stir continuously during the addition process. After the rhCol-Ⅲ solution is added dropwise, continue stirring for 30 minutes to mix the two solutions evenly. The RSF-rhCol mixed solution is divided into six-well cell culture plates and placed in a freeze dryer for freeze drying.

[0073] Step S2: After the freeze-drying process is completed, the freeze-dried 6RSF-rhCol block material is taken out, immersed in anhydrous ethanol solution, stirred and cleaned for 1 hour, immersed in ethanol solution again, stirred and cleaned for 1 hour, and finally immersed in ultrapure water, stirred and cleaned for 3 times, each time for 1 hour. After the soaking and cleaning, the 6RSF-rhCol block material is repacked in a six-well cell culture plate and placed in a freeze dryer for freeze drying.

[0074] Step S3: After freeze-drying, the 6RSF-rhCol material is mechanically crushed using an electric mill, and the crushed particles are 6RSF-rhCol particles.

[0075] Example 3

[0076] A 3RSF-rhCol composite particle is prepared by the following steps:

[0077] Step S1: Dissolve 472.5 mg of RSF in 3.15 ml of ultrapure water, stir at room temperature for 2 hours, and obtain a RSF solution with a concentration of 150 mg / ml after the dissolution is completed; dissolve 157.5 mg of rhCol-Ⅲ in 1.05 ml of ultrapure water, and ultrasonically dissolve to obtain a rhCol-Ⅲ solution with a concentration of 150 mg / ml. Use a dropper to draw the rhCol-Ⅲ solution and add it dropwise to the RSF solution. Stir continuously during the addition process. After the rhCol-Ⅲ solution is added dropwise, continue to stir for 30 minutes to mix the two solutions evenly. The RSF-rhCol mixed solution is divided into six-well cell culture plates and placed in a freeze dryer for freeze drying.

[0078] Step S2: After the freeze-drying process is completed, the freeze-dried 3RSF-rhCol block material is taken out, immersed in anhydrous ethanol solution, stirred and cleaned for 1 hour, immersed in ethanol solution again, stirred and cleaned for 1 hour, and finally immersed in ultrapure water, stirred and cleaned for 3 times, each time for 1 hour. After the soaking and cleaning, the 3RSF-rhCol block material is repacked in a six-well cell culture plate and placed in a freeze dryer for freeze drying.

[0079] Step S3: After freeze-drying, the 3RSF-rhCol material is mechanically crushed using an electric mill, and the crushed particles are 3RSF-rhCol particles.

[0080] Example 4

[0081] A 2RSF-rhCol composite particle is prepared by the following steps:

[0082] Step S1: Dissolve 420 mg of RSF in 2.8 ml of ultrapure water, stir at room temperature for 2 hours, and obtain a RSF solution with a concentration of 150 mg / ml after the dissolution is completed; dissolve 210 mg of rhCol-Ⅲ in 1.4 ml of ultrapure water, and ultrasonically dissolve to obtain a rhCol-Ⅲ solution with a concentration of 150 mg / ml. Use a dropper to draw the rhCol-Ⅲ solution and add it dropwise to the RSF solution. Stir continuously during the addition process. After the rhCol-Ⅲ solution is added dropwise, continue stirring for 30 minutes to mix the two solutions evenly. The RSF-rhCol mixed solution is divided into six-well cell culture plates, placed in a freeze dryer for freeze drying, and the freeze drying process is completed.

[0083] Step S2: After the freeze-drying process is completed, the freeze-dried 2RSF-rhCol block material is taken out, immersed in anhydrous ethanol solution, stirred and cleaned for 1 hour, immersed in ethanol solution again, stirred and cleaned for 1 hour, and finally immersed in ultrapure water, stirred and cleaned for 3 times, each time for 1 hour. After the soaking and cleaning, the 2RSF-rhCol block material is repacked in a six-well cell culture plate and placed in a freeze dryer for freeze drying.

[0084] Step S3: After freeze-drying, the 2RSF-rhCol material is mechanically crushed using an electric mill, and the crushed particles are 2RSF-rhCol particles.

[0085] Example 5

[0086] A composite gel for osteochondral repair is prepared by the following steps:

[0087] Step S1: 20 mg of HA was slowly added to 1 ml of PBS and stirred at room temperature until HA was completely dissolved. 200 mg of RSF microparticles were added to 1 ml of HA solution and the microparticles were completely and evenly dispersed in HA by magnetic stirring to obtain RSF gel. The RSF gel was then sterilized by wet heat at 121°C for 12 min. After sterilization, it was dispensed into 1 ml syringes on a sterile operating table and stored at 4°C.

[0088] Step S2: Add 0.2 ml of sterile calcium chloride solution to 1000 U of thrombin and dissolve it by ultrasonication. The concentration of calcium chloride solution is 50 mg / ml. In a sterile operating table, 0.8 ml of RSF gel and 0.2 ml of thrombin solution are mixed evenly through a two-way connecting tube, and the mixed materials are loaded into a 2 ml syringe for standby use. Weigh 20 mg of fibrinogen and put it into a sterile centrifuge tube, add 1 ml of sterile enzyme-free water, and dissolve it by ultrasonication. Pay attention to the temperature of the water in the ultrasonic machine during the dissolution process. When the temperature is slightly high, the water needs to be changed in time. After the dissolution is completed, the fibrinogen solution is loaded into a 2 ml syringe. Install the two syringes on the double-mixing device, push the syringe, and the two materials are pushed out at the same time. The RSF gel preparation is formed immediately after the solution contacts.

[0089] Example 6

[0090] A composite gel for osteochondral repair is prepared by the following steps:

[0091] Step S1: 20 mg of HA was slowly added to 1 ml of PBS and stirred until HA was completely dissolved. 200 mg of 6RSF-rhCol microparticles were added to 1 ml of HA solution and the microparticles were completely and evenly dispersed in HA by magnetic stirring to obtain 6RSF-rhCol gel. The 6RSF-rhCol gel was then sterilized by wet heat at 121°C for 12 min. After sterilization, it was dispensed into 1 ml syringes on a sterile operating table and stored at 4°C.

[0092] Step S2: add 0.2ml sterile calcium chloride solution to 1000U thrombin and dissolve it ultrasonically, the concentration of calcium chloride solution is 50mg / ml. In the sterile operating table, 0.8ml of 6RSF-rhCol gel and 0.2ml thrombin solution are mixed evenly through a two-way connecting tube, and the mixed materials are loaded into a 2ml syringe for standby use. Weigh 20mg of fibrinogen and put it into a sterile centrifuge tube, add 1ml sterile enzyme-free water, and dissolve it ultrasonically. Pay attention to the temperature of the water in the ultrasonic machine during the dissolution process. When the temperature is slightly high, change the water in time. After the dissolution is completed, load the fibrinogen solution into a 2ml syringe. Install the two syringes on the double-mixing device, push the syringe, and the two materials are pushed out at the same time. After the solution contacts, a 6RSF-rhCol composite gel preparation is formed immediately.

[0093] Example 7

[0094] A composite gel for osteochondral repair is prepared by the following steps:

[0095] Step S1: Slowly add 20 mg of HA to 1 ml of PBS and stir until HA is completely dissolved. Add 200 mg of 3RSF-rhCol microparticles to 1 ml of HA solution and use magnetic stirring to make the microparticles completely and evenly dispersed in HA to obtain 3RSF-rhCol gel. Then, sterilize the 3RSF-rhCol gel with wet heat at 121°C for 12 minutes. After sterilization, dispense it into 1 ml syringes on a sterile operating table and store it at 4°C.

[0096] Step S2: Add 0.2ml sterile calcium chloride solution to 1000U of thrombin and dissolve it ultrasonically, and the concentration of calcium chloride solution is 50mg / ml. In the sterile operating table, 0.8ml of 3RSF-rhCol gel and 0.2ml of thrombin solution are mixed evenly through a two-way connecting tube, and the mixed materials are loaded into a 2ml syringe for standby use. Weigh 20mg of fibrinogen and put it into a sterile centrifuge tube, add 1ml sterile enzyme-free water, and dissolve it ultrasonically. Pay attention to the temperature of the water in the ultrasonic machine during the dissolution process. When the temperature is slightly high, change the water in time. After the dissolution is completed, load the fibrinogen solution into a 2ml syringe. Install the two syringes on the double-mixing device, push the syringe, and the two materials are pushed out at the same time. The 3RSF-rhCol composite gel preparation is formed immediately after the solution contacts.

[0097] Example 8

[0098] A composite gel for osteochondral repair is prepared by the following steps:

[0099] Step S1: Slowly add 20 mg of HA into 1 ml of PBS and stir until HA is completely dissolved. Add 200 mg of 2RSF-rhCol microparticles into 1 ml of HA solution and use magnetic stirring to make the microparticles completely and evenly dispersed in HA to obtain 2RSF-rhCol gel. Then, sterilize the 2RSF-rhCol gel with wet heat at 121°C for 12 minutes. After sterilization, dispense it into 1 ml syringes on a sterile operating table and store it at 4°C.

[0100] Step S2: add 0.2ml sterile calcium chloride solution to 1000U thrombin for ultrasonic dissolution, and the concentration of calcium chloride solution is 50mg / ml. In the sterile operating table, 0.8ml of 2RSF-rhCol gel is mixed with 0.2ml thrombin solution through a two-way connecting tube, and the mixed material is loaded into a 2ml syringe for standby use. Weigh 20mg of fibrinogen and put it into a sterile centrifuge tube, add 1ml sterile enzyme-free water, and ultrasonically dissolve it. Pay attention to the temperature of the water in the ultrasonic machine during the dissolution process. When the temperature is slightly high, change the water in time. After the dissolution is completed, load the fibrinogen solution into a 2ml syringe. Install the two syringes on the double-mixing device, push the syringe, and the two materials are pushed out at the same time. After the solution contacts, a 2RSF-rhCol composite gel preparation is formed immediately.

[0101] RSF-rhCol composite gel performance analysis:

[0102] Figure 3 The fluorescence image of 2RSF-rhCol composite microparticles is shown. rhCol-Ⅲ was labeled with fluorescein isothiocyanate, and the prepared 2RSF-rhCol composite microparticles were observed under a laser confocal microscope. The results showed that the labeled rhCol-Ⅲ exhibited green fluorescence and was evenly dispersed in the microparticles, indicating the successful complexation of RSF and rhCol-Ⅲ.

[0103] Figure 4 The scanning electron microscope image of the RSF-rhCol composite particles is shown. As can be seen from the figure, the prepared particles are relatively evenly dispersed in size, with a particle size distribution of 80 to 100 μm.

[0104] Figure 5 The particle size diagram of 2RSF-rhCol composite particles is shown. The size of the composite particles is roughly dispersed in 100 μm, which is similar to the results of scanning electron microscopy.

[0105] Figure 6 The preparation appearance of the 2RSF-rhCol composite gel is shown. The composite gel can be squeezed out from a needle and is injectable. The composite gel can be molded to adapt to various shapes, indicating that the gel can adapt to the defect location.

[0106] Figure 7 The scanning electron micrograph of the prepared RSF-rhCol composite gel is shown. From the figure, it can be observed that the gel has a porous structure, which can transport nutrients and metabolic waste in the body, creating a good environment for cartilage regeneration.

[0107] Figure 8The rheological diagram of the prepared RSF-rhCol composite gel is shown, which shows that in the frequency range of 0.1 to 100 Hz, the storage modulus of the RSF-rhCol gel is always greater than the loss modulus, which indicates that the prepared RSF-rhCol gel is in a gel state.

[0108] Fig. 9 The cytotoxicity data of RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels prepared in Examples 5 to 8 on bone marrow mesenchymal stem cells (BMSC) are shown. At 37°C, the prepared RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels were extracted in α-MEM complete medium at a concentration of 0.2 g / mL for 24 hours. BMSC cells were evenly plated in a 96-well plate, with approximately 5,000 cells per well. After culturing for 24 hours, the cells were completely attached to the wall, and different groups of hydrogel extracts were added. The control group was added with α-MEM complete medium, and continued to be cultured for 24 hours. 100 μL of α-MEM complete medium containing 10% CCK-8 was added to each well. After incubation in the incubator for 1 hour, the cell absorbance was detected by an ELISA instrument to calculate the cell survival rate. The results showed that compared with the control group, the cell survival rate in the material group was improved, indicating that the prepared hydrogel has good cell compatibility and can promote cell proliferation.

[0109] Fig.10 Crystal violet staining photos and cell migration rate statistics of RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels prepared in Examples 5 to 8 are shown. An equal amount of hydrogel material was added to a 24-well plate, and 500 μL of serum-containing culture medium was added. BMSC cells were cultured at 3×10 4 The density of cells / mL was dispersed in serum-free culture medium, 200μL of cell suspension was added above the Transwell chamber, and the Transwell chamber was placed in a 24-well plate. After culturing in an incubator for 24 hours, crystal violet staining was used, and photos were taken and the cell migration rate was calculated. The results showed that compared with the control group (no material was added), the number of cell migration in the material group increased significantly, and with the increase of rhCol-Ⅲ content, the migration rate increased, indicating that RSF-rhCol can promote cell migration. The cell density and metabolic activity in the cartilage are low, and the increase in cell migration ability helps BMSC cells to gather at the defect site, creating a favorable environment for cartilage regeneration after stem cell chondrogenic differentiation. Among them, the effect of 3RSF-rhCol and 2RSF-rhCol in promoting cell migration was significantly better than that of the control group, RSF, and 6RSF-rhCol groups; 2RSF-rhCol was the best (P<0.0001).

[0110] Fig.11 The graphs show the effects of RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels prepared in Examples 5 to 8 on chondrogenic differentiation of BMSC cells. 4 / mL of cell suspension was evenly spread in a 24-well plate, 500μL of cell suspension was added to each well, and after the cells in the 24-well plate were completely grown, they were placed in a Transwell chamber, and RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels were added to the chamber for co-incubation. No material was added to the control group, and chondrogenic induction medium was used for chondrogenic induction. The chondrogenic induction medium was replaced every 2 days. After 14 days of chondrogenic induction, the cell RNA was extracted using the RNAeasyTM Animal RNA Extraction Kit (centrifugal column type), and the expression of related genes in RNA was detected using the BeyoFastTM SYBRGreen One-Step qRT-PCR Kit. The results showed that 2RSF-rhCol composite gel could significantly increase the expression of SOX9, COLⅡ, and ACAN related chondrogenic genes, indicating that 2RSF-rhCol composite gel could promote the chondrogenic differentiation of BMSC cells, which was significantly better than the control group, RSF, 3RSF-rhCol, and 6RSF-rhCol groups (P<0.01).

[0111] Fig.12The treatment pictures of RSF, 6RSF-rhCol and 2RSF-rhCol composite gels prepared in Examples 5-8 on cartilage defect repair are shown. Because the effect of rhCol-Ⅲ content was verified in the in vitro cell experiment, the sham group (Sham), control group (Control), RSF group (collagen-free group), 6RSF-rhCol (low collagen group) and 2RSF-rhCol (high collagen group) were set up in the subsequent animal experiment. Male SD rats aged 8 weeks were selected, the rats were anesthetized, the left leg of the rats was depilated and disinfected, and a defect with a diameter of 2mm and a depth of 1.5mm was established in the distal femoral trochlea of ​​the rats using a vulgar dental drill. RSF, 6RSF-rhCol and 2RSF-rhCol gels were injected at the defect site. The sham group only performed skin incision and suture treatment. The control group did not fill the material after the defect was established. The rat status was observed after surgery to prevent wound infection and death. The rats were killed one month and two months after surgery, and the repair of the defect was photographed and counted. The knee joints were collected and fixed in 4% paraformaldehyde. The visual photos of defect repair showed that one month after repair, part of the fibrocartilage tissue regenerated and the material failed to completely degrade. Two months later, the defect in the 2RSF-rhCol material group was basically filled with new cartilage tissue, which was well integrated with the surrounding cartilage tissue, and the defect repair effect was good.

[0112] Fig.13 The micro-CT images of cartilage repair are shown, and the results show that the defect location of each group was clearly visible in the first month. After two months, the cartilage defects in each group were repaired to a certain extent. The defect area in the control group was more obvious, the defect in the RSF group and the 6RSF-rhCol group recovered well, but the defect area was still visible, and the defect area in the 2RSF-rhCol group almost completely disappeared, indicating that the 2RSF-rhCol gel has a good therapeutic effect on the repair of osteochondral defects.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A composite gel for osteochondral repair, characterized in that: The composite gel is RSF-rhCol gel, which comprises RSF particles made of regenerated silk fibroin (RSF) and recombinant humanized type III collagen (rhCol-III), sodium hyaluronate, fibrinogen and thrombin.

2. A composite gel for osteochondral repair as claimed in claim 1, characterized in that: The mass ratio of regenerated silk fibroin (RSF) and recombinant humanized type III collagen (rhCol-III) in the RSF particles in the composite gel is 2-6:

1.

3. A composite gel for osteochondral repair according to claim 1, characterized in that: The mass ratio of the regenerated silk fibroin to the recombinant humanized type III collagen in the RSF microparticles in the composite gel is 2 to 3:1, preferably 2:

1.

4. A composite gel for osteochondral repair according to any one of claims 1 to 3, characterized in that: The mass ratio of the RSF microparticles to sodium hyaluronate is 5 to 20:1, preferably 10:

1.

5. A composite gel for osteochondral repair according to any one of claims 1 to 4, characterized in that: The dosage ratio of the RSF microparticles to thrombin is: 1 mg: 2-10 U, preferably 1 mg: 5 U.

6. A composite gel for osteochondral repair according to any one of claims 1 to 5, characterized in that: The mass ratio of the RSF microparticles to fibrinogen is 5 to 20:1, preferably 10:

1.

7. A composite gel for osteochondrium repair according to any one of claims 1 to 6, wherein the preparation method of the composite gel is as follows: Step 1: preparing a composite material: dissolving RSF and rhCol-Ⅲ in ultrapure water to a concentration of 100-200 mg / ml (preferably 150 mg / ml), respectively, mixing the two solutions in a mass ratio of RSF to rhCol-Ⅲ of 2-6:1 (preferably 2-3:1), stirring evenly and then freeze-drying; Step 2: Preparation of insoluble materials: After freeze-drying, the freeze-dried RSF-rhCol-Ⅲ block material is washed with alcohol and water in sequence, and freeze-dried again; Step 3: Preparation of composite microparticles: Mechanically crushing the freeze-dried material to obtain RSF-rhCol composite microparticles; Step 4: preparing a composite gel: dissolving sodium hyaluronate (HA) in a PBS solution, wherein the concentration of the sodium hyaluronate solution is 10 to 50 mg / ml, preferably 20 mg / ml after complete dissolution; uniformly dispersing and mixing the prepared RSF-rhCol-III composite microparticles in the HA solution, wherein the concentration of the RSF-rhCol composite microparticles is 100 to 500 mg / ml, preferably 200 mg / ml; preparing an RSF-rhCol-III composite gel; Step 5: Prepare an RSF-rhCol-III composite gel preparation for osteochondrium repair: dissolve thrombin in a calcium chloride solution, wherein the concentration of the thrombin is 2000-7000 U / ml (preferably 5000 U / ml), and dissolve fibrinogen in enzyme-free water by ultrasound, wherein the concentration of the fibrinogen is 10-50 mg / ml, preferably 20 mg / ml; mix the RSF-rhCol-III composite gel prepared in step 4 with the thrombin solution through a two-way connecting tube, and then inject the RSF-rhCol composite gel preparation with the fibrinogen solution through a double drug mixing device.

8. A method for preparing a composite gel for osteochondral repair according to claim 1, characterized in that: The following steps are involved: Step 1: preparing a composite material: dissolving RSF and rhCol-Ⅲ in ultrapure water to a concentration of 100-200 mg / ml (preferably 150 mg / ml), respectively, mixing the two solutions in a mass ratio of RSF to rhCol-Ⅲ of 2-6:1 (preferably 2-3:1), stirring evenly and then freeze-drying; Step 2: Preparation of insoluble materials: After freeze-drying, the freeze-dried RSF-rhCol-Ⅲ block material is washed with alcohol and water in sequence, and freeze-dried again; Step 3: Preparation of composite microparticles: mechanically crushing the freeze-dried material to obtain RSF-rhCol composite microparticles; Step 4: preparing a composite gel: dissolving sodium hyaluronate (HA) in a PBS solution, wherein the concentration of the sodium hyaluronate solution is 10 to 50 mg / ml, preferably 20 mg / ml after complete dissolution; uniformly dispersing and mixing the prepared RSF-rhCol-III composite microparticles in the HA solution, wherein the concentration of the RSF-rhCol composite microparticles is 100 to 500 mg / ml, preferably 200 mg / ml; preparing an RSF-rhCol-III composite gel; Step 5: Prepare an RSF-rhCol-III composite gel preparation for osteochondrium repair: dissolve thrombin in a calcium chloride solution, wherein the concentration of the thrombin is 2000-7000 U / ml (preferably 5000 U / ml), and dissolve fibrinogen in enzyme-free water by ultrasound, wherein the concentration of the fibrinogen is 10-50 mg / ml, preferably 20 mg / ml; mix the RSF-rhCol-III composite gel prepared in step 4 with the thrombin solution through a two-way connecting tube, and then inject the RSF-rhCol composite gel preparation with the fibrinogen solution through a double drug mixing device.

9. Use of the composite gel for osteochondral repair according to any one of claims 1 to 8 in the preparation of a drug for treating osteoarthritis.

10. The use according to claim 9, characterized in that Used to treat cartilage damage in osteoarthritis.

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