Cartilage extracellular matrix and acellular small intestinal submucosa composite scaffold, and preparation method and application thereof

Abstract

The application belongs to the technical field of biological materials, and particularly relates to a kind of cartilage extracellular matrix and acellular small intestine submucosa composite scaffold and its preparation method and application, the scaffold is composed of 5~95wt% of cartilage ECM particles and acellular SIS matrix, and is a uniform or gradient composite structure, and in the gradient structure, one layer of cartilage ECM accounts for 50~100wt%, and the other layer accounts for 0~50wt%. Its preparation includes cartilage and SIS decellularization, particle preparation, mixing and dispersion, and freeze-drying or 3D printing forming. The application is composed of natural materials, retains type II collagen and other active ingredients of cartilage ECM, and improves mechanical properties with the help of SIS, without introducing synthetic polymers, and is safe in degradation and good in biocompatibility. The scaffold can flexibly regulate mechanical and biological properties, is suitable for weight-bearing joint cartilage and osteochondral defect repair of knee joint, ankle joint and the like, can be used alone or in combination with cells, and has good clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa, its preparation method and application. Background Technology

[0002] Tissue-engineered scaffolds have significant application value in the field of cartilage regeneration and repair. Especially in the repair of articular cartilage defects, scaffolds not only need to provide a microenvironment for cell adhesion, proliferation, and differentiation, but also need to possess certain mechanical properties to withstand joint loads and maintain structural stability, thereby supporting the long-term repair of cartilage tissue. Studies have shown that the mechanical properties of scaffolds (such as compressive modulus, elastic resilience, and fatigue strength) directly affect the quality of cartilage regeneration and functional recovery.

[0003] Currently, decellularized chondrocyte extracellular matrix (ECM) scaffolds exhibit good chondrogenic capacity due to the retention of abundant type II collagen, glycoglucan, and chondrogenic active factors. However, their mechanical strength is generally low, making them prone to disintegration, displacement, and detachment under load, and difficult to maintain their shape and position in heavy-duty environments. To improve mechanical properties, existing technologies often employ methods such as polymer blending, nanofiller reinforcement, or chemical crosslinking. However, these methods often introduce non-natural synthetic materials and chemical reagents such as crosslinking agents, potentially leading to issues related to biocompatibility and the safety of degradation products.

[0004] Submucosal layer of the small intestine (SIS) is a natural decellularized matrix material mainly composed of collagen. It possesses good mechanical strength and biocompatibility and has been widely used in soft tissue repair. However, SIS lacks cartilage-specific components, and its ability to induce cartilage regeneration is limited, making it difficult to meet the tissue-specific requirements for cartilage defect repair.

[0005] Although existing studies have attempted to improve scaffold performance through composite material strategies, there are currently no publicly available reports on the construction of a pure bio-based composite scaffold that combines cartilage ECM particles with decellularized SIS matrix to create a scaffold that combines cartilage-inducing activity and enhanced mechanical properties, and on the systematic optimization for the repair of weight-bearing cartilage defects.

[0006] Therefore, there is still a lack of a composite scaffold material in the current technology that can improve the overall mechanical properties of the scaffold while maintaining the specific bioactivity of cartilage tissue and is suitable for repairing cartilage defects under load. Summary of the Invention

[0007] To address the problems mentioned in the background art, this invention proposes a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa, its preparation method, and its application. By combining cartilage ECM particles with decellularized SIS matrix, the compressive modulus and structural stability of the scaffold are significantly improved, enabling it to better bear joint loads and providing reliable mechanical support for cartilage regeneration.

[0008] The technical solution adopted by this invention to solve its technical problem is as follows: A composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa is provided, comprising chondrocyte ECM particles and decellularized SIS matrix. The chondrocyte ECM particles are mixed with the decellularized SIS matrix at a mass fraction of 5-95 wt%, and the average particle size of the chondrocyte ECM particles is 20-500 μm. ECM refers to chondrocyte extracellular matrix, and SIS refers to small intestinal submucosa.

[0009] Furthermore, the composite scaffold is a uniform composite structure or a gradient composite structure.

[0010] Furthermore, when it is a uniform composite structure, the particle size of the cartilage ECM particles is 20~500μm, and the mass ratio of decellularized SIS matrix to cartilage ECM particles is 1:19 to 19:1.

[0011] Furthermore, when it is a gradient composite structure, it includes two layers, wherein the mass fraction of cartilage ECM particles in one layer is 50~100wt% and the mass fraction of SIS is 0~50wt%, and the mass fraction of cartilage ECM particles in the other layer is 0~50wt% and the mass fraction of SIS is 50~100wt%.

[0012] This invention also provides a method for preparing a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa, comprising the following steps:

[0013] S1. Decellularization and Granule Preparation of Cartilage: Cartilage tissue was selected and cut into pieces, and the cartilage was subjected to repeated freeze-thaw cycles; then it was treated in a surfactant solution; nucleic acids were removed by enzymatic hydrolysis with DNase and RNase; finally, it was freeze-dried, pulverized and sieved to obtain cartilage ECM granules;

[0014] S2, SIS decellularization treatment: Small intestinal tissue was selected, the small intestinal mucosa and muscle layer were peeled off, and the submucosal tissue of the small intestine was taken; after degreasing treatment and treatment with surfactant, decellularization was performed, and after pulverization, SIS matrix suspension was prepared;

[0015] S3. Mixing and dispersing particles with SIS: Mix the chondrocyte extracellular matrix (ECM) particles with the SIS matrix suspension according to the preset mass ratio, and stir to disperse to obtain a uniform suspension.

[0016] S4. Molding: The suspension is injected into the mold, pre-frozen, and then freeze-dried or 3D printed to obtain the composite scaffold.

[0017] Further, in step S1, the number of freeze-thaw cycles is 1-4, the surfactants include but are not limited to 0.1%~2.0% Triton X-100 solution, 0.5%~3.0% sodium deoxycholate SDC or 0.5%~3.0% sodium dodecyl sulfate SDS, the treatment time is 2-24h, and the enzymatic hydrolysis uses DNase and RNase.

[0018] Furthermore, in step S4, when preparing a uniform composite structure scaffold, the solid content of the suspension is 2-90%, the pre-freezing temperature is -80℃ to -20℃, the pre-freezing time is 1-6h, and the freeze-drying time is 5-100h.

[0019] Further, in step S4, when preparing the gradient composite structure scaffold, a monolayer suspension made of 0-50 wt% cartilage ECM particles and 50-100 wt% SIS matrix is ​​first injected into a mold for pre-freezing. Then, a surface suspension made of 50-100 wt% cartilage ECM particles and 0-50 wt% SIS matrix is ​​added to its surface for pre-freezing, followed by freeze-drying.

[0020] The application of a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa in the preparation of tissue-engineered implants for cartilage defect repair, wherein the implants are used for the repair of cartilage defects or osteochondral defects in weight-bearing joints, and the composite scaffold can be used alone or in combination with chondrocytes or stem cells.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] (1) By combining cartilage ECM particles with decellularized SIS matrix, the present invention significantly improves the compressive modulus and structural stability of the scaffold, enabling it to better bear joint loads and providing reliable mechanical support for cartilage regeneration.

[0023] (2) The composite scaffold of the present invention retains the key active components in cartilage ECM (such as type II collagen and glycosaminoglycans), has excellent cartilage induction ability, and can effectively promote the adhesion, proliferation and functional expression of chondrocytes.

[0024] (3) The scaffold of the present invention is made entirely of decellularized natural biomaterials, without the introduction of synthetic polymers, and the degradation products are safe and have excellent biocompatibility, which reduces the risk of inflammatory response and is conducive to clinical translation.

[0025] (4) By adjusting the particle ratio, particle size and adopting a gradient structure design, the mechanical properties, pore structure and bioactivity of the scaffold can be flexibly controlled to meet the repair needs of different defect locations and depths. Attached Figure Description

[0026] Figure 1 This is a SEM image of the cartilage ECM-SIS composite scaffold from Example 1.

[0027] Figure 2 The stress-strain curve of the composite stent in Example 1 is shown in the compression test diagram.

[0028] Figure 3 SEM images of cartilage ECM scaffolds for comparison.

[0029] Figure 4 The stress-strain curves for compression testing of a pure cartilage ECM scaffold are shown as a comparative example. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1:

[0032] 1. Source of materials

[0033] Cartilage: Transparent cartilage from the knee joints of fresh pigs from the slaughterhouse;

[0034] SIS: Fresh, healthy pig small intestines from the slaughterhouse.

[0035] 2. Preparation steps

[0036] Preparation of cartilage ECM particles: hyaline cartilage from pig knee joints was peeled, cut into small pieces, and subjected to one freeze-thaw cycle. Then, it was transferred to 1.0% Triton X-100 solution and treated with a gentle shaker for 24 hours. After removing nucleic acids by enzymatic hydrolysis with DNase and RNase, it was repeatedly washed with purified water until the washing solution was clear. After freeze-drying, it was pulverized and sieved through a standard sieve to collect cartilage ECM particles with a particle size of 100~150μm. The particles were then sealed for later use.

[0037] Preparation of SIS matrix suspension: Fresh pig small intestine was taken, and the mucosa and muscle layer were removed by mechanical dissection, while the submucosal tissue of the small intestine was retained. After defatting the tissue, 0.5% Triton x-100 solution was added for decellularization to fully remove cellular components and obtain decellularized SIS matrix. After freeze-drying, it was pulverized with a grinder and prepared into SIS matrix suspension with purified water.

[0038] Particles and SIS Mixing and Dispersion: Cartilage ECM particles account for 80 wt% of the total solid mass. They are mixed with the above-mentioned SIS matrix suspension and fully dispersed by magnetic stirring to ensure that the cartilage ECM particles are evenly distributed in the SIS matrix, forming a uniform suspension without obvious agglomeration.

[0039] Molding: The homogeneous suspension was injected into a metal mold with dimensions of 40mm×30mm×3mm, pre-frozen at −30℃ for 2 hours, then transferred to a vacuum freeze dryer for continuous freeze-drying for 48 hours. After removal, it was aseptically treated to obtain a porous composite scaffold. Figure 1 As shown, this is a SEM image of the cartilage ECM-SIS composite scaffold from Example 1, with the SIS highlighted in red; as... Figure 2 As shown, this is a stress-strain curve of the composite stent in Example 1 under compression test.

[0040] Example 2:

[0041] 1. Source of materials

[0042] Cartilage: Healthy cow knee joint cartilage;

[0043] SIS: Healthy pig small intestine segment.

[0044] 2. Preparation steps

[0045] Preparation of cartilage ECM particles: Cartilage from the knee joint of bovine patients was peeled, cut into 3mm×3mm×3mm pieces, and subjected to repeated freeze-thaw cycles at -80℃ / room temperature 3 times; then transferred to 2.0% Triton X-100 solution and treated with a gentle shaker at 37℃ for 20h; after washing 3 times with PBS buffer, a mixed enzyme solution of 50U / mL DNase and 2U / mL RNase was added, and the mixture was enzymatically digested at 37℃ for 4h to remove nucleic acids; after freeze-drying, the cartilage ECM particles were pulverized using a high-speed grinder and sieved through a standard sieve to collect 200-300μm cartilage ECM particles for later use.

[0046] Preparation of SIS matrix suspension: The mucosa and muscle layer of porcine small intestine were peeled off, and the submucosal tissue of small intestine was taken and treated with 0.05% trypsin solution at 37℃ for 30 min to remove fat; it was then transferred to 0.5% Triton X-100 solution and treated on a shaker for 18 h, with the solution being changed twice during the process; after washing 5 times with PBS buffer, it was freeze-dried and ground, and then prepared into a 5% (w / w) SIS matrix suspension with deionized water.

[0047] Particles and SIS mixed and dispersed: Mix SIS matrix and cartilage ECM particles at a mass ratio of 1:5, add deionized water to adjust the solid content of the suspension to 9%, and magnetically stir for 30 minutes (300 r / min) to ensure uniform dispersion of particles without agglomeration.

[0048] Molding: The suspension was injected into a cylindrical mold (8 mm in diameter and 5 mm in height), pre-frozen at -50°C for 5 hours, and then transferred to a freeze dryer for freeze drying at -55°C and 10 Pa vacuum for 40 hours. After removal, it was aseptically packaged to obtain a uniform composite structure scaffold.

[0049] Example 3:

[0050] Cartilage: Hyaluronic acid cartilage from the knee joint of healthy sheep;

[0051] SIS: Healthy sheep small intestine segment (weight 15-20kg).

[0052] 2. Preparation steps

[0053] Preparation of cartilage ECM particles: Following the decellularization process in Example 2, the hyaline cartilage of sheep knee joint was cut into pieces, frozen and thawed three times, treated with 0.5% Triton X-100 for 22 hours, enzymatically hydrolyzed for 3 hours, pulverized, and sieved to obtain cartilage ECM particles of 20-50 μm.

[0054] Preparation of SIS matrix suspension: Following the decellularization process in Example 2, the submucosal tissue of sheep small intestine was degreased and treated with 0.5% Triton X-100 for 20 hours to prepare a SIS matrix suspension with a mass concentration of 6%. 0.5% trehalose (thickening agent) was added to adjust the viscosity to 5000-8000 mPa·s to meet the requirements of 3D printing.

[0055] Gradient suspension preparation:

[0056] Bottom suspension: 15wt% cartilage ECM particles + 85wt% SIS matrix, solid content 10%, stirred evenly and then ultrasonically dispersed for 20 min;

[0057] Surface suspension: 70wt% cartilage ECM particles + 30wt% SIS matrix, solid content 9%, stirred evenly and then ultrasonically dispersed for 20min.

[0058] 3D printing process: A bio-3D printer (nozzle diameter 0.4mm) was used to print the bottom suspension first at a printing speed of 5mm / s, a layer thickness of 0.2mm, and a printing height of 3mm. Then, a top suspension was printed on the bottom surface at a printing height of 2mm. The overall support dimensions were 10mm×10mm×5mm. After printing, the support was pre-frozen at -50℃ for 6 hours, freeze-dried for 48 hours, and then sterilized before use.

[0059] Example 4:

[0060] 1. Source of materials

[0061] Cartilage: Healthy pig nasal cartilage;

[0062] SIS: Healthy pig small intestine segment;

[0063] Mesenchymal stem cells: Rabbit bone marrow mesenchymal stem cells (third generation, concentration 1×10⁻⁶) 6 (cells / mL).

[0064] 2. Preparation steps

[0065] Preparation of cartilage ECM particles: Using porcine nasal cartilage as raw material, connective tissue was removed, and after washing and removing grease, the cartilage was separated and cut into pieces. The pieces were then subjected to repeated freeze-thaw cycles at -80℃ / 25℃ for 4 times. After treatment in 1.0% Triton X-100 solution for 24 hours, the cartilage was washed and then enzymatically hydrolyzed for 5 hours to remove nucleic acids. The particles were then pulverized and sieved to obtain cartilage ECM particles of 150-200μm.

[0066] Preparation of SIS matrix suspension: After defatting the submucosa of porcine small intestine, the SIS matrix suspension was prepared by treating it with 0.5% Triton X-100 for 22 hours and then ultrasonically dispersed for 25 minutes.

[0067] Gradient suspension preparation:

[0068] The underlying suspension consists of 10 wt% cartilage ECM particles + 90 wt% SIS matrix, with a solid content of 10%.

[0069] Surface suspension: 65wt% cartilage ECM particles + 35wt% SIS matrix, solid content 9%.

[0070] Molding and Cell Loading: First, the bottom suspension was injected into the mold and pre-frozen at -48℃ for 6 hours; then, the top suspension was laid on top and pre-frozen at -48℃ for 4 hours; after freeze-drying for 49 hours, the mesenchymal stem cell suspension was loaded into the scaffold using a negative pressure osmosis method (loading capacity 1×10⁻⁶). 6 cells / cm 2 ), incubated at 37℃ for 2 hours to obtain cell-loaded gradient composite scaffolds.

[0071] Example 5:

[0072] 1. Source of materials

[0073] Cartilage: Healthy hyaline cartilage in the knee joint (age 1-2 years);

[0074] SIS: Healthy bovine small intestine segment (weighing 300-350kg).

[0075] 2. Preparation steps

[0076] Preparation of cartilage ECM particles: Bovine cartilage was cut into blocks and freeze-thawed three times. The blocks were then treated with 1.5% Triton X-100 solution for 18 hours, followed by enzymatic hydrolysis for 3 hours to remove nucleic acids. After pulverization, the cartilage ECM particles of 50-100 μm were collected by sieving.

[0077] Preparation of SIS matrix suspension: After defatting the SIS tissue, it was treated with 0.5% Triton X-100 for 19 h to prepare a SIS matrix suspension with a mass concentration of 4%, and magnetically stirred for 40 min until uniformly dispersed.

[0078] Particles and SIS mixed and dispersed: SIS matrix and cartilage ECM particles were mixed at a mass ratio of 1:6, the solid content of the suspension was adjusted to 8%, and ultrasonically dispersed for 30 minutes (power 100W) to eliminate micro-agglomeration.

[0079] Molding: The suspension was injected into a square mold (10mm×10mm×3mm), pre-frozen at -48℃ for 5h, freeze-dried for 50h, and sterilized by 25kGy dose of sterile irradiation to obtain a bovine ECM uniform composite scaffold.

[0080] Example 6:

[0081] 1. Source of materials

[0082] Cartilage: Healthy pig ear cartilage;

[0083] SIS: Fresh and healthy pork small intestine segments.

[0084] 2. Preparation steps

[0085] Preparation of cartilage ECM particles: Using pig ear cartilage as raw material, the skin, subcutaneous tissue and cartilage membrane were removed, the cartilage was cut into pieces and frozen and thawed twice, treated with 1.0% sodium deoxycholate SDC for 16 h, enzymatically hydrolyzed for 4 h to remove nucleic acid, and then crushed and sieved to obtain cartilage ECM particles of 300-400 μm.

[0086] Preparation of SIS matrix suspension: After 21 hours of defatting and post-treatment of SIS tissue, a SIS matrix suspension with a mass concentration of 6% was prepared.

[0087] Gradient suspension preparation:

[0088] The base layer (bone side): 20wt% cartilage ECM particles + 80wt% SIS matrix, with a solid content of 10%;

[0089] Surface layer (cartilage side): 80wt% cartilage ECM particles + 20wt% SIS matrix, solid content 9%.

[0090] Molding and cell seeding: First, inject the bottom suspension into the mold and pre-freeze at -50℃ for 3.0 h; then add the top suspension and pre-freeze at -50℃ for 3.0 h; after freeze-drying for 48 h, seed with chondrocytes (concentration 2×10⁻⁶). 6 cells / cm 2 The scaffold for cartilage repair was obtained by culturing at 37℃ and 5% CO2 for 48 hours.

[0091] Comparative Example 1: Pure cartilage ECM scaffold

[0092] 1. Source of materials

[0093] Cartilage: Same as in Example 1, i.e., hyaline cartilage from the knee joint of fresh pigs from the slaughterhouse.

[0094] 2. Preparation steps

[0095] Cartilage processing: The transparent cartilage of the pig knee joint was peeled off, cut into small pieces, and placed in purified water for repeated freeze-thaw cycles 3 times. Then it was transferred to 1.0% Triton X-100 solution and treated on a gentle shaker for 24 hours. After removing nucleic acids by enzymatic hydrolysis with DNase and RNase, it was washed repeatedly with purified water until the washing solution was clear.

[0096] Molding: The processed cartilage ECM tissue was pulverized, suspended in physiological saline, stirred evenly, and then poured into a 40mm×30mm×3mm metal mold. It was pre-frozen at −80℃ for 5 hours, then transferred to a vacuum freeze dryer for continuous freeze-drying for 48 hours. After removal, it underwent aseptic treatment to obtain a pure cartilage ECM porous scaffold. Figure 3 The image shown is a comparative SEM image of a cartilage ECM scaffold; as shown... Figure 4 As shown, this is a stress-strain curve of a pure cartilage ECM scaffold under compression test, representing a comparative example.

[0097]

[0098] In summary, the six embodiments of this invention systematically verified the feasibility and superiority of the cartilage ECM and decellularized SIS composite scaffold through diverse natural biomaterial sources (covering cartilage and small intestinal tissues from different species such as pigs, sheep, dogs, and cattle), flexible and adjustable preparation parameters (cartilage ECM particle size of 20-500 μm to adapt to different repair needs, and precise control of SIS matrix concentration and suspension solid content), differentiated structural design (targeted optimization of uniform and gradient structures), and multi-factor molding processes (combination of freeze-drying and 3D printing). In the embodiments, the composite scaffolds achieved a synergistic improvement in mechanical properties and bioactivity—the compressive modulus was increased by 2-3 times compared to pure cartilage ECM scaffolds, the elastic recovery rate was maintained above 84%, and the active components such as type II collagen of cartilage ECM were completely preserved. Furthermore, the gradient structural design achieved functional zoning of the surface cartilage induction and the underlying mechanical support. Furthermore, the scaffold can be loaded with stem cells, chondrocytes, etc. to further enhance the repair effect, making it suitable for cartilage and osteochondral defects in different weight-bearing joints such as the knee, hip, and ankle. Its all-natural biological composition, controllable preparation process, and personalized customization capabilities not only solve the core problems of insufficient mechanical strength and biocompatibility risks in existing technologies, but also provide a standardized and scalable tissue engineering scaffold solution for clinical translation, showing broad application prospects.

[0099] In summary, this invention, through a composite strategy of cartilage ECM particles and decellularized SIS matrix, successfully solves the problems in existing technologies, such as the difficulty in balancing the mechanical strength and bioactivity of the scaffold and the poor safety of the synthetic materials. Furthermore, the preparation process is highly scalable and the parameters can be flexibly adjusted, allowing for individualized design based on different defect locations, defect sizes, and weight-bearing requirements. This provides a tissue engineering scaffold solution with both practicality and clinical translational potential for the repair of weight-bearing joint cartilage defects and osteochondral defects.

[0100] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa, characterized in that, It includes cartilage ECM particles and decellularized SIS matrix, wherein the cartilage ECM particles are mixed with the decellularized SIS matrix at a mass fraction of 5-95 wt%, and the average particle size of the cartilage ECM particles is 20-500 μm.

2. The composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa according to claim 1, characterized in that, The composite scaffold is a uniform composite structure or a gradient composite structure.

3. The composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa according to claim 2, characterized in that, When it is a uniform composite structure, the particle size of the cartilage ECM particles is 20~500μm, and the mass ratio of decellularized SIS matrix to cartilage ECM particles is 1:19 to 19:

1.

4. The composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa according to claim 2, characterized in that, When it is a gradient composite structure, the mass fraction of cartilage ECM particles in one layer is 50~100wt% and the mass fraction of SIS is 0~50wt%, and the mass fraction of cartilage ECM particles in the other layer is 0~50wt% and the mass fraction of SIS is 50~100wt%.

5. A method for preparing a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Decellularization and Granule Preparation of Cartilage: Cartilage tissue was selected and cut into pieces, and the cartilage was subjected to freeze-thaw treatment; then it was treated with a surfactant solution. Nucleic acid was removed by enzymatic hydrolysis with DNase and RNase; finally, the cartilage ECM particles were obtained by freeze-drying, pulverizing and sieving. S2, SIS decellularization treatment: Small intestinal tissue was selected, the small intestinal mucosa and muscle layer were separated, and the submucosal tissue of the small intestine was taken; After degreasing and decellularization with surfactant, a SIS matrix suspension was prepared. S3. Mixing and dispersing particles with SIS: Mix cartilage ECM particles with SIS matrix suspension according to the preset mass ratio, and stir to disperse to obtain a uniform suspension; S4. Molding: The suspension is injected into the mold, pre-frozen, and then freeze-dried or 3D printed to obtain the composite scaffold.

6. The method for preparing a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa according to claim 5, characterized in that, In step S1, the freeze-thaw cycles are 1-4 times, and the surfactants include, but are not limited to, 0.1%~2.0% Triton X-100 solution, 0.5%~3.0% sodium deoxycholate or 0.5%~3.0% sodium dodecyl sulfate. The treatment time is 2-24 hours, and the enzymatic hydrolysis uses DNase and RNase.

7. The method for preparing a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa according to claim 5, characterized in that, In step S4, when preparing a uniform composite structure scaffold, the solid content of the suspension is 2-90%, the pre-freezing temperature is -80℃ to -20℃, the pre-freezing time is 1-6h, and the freeze-drying time is 5-100h.

8. The method for preparing a composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa according to claim 5, characterized in that, In step S4, when preparing the gradient composite structure scaffold, the bottom suspension, which is made by mixing 0-50 wt% cartilage ECM particles and 50-100 wt% SIS matrix, is first injected into the mold for pre-freezing. Then, a surface suspension, which is made by mixing 50-100 wt% cartilage ECM particles and 0-50 wt% SIS matrix, is added to its surface for pre-freezing and freeze-drying.

9. The application of the composite scaffold of chondrocyte extracellular matrix and decellularized small intestinal submucosa as described in any one of claims 1-4 in the preparation of tissue-engineered implants for cartilage defect repair, characterized in that, The implant is used to repair cartilage defects or osteochondral defects in weight-bearing joints. The composite scaffold can be used alone or in combination with chondrocytes or stem cells.

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