Shapeable and self-adhesive cartilage repair material and preparation method thereof

Abstract

The invention provides a moldable and self-adhesive cartilage repair material and a preparation method thereof, and the preparation method comprises the following steps: (1) uniformly mixing decellularized cartilage particles obtained by treating fresh articular cartilage tissue, a decellularized fibrous dermal matrix obtained by treating fresh skin tissue, mussel protein and purified water to obtain composite slurry; the composite slurry is prepared from the following components in percentage by mass: 1 to 30 weight percent of decellularized cartilage particles, 55 to 73 weight percent of decellularized fibrous dermal matrix, 2 to 5 weight percent of mussel protein and 5 to 21 weight percent of purified water; and (2) carrying out freeze-drying treatment on the composite slurry to obtain the moldable and self-adhesive cartilage repair material. The cartilage repair material provided by the invention retains main components of cartilage, has plasticity and self-adhesion performance, and can effectively simulate a cartilage regeneration repair microenvironment.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a malleable, self-adhesive cartilage repair material and its preparation method. Background Technology

[0002] Articular cartilage, as the connection point of the human limb skeleton, primarily functions to bear and transmit loads and provide excellent load-bearing lubrication for the joint surfaces. Joint defects and pathological degeneration caused by physiological or pathological injuries, such as sports injuries and accidents, especially cartilage damage due to arthritis, are increasingly common, seriously harming human health. Articular cartilage lacks blood vessels and cells, making it difficult for it to repair itself and restore its physiological function once damaged; surgical intervention is usually required.

[0003] Clinically, the treatment of cartilage damage can be mainly divided into traditional surgical treatment, autologous cartilage transplantation, and tissue-engineered scaffold treatment. Traditional surgical treatment primarily involves arthroscopic microfracture. This technique involves removing the damaged cartilage and then using an articular cone to create multiple micropores 2-4 mm deep at 3-4 mm intervals in the subchondral bone plate. These micropores promote blood clotting from the medullary cavity at the damaged cartilage site, providing bone marrow mesenchymal stem cells and nutrients for cartilage repair. This technique is simple to perform and can repair damage to a certain extent, but it primarily repairs fibrous cartilage, whose structure and mechanical properties differ significantly from normal cartilage tissue. Autologous cartilage transplantation mainly involves harvesting cartilage tissue from non-weight-bearing areas of the patient's own body. However, it suffers from limited availability and complex procedures. In recent years, with advancements in tissue engineering technology, a series of tissue-engineered scaffolds for cartilage defects have been developed.

[0004] Currently available cartilage repair scaffolds, such as ChondroGide® and COLTRIX CartiRegen, are available in membrane and gel forms, respectively, with type I and type III collagen as their main components. Normal adult cartilage is 2-3 mm thick, and damage primarily results in irregular defects. Existing cartilage repair scaffold materials are difficult to effectively adhere and fix to the defect site, requiring the use of fibrin adhesives and sutures, thus increasing surgical complexity and treatment costs. Furthermore, natural cartilage is mainly composed of type II collagen and polysaccharides, while existing cartilage repair materials are primarily type I and type III collagen, which differ from the main components of cartilage and cannot provide the necessary microenvironment for cartilage regeneration and repair.

[0005] Therefore, there is an urgent need for a malleable, self-adhesive cartilage repair material and its preparation method. Summary of the Invention

[0006] This invention provides a malleable, self-adhesive cartilage repair material and its preparation method. The provided cartilage repair material retains the main components of cartilage and has both malleability and self-adhesive properties, effectively simulating the microenvironment for cartilage regeneration and repair.

[0007] In a first aspect, the present invention provides a method for preparing a malleable, self-adhesive cartilage repair material, the method comprising: (1) A composite slurry is prepared by mixing decellularized cartilage particles obtained from fresh articular cartilage tissue, decellularized fibrous dermal matrix obtained from fresh skin tissue, mussel protein, and purified water. The composite slurry comprises the following components in the following mass fractions: decellularized cartilage particles 1wt%~30wt%, decellularized fibrous dermal matrix 55wt%~73wt%, mussel protein 2wt%~5wt%, and purified water 5wt%~21wt%. (2) The composite slurry is freeze-dried to obtain the malleable, self-adhesive cartilage repair material.

[0008] Preferably, the decellularized cartilage particles are obtained by the following method: Fresh articular cartilage tissue was subjected to a series of processes, including grinding, treatment with peracetic acid solution, washing, treatment with Triton solution, washing, treatment with DNase enzyme solution, and washing, to obtain the decellularized cartilage particles.

[0009] Preferably, the decellularized fibrous dermal matrix is ​​obtained by the following method: Fresh skin tissue is peeled to obtain dermal tissue; the dermal tissue is then ground, treated with peracetic acid solution, washed, treated with Triton solution, and washed again to obtain the decellularized fibrous dermal matrix.

[0010] Preferably, the fresh articular cartilage tissue and the fresh skin tissue are derived from at least one of human, pig, cow, sheep, horse, donkey and monkey.

[0011] Preferably, the concentration of the peracetic acid solution is 0.1% to 0.4%.

[0012] More preferably, the concentration of the Triathon solution is 0.2% to 2%.

[0013] Preferably, the decellularized cartilage particles are obtained by the following method: Fresh articular cartilage tissue was ground to obtain cartilage particles with a particle size of less than 500µm; The cartilage particles were soaked in the peracetic acid solution for 0.5 to 2 hours, and then the peracetic acid was removed by washing to obtain the first cartilage particles. The first cartilage particles were added to the Triton solution and shaken for 4-24 hours. Then, the Triton was removed by washing to obtain the second cartilage particles. The second cartilage particles were added to a DNase enzyme solution and shaken for 2-24 hours. Then, the DNase enzyme was removed by washing to obtain the decellularized cartilage particles.

[0014] More preferably, the concentration of the DNase enzyme solution is 2~50 U / mL.

[0015] Preferably, the decellularized fibrous dermal matrix is ​​obtained by the following method: The dermis and subcutaneous fat of fresh skin tissue are removed using a peeling machine to obtain dermal tissue. The dermal tissue was ground to obtain a fibrous dermal matrix; The fibrous dermal matrix is ​​immersed in the peracetic acid solution for 0.5-2 hours, and then the peracetic acid is removed by washing to obtain the first matrix; The first matrix is ​​added to the Triton solution and shaken for 6-24 hours. Then, the Triton is removed by washing to obtain the decellularized fibrous dermal matrix.

[0016] Preferably, step (1) further includes adding an inducing agent to the composite slurry.

[0017] More preferably, the concentration of the inducing agent in the composite slurry is 200~1000nM.

[0018] Preferably, the composite slurry comprises the following components in the following mass fractions: 20wt%~30wt% decellularized cartilage particles, 55wt%~73wt% decellularized fibrous dermal matrix, 2wt%~5wt% mussel protein, and 5wt%~10wt% purified water.

[0019] Preferably, in step (2): the freeze-drying process includes pre-freezing and vacuum drying.

[0020] Preferably, the pre-freezing treatment is performed at a temperature of -80 to -20°C for 2 to 8 hours.

[0021] Preferably, the vacuum drying process is performed at a temperature of -20 to -3°C for 24 to 72 hours.

[0022] In a second aspect, the present invention provides a malleable, self-adhesive cartilage repair material prepared by the preparation method described in the first aspect above.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The malleable, self-adhesive cartilage repair material provided by this invention is composed of decellularized cartilage particles, fibrous decellularized dermal matrix, and mussel protein. The decellularized cartilage particles retain the main components of natural cartilage (II collagen and polysaccharides), which can provide a similar tissue microenvironment for cartilage repair. Moreover, the fibrous decellularized dermal matrix has a higher specific surface area than traditional membranes, which is more conducive to promoting the adhesion and proliferation of stem cells inside the scaffold after microfracture surgery. At the same time, the entanglement between the fibrous structures endows the cartilage repair material with shaping ability, allowing it to be shaped and filled according to the irregular defect shape. Mussel protein, as a material with excellent wet adhesion, also has anti-inflammatory and antioxidant effects. After the composite scaffold prepared by this cartilage repair material absorbs blood from the microfracture, it can quickly exert its adhesive effect and fix it to the cartilage defect site. Compared with traditional cartilage repair materials, there is no need to use biological adhesives, which further reduces the complexity of surgery and treatment costs.

[0024] (2) The present invention also introduces an inducer (KGN) as a small molecule compound into the cartilage repair material, thereby promoting the differentiation of stem cells adhering to the composite scaffold into chondrocytes through KGN, so as to better realize the regeneration and repair of cartilage defects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is an immunohistochemical image of type II collagen in decellularized cartilage particles provided in Embodiment 1 of the present invention; Figure 2 This is an Alcian blue staining image of decellularized cartilage particles provided in Embodiment 1 of the present invention; Figure 3 This is a microscope image of a decellularized fibrous dermal matrix provided in Embodiment 1 of the present invention; Figure 4 This is a macroscopic view of a composite stent provided in Embodiment 1 of the present invention; Figure 5 This is a macroscopic view of a composite scaffold after shaping, provided in Embodiment 1 of the present invention; Figure 6 This is an image showing the effect of a composite scaffold provided in Embodiment 1 of the present invention on the adhesion of defective cartilage tissue. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but 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.

[0028] This invention provides a method for preparing a malleable, self-adhesive cartilage repair material, the method comprising: (1) A composite slurry is prepared by mixing decellularized cartilage particles obtained from fresh articular cartilage tissue, decellularized fibrous dermal matrix obtained from fresh skin tissue, mussel protein, and purified water. The composite slurry comprises the following components in the following mass fractions: decellularized cartilage particles 1wt%~30wt% (e.g., 1 wt%, 2 wt%, 5 wt%, 8 wt%, 10 wt%, 15 wt%, 18 wt%, 20 wt%, 22 wt%, 24 wt%, 25 wt%, 26 wt%, 28 wt%, or 30 wt%), decellularized fibrous dermal matrix 55wt%~73wt% (e.g., 55 wt%, 58 wt%, 60 wt%, 62 wt%, 65 wt%, 68 wt%, 70 wt%, or 73 wt%), and mussel protein 2wt%~5wt% (e.g., 2 wt%, 3 wt%, 4 wt%). 5 wt% or 5 wt%), purified water 5 wt% to 21 wt% (for example, it can be 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 12 wt%, 15 wt%, 16 wt%, 18 wt% or 21 wt%). (2) The composite slurry was freeze-dried to obtain a plastic, self-adhesive cartilage repair material.

[0029] In this embodiment of the invention, the cartilage repair material is composed of decellularized cartilage particles, fibrous decellularized dermal matrix, and mussel protein. The decellularized cartilage particles retain the main components of natural cartilage (II collagen and polysaccharides), providing a similar tissue microenvironment for cartilage repair. Furthermore, the fibrous decellularized dermal matrix has a higher specific surface area than traditional membranes, which is more conducive to promoting the adhesion and proliferation of stem cells within the scaffold after microfracture surgery. Simultaneously, the entanglement between the fibrous structures endows the cartilage repair material with shaping ability, allowing it to be molded and filled according to the shape of irregular defects. Mussel protein, as a material with excellent wet adhesion and anti-inflammatory and antioxidant effects, enables the composite scaffold prepared from this cartilage repair material to quickly exert its adhesive effect after absorbing blood from the microfracture, fixing it to the cartilage defect site. Compared to traditional cartilage repair materials, no biological adhesives are needed, further reducing surgical complexity and treatment costs.

[0030] In this embodiment of the invention, experiments have confirmed that, with other components remaining constant, the more decellularized cartilage particles there are, the closer the material is to the original tissue. However, if the amount of these particles exceeds 30 wt%, the other components are insufficient, and the excessive amount of decellularized cartilage particles cannot be completely coated, affecting adhesion performance and potentially causing the particles to detach. With other components remaining constant, if the amount of decellularized fibrous dermal matrix is ​​less than 55 wt%, it cannot effectively encapsulate the loaded decellularized cartilage particles; however, if its content is higher than 73 wt%, the amount of decellularized cartilage particles will be too low, failing to effectively provide the microenvironment required for cartilage regeneration and repair. With other components remaining constant, if the amount of mussel protein is less than 2 wt%, the amount is too low, resulting in poor adhesion; however, if its amount is higher than 5 wt%, the amount is too high, leading to excessive viscosity and affecting the uniformity of mixing of the components.

[0031] In some preferred embodiments, the decellularized cartilage particles are obtained by the following method: Fresh articular cartilage tissue was subjected to a series of processes, including grinding, treatment with peracetic acid solution, washing, treatment with Triton solution, washing, treatment with DNase enzyme solution, and washing, to obtain decellularized cartilage particles.

[0032] In some preferred embodiments, the decellularized fibrous dermal matrix is ​​obtained by the following method: Fresh skin tissue was peeled to obtain dermal tissue; the dermal tissue was then ground, treated with peracetic acid solution, washed, treated with Triton solution, and washed again to obtain decellularized fibrous dermal matrix.

[0033] In some preferred embodiments, the fresh articular cartilage tissue and fresh skin tissue are derived from at least one of human, pig, cow, sheep, horse, donkey and monkey.

[0034] In some preferred embodiments, the concentration of the peracetic acid solution is 0.1% to 0.4% (e.g., it can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35% or 0.4%).

[0035] In some more preferred embodiments, the concentration of the Triton solution is 0.2% to 2% (e.g., it can be 0.2%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.6%, 1.8% or 2%).

[0036] In some preferred embodiments, the decellularized cartilage particles are obtained by the following method: Fresh articular cartilage tissue was ground to obtain cartilage particles with a particle size of less than 500µm; The cartilage particles are soaked in a peracetic acid solution for 0.5 to 2 hours (e.g., 0.5 hours, 1 hour, 1.5 hours, or 2 hours), and then the peracetic acid is removed by washing to obtain the first cartilage particles. The first cartilage particles are added to a Triton solution and shaken for 4 to 24 hours (e.g., 4 hours, 6 hours, 10 hours, 15 hours, 18 hours, or 20 hours), and then the Triton is removed by washing to obtain the second cartilage particles. The second cartilage particles are added to a DNase enzyme solution and shaken for 2 to 24 hours (e.g., 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 24 hours). Then, the DNase enzyme is removed by washing to obtain decellularized cartilage particles.

[0037] In some preferred embodiments, the concentration of the DNase enzyme solution is 2 to 50 U / mL (e.g., it can be 2 U / mL, 5 U / mL, 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, 30 U / mL, 35 U / mL, 40 U / mL, 45 U / mL or 50 U / mL).

[0038] In this embodiment of the invention, peracetic acid works by oxidizing and destroying the cell membrane, enzyme system, and nucleic acid structure of microorganisms, creating conditions for subsequent enzymatic hydrolysis or cell separation; at the same time, it effectively kills bacteria, fungi, and other microorganisms attached to the surface of cartilage particles, preventing subsequent experimental contamination. Triton solution is then used to further disrupt the cell membrane and intracellular lipid structure, dissolving cell membrane lipids and thoroughly removing residual cell debris; then DNase enzyme is used to remove nucleic acid residues. In this way, the extracellular matrix of cartilage cells is preserved while cellular components are thoroughly removed, resulting in decellularized cartilage particles containing collagen II and polysaccharides.

[0039] In some preferred embodiments, the decellularized fibrous dermal matrix is ​​obtained by the following method: The dermis and subcutaneous fat of fresh skin tissue are removed using a peeling machine to obtain dermal tissue. The dermal tissue was ground to obtain a fibrous dermal matrix; The fibrous dermal matrix is ​​immersed in a peracetic acid solution for 0.5 to 2 hours (e.g., 0.5 hours, 1 hour, 1.5 hours, or 2 hours), and then the peracetic acid is removed by washing to obtain the first matrix. The first matrix is ​​added to the Triton solution and shaken for 6 to 24 hours (e.g., 6 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 24 hours), and then the Triton is removed by washing to obtain acellular fibrous dermal matrix.

[0040] In this embodiment of the invention, peracetic acid solution denatures proteins through strong oxidation, thereby destroying cell structure to treat membrane proteins, intracellular proteins, and pathogens; Triton solution dissolves the lipid bilayer, removing membrane protein fragments, treating lipid components and cell membrane debris, while washing removes any residual reagents each time. In this way, cells and genetic material are thoroughly removed, while preserving the structural and functional components of the extracellular matrix to the greatest extent possible, and collagen fiber bundles are completely preserved, resulting in a decellularized fibrous dermal matrix.

[0041] In some preferred embodiments, step (1) further includes adding an inducing agent to the composite slurry.

[0042] In some preferred embodiments, the concentration of the inducer in the composite slurry is 200-1000 nM (e.g., 200 nM, 250 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1000 nM).

[0043] In this embodiment of the invention, an inducer (KGN) as a small molecule compound is also introduced into the cartilage repair material. This KGN promotes the differentiation of stem cells adhered to the composite scaffold into chondrocytes, thereby better achieving the regeneration and repair of cartilage defects. If the concentration of the inducer in the composite slurry is too low, it will be difficult to fully exert its effect; however, if the concentration is too high, it will make the cartilage repair material toxic.

[0044] In some preferred embodiments, the composite slurry comprises the following components in the following mass fractions: 20 wt% to 30 wt% decellularized cartilage particles (e.g., 20 wt%, 22 wt%, 24 wt%, 25 wt%, 26 wt%, 28 wt%, or 30 wt%), 55 wt% to 73 wt% decellularized fibrous dermal matrix (e.g., 55 wt%, 58 wt%, 60 wt%, 62 wt%, 65 wt%, 68 wt%, 70 wt%, or 73 wt%), 2 wt% to 5 wt% mussel protein (e.g., 2 wt%, 3 wt%, 4 wt%, or 5 wt%), and 5 wt% to 10 wt% purified water (e.g., 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, or 10 wt%).

[0045] In some preferred embodiments, in step (2): the freeze-drying process includes pre-freezing and vacuum drying.

[0046] In some preferred embodiments, the pre-freezing temperature is -80 to -20°C (e.g., -80°C, -60°C, -50°C, -40°C, -30°C, -25°C, or -20°C), and the time is 2 to 8 hours (e.g., 2 hours, 4 hours, 6 hours, or 8 hours).

[0047] In some preferred embodiments, the vacuum drying process is performed at a temperature of -20 to -3°C (e.g., -20°C, -15°C, -10°C, -8°C, -6°C, or -3°C) for a time of 24 to 72 hours (e.g., 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 50 hours, 60 hours, 66 hours, or 72 hours).

[0048] In this embodiment of the invention, freeze-drying is performed at low temperatures, which effectively preserves bioactive components and avoids structural damage and functional loss caused by high temperatures. Furthermore, by pre-freezing and fixing the microstructure of the composite slurry, followed by vacuum drying, the material's natural porous network and mechanical support framework are preserved, maintaining the integrity of its three-dimensional structure and providing a suitable microenvironment for subsequent cell growth and tissue regeneration. This porous network not only facilitates cell infiltration and nutrient exchange but also regulates the material's mechanical properties and degradation rate through porosity. Simultaneously, moisture is further removed, significantly reducing the risk of material degradation and microbial contamination, enabling long-term stable storage of the cartilage repair material, facilitating transportation and clinical use. Thus, freeze-drying preserves the nanofiber structure and growth factor binding sites of the natural cartilage extracellular matrix, while maintaining the orientation and mechanical gradient of collagen fibers in the decellularized fibrous dermal matrix and solidifying the spatial distribution of mussel protein adhesion active groups, achieving synergistic optimization of the structure-function of the cartilage repair material. More importantly, the freeze-dried cartilage repair material can rapidly recover its initial rheological properties after rehydration, and its porous structure promotes shaping and fixation during surgical implantation.

[0049] The present invention also provides a malleable, self-adhesive cartilage repair material, which is prepared by any of the preparation methods described above.

[0050] Unless otherwise specified, the raw materials used in this invention can be commercially available products or synthesized using existing methods.

[0051] In this invention, the use of "and / or" between multiple technical features indicates that these technical features are connected by an "and / or" relationship, meaning that it can be any one of these technical features, or any combination of two or more of these technical features. In the description of the embodiments of this invention, unless otherwise expressly specified and limited, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance; unless otherwise specified or stated, the term "multiple" refers to two or more. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0052] To more clearly illustrate the technical solution and advantages of the present invention, the following describes in detail a malleable, self-adhesive cartilage repair material and its preparation method through several embodiments.

[0053] Example 1 A method for preparing a malleable, self-adhesive cartilage repair material includes: (1) Fresh pig joint cartilage tissue was taken and ground with liquid nitrogen grinder to obtain cartilage particles with a particle size of less than 500µm; (2) The cartilage particles were added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the cartilage particles were washed with physiological saline to remove the peracetic acid and the first cartilage particles were obtained. (3) The first cartilage particles were added to a 0.5% Triton solution at a ratio of 1g:5mL and shaken for 4h. Then, the Triton solution was washed with physiological saline to remove the Triton solution, and the second cartilage particles were obtained. Subsequently, the second cartilage particles were added to a 20U / mL DNase enzyme solution at a ratio of 1g:5mL and shaken for 8h. Then, the DNase enzyme was washed with physiological saline to remove the DNase enzyme, and decellularized cartilage particles were obtained. (4) Take fresh pig skin tissue, remove the dermis and subcutaneous fat using a peeling machine to obtain dermal tissue, cut it into small pieces of about 1cm×1cm, and grind it using a rubber grinder to obtain fibrous dermal matrix; (5) The fibrous dermal matrix obtained in (4) was added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the peracetic acid was removed by washing with physiological saline. (6) Add the fibrous dermal matrix treated in (5) to a 1% Triton solution at a ratio of 1g:5mL, shake for 6h, and then wash with physiological saline to remove Triton to obtain decellularized fibrous dermal matrix. (7) The decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry; according to the mass calculation, the decellularized cartilage particles in the composite slurry are 30wt%, the decellularized fibrous dermal matrix is ​​60wt%, the mussel protein is 3wt%, and the purified water is 7wt%; the concentration of KGN in the composite slurry is 500nM; (8) The composite slurry prepared in (7) is freeze-dried to obtain a composite scaffold (i.e., a shape-forming, self-adhesive cartilage repair material); wherein, the freeze-drying treatment includes pre-freezing treatment and vacuum drying treatment, the temperature of the pre-freezing treatment is -20℃ and the time is 4h; the temperature of the vacuum drying treatment is -3℃ and the time is 24h.

[0054] Example 2 A method for preparing a malleable, self-adhesive cartilage repair material includes: (1) Fresh bovine articular cartilage tissue was taken and ground with liquid nitrogen to obtain cartilage particles with a particle size of less than 500µm; (2) The cartilage particles were added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the cartilage particles were washed with physiological saline to remove the peracetic acid and the first cartilage particles were obtained. (3) The first cartilage particles were added to a 0.5% Triton solution at a ratio of 1g:5mL and shaken for 4h. Then, the Triton solution was washed with physiological saline to remove the Triton solution, and the second cartilage particles were obtained. Subsequently, the second cartilage particles were added to a 20U / mL DNase enzyme solution at a ratio of 1g:5mL and shaken for 8h. Then, the DNase enzyme was washed with physiological saline to remove the DNase enzyme, and decellularized cartilage particles were obtained. (4) Take fresh bovine skin tissue, remove the dermis and subcutaneous fat using a peeling machine to obtain dermal tissue, cut it into small pieces of about 1cm×1cm, and grind it using a rubber grinder to obtain fibrous dermal matrix; (5) The fibrous dermal matrix obtained in (4) was added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the peracetic acid was removed by washing with physiological saline. (6) Add the fibrous dermal matrix treated in (5) to a 1% Triton solution at a ratio of 1g:5mL, shake for 6h, and then wash with physiological saline to remove Triton to obtain decellularized fibrous dermal matrix. (7) The decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry; according to the mass calculation, the decellularized cartilage particles in the composite slurry are 30wt%, the decellularized fibrous dermal matrix is ​​60wt%, the mussel protein is 3wt%, and the purified water is 7wt%; the concentration of KGN in the composite slurry is 500nM; (8) The composite slurry prepared in (7) is freeze-dried to obtain a composite scaffold (i.e., a shape-forming, self-adhesive cartilage repair material); wherein, the freeze-drying treatment includes pre-freezing treatment and vacuum drying treatment, the temperature of the pre-freezing treatment is -20℃ and the time is 4h; the temperature of the vacuum drying treatment is -3℃ and the time is 24h.

[0055] Example 3 A method for preparing a malleable, self-adhesive cartilage repair material includes: (1) Fresh pig joint cartilage tissue was taken and ground with liquid nitrogen grinder to obtain cartilage particles with a particle size of less than 500µm; (2) The cartilage particles were added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the cartilage particles were washed with physiological saline to remove the peracetic acid and the first cartilage particles were obtained. (3) The first cartilage particles were added to a 2% Triton solution at a ratio of 1g:5mL and shaken for 2h. Then, the Triton solution was washed with physiological saline to remove the Triton solution, and the second cartilage particles were obtained. Subsequently, the second cartilage particles were added to a 40U / mL DNase enzyme solution at a ratio of 1g:5mL and shaken for 4h. Then, the DNase enzyme was washed with physiological saline to remove the DNase enzyme, and decellularized cartilage particles were obtained. (4) Take fresh pig skin tissue, remove the dermis and subcutaneous fat using a peeling machine to obtain dermal tissue, cut it into small pieces of about 1cm×1cm, and grind it using a rubber grinder to obtain fibrous dermal matrix; (5) The fibrous dermal matrix obtained in (4) was added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the peracetic acid was removed by washing with physiological saline. (6) Add the fibrous dermal matrix treated in (5) to a 1% Triton solution at a ratio of 1g:5mL, shake for 6h, and then wash with physiological saline to remove Triton to obtain decellularized fibrous dermal matrix. (7) The decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry; according to the mass calculation, the decellularized cartilage particles in the composite slurry are 30wt%, the decellularized fibrous dermal matrix is ​​60wt%, the mussel protein is 3wt%, and the purified water is 7wt%; the concentration of KGN in the composite slurry is 500nM; (8) The composite slurry prepared in (7) is freeze-dried to obtain a composite scaffold (i.e., a shape-forming, self-adhesive cartilage repair material); wherein, the freeze-drying treatment includes pre-freezing treatment and vacuum drying treatment, the temperature of the pre-freezing treatment is -20℃ and the time is 4h; the temperature of the vacuum drying treatment is -3℃ and the time is 24h.

[0056] Example 4 A method for preparing a malleable, self-adhesive cartilage repair material includes: (1) Fresh bovine articular cartilage tissue was taken and ground with liquid nitrogen to obtain cartilage particles with a particle size of less than 500µm; (2) The cartilage particles were added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the cartilage particles were washed with physiological saline to remove the peracetic acid and the first cartilage particles were obtained. (3) The first cartilage particles were added to a 0.5% Triton solution at a ratio of 1g:5mL and shaken for 4h. Then, the Triton solution was washed with physiological saline to remove the Triton solution, and the second cartilage particles were obtained. Subsequently, the second cartilage particles were added to a 20U / mL DNase enzyme solution at a ratio of 1g:5mL and shaken for 8h. Then, the DNase enzyme was washed with physiological saline to remove the DNase enzyme, and decellularized cartilage particles were obtained. (4) Take fresh bovine skin tissue, remove the dermis and subcutaneous fat using a peeling machine to obtain dermal tissue, cut it into small pieces of about 1cm×1cm, and grind it using a rubber grinder to obtain fibrous dermal matrix; (5) The fibrous dermal matrix obtained in (4) was added to a 0.2% peracetic acid solution at a ratio of 1g:5mL and soaked for 0.5h. Then, the peracetic acid was removed by washing with physiological saline. (6) Add the fibrous dermal matrix treated in (5) to a 1% Triton solution at a ratio of 1g:5mL, shake for 6h, and then wash with physiological saline to remove Triton to obtain decellularized fibrous dermal matrix. (7) The decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry; according to the mass calculation, the decellularized cartilage particles in the composite slurry are 25wt%, the decellularized fibrous dermal matrix is ​​65wt%, the mussel protein is 5wt%, and the purified water is 5wt%; the concentration of KGN in the composite slurry is 1000nM; (8) The composite slurry prepared in (7) is freeze-dried to obtain a composite scaffold (i.e., a shape-forming, self-adhesive cartilage repair material); wherein, the freeze-drying treatment includes pre-freezing treatment and vacuum drying treatment, the temperature of the pre-freezing treatment is -20℃ and the time is 4h; the temperature of the vacuum drying treatment is -3℃ and the time is 24h.

[0057] Example 5 Example 5 is basically the same as Example 1, except that the mass fraction of each component in the composite slurry is different. Specifically, in step (7), the decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry. According to the mass calculation, the decellularized cartilage particles in the composite slurry are 20wt%, the decellularized fibrous dermal matrix is ​​73wt%, the mussel protein is 2wt%, and the purified water is 5wt%. The concentration of KGN in the composite slurry is 500nM.

[0058] Example 6 Example 6 is basically the same as Example 1, except that the mass fraction of each component in the composite slurry is different. Specifically, in step (7), the decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry. According to the mass calculation, the decellularized cartilage particles in the composite slurry are 30wt%, the decellularized fibrous dermal matrix is ​​55wt%, the mussel protein is 5wt%, and the purified water is 10wt%. The concentration of KGN in the composite slurry is 500nM.

[0059] Example 7 Example 7 is basically the same as Example 1, except that the mass fraction of each component in the composite slurry is different. Specifically, in step (7), the decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry. According to the mass calculation, the composite slurry contains 1 wt% decellularized cartilage particles, 73 wt% decellularized fibrous dermal matrix, 5 wt% mussel protein, and 21 wt% purified water. The concentration of KGN in the composite slurry is 500 nM.

[0060] Comparative Example 1 Comparative Example 1 is basically the same as Example 1, except that decellularized cartilage particles were not added to the composite slurry. Specifically, in step (7), the decellularized fibrous dermal matrix, mussel protein, inducer (KGN) and purified water prepared in (6) are mixed evenly using a homogenizer to obtain a composite slurry; according to the mass calculation, the composite slurry contains 60wt% decellularized fibrous dermal matrix, 3wt% mussel protein and 37wt% purified water; wherein the concentration of KGN in the composite slurry is 500nM.

[0061] Comparative Example 2 Comparative Example 2 is basically the same as Example 1, except that the mass fraction of each component in the composite slurry is different. Specifically, in step (7), the decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), mussel protein, inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry. According to the mass calculation, the decellularized cartilage particles in the composite slurry are 35wt%, the decellularized fibrous dermal matrix is ​​55wt%, the mussel protein is 3wt%, and the purified water is 7wt%. The concentration of KGN in the composite slurry is 500nM.

[0062] Comparative Example 3 Comparative Example 3 is basically the same as Example 1, except that: no decellularized fibrous dermal matrix was added to the composite slurry; Specifically, in step (7), the decellularized cartilage particles, mussel protein, inducer (KGN) and purified water prepared in (3) are mixed evenly using a homogenizer to obtain a composite slurry; according to the mass calculation, the decellularized cartilage particles in the composite slurry are 30wt%, the mussel protein is 3wt%, and the purified water is 67wt%; wherein the concentration of KGN in the composite slurry is 500nM.

[0063] Comparative Example 4 Comparative Example 4 is basically the same as Example 1, except that mussel protein was not added to the composite slurry; Specifically, in step (7), the decellularized cartilage particles prepared in (3), the decellularized fibrous dermal matrix prepared in (6), the inducer (KGN) and purified water are homogenized and mixed evenly to obtain a composite slurry. According to the mass calculation, the decellularized cartilage particles in the composite slurry are 30wt%, the decellularized fibrous dermal matrix is ​​60wt%, and the purified water is 10wt%. The concentration of KGN in the composite slurry is 500nM.

[0064] The decellularized cartilage particles and decellularized fibrous dermal matrix prepared in the above embodiments were tested for residual DNA. The testing was performed according to the method specified in YY / T 1876-2023 "Determination of DNA Residual in Animal-Derived Biological Materials for Tissue-Engineered Medical Products: Fluorescent Staining Method". The results showed that the residual DNA in the decellularized cartilage particles prepared in this embodiment was 15.5±1.4 ng / mg, and the residual DNA in the decellularized fibrous dermal matrix was 8.5±0.7 ng / mg, both below 50 ng / mg. This meets the requirement of less than 50 ng / mg for residual DNA in animal-derived medical devices, demonstrating that the method of this application can effectively remove residual DNA and has low immunogenicity.

[0065] Taking the composite scaffold prepared in Example 1 as an example, the immunohistochemical data of type II collagen in the decellularized cartilage particles prepared in Example 1 are as follows: Figure 1 As shown, this demonstrates that the decellularized cartilage particles prepared by this method can effectively retain type II collagen in cartilage. Simultaneously, the decellularized cartilage particles prepared in Example 1 were stained with alexandrite blue, yielding the following results: Figure 2 The image shown is a graph of alcian blue staining data for decellularized cartilage granules, created by... Figure 2 It can be seen that the polysaccharide components in the decellularized cartilage granules were effectively preserved. Microscopic observation of the decellularized fibrous dermal matrix prepared in Example 1 revealed... Figure 3As shown, the microscopic state of the decellularized fibrous dermal matrix exhibits a fibrous band-like structure. The entanglement between the fibrous structures in the composite scaffold further endows the composite scaffold product with shaping capabilities; simultaneously, the fibrous structure has a higher specific surface area, which can promote cell adhesion and proliferation. Figure 4 As shown in the macroscopic diagram of the composite scaffold, the composite scaffold prepared in Example 1 is a blocky white solid. Furthermore, after completely immersing the composite scaffold prepared in physiological saline, it can be kneaded and shaped as needed to obtain the desired result. Figure 5 The shaped spherical solid shown is used to fill irregular defects, which further demonstrates the malleability of the composite scaffold. Figure 6 The diagram shows the effect of the composite scaffold prepared in Example 1 on the adhesion of defective cartilage tissue. Figure 6 It can be seen that by verifying the adhesion effect of the material through an in vitro cartilage defect model, the composite scaffold prepared in Example 1 can effectively adhere to the cartilage defect site by relying on the adhesion of its own mussel protein, thus avoiding the use of fibrin adhesive-type biological glues during surgery.

[0066] Compared with Example 1, Comparative Example 1, due to the absence of decellularized cartilage particles, resulted in a composite scaffold that differed from natural cartilage components, lacking type II collagen and thus failing to provide the necessary microenvironment for cartilage regeneration and repair. Comparative Example 2, due to the addition of excessive decellularized cartilage particles, caused them to easily detach, thereby affecting the adhesion performance of the composite scaffold. Comparative Example 3, due to the absence of decellularized fibrous dermal matrix, could not effectively encapsulate the loaded decellularized cartilage particles, leading to their easy detachment and affecting the adhesion performance of the composite scaffold. Comparative Example 4, lacking mussel protein, resulted in the prepared composite scaffold failing to adhere, necessitating the use of adhesive-type bio-glue.

[0067] In summary, this invention utilizes decellularized cartilage particles obtained through a decellularization process of natural cartilage, preserving the main components of cartilage. The composite scaffold prepared based on this process provides a microenvironment similar to that for cartilage regeneration and repair. For irregular cartilage defects, existing scaffolds require adhesive fixation and have poor shaping ability, increasing surgical complexity and treatment costs. The malleable, self-adhesive cartilage repair material provided in this invention is prepared from decellularized cartilage particles, decellularized fibrous dermal matrix, mussel protein, and a cartilage tissue formation inducer. The decellularized fibrous dermal matrix can be arbitrarily shaped due to fiber entanglement, and the mussel protein, after absorbing microfracture exudate, exerts its own adhesive effect, adhering and fixing the scaffold to the cartilage defect site. The decellularized cartilage particles and KGN provide a similar favorable microenvironment for in-situ cartilage repair, better achieving regeneration and repair of cartilage defects.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method of preparing a shapeable, self-adhesive cartilage repair material, characterized in that, Comprise: (1) mixing the decellularized cartilage granules obtained by treating fresh articular cartilage tissue, the decellularized fibrous dermal matrix obtained by treating fresh skin tissue, mussel protein and purified water to obtain a composite slurry; the composite slurry comprises the following components by mass fraction: decellularized cartilage granules 1wt%-30wt%, decellularized fibrous dermal matrix 55wt%-73wt%, mussel protein 2wt%-5wt%, and purified water 5wt%-21wt%; (2) freeze-drying the composite slurry to obtain the shapeable, self-adhesive cartilage repair material.

2. The production method according to claim 1, characterized by, The decellularized cartilage granules are obtained by the following method: Grinding the fresh articular cartilage tissue in sequence, treating with peracetic acid solution, washing, treating with triton solution, washing, treating with DNase enzyme solution and washing to obtain the decellularized cartilage granules; And / or, The decellularized fibrous dermal matrix is obtained by the following method: The fresh skin tissue is subjected to dermal treatment to obtain dermal tissue; the dermal tissue is ground, treated with peracetic acid solution, washed, treated with triton solution and washed to obtain the decellularized fibrous dermal matrix.

3. The preparation method according to claim 2, characterized in that, The fresh articular cartilage tissue and the fresh skin tissue are derived from at least one of human, pig, cow, sheep, horse, donkey and monkey; and / or, The concentration of the peracetic acid solution is 0.1%-0.4%; preferably, the concentration of the triton solution is 0.2%-2%.

4. The production method according to claim 2, characterized by, The decellularized cartilage granules are obtained by the following method: Grinding the fresh articular cartilage tissue to obtain cartilage granules with a particle size of less than 500µm; Soaking the cartilage granules in the peracetic acid solution for 0.5-2h, and then removing the peracetic acid by washing to obtain first cartilage granules; Shaking the first cartilage granules in the triton solution for 4-24h, and then removing the triton by washing to obtain second cartilage granules; Shaking the second cartilage granules in the DNase enzyme solution for 2-24h, and then removing the DNase enzyme by washing to obtain the decellularized cartilage granules; preferably, the concentration of the DNase enzyme solution is 2-50U / mL.

5. The preparation method according to claim 2, characterized in that, The decellularized fibrous dermal matrix is obtained by the following method: Using a dermal peeling machine to remove the dermal layer and subcutaneous fat of the fresh skin tissue to obtain dermal tissue; Grinding the dermal tissue to obtain fibrous dermal matrix; Soaking the fibrous dermal matrix in the peracetic acid solution for 0.5-2h, and then removing the peracetic acid by washing to obtain first matrix; Shaking the first matrix in the triton solution for 6-24h, and then removing the triton by washing to obtain the decellularized fibrous dermal matrix.

6. The method of claim 1, wherein, In step (1), further comprising: Adding an inducer to the composite slurry; preferably, the concentration of the inducer in the composite slurry is 200-1000nM.

7. The preparation method according to claim 1, characterized in that, The composite slurry comprises the following components by mass fraction: 20wt%-30wt% of the decellularized cartilage particles, 55wt%-73wt% of the decellularized fibrous dermal matrix, 2wt%-5wt% of the mussel protein, and 5wt%-10wt% of purified water.

8. The production method according to any one of claims 1 to 7, characterized by, In step (2): The freeze-drying treatment comprises a pre-freezing treatment and a vacuum drying treatment.

9. The production method according to claim 8, characterized by, The pre-freezing treatment has a temperature of -80--20 ℃ and a time of 2-8 h; and / or, The vacuum drying treatment has a temperature of -20--3 ℃ and a time of 24-72 h.

10. A shapeable, self-adhesive cartilage repair material prepared by the preparation method according to any one of claims 1-9.

Images