A composite gel for osteochondral repair, its preparation method and application

Abstract

This invention discloses a composite gel for osteochondral repair, its preparation method, and its applications. The main raw materials of the composite gel are regenerated silk fibroin (RSF) and recombinant humanized type III collagen (rhCol-III), with sodium hyaluronate (HA), fibrinogen, and thrombin as auxiliary raw materials. In this invention, RSF solution and rhCol-III solution are first mixed, and RSF-rhCol microparticles are obtained through freeze-drying and mechanical pulverization. The microparticles are then dispersed in HA solution to obtain the composite gel. The gel is then mixed with thrombin solution, where thrombin catalyzes the cleavage of fibrinogen into fibrin. The fibrin monomers are then cross-linked to obtain an injectable composite gel formulation that can immediately gel. The RSF-rhCol composite gel prepared by this invention can adapt to defect areas and has a certain degree of adhesion. It also exhibits excellent biocompatibility, promotes the migration of bone marrow mesenchymal stem cells, induces cell differentiation, and promotes cartilage tissue regeneration.

Description

Technical Field

[0001] This invention relates to a composite gel for osteochondral repair, its preparation method, and its application, belonging to the field of biomedical technology. Background Technology

[0002] Osteoarthritis is a degenerative disease that severely impacts daily life, typically caused by excessive mechanical stress 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, and the low metabolic activity and migration capacity of chondrocytes, the self-healing ability of articular cartilage is limited. Current treatment strategies for cartilage repair include microfractures, autologous chondrocyte transplantation, and allogeneic / autologous cartilage transplantation; however, these strategies may cause secondary trauma and immune rejection. Hydrogels, with their porous three-dimensional network structure, possess high water retention, adhesion, and good biocompatibility, making them suitable for the treatment of cartilage defects. Hydrogels incorporating specific active substances or loaded with tissue-engineered cells can be applied to treat cartilage defects.

[0003] Regenerated silk fibroin is a natural high-molecular-weight protein material extracted from silkworm silk. It has good biocompatibility, biodegradability, and thermal stability. However, regenerated silk fibroin has low bioactivity and low cell adhesion, requiring its use in combination with other biomaterials. Recombinant humanized collagen is obtained through bio-fermentation technology by optimizing the recombinant expression of the original gene sequence of human skin collagen using genetic engineering techniques. Recombinant humanized type III collagen exhibits characteristics and stability extremely similar to natural human collagen, with no viral risks, excellent biocompatibility and efficacy, and low immunogenicity. Recombinant humanized type III collagen can promote cell adhesion, proliferation, and migration, as well as extracellular matrix remodeling, thereby accelerating tissue repair and regeneration. However, recombinant humanized type III collagen is metabolized relatively quickly and may not be able to adapt to the recovery cycle of cartilage defects.

[0004] This invention combines regenerated silk fibroin and recombinant humanized collagen through the cross-linking of fibrinogen and thrombin, thereby leveraging the specific advantages of both biomaterials to prepare a composite gel material that can promote the repair of osteochondral defects. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a composite gel for osteochondral repair, its preparation method, and its application, which can solve the problem of difficult cartilage regeneration.

[0006] This invention provides a composite gel for osteochondral repair, characterized in that the composite gel is an RSF-rhCol gel, which 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) to recombinant humanized type III collagen (rhCol-III) in the RSF microparticles of the composite gel is 2 to 6:1; preferably 2 to 3:1; and 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 ratio of RSF microparticles to thrombin is 1 mg: 2 to 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 osteochondral repair, wherein the specific steps of the method for preparing the composite gel for cartilage repair are as follows:

[0012] Step 1: Preparation of composite material: Dissolve RSF and rhCol-Ⅲ separately in ultrapure water, mix the two solutions, stir evenly, and then freeze-dry.

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

[0014] Step 3: Preparation of composite microparticles: The final material is mechanically pulverized to obtain RSF-rhCol-Ⅲ composite microparticles;

[0015] Step 4: Preparation of RSF-rhCol-Ⅲ composite gel: Sodium hyaluronate is dissolved in PBS solution. After complete dissolution, the RSF-rhCol-Ⅲ composite microparticles obtained in step 3 are uniformly dispersed and mixed in sodium hyaluronate solution to obtain RSF-rhCol-Ⅲ gel.

[0016] Step 5: Preparation of RSF-rhCol-Ⅲ composite gel formulation: Fibrinogen is dissolved in enzyme-free water by ultrasound, and thrombin is dissolved in calcium chloride solution. The RSF-rhCol-Ⅲ gel obtained in step 4 is mixed evenly with the thrombin solution through a two-way connector tube, and then injected with the fibrinogen solution through a dual-combination device to form the RSF-rhCol-Ⅲ composite gel formulation.

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

[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, wherein 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 RSF-rhCol-III gel / thrombin solution to the fibrinogen solution is 1:0.5 to 3, preferably 1:1.

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

[0029] Step 1: Preparation of composite material: Dissolve RSF and rhCol-III separately in ultrapure water to a concentration of 100-200 mg / ml (preferably 150 mg / ml). Mix the two solutions at a mass ratio of RSF to rhCol-III of 2-6:1 (preferably 2-3:1; most preferably 2:1), stir evenly, and then freeze-dry.

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

[0031] Step 3: Preparation of composite microparticles: RSF-rhCol composite microparticles are obtained by mechanically pulverizing the freeze-dried material;

[0032] Step 4: Preparation of composite gel: Sodium hyaluronate (HA) is dissolved in PBS solution. After complete dissolution, the concentration of the sodium hyaluronate solution is 10-50 mg / ml, preferably 20 mg / ml. The prepared RSF-rhCol-III composite microparticles are uniformly dispersed and mixed in the sodium hyaluronate solution. The concentration of the RSF-rhCol composite microparticles is 100-500 mg / ml, preferably 200 mg / ml. The RSF-rhCol-III composite gel is thus obtained.

[0033] Step 5: Preparation of RSF-rhCol-III composite gel formulation for osteochondral repair: Thrombin is dissolved in calcium chloride solution at a concentration of 2000–7000 U / ml (preferably 5000 U / ml). Fibrinogen is dissolved in enzyme-free water by ultrasound at a concentration of 10–50 mg / ml (preferably 20 mg / ml). The RSF-rhCol-III composite gel prepared in Step 4 is mixed evenly with the thrombin solution through a two-way connector tube, and then injected with the fibrinogen solution using a dual-combination device to produce the RSF-rhCol composite gel formulation.

[0034] The regenerated silk fibroin of this invention is a natural high-molecular-weight fibrous protein with excellent mechanical properties, good biocompatibility, biodegradability, and multifunctionality in structural adjustment, showing broad application prospects. Recombinant humanized type III collagen can promote cell adhesion and migration, as well as extracellular matrix remodeling, creating a favorable repair environment, thereby accelerating tissue repair and regeneration.

[0035] The beneficial effects of this invention are:

[0036] 1. This invention utilizes the catalytic cleavage reaction of thrombin on fibrinogen, converting fibrinogen into fibrin. The fibrin monomers cross-link to form a stable gel network structure. The composite gel can be directly injected into the defect site by parallel injection using a dual-drug syringe.

[0037] 2. By washing RSF-rhCol material with anhydrous ethanol, the protein material is induced to transform into a β-sheet conformation, forming an insoluble and more stable composite material.

[0038] 3. The three-dimensional network structure of hydrogels provides a physiologically simulated environment for cell metabolism and facilitates the transport of nutrients, thus creating a favorable environment for cartilage repair. Both regenerated silk fibroin and recombinant humanized type III collagen exhibit 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 in this invention can promote cell migration, inhibit inflammatory response, promote cartilage tissue regeneration, and has a significant therapeutic effect on cartilage defects. Attached Figure Description

[0040] Figure 1 A schematic flowchart illustrating the preparation method of RSF-rhCol composite gel.

[0041] Figure 2 A schematic structural flowchart of the method for preparing RSF-rhCol composite gel.

[0042] Figure 3 The distribution diagram of rhCol-Ⅲ in 2RSF-rhCol.

[0043] Figure 4 This is a scanning electron microscope image of RSF-rhCol composite microparticles.

[0044] Figure 5 The particle size distribution of the 2RSF-rhCol composite microparticles is shown.

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

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

[0047] Figure 8 The rheological diagram is shown for the RSF-rhCol composite gel.

[0048] Figure 9 This is a graph showing the cytotoxicity data of RSF-rhCol composite gel on bone marrow mesenchymal stem cells.

[0049] Figure 10 A statistical graph showing the effect of RSF-rhCol composite gel on the migration of bone marrow mesenchymal stem cells.

[0050] Figure 11A diagram illustrating the effect of RSF-rhCol composite gel on the expression of cartilage-related genes in cells.

[0051] Figure 12 Photographs of cartilage samples taken from rats treated with RSF-rhCol composite gel for cartilage defects.

[0052] Figure 13 Micro-CT scan of rats with cartilage defects treated with RSF-rhCol composite gel. Detailed Implementation

[0053] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

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

[0055] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0056] Regenerated silk fibroin (RSF) was purchased from Nanjing Siyuan Medical Technology Co., Ltd.; recombinant humanized type III collagen (rhCol-III) was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.; sodium hyaluronate (HA) was purchased from Bloomage Biotechnology Co., Ltd.; fibrinogen was purchased from Beijing Solarbio 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 includes 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 is washed twice with alcohol, three times with water, and then freeze-dried again;

[0060] Step 3: Mechanically pulverize to obtain RSF-rhCol composite microparticles;

[0061] Step 4: Dissolve HA and disperse the RSF-rhCol composite microparticles in HA to obtain RSF-rhCol gel;

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

[0063] In this invention, in order to obtain a sterile RSF-rhCol composite gel for use in in vitro and in vivo experiments, the prepared RSF-rhCol gel is first sterilized by moist heat at 121°C for 12 minutes; fibrinogen and thrombin are dissolved using sterile reagents, and the gel formation is carried out under sterile conditions.

[0064] The following detailed description uses specific examples:

[0065] Example 1

[0066] An RSF composite microparticle is prepared by the following steps:

[0067] Step S1: Dissolve 630 mg of RSF in 4.2 ml of ultrapure water and stir at room temperature for 2 hours. After dissolution, an RSF solution with a concentration of 150 mg / ml is obtained. Aliquot the RSF solution into six-well cell culture plates and freeze-dry them.

[0068] Step S2: After the freeze-drying process is complete, remove the freeze-dried RSF block material and soak it in anhydrous ethanol solution with stirring for 1 hour. Then soak it in ethanol again with stirring for 1 hour, and finally soak it in ultrapure water with stirring for 1 hour each time. After soaking and washing, repackage the RSF block material into six-well cell culture plates and freeze-dry it.

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

[0070] Example 2

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

[0072] Step S1: Dissolve 540 mg of RSF in 3.6 ml of ultrapure water and stir at room temperature for 2 hours. After dissolution, an RSF solution with a concentration of 150 mg / ml is obtained. Dissolve 90 mg of rhCol-III in 0.6 ml of ultrapure water and sonicate to obtain an rhCol-III solution with a concentration of 150 mg / ml. Add the rhCol-III solution dropwise to the RSF solution using a dropper, stirring continuously during the addition. After the rhCol-III solution is added, continue stirring for 30 minutes to ensure the two solutions are thoroughly mixed. Aliquot the RSF-rhCol mixture into six-well cell culture plates and freeze-dry them.

[0073] Step S2: After the freeze-drying process is complete, remove the freeze-dried 6RSF-rhCol block material and soak it in anhydrous ethanol solution with stirring for 1 hour. Then soak it in ethanol again with stirring for 1 hour, and finally soak it in ultrapure water with stirring for 1 hour each time. After soaking and washing, repackage the 6RSF-rhCol block material into six-well cell culture plates and freeze-dry it.

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

[0075] Example 3

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

[0077] Step S1: Dissolve 472.5 mg of RSF in 3.15 ml of ultrapure water and stir at room temperature for 2 hours. After dissolution, an RSF solution with a concentration of 150 mg / ml is obtained. Dissolve 157.5 mg of rhCol-III in 1.05 ml of ultrapure water and sonicate to obtain an rhCol-III solution with a concentration of 150 mg / ml. Add the rhCol-III solution dropwise to the RSF solution using a dropper, stirring continuously during the addition. After the rhCol-III solution is added, continue stirring for 30 minutes to ensure the two solutions are thoroughly mixed. Aliquot the RSF-rhCol mixture into six-well cell culture plates and freeze-dry them.

[0078] Step S2: After the freeze-drying process is complete, remove the freeze-dried 3RSF-rhCol block material and soak it in anhydrous ethanol solution with stirring for 1 hour. Then soak it in ethanol again with stirring for 1 hour, and finally soak it in ultrapure water with stirring for 1 hour each time. After soaking and washing, repackage the 3RSF-rhCol block material into six-well cell culture plates and freeze-dry it.

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

[0080] Example 4

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

[0082] Step S1: Dissolve 420 mg of RSF in 2.8 ml of ultrapure water and stir at room temperature for 2 hours. After dissolution, an RSF solution with a concentration of 150 mg / ml is obtained. Dissolve 210 mg of rhCol-III in 1.4 ml of ultrapure water and sonicate to obtain an rhCol-III solution with a concentration of 150 mg / ml. Add the rhCol-III solution dropwise to the RSF solution using a dropper, stirring continuously during the addition. After the rhCol-III solution is added, continue stirring for 30 minutes to ensure the two solutions are thoroughly mixed. Aliquot the RSF-rhCol mixture into six-well cell culture plates and freeze-dry them. After the freeze-drying process is complete...

[0083] Step S2: After the freeze-drying process is complete, remove the freeze-dried 2RSF-rhCol block material and soak it in anhydrous ethanol solution for 1 hour with stirring. Then soak it in ethanol solution again for 1 hour with stirring. Finally, soak it in ultrapure water for 1 hour each time. After soaking and washing, repackage the 2RSF-rhCol block material into six-well cell culture plates and freeze-dry it.

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

[0085] Example 5

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

[0087] Step S1: Slowly add 20 mg of HA to 1 ml of PBS and stir at room temperature until the HA is completely dissolved. Add 200 mg of RSF microparticles to 1 ml of HA solution and use magnetic stirring to completely and uniformly disperse the microparticles in the HA to obtain an RSF gel. Then, sterilize the RSF gel by moist heat at 121°C for 12 min. After sterilization, aliquot the gel into 1 ml syringes in a sterile operating table and store at 4°C.

[0088] Step S2: Dissolve 0.2 ml of sterile calcium chloride solution (50 mg / ml) in 1000 U of thrombin using ultrasonication. In a sterile operating room, mix 0.8 ml of RSF gel with 0.2 ml of thrombin solution using a two-way connector. Transfer the mixture to a 2 ml syringe for later use. Weigh 20 mg of fibrinogen into a sterile centrifuge tube, add 1 ml of sterile, enzyme-free water, and dissolve using ultrasonication. Monitor the water temperature during dissolution; change the water if it becomes too hot. After dissolution, transfer the fibrinogen solution to a 2 ml syringe. Attach both syringes to a dual-mixing device and push the syringes; both materials will be ejected simultaneously, forming an RSF gel upon contact.

[0089] Example 6

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

[0091] Step S1: Slowly add 20 mg of HA to 1 ml of PBS, stirring until the HA is completely dissolved. Add 200 mg of 6RSF-rhCol microparticles to 1 ml of HA solution, and use magnetic stirring to completely and uniformly disperse the microparticles in the HA to obtain 6RSF-rhCol gel. Then, sterilize the 6RSF-rhCol gel by moist heat at 121°C for 12 min. After sterilization, aliquot it into 1 ml syringes in a sterile operating table and store at 4°C.

[0092] Step S2: Dissolve 0.2 ml of sterile calcium chloride solution (50 mg / ml) in 1000 U of thrombin using ultrasonication. In a sterile operating room, mix 0.8 ml of 6RSF-rhCol gel with 0.2 ml of thrombin solution using a two-way connector. Transfer the mixture to a 2 ml syringe for later use. Weigh 20 mg of fibrinogen into a sterile centrifuge tube, add 1 ml of sterile, enzyme-free water, and dissolve using ultrasonication. Monitor the water temperature during dissolution; replace the water if it becomes too hot. After dissolution, transfer the fibrinogen solution to a 2 ml syringe. Attach both syringes to a dual-mixing device and push the syringes; both materials will be ejected simultaneously, forming a 6RSF-rhCol composite gel upon contact.

[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, stirring until the HA is completely dissolved. Add 200 mg of 3RSF-rhCol microparticles to 1 ml of HA solution, and use magnetic stirring to completely and uniformly disperse the microparticles in the HA to obtain 3RSF-rhCol gel. Then, sterilize the 3RSF-rhCol gel by moist heat at 121°C for 12 min. After sterilization, aliquot it into 1 ml syringes in a sterile operating table and store at 4°C.

[0096] Step S2: Dissolve 0.2 ml of sterile calcium chloride solution (50 mg / ml) in 1000 U of thrombin using ultrasonication. In a sterile operating room, mix 0.8 ml of 3RSF-rhCol gel with 0.2 ml of thrombin solution using a two-way connector. Transfer the mixture to a 2 ml syringe for later use. Weigh 20 mg of fibrinogen into a sterile centrifuge tube, add 1 ml of sterile, enzyme-free water, and dissolve using ultrasonication. Monitor the water temperature during dissolution; replace the water if it becomes too hot. After dissolution, transfer the fibrinogen solution to a 2 ml syringe. Attach both syringes to a dual-mixing device and push the syringes; both materials will be ejected simultaneously, forming a 3RSF-rhCol composite gel upon contact.

[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 to 1 ml of PBS, stirring until the HA is completely dissolved. Add 200 mg of 2RSF-rhCol microparticles to 1 ml of HA solution, and use magnetic stirring to completely and uniformly disperse the microparticles in the HA to obtain 2RSF-rhCol gel. Then, sterilize the 2RSF-rhCol gel by moist heat at 121°C for 12 min. After sterilization, aliquot it into 1 ml syringes in a sterile operating table and store at 4°C.

[0100] Step S2: Dissolve 0.2 ml of sterile calcium chloride solution (50 mg / ml) in 1000 U of thrombin using ultrasonication. In a sterile operating room, mix 0.8 ml of 2RSF-rhCol gel with 0.2 ml of thrombin solution using a two-way connector. Transfer the mixture to a 2 ml syringe for later use. Weigh 20 mg of fibrinogen into a sterile centrifuge tube, add 1 ml of sterile, enzyme-free water, and dissolve using ultrasonication. Monitor the water temperature during dissolution; replace the water if it becomes too hot. After dissolution, transfer the fibrinogen solution to a 2 ml syringe. Attach both syringes to a dual-syringe device and push the syringes; both materials will be ejected simultaneously, forming a 2RSF-rhCol composite gel upon contact.

[0101] Performance analysis of RSF-rhCol composite gel:

[0102] Figure 3 The fluorescence image of the 2RSF-rhCol composite microparticles is shown. rhCol-III was labeled with fluorescein isothiocyanate. 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 uniformly dispersed in the microparticles, indicating that RSF and rhCol-III were successfully composited.

[0103] Figure 4 The image shows a scanning electron microscope (SEM) image of the RSF-rhCol composite microparticles. As can be seen from the figure, the prepared microparticles are relatively uniformly dispersed in size, with a particle size distribution of 80–100 μm.

[0104] Figure 5 The particle size distribution of the 2RSF-rhCol composite microparticles is shown. The composite microparticles are approximately dispersed at a size of 100 μm, which is similar to the results obtained by scanning electron microscopy.

[0105] Figure 6 The image shows the appearance of the 2RSF-rhCol composite gel. The composite gel can be extruded from the needle tip, making it injectable. Furthermore, the composite gel can be molded to fit various shapes, indicating that the gel can adapt to defect locations.

[0106] Figure 7 The image shows a scanning electron microscope (SEM) image of the prepared RSF-rhCol composite gel. The image shows that the gel has a porous structure, which can transport nutrients and metabolic waste in vivo, creating a favorable environment for cartilage regeneration.

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

[0108] Figure 9 The cytotoxicity data of the RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels prepared in Examples 5-8 on bone marrow mesenchymal stem cells (BMSCs) are shown. 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 h at 37°C. BMSC cells were evenly seeded in 96-well plates, approximately 5000 cells per well. After culturing for 24 h, the cells were fully adherent. Hydrogel extraction solutions for different groups were added, while the control group was added to α-MEM complete medium. Culture was continued for another 24 h, and then 100 μL of α-MEM complete medium containing 10% CCK-8 was added to each well. After incubation for 1 h, cell absorbance was measured using a microplate reader, and cell viability was calculated. The results showed that the cell survival rate of the material group was improved compared with the control group, indicating that the prepared hydrogel has good cell compatibility and can promote cell proliferation.

[0109] Figure 10 Crystal violet staining images and cell migration rate graphs of the effects of RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels prepared in Examples 5-8 on BMSC cell migration are shown. Equal volumes of hydrogel material and 500 μL of serum-containing culture medium were added to 24-well plates. BMSC cells were cultured at 3 × 10⁻⁶ cells / well. 4 Cells were dispersed at a density of 100 cells / mL in serum-free culture medium. 200 μL of cell suspension was added above each Transwell chamber, which was then placed in a 24-well plate. After culturing for 24 h, crystal violet staining was performed, and images were taken to calculate cell migration rate. Results showed that compared to the control group (no added material), the number of migrating cells in the material group was significantly increased, and the migration rate increased with increasing rhCol-III content, indicating that RSF-rhCol promotes cell migration. The low cell density and metabolic activity at cartilage sites, coupled with increased cell migration ability, facilitate the aggregation of BMSCs at the defect site, creating a favorable environment for cartilage regeneration after stem cell cartilage differentiation. Among these, 3RSF-rhCol and 2RSF-rhCol showed significantly better cell migration promotion effects than the control group, RSF, and 6RSF-rhCol groups; 2RSF-rhCol showed the best effect (P<0.0001).

[0110] Figure 11 The diagram illustrates the effect of the RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol composite gels prepared in Examples 5-8 on chondrogenic differentiation of BMSC cells. The cell concentration was 4 × 10⁻⁶ cells / year. 4 500 μL of cell suspension was evenly spread into each well of a 24-well plate. After the cells were fully confluent, the plate was placed in a Transwell chamber. A composite gel containing RSF, 6RSF-rhCol, 3RSF-rhCol, and 2RSF-rhCol was added to the chamber for co-incubation. The control group was not incubated with any materials, but instead used chondrogenic medium for chondrogenic induction. The chondrogenic medium was changed every 2 days. After 14 days of chondrogenic induction, RNA was extracted from the cells using the RNAeasy™ Animal RNA Extraction Kit (centrifugal column method). The expression of related genes in the RNA was detected using the BeyoFast™ SYBRGreen One-Step qRT-PCR Kit. The results showed that the 2RSF-rhCol composite gel significantly increased the expression of SOX9, COLII, and ACAN-related chondrogenesis genes, indicating that the 2RSF-rhCol composite gel could promote chondrogenesis of BMSC cells, which was significantly better than the control group, RSF, 3RSF-rhCol, and 6RSF-rhCol groups (P<0.01).

[0111] Figure 12Images show the therapeutic effects of the RSF, 6RSF-rhCol, and 2RSF-rhCol composite gels prepared in Examples 5-8 on cartilage defect repair. Because the effect of rhCol-III content was verified in in vitro cell experiments, subsequent animal experiments included a sham-operated group (Sham), a control group (Control), an RSF group (no collagen), a 6RSF-rhCol group (low collagen), and a 2RSF-rhCol group (high collagen). Eight-week-old male SD rats were selected. After anesthesia and disinfection of the left leg, a 2mm diameter and 1.5mm depth defect was created at the distal femoral trochlea using a low-profile dental drill. RSF, 6RSF-rhCol, and 2RSF-rhCol gels were injected into the defect site. The sham-operated group only underwent skin incision and suturing, while the control group did not receive material filling after defect creation. The rats' condition was observed post-operatively to prevent wound infection and death. Rats were sacrificed one and two months post-surgery. The repair status of the defect was photographed and statistically analyzed. Rats' knee joints were collected and fixed in 4% paraformaldehyde. Visual images of the repaired defect showed that one month after repair, some fibrocartilage tissue regenerated, and the material did not completely degrade. Two months later, the defect in the 2RSF-rhCol material group was basically filled with newly formed cartilage tissue and fused well with the surrounding cartilage tissue, indicating a good defect repair effect.

[0112] Figure 13 Micro-CT images of cartilage repair are shown. Results indicate that in the first month, the defect locations were clearly visible in each group. After two months, all groups showed some degree of cartilage defect repair. The defect area was more obvious in the control group. The RSF group and the 6RSF-rhCol group showed good defect recovery, but the defect area was still visible. The defect area in the 2RSF-rhCol group almost completely disappeared, indicating the good therapeutic effect of 2RSF-rhCol gel on osteocartilage defect repair.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate and not 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 modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A composite gel for osteochondral repair, characterized in that, The composite gel is an RSF-rhCol gel, which includes RSF microparticles made from regenerated silk fibroin (RSF) and recombinant humanized type III collagen (rhCol-III), sodium hyaluronate, fibrinogen, and thrombin. The RSF in the RSF-rhCol is in the β-sheet conformation.

2. A composite gel for osteochondral repair as described 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 microparticles of the composite gel is 2~6:

1.

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

1.

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

1.

5. A composite gel for osteochondral repair as described in claim 3, characterized in that, The mass ratio of RSF microparticles to sodium hyaluronate is 5~20:

1.

6. The composite gel for osteochondral repair according to claim 5, characterized in that, The mass ratio of RSF microparticles to sodium hyaluronate is 10:

1.

7. A composite gel for osteochondral repair as described in claim 5, characterized in that, The ratio of RSF microparticles to thrombin is 1 mg : 2~10 U.

8. The composite gel for osteochondral repair according to claim 7, characterized in that, The ratio of RSF microparticles to thrombin is 1 mg : 5 U.

9. A composite gel for osteochondral repair as described in claim 7, characterized in that, The mass ratio of RSF microparticles to fibrinogen is 5~20:

1.

10. The composite gel for osteochondral repair according to claim 9, characterized in that, The mass ratio of RSF microparticles to fibrinogen is 10:

1.

11. A composite gel for osteochondral repair as described in any one of claims 1-10, said composite gel being prepared by the following method: Step 1: Preparation of composite material: Dissolve RSF and rhCol-III separately in ultrapure water to a concentration of 100~200mg / mL. Mix the two solutions at a mass ratio of RSF to rhCol-III of 2~6:1, stir evenly, and then freeze-dry. Step 2: Preparation of insoluble material: After freeze-drying, the freeze-dried RSF-rhCol-Ⅲ block material is sequentially washed with alcohol and water, and then freeze-dried again; Step 3: Preparation of composite microparticles: RSF-rhCol composite microparticles are obtained by mechanically pulverizing the freeze-dried material; Step 4: Preparation of composite gel: Sodium hyaluronate (HA) is dissolved in PBS solution. After complete dissolution, the concentration of the sodium hyaluronate solution is 10~50 mg / mL. The prepared RSF-rhCol-III composite microparticles are uniformly dispersed and mixed in the HA solution. The concentration of the RSF-rhCol composite microparticles is 100~500 mg / mL. The RSF-rhCol-III composite gel is thus prepared. Step 5: Preparation of RSF-rhCol-III composite gel formulation for osteochondral repair: Thrombin is dissolved in calcium chloride solution at a concentration of 2000-7000 U / mL; fibrinogen is dissolved in enzyme-free water by ultrasound at a concentration of 10-50 mg / mL; the RSF-rhCol-III composite gel prepared in Step 4 is mixed evenly with the thrombin solution through a two-way connector, and then injected with the fibrinogen solution through a dual-combination device to form the RSF-rhCol composite gel formulation.

12. The composite gel for osteochondral repair according to claim 11, wherein the concentration in step 1 is 150 mg / mL and the mass ratio is 2-3:1; the concentration of sodium hyaluronate solution in step 4 is 20 mg / mL and the concentration of RSF-rhCol composite microparticles is 200 mg / mL; and the concentration of thrombin in step 5 is 5000 U / mL and the concentration of fibrinogen is 20 mg / mL.

13. A method for preparing a composite gel for osteochondral repair as described in claim 1, characterized in that, Includes the following steps: Step 1: Preparation of composite material: Dissolve RSF and rhCol-III separately in ultrapure water to a concentration of 100~200mg / mL. Mix the two solutions at a mass ratio of RSF to rhCol-III of 2~6:1, stir evenly, and then freeze-dry. Step 2: Preparation of insoluble material: After freeze-drying, the freeze-dried RSF-rhCol-Ⅲ block material is sequentially washed with alcohol and water, and then freeze-dried again; Step 3: Preparation of composite microparticles: RSF-rhCol composite microparticles are obtained by mechanically pulverizing the freeze-dried material; Step 4: Preparation of composite gel: Sodium hyaluronate (HA) is dissolved in PBS solution. After complete dissolution, the concentration of the sodium hyaluronate solution is 10~50 mg / mL. The prepared RSF-rhCol-III composite microparticles are uniformly dispersed and mixed in the HA solution. The concentration of the RSF-rhCol composite microparticles is 100~500 mg / mL. The RSF-rhCol-III composite gel is thus prepared. Step 5: Preparation of RSF-rhCol-III composite gel formulation for osteochondral repair: Thrombin is dissolved in calcium chloride solution at a concentration of 2000-7000 U / mL; fibrinogen is dissolved in enzyme-free water by ultrasound at a concentration of 10-50 mg / mL; the RSF-rhCol-III composite gel prepared in Step 4 is mixed evenly with the thrombin solution through a two-way connector, and then injected with the fibrinogen solution through a dual-combination device to form the RSF-rhCol composite gel formulation.

14. The method for preparing the composite gel for osteochondral repair according to claim 13, wherein the concentration in step 1 is 150 mg / mL and the mass ratio is 2~3:1; the concentration of sodium hyaluronate solution in step 4 is 20 mg / mL and the concentration of RSF-rhCol composite microparticles is 200 mg / mL; and the concentration of thrombin in step 5 is 5000 U / mL and the concentration of fibrinogen is 20 mg / mL.

15. Use of the composite gel for osteochondral repair according to any one of claims 1 to 12 in the preparation of a medicament for treating osteoarthritis.

16. Use of the composite gel for osteochondral repair according to any one of claims 1 to 12 in the preparation of a medicament for treating osteoarthritis with cartilage damage.

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