Collagen-fibrin composite gel for cartilage defect repair and preparation method thereof

Abstract

The invention discloses collagen-fibrin composite gel for cartilage defect repair and a preparation method of the collagen-fibrin composite gel, and belongs to the technical field of gel preparation. The preparation method of the collagen-fibrin composite gel comprises the following steps: mixing a collagen solution with a thrombin solution to obtain a collagen-thrombin mixed solution; preparing a fibrinogen solution; and respectively filling the collagen-thrombin mixed solution and the fibrinogen solution into a duplex syringe, synchronously extruding and mixing to obtain a composite gel precursor, and standing to obtain the collagen-fibrin composite gel. The prepared gel has good mechanical properties, can induce bone marrow mesenchymal stem cells and surrounding autologous cartilage cells to migrate inwards, enables the cells to be amplified according to the three-dimensional structure of the gel, and further can be used as a raw material for repairing defective cartilage to achieve repairing of the defective cartilage.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gel preparation, in particular to a collagen-fibrin composite gel for repairing cartilage defects and a preparation method thereof. BACKGROUND

[0002] Collagen is a natural protein with a wide range of sources, mainly existing in the connective tissue of animals. Due to the biocompatibility, bioactivity and low immunogenicity of collagen, it has become one of the natural materials favored in the field of tissue engineering. Collagen is also the main component of human joint cartilage. Studies have shown that both type I collagen and type II collagen are conducive to the adhesion, proliferation and differentiation of chondrocytes. The biological activity of type I collagen is suitable for developing and applying to biomaterials for cartilage repair. In addition, type I collagen has a more uniform pore structure than type II collagen, and the degradation rate of type I collagen in the body is slower than that of type II collagen, which can provide a more persistent and stable three-dimensional space structure for the proliferation and differentiation of chondrocytes. In addition, the most important advantage of type I collagen is that it can accurately induce chondrocytes around the defect area into the defect area. At the same time, type I collagen is widely available, has a clear structure, and has been well researched. Compared with type II collagen, it is easier to obtain, so type I collagen can be used as the main matrix of cartilage repair materials.

[0003] Chinese invention patent CN116999619A discloses a collagen gel for cartilage and a preparation method thereof. The method extracts type I collagen solution from bovine Achilles tendon to prepare collagen gel. The gel can be anchored in the cartilage defect site and applied to cartilage repair. However, the mechanical properties of the product are relatively poor, and it cannot fully support cartilage repair.

[0004] Therefore, it is urgent to study a new gel material that has good mechanical properties and can be used as a cartilage repair material. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application provides a collagen-fibrin composite gel for repairing cartilage defects and a preparation method thereof. The collagen-fibrin composite gel is obtained by introducing fibrin and using double injection. The prepared gel has good mechanical properties and can induce bone marrow mesenchymal stem cells and surrounding autologous chondrocytes to migrate inward, and make the cells expand according to the three-dimensional structure of the gel. The expanded chondrocytes absorb the degradation products of type I collagen as nutrients to synthesize and secrete type II collagen, forming new cartilage-like tissue with special morphology and function of in-situ tissue cells, and then repairing the defective cartilage.

[0006] The technical scheme of the present application is as follows: The first aspect of the present application protects a preparation method of a collagen-fibrin composite gel, comprising the following steps: S1: mixing a collagen solution with a thrombin solution to obtain a collagen-thrombin mixed solution; S2: preparing a fibrinogen solution; S3: loading the collagen-thrombin mixed solution of step S1 and the fibrinogen solution of step S2 into a double syringe respectively, synchronously extruding, mixing to obtain a composite gel precursor, and standing to obtain a collagen-fibrin composite gel.

[0007] Preferably, in step S1, the collagen solution comprises a type I collagen solution.

[0008] Preferably, in step S1, the concentration of the collagen solution is 20-40 mg / mL.

[0009] Preferably, in step S1, the concentration of the thrombin solution is 400-600 IU / mL.

[0010] Preferably, in step S1, the volume ratio of the collagen solution to the thrombin solution is 3-4: 1-2.

[0011] Preferably, in step S2, the fibrinogen solution is obtained by dissolving fibrinogen in water. The concentration of the fibrinogen solution is 40-60 mg / mL.

[0012] Preferably, in step S3, the extrusion speed is 5-10 mm / s.

[0013] Preferably, in step S3, the standing temperature is 25-37℃, and the standing time is 3-5 min.

[0014] The second aspect of the present application protects a collagen-fibrin composite gel prepared by the preparation method of the first aspect.

[0015] The third aspect of the present application protects the use of the collagen-fibrin composite gel prepared by the preparation method of the first aspect or the collagen-fibrin composite gel of the second aspect, for preparing a material for repairing cartilage defects.

[0016] The present application has the beneficial technical effects of: This invention introduces fibrin and uses a dual-injection method to obtain a collagen-fibrin composite gel. By introducing fibrin and thrombin, a covalently cross-linked three-dimensional structure can be formed, improving the mechanical properties of the prepared gel. Furthermore, the type I collagen contained in the composite gel can induce bone marrow mesenchymal stem cells and surrounding autologous chondrocytes to migrate inward, and the cells expand according to the gel's three-dimensional structure. In addition, the expanded chondrocytes can absorb the degradation products of type I collagen as nutrients, synthesize and secrete type II collagen, thereby forming new cartilage-like tissue with the special morphology and function of in situ tissue cells, thus repairing damaged cartilage.

[0017] This invention further optimizes the ratio of raw materials and the preparation process, which significantly improves the mechanical properties and chondrocyte growth promotion properties of the collagen-fibrin composite gel, thereby further enhancing the gel's cartilage defect repair effect. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the double-unit mixing device used in this invention.

[0019] In the diagram: 1. Needle; 2. Collagen-thrombin mixed solution syringe; 3. Fibrinogen solution syringe; 4. Double frame; 5. Double plunger; 6. Y-connector. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the embodiments.

[0021] To address the shortcomings of existing type I collagen, this invention provides a collagen-fiber composite gel for cartilage defect repair and its preparation method, thereby solving the aforementioned technical problems.

[0022] The first aspect of this invention protects a method for preparing a collagen-fibrin composite gel, comprising the following steps; S1: Mix collagen solution with thrombin solution to obtain collagen-thrombin mixed solution; S2: Preparation of fibrinogen solution; S3: The collagen-thrombin mixture from step S1 and the fibrinogen solution from step S2 are respectively loaded into a dual syringe, squeezed out simultaneously, mixed, and the composite gel precursor is obtained. After standing, the collagen-fibrin composite gel is obtained.

[0023] It is understood that the collagen-fibrin composite gel for cartilage defect repair of the present invention is prepared using collagen solution, thrombin solution, and fibrin solution. The collagen-thrombin mixed solution is prepared, and then the solution and fibrin solution are dispensed into a double syringe, extruded simultaneously, and allowed to stand. Fibrinogen, a precursor of fibrin and a natural glycoprotein isolated from blood, can form a fibrin gel network under the action of thrombin, effectively adhering to the wound and achieving good hemostasis and promoting wound healing. By introducing fibrinogen and thrombin into type I collagen to form a collagen-fibrin composite gel, the bioactivity of collagen and fibrin is preserved. Furthermore, the introduction of fibrin and thrombin to form a covalently cross-linked three-dimensional network structure greatly improves the material's ductility and extends its degradation period. Collagen provides stiffness to the composite gel; the synergistic effect of the two substances results in a composite gel with a high elastic modulus, capable of withstanding certain pressure. In addition, collagen can provide a stable environment for the proliferation and migration of chondrocytes, inducing autologous bone marrow mesenchymal stem cells and surrounding autologous chondrocytes to migrate and expand into it. Chondrocytes can obtain sufficient nutrients from it, carry out nutrient and waste exchange, and expand according to a pre-designed three-dimensional morphological scaffold. After expansion, the chondrocytes continue to proliferate as type I collagen is gradually degraded, and secrete extracellular matrix such as type II collagen and proteoglycans, forming new cartilage-like tissue with the special morphology and function of in situ tissue cells, thus achieving the effect of filling and repairing cartilage tissue and replacing cartilage function.

[0024] In some embodiments, in step S1, the collagen solution includes a type I collagen solution.

[0025] In some embodiments, the collagen is type I collagen extracted from animal tissue.

[0026] In some embodiments, the collagen is type I collagen extracted from animal skin or animal Achilles tendon.

[0027] In some embodiments, the collagen is extracted by the following method: (1) Cut the animal tissue into small pieces, wash with water, defatted, remove non-collagenous components, and finally wash with sterile distilled water to obtain defatted tissue.

[0028] (2) Soak the defatted tissue from (1) in an acid solution, add pepsin for enzymatic hydrolysis, and obtain the hydrolysis product.

[0029] (3) Centrifuge the enzymatic hydrolysis product from (2), take the supernatant and adjust the pH to about 7. Then add solid NaCl to make the final NaCl concentration 4 mol / L, let it stand, centrifuge to remove the supernatant, and you will get the crude collagen product.

[0030] (4) Dissolve the crude collagen product in (3) with acid solution, centrifuge, take the supernatant and put it into a dialysis bag (14000 kDa), dialyze in acid solution for 2-3 days, change the dialysate every 5 h to obtain type I collagen solution.

[0031] In some embodiments, in step S1, the collagen solution is obtained by diluting collagen with PBS buffer.

[0032] In some embodiments, in step S1, the concentration of the collagen solution is 20-40 mg / mL.

[0033] Understandably, too high a concentration of collagen solution will lead to a decrease in the mechanical properties of the composite gel, while too low a concentration will reduce cell proliferation capacity.

[0034] In some embodiments, the thrombin solution is obtained by dissolving thrombin in a 40 mM calcium chloride solution and stirring thoroughly until completely dissolved.

[0035] Preferably, the thrombin of the present invention is purchased from Shanghai RAAS Blood Products Co., Ltd.

[0036] In some embodiments, in step S1, the concentration of the thrombin solution is 400-600 IU / mL.

[0037] Understandably, a high concentration of thrombin solution accelerates the gelation of the composite gel, which is detrimental to clinical surgical procedures, while a low concentration fails to promote complete gelation of the composite gel.

[0038] In some embodiments, in step S1, the volume ratio of the collagen solution to the thrombin solution is 3-4:1-2.

[0039] Understandably, using too little collagen solution compared to thrombin solution can lead to both an increased thrombin content causing the composite gel to form too quickly, which is not conducive to clinical operation, and a decreased collagen content causing a reduction in cell proliferation rate.

[0040] In some embodiments, in step S2, the fibrinogen solution is obtained by dissolving fibrinogen in water.

[0041] Preferably, the fibrinogen of the present invention is purchased from Shanghai RAAS Blood Products Co., Ltd.

[0042] In some embodiments, the concentration of the fibrinogen solution is 40-60 mg / mL.

[0043] In some embodiments, the syringe used in this invention is not limited to a dual syringe; any device that can achieve mixing after extrusion is within the scope of protection of this invention.

[0044] Figure 1 A schematic diagram of the dual syringe used in this invention is provided. In the diagram: 1 is the needle; 2 is the collagen-thrombin mixed solution syringe; 3 is the fibrinogen solution syringe; 4 is the dual frame; 5 is the dual plunger; 6 is the Y-connector. In use, the collagen-thrombin mixed solution and the fibrinogen solution are respectively loaded into their corresponding syringes, and then the dual plunger is pushed to mix the two solutions at the Y-connector. The mixture is then extruded through the needle 1 to obtain the gel precursor.

[0045] In step S3, the collagen-thrombin mixture from step S1 and the fibrinogen solution from step S2 are respectively loaded into the dual syringes. Specifically, the collagen-thrombin mixture from step S1 and the fibrinogen solution from step S2 are respectively loaded into the two corresponding syringes of the dual syringes.

[0046] In some embodiments, the extrusion speed in step S3 is 5-10 mm / s.

[0047] Understandably, if the extrusion speed is too fast, it will lead to uneven mixing of fibrinogen and collagen / thrombin; if the extrusion speed is too slow, it will cause needle blockage, which is not conducive to clinical operation.

[0048] In some implementations, in step S3, the settling temperature is 25-37°C and the settling time is 3-5 minutes.

[0049] It is understood that the gel precursor of the present invention, after standing for 3-5 minutes, can form a structurally complete solid in situ and can anchor itself to the cartilage defect site for repair. If the standing time is too short, the composite gel network will not be fully formed; if the standing time is too long, it will lead to gel structure shrinkage and dehydration.

[0050] A second aspect of this invention protects a collagen-fibrin composite gel prepared by the preparation method described in the first aspect above.

[0051] The third aspect of this invention protects a collagen-fiber composite gel prepared by the preparation method described in the first aspect above, or the application of the collagen-fiber composite gel described in the second aspect above, for the preparation of materials for cartilage defect repair.

[0052] In this invention, the thrombin used in the embodiments is topical human thrombin from the human fibrin adhesive of Shanghai RAAS Blood Products Co., Ltd.; the fibrinogen used is topical human fibrinogen from the human fibrin adhesive of Shanghai RAAS Blood Products Co., Ltd. Components not explicitly specified in this invention can be considered commercially available products.

[0053] The present invention will be further described below through examples and other means.

[0054] Example 1 The preparation method of the collagen-fibrin composite gel includes the following steps: a. Collagen extraction Type I collagen solution was extracted from pig skin.

[0055] (1) Pretreatment of pig skin: The pig skin is crushed into small pieces and washed with clean water. 8% Na2CO3 solution is added at a ratio of 1:5 to the weight of the pig skin to degrease the pig skin. Then 5% NaCl solution is added to remove non-collagenous components. Finally, the pig skin is washed with sterile distilled water to obtain degreased pig skin.

[0056] (2) Extraction: The defatted pig skin in (1) was soaked in 0.1M pH 2.5 acetic acid solution, 1% pepsin was added, and enzymatic hydrolysis was carried out at 4℃ for 24 h to obtain the enzymatic hydrolysis product.

[0057] (3) Separation: Centrifuge the enzymatic hydrolysis product from (2) at 10,000 rpm, take the supernatant and adjust the pH to about 7 with 1M NaOH. Then add solid NaCl to make the final NaCl concentration 4 mol / L, let stand for 24 h, centrifuge to remove the supernatant, and obtain the crude collagen product.

[0058] (4) Purification: Dissolve the crude collagen product in (3) with 0.1 M pH 2.5 acetic acid solution, centrifuge at 10000 rpm, take the supernatant and put it into a dialysis bag (14000 kDa), dialyze in 0.05 M pH 2.5 acetic acid solution for 2-3 days, change the dialysate every 5 h to obtain type I collagen solution, dilute the type I collagen solution with PBS buffer solution to 30 mg / mL, and finally store it in an environment of 4℃.

[0059] b. Preparation of thrombin solution To prepare a thrombin solution with a concentration of 500 IU / mL: Dissolve thrombin in 40 mM calcium chloride solution and stir thoroughly until completely dissolved to obtain a thrombin solution with a concentration of 500 IU / mL.

[0060] c. Preparation of collagen-thrombin mixed solution Preparation of collagen-thrombin mixed solution: Mix the type I collagen solution from step a and the thrombin solution from step b at a volume ratio of 4:1 to obtain the collagen-thrombin mixed solution.

[0061] d. Preparation of fibrinogen To prepare a fibrinogen solution with a concentration of 50 mg / mL: Dissolve fibrinogen in sterile water and stir until all fibrinogen is dissolved to obtain a 50 mg / mL fibrinogen solution.

[0062] e. Preparation of collagen-fibrin composite gel The collagen-thrombin mixture and fibrin solution prepared in steps c and d were respectively placed in a collagen-thrombin mixture syringe and a fibrinogen syringe, as follows: Figure 1 As shown, the collagen-thrombin solution syringe and the fibrinogen solution syringe are assembled using a double frame, a Y-connector, a double plunger, and a needle. The double plunger is assembled with the syringe. The 22 G needle is assembled and the double plunger is pushed at 5 mm / s to expel the collagen-thrombin mixture and fibrinogen solution, thus obtaining the collagen-fibrinogen composite gel precursor.

[0063] The collagen-fibrin composite gel precursor was placed at 37°C and allowed to stand for 5 min to obtain the collagen-fibrin composite gel.

[0064] The state of the composite gel prepared in this embodiment was observed, the compressive stress-strain curve and elastic modulus of the composite gel were detected, and the cytotoxicity of the composite gel was detected according to the in vitro cytotoxicity test method of GB / T 16886.5-2017. The results are shown in Tables 1-2.

[0065] Example 2 This example is essentially the same as Example 1, except that the source of type I collagen extraction has been changed. In this example, type I collagen solution is extracted from bovine hide.

[0066] Example 3 This example is essentially the same as Example 1, except that the source of type I collagen extraction has been changed. In this example, type I collagen solution is extracted from bovine Achilles tendon.

[0067] Example 4 The method is basically the same as in Example 1, except that the concentration of the type I collagen solution is changed. In this example, the concentration of the type I collagen solution is 20 mg / mL.

[0068] Example 5 The method is basically the same as in Example 1, except that the concentration of the type I collagen solution is changed. In this example, the concentration of the type I collagen solution is 25 mg / mL.

[0069] Example 6 The example is basically the same as Example 1, except that the concentration of the thrombin solution is changed. In this example, the concentration of the thrombin solution is 400 IU / mL.

[0070] Example 7 The example is basically the same as Example 1, except that the concentration of the thrombin solution is changed. In this example, the concentration of the thrombin solution is 600 IU / mL.

[0071] Example 8 The study was essentially the same as in Example 1, except that the concentration of the fibrinogen solution was changed. In this example, the concentration of the fibrinogen solution was 40 mg / mL.

[0072] Example 9 The study was essentially the same as in Example 1, except that the concentration of the fibrinogen solution was changed. In this example, the concentration of the fibrinogen solution was 60 mg / mL.

[0073] Example 10 The method is basically the same as in Example 1, except that the mixing ratio of type I collagen solution and thrombin solution is changed. In this example, the mixing ratio of type I collagen solution and thrombin solution is 3:2.

[0074] Example 11 The process is basically the same as in Example 1, except that the extrusion speed of the mixed solution is changed. In this example, the extrusion speed of the mixed solution is 8 mm / s.

[0075] Example 12 The process is basically the same as in Example 1, except that the extrusion speed of the mixed solution is changed. In this example, the extrusion speed of the mixed solution is 10 mm / s.

[0076] Example 13 The example is basically the same as Example 1, except that the static temperature of the collagen-fiber composite gel precursor was changed. In this example, the static temperature of the collagen-fiber composite gel precursor was 25°C.

[0077] Example 14 This is basically the same as Example 1, except that the static temperature of the collagen-fiber composite gel precursor was changed. In this example, the static temperature of the collagen-fiber composite gel precursor was 30°C.

[0078] Example 15 The example is basically the same as Example 1, except that the settling time of the collagen-fiber composite gel precursor was changed. In this example, the settling time of the collagen-fiber composite gel precursor was 3 min.

[0079] Comparative Example 1 The method is basically the same as in Example 1, except that the concentration of the type I collagen solution is changed. In this example, the concentration of the type I collagen solution is 10 mg / mL.

[0080] Comparative Example 2 The method is basically the same as in Example 1, except that the concentration of the type I collagen solution is changed. In this example, the concentration of the type I collagen solution is 50 mg / mL.

[0081] Comparative Example 3 The example is basically the same as Example 1, except that the concentration of the thrombin solution is changed. In this example, the concentration of the thrombin solution is 300 IU / mL.

[0082] Comparative Example 4 The example is basically the same as Example 1, except that the concentration of the thrombin solution is changed. In this example, the concentration of the thrombin solution is 700 IU / mL.

[0083] Comparative Example 5 The study was essentially the same as in Example 1, except that the concentration of the fibrinogen solution was changed. In this example, the concentration of the fibrinogen solution was 30 mg / mL.

[0084] Comparative Example 6 The study was essentially the same as in Example 1, except that the concentration of the fibrinogen solution was changed. In this example, the concentration of the fibrinogen solution was 70 mg / mL.

[0085] Comparative Example 7 The method is basically the same as in Example 1, except that the mixing ratio of type I collagen solution and thrombin solution is changed. In this example, the mixing ratio of type I collagen solution and thrombin solution is 1:1.

[0086] Comparative Example 8 The process is basically the same as in Example 1, except that the extrusion speed of the mixed solution is changed. In this example, the extrusion speed of the mixed solution is 3 mm / s.

[0087] Comparative Example 9 The process is basically the same as in Example 1, except that the extrusion speed of the mixed solution is changed. In this example, the extrusion speed of the mixed solution is 15 mm / s.

[0088] Comparative Example 10 The example is basically the same as Example 1, except that the static temperature of the collagen-fiber composite gel precursor was changed. In this example, the static temperature of the collagen-fiber composite gel precursor was 20°C.

[0089] Comparative Example 11 The example is basically the same as Example 1, except that the settling time of the collagen-fiber composite gel precursor was changed. In this example, the settling time of the collagen-fiber composite gel precursor was 1 min.

[0090] Test case The mechanical properties of the collagen-fiber composite gels of the embodiments and comparative examples of this invention were determined at 50% compression deformation, and the cytotoxicity of the collagen-fiber composite gels of the embodiments and comparative examples was determined according to the in vitro cytotoxicity test method of GB / T 16886.5-2017. The cytotoxicity test method was as follows: extracts of the composite gels prepared in the embodiments and comparative examples were prepared, and human bone marrow mesenchymal stem cells (hBMSCs) were seeded into culture dishes and cultured until near confluence. Experimental groups and blank groups were set up. In the experimental group, the cell culture medium was discarded, and the extracts of the materials from each embodiment and comparative example were added for culture; in the blank group, only cell culture medium was used for culture. Cell viability was determined using the CCK-8 assay. The results are shown in Tables 1 and 2.

[0091] Table 1. Mechanical properties of the composite gels in the examples and comparative examples

[0092] Table 2. Comparison of absorbance for cytotoxicity detection of each composite gel in the examples and comparative examples.

[0093] As shown in the table above, there were no significant differences in the mechanical properties and cytotoxicity of the collagen-fibrin composite gels prepared in Examples 1-15. Compared with the blank group, the collagen-fibrin composite gels prepared in the examples and comparative examples did not exhibit cytotoxicity; on the contrary, they promoted cell growth, although the cell proliferation rates differed. The composite gel in Comparative Example 1 did not show significant differences in mechanical properties compared to the composite gel in Example 1, but due to its low collagen concentration, it resulted in insufficient nutrient supply to the cells, leading to reduced cell proliferation. The composite gel in Comparative Example 2, due to its high collagen concentration, experienced a slower gelation process and decreased mechanical properties, but cell proliferation increased. The composite gel in Comparative Example 3, due to its low thrombin concentration, had difficulty forming gels, resulting in decreased mechanical properties. The composite gel in Comparative Example 4, with its excessively high thrombin concentration, had a short gelation time, which was unfavorable for actual surgical procedures. The composite gel in Comparative Example 5, with its excessively low fibrin concentration, resulted in a poor gel state and decreased mechanical properties. Comparative Example 6: The high fibrin concentration in the composite gel led to excessively rapid gelation, which was also detrimental to the actual surgical procedure. Comparative Example 7: The reduced collagen and increased thrombin ratio in the composite gel resulted in reduced cellular nutrient supply. Comparative Example 8: The excessively slow extrusion speed of the composite gel caused gel blockage of the Y-joint during the procedure. Comparative Example 9: The excessively fast extrusion speed of the composite gel resulted in uneven mixing of the collagen / thrombin solution and fibrin solution. Comparative Example 10: The excessively low settling temperature of the composite gel was detrimental to gel formation. Comparative Example 11: The excessively short settling time of the composite gel also resulted in poor gel condition.

[0094] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for preparing collagen-fibrin composite gel, characterized in that, Includes the following steps: S1: Mix collagen solution with thrombin solution to obtain collagen-thrombin mixed solution; S2: Preparation of fibrinogen solution; S3: The collagen-thrombin mixed solution from step S1 and the fibrinogen solution from step S2 are respectively loaded into a dual syringe, squeezed out simultaneously, mixed, and the composite gel precursor is obtained. After standing, the collagen-fibrin composite gel is obtained.

2. The preparation method according to claim 1, characterized in that, In step S1, the collagen solution includes a type I collagen solution.

3. The preparation method according to any one of claims 1-2, characterized in that, In step S1, the concentration of the collagen solution is 20-40 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the thrombin solution is 400-600 IU / mL.

5. The preparation method according to claim 1, characterized in that, In step S1, the volume ratio of the collagen solution to the thrombin solution is 3-4:1-2.

6. The preparation method according to claim 1, characterized in that, In step S2, the fibrinogen solution is obtained by dissolving fibrinogen in water; The concentration of the fibrinogen solution is 40-60 mg / mL.

7. The preparation method according to claim 1, characterized in that, In step S3, the extrusion speed is 5-10 mm / s.

8. The preparation method according to claim 1, characterized in that, In step S3, the settling temperature is 25-37℃ and the settling time is 3-5 min.

9. A collagen-fibrin composite gel prepared by the preparation method according to any one of claims 1-8.

10. The application of a collagen-fibrin composite gel prepared by the preparation method according to any one of claims 1-8, or the collagen-fibrin composite gel according to claim 9, characterized in that, Materials used to prepare cartilage defect repair materials.

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