A decellularized cartilage matrix and methods of making and using the same

Abstract

The application provides a kind of decellularized cartilage matrix and its preparation method and use, belong to medical biomaterial technical field.Aiming at the problems that the prior art is difficult to effectively decellularize thicker cartilage tissue, and the DNA residue is high after processing, and the extracellular matrix is easy to lose, the application provides an optimized preparation method.The method can directly process the cartilage tissue with a thickness of 500-1000 μm efficiently, and by using a specific combination of decellularization reagents, a large amount of cell components can be removed while the extracellular matrix such as glycosaminoglycan is maximally retained.The DNA residue of the obtained decellularized cartilage matrix can be <0.78 ng / mg, the immunogenicity is significantly reduced, and the biocompatibility is excellent.The application expands the application of knee joint cartilage, laryngeal cartilage, tracheal cartilage and cartilage from various sources, and the prepared matrix can be used in the field of regenerative medicine such as cartilage defect repair and tissue engineering scaffold.

Description

Technical Field

[0001] This invention belongs to the field of medical biomaterials technology, specifically relating to a decellularized cartilage matrix, its preparation method, and its uses. Background Technology

[0002] Decellularized extracellular matrix (dECM) materials are attracting increasing attention due to their inherent structure, high bioactivity, low immunogenicity, and good biodegradability, making them difficult to imitate with synthetic materials. Decellularized cartilage matrix is ​​a bio-derived material that retains its extracellular matrix structure and bioactive components by removing cellular components. It possesses natural tissue structure and biocompatibility, and can be widely used in cartilage repair, tissue engineering scaffolds, and regenerative medicine. Currently, the main sources of raw materials for decellularized cartilage matrix include articular cartilage and rib cartilage. To further expand its application potential, developing matrix materials from more sources (such as knee cartilage, laryngeal cartilage, and tracheal cartilage) is of great value.

[0003] In terms of preparation methods, existing technologies typically employ physical, chemical, or enzymatic methods for decellularization. However, due to the dense structure of cartilage tissue, a single method is often insufficient to completely remove cellular components. Therefore, a combined physical and chemical treatment strategy is often used. Nevertheless, these methods still have significant limitations: on the one hand, to promote the penetration of decellularization reagents, the cartilage tissue often needs to be pre-crushed or cut into extremely thin sheets (usually less than 50 micrometers). This process easily leads to the loss of a large amount of important extracellular matrix components such as collagen and glycosaminoglycans, affecting the structural and functional integrity of the matrix; on the other hand, even after treatment, the decellularization effect of the resulting product is still unsatisfactory, with generally high levels of residual DNA. For example, in existing patented technologies, the method disclosed in patent CN116173303B, after processing cartilage into cartilage membranes of 50-500 μm and then treating them with cross-linking and decellularization reagents, still results in a residual DNA content of 1.5 ng / mg. Another patent, CN118995587B, uses sodium borohydride and sodium dodecyl sulfate (SDS) to treat 300 μm thick cartilage, resulting in a residual DNA content of 20-40 ng / mg. Yet another patent, CN114796615B, uses a non-ionic reagent treatment method, with residual DNA content also between 5-10 ng / mg. This residual DNA may trigger an immune response, limiting the clinical application of the material. Furthermore, existing methods are difficult to apply to cartilage tissues of greater thickness or volume, resulting in limitations in the morphological and mechanical adaptability of the prepared matrix materials.

[0004] Therefore, how to achieve efficient and deep decellularization of thicker cartilage tissue while maintaining the integrity of the extracellular matrix and significantly reducing DNA residue has become a key technical problem that urgently needs to be solved in the field of tissue engineering and regenerative medicine. Summary of the Invention

[0005] In view of the problems existing in the prior art, such as difficulty in decellularizing thick cartilage tissue, high DNA residue, and easy loss of extracellular matrix components during the processing, the purpose of this invention is to provide a decellularized cartilage matrix, its preparation method, and its uses.

[0006] To achieve the above objectives, the specific technical solution provided by the present invention is as follows: This invention provides a method for preparing decellularized cartilage matrix, the method comprising the following steps: (1) Clean the cartilage tissue, slice it, and the thickness range is 500um~1000um; (2) The sliced ​​cartilage tissue was subjected to freeze-thaw cycles; (3) The cartilage tissue subjected to freeze-thaw cycles is subjected to multi-stage chemical decellularization treatment, wherein the multi-stage chemical decellularization treatment includes: soaking and washing with at least three decellularization treatment solutions in sequence; (4) The tissue treated in step (3) is washed to obtain decellularized cartilage matrix; The at least three decellularization treatment solutions are selected from at least three of the following: nonionic detergent solutions containing buffer, nuclease treatment solutions, ionic detergent solutions, and ionic detergent solutions containing buffer.

[0007] Further, in step (3), the decellularization treatment solutions are, in sequence: Triton X-100 solution containing Tris, TNE solution, and SDS solution containing Tris.

[0008] Further, in step (3), the Triton X-100 solution containing Tris has a Triton X-100 mass-volume concentration of 0.5%~2% and a Tris mass-volume concentration of 0.01%~0.05%; the TNE solution has a TNE mass-volume concentration of 0.1%~0.5%; and the SDS solution containing Tris has an SDS mass-volume concentration of 1%~3% and a Tris mass-volume concentration of 0.01%~0.05%. The different decellularization treatment solutions were used for immersion treatment at temperatures ranging from 4°C to 37°C for 24 to 96 hours.

[0009] Further, in step (3), the Triton X-100 solution containing Tris has a Triton X-100 mass-volume concentration of 1% and a Tris mass-volume concentration of 0.02%; the TNE solution has a TNE mass-volume concentration of 0.25%; and the SDS solution containing Tris has an SDS mass-volume concentration of 2% and a Tris mass-volume concentration of 0.02%. The Tris-containing Triton X-100 solution was soaked at a temperature of 37°C for 48 hours. The TNE solution was used for immersion treatment at a temperature of 4°C for 24 hours. The Tris-containing SDS solution was soaked at a temperature of 37°C for 96 hours.

[0010] Further, in step (1), the slicing is the cutting of cartilage tissue into slices with a thickness of 500~1000μm.

[0011] Further, in step (2), the freeze-thaw cycle treatment includes: freezing the tissue in liquid nitrogen for 1 to 10 minutes, and then thawing it at 30 to 45°C for 20 to 40 minutes; the number of freeze-thaw cycles is 2 to 5.

[0012] Further, in step (2), the freeze-thaw cycle treatment includes: freezing the tissue in liquid nitrogen for 5 minutes, and then thawing it at 37°C for 30 minutes; the freeze-thaw cycle is repeated 3 times.

[0013] Furthermore, after step (3) is completed, the procedure also includes: soaking the tissue treated in step (3) in water at 2~6°C for 20~30 hours.

[0014] Furthermore, after step (3) is completed, the procedure also includes: soaking the tissue treated in step (3) in water at 4°C for 24 hours.

[0015] Furthermore, the cartilage tissue is derived from hyaline cartilage of the knee joint, laryngeal cartilage, or tracheal cartilage of a pig.

[0016] The present invention also provides a decellularized cartilage matrix prepared by the above-described preparation method.

[0017] The present invention also provides the application of the above-mentioned decellularized cartilage matrix in the preparation of tissue engineering products, wherein the tissue engineering products are selected from scaffolds, hydrogels, bio-inks, filler materials or patches.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) Achieved efficient decellularization of thicker cartilage tissue: The method of the present invention can be directly applied to intact cartilage sheets with a thickness of 500~1000μm without prior crushing or preparation of ultrathin sections, effectively avoiding the loss of a large amount of extracellular matrix caused by excessive physical processing, and better preserving the complete three-dimensional structure and mechanical properties of natural cartilage matrix.

[0019] (2) Significantly reduced immunogenicity: Through optimized decellularization process, the residual DNA content of the decellularized cartilage matrix obtained is <0.78 ng / mg, which is far lower than the level reported in existing technologies. This extremely low residual DNA content greatly reduces the risk of immune rejection that may be caused after material implantation and improves the safety of clinical use.

[0020] (3) The active components of the extracellular matrix are well preserved: While removing a large number of cellular components, this method can retain the bioactive components such as glycosaminoglycans and TGFβ3, which are crucial for cartilage regeneration, to the greatest extent possible, providing a good microenvironment for cell adhesion, proliferation and differentiation.

[0021] (4) Expanded raw material sources and application potential: The preparation method described herein is applicable to various cartilage tissues such as knee joint cartilage, laryngeal cartilage, and tracheal cartilage, thus broadening the raw material sources for decellularized cartilage matrix. The prepared matrix can be used in multiple fields such as cartilage defect repair, tissue engineering scaffold construction, and biomaterial development, with broad application prospects.

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0024] Figure 1 A macroscopic morphological comparison of natural cartilage and decellularized cartilage matrix.

[0025] Figure 2 Comparison of the histological structures of natural cartilage and decellularized cartilage matrix: (Top) HE staining images of natural cartilage and decellularized cartilage matrix prepared in Examples 1-4; (Bottom) HE staining comparison of Examples 1 and 2 after treatment in key decellularization steps (1)-(3).

[0026] Figure 3A comparison of the DNA, xenogeneic α-galactosyl antigen (α-Gal), endotoxin, and glycosaminoglycan (GAG) content of the decellularized cartilage matrix prepared in Examples 1-2 compared to natural cartilage.

[0027] Figure 4 The comparison of the contents of epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF) in the decellularized cartilage matrix prepared in Examples 1-2 compared with natural cartilage.

[0028] Figure 5 HE staining comparison of natural cartilage of the larynx and trachea and their decellularized matrix.

[0029] Figure 6 This study compares the DNA content of natural cartilage with that of decellularized cartilage matrix from the larynx and trachea.

[0030] Figure 7 To observe the thermosensitive self-assembly behavior of decellularized cartilage matrix. Detailed Implementation

[0031] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0032] In this embodiment of the invention, the percentage of solution concentration "%" represents the mass-volume ratio. For example, a 1% Triton X-100 solution of 0.02% Tris means that the mass-volume concentration of Triton X-100 in the solution is 1% and the mass-volume concentration of Tris is 0.02%.

[0033] Example 1: Method for preparing decellularized cartilage matrix This embodiment provides a specific method for preparing decellularized cartilage matrix using porcine knee joint cartilage tissue (hyaluronic cartilage).

[0034] The specific steps are as follows: (1) Take fresh pig knee joint cartilage tissue, rinse it thoroughly with pure water several times to remove surface deposits, and cut the cartilage tissue into slices with a thickness of 500~1000μm. Figure 1 ),spare; (2) Place the above tissue sections in liquid nitrogen for rapid freezing for 5 minutes, then transfer them to 37°C for 30 minutes to thaw. Repeat the freeze-thaw cycle three times. (3) Immerse the freeze-thawed cartilage tissue in 400 ml of 1% Triton X-100 solution containing 0.02% Tris and place it on a shaker at 37°C for 48 h. Change the solution every 24 h, and rinse the tissue repeatedly with pure water before changing the solution; (4) Discard the above solution, rinse the tissue thoroughly with pure water, add 400 ml of 0.25% trypsin (TNE) solution, and place at 4°C for 24 h; (5) Discard the SDS solution, rinse the tissue, add 400 ml of 2% sodium dodecyl sulfate (SDS) solution containing 0.02% Tris, and incubate at 37°C on a shaker for 96 h. Replace the solution with fresh solution every 24 h, rinsing the tissue repeatedly with pure water before each replacement. (6) Discard the solution, rinse the tissue repeatedly with pure water, add 400ml of pure water, and place at 4℃ for 24h; (7) Discard the pure water and rinse the tissue repeatedly with plenty of pure water until no more foam is produced in the rinsing solution, thus obtaining decellularized cartilage matrix. Figure 1 ).

[0035] Example 2: Method for preparing decellularized cartilage matrix This embodiment provides a specific method for preparing decellularized cartilage matrix using porcine knee joint cartilage tissue (hyaluronic cartilage).

[0036] The specific steps are as follows: (1) Take fresh pig knee joint cartilage tissue, rinse it thoroughly with pure water several times to remove surface attachments, cut the cartilage tissue into slices with a thickness of 500~1000μm, and set aside. (2) Place the above tissue sections in liquid nitrogen for rapid freezing for 5 minutes, then transfer them to 37°C for 30 minutes to thaw. Repeat the freeze-thaw cycle three times. (3) Immerse the freeze-thawed cartilage tissue in 400 ml of 2% SDS solution containing 0.02% Tris and place it on a shaker at 37°C for 48 h. Change the solution every 24 h, and rinse the tissue repeatedly with pure water before changing the solution; (4) Discard the above solution, rinse the tissue thoroughly with pure water, add 400 ml of 1% Triton X-100 solution containing 0.02% Tris, and place on a shaker at 37°C for 48 h. During this period, change the solution every 24 h, rinsing the tissue repeatedly with pure water before changing the solution; (5) Discard the above solution, rinse with pure water, add 400ml of pure water, and place at 4℃ for 24h; (6) Discard the pure water and rinse the tissue repeatedly with a large amount of pure water until no more foam is produced in the rinsing solution, and obtain decellularized cartilage matrix.

[0037] Example 3: Method for preparing decellularized cartilage matrix This embodiment provides a specific method for preparing decellularized cartilage matrix using porcine knee joint cartilage tissue (hyaluronic cartilage).

[0038] The specific steps are as follows: (1) Take fresh pig knee joint cartilage tissue, rinse it thoroughly with pure water several times to remove surface attachments, cut the cartilage tissue into slices with a thickness of 500~1000μm, and set aside. (2) Place the above tissue sections in liquid nitrogen for rapid freezing for 5 minutes, then transfer them to 37°C for 30 minutes to thaw. Repeat the freeze-thaw cycle three times. (3) Immerse the freeze-thawed cartilage tissue in 400 ml of 0.02% Tris solution and place it on a shaker at 4°C for 48 h. Change the solution every 24 h, and rinse the tissue repeatedly with pure water before changing the solution; (4) Discard the above solution, rinse the tissue thoroughly with pure water, add 400 ml of 1% Triton X-100 solution containing 0.02% Tris, and place on a shaker at 37°C for 48 h. During this period, change the solution every 24 h, rinsing the tissue repeatedly with pure water before changing the solution; (5) Discard the above solution, rinse the tissue thoroughly with pure water, add 400 ml of 0.25% TNE solution, and place at 4°C for 24 h; (6) Discard the TNE solution, rinse with pure water, add 400 ml of 0.02% Tris solution, and incubate on a shaker at 4°C for 48 h. Replace with fresh 0.02% Tris solution every 24 h during this period; (7) Discard the solution, rinse the tissue repeatedly with pure water, add 400ml of pure water, and place at 4℃ for 24h; (8) Discard the pure water and rinse the tissue repeatedly with a large amount of pure water until no more foam is produced in the rinsing solution, and obtain decellularized cartilage matrix.

[0039] Example 4: Method for preparing decellularized cartilage matrix This embodiment provides a specific method for preparing decellularized cartilage matrix using porcine knee joint cartilage tissue (hyaluronic cartilage).

[0040] The specific steps are as follows: (1) Take fresh pig knee joint cartilage tissue, rinse it thoroughly with pure water several times to remove surface attachments, cut the cartilage tissue into slices with a thickness of 500~1000μm, and set aside. (2) Place the above tissue sections in liquid nitrogen for rapid freezing for 5 minutes, then transfer them to 37°C for 30 minutes to thaw. Repeat the freeze-thaw cycle three times. (3) Immerse the freeze-thawed cartilage tissue in 400 ml of 2% SDS solution containing 0.02% Tris and place it on a shaker at 37°C for 24 h. Replace with fresh solution after 24 h, and rinse the tissue repeatedly with pure water before replacement; (4) Repeat step (3) 7 times; (5) Discard the solution, rinse the tissue repeatedly with pure water, add 400ml of pure water, and place at 4℃ for 24h; (6) Discard the pure water and rinse the tissue repeatedly with a large amount of pure water until no more foam is produced in the rinsing solution, and obtain decellularized cartilage matrix.

[0041] Example 5: Method for preparing decellularized cartilage matrix The method of Example 1 is referenced, except that the porcine knee joint cartilage tissue (hyaluronic cartilage) is replaced with porcine laryngeal cartilage (hyaluronic cartilage, elastic cartilage and fibrocartilage) or porcine tracheal cartilage (hyaluronic cartilage) to prepare the corresponding decellularized cartilage matrix.

[0042] The following experimental examples demonstrate the beneficial effects of the present invention.

[0043] Experiment 1: Characterization and compositional analysis of different decellularized cartilage matrices 1. Experimental Methods (1) Organizational structure observation The histological examination of natural cartilage and decellularized cartilage matrix prepared in Examples 1-4 was performed using hematoxylin-eosin (HE) staining to assess cellular residue and matrix structural integrity. Figure 2 ).

[0044] (2) Evaluation of decellularization effect HE staining was used to observe residual cell nuclei; the amount of residual DNA in the decellularized material was detected using the Quant-iT™ PicoGreen™ dsDNA kit (catalog number P11496); the residual α-Gal was determined by enzyme-linked immunosorbent assay (ELISA) using the Zhuocai (catalog number ZC-39391) porcine α-galactosyl antigen (α-Gal) kit; and the endotoxin content was determined by enzyme-linked immunosorbent assay (ELISA) using the Beyotime (catalog number C0276S) endotoxin detection kit. Figure 3 ).

[0045] (3) Detection of extracellular matrix components and growth factor retention 1) Glycosaminoglycan (GAG) content: The content was quantitatively detected using the enzyme-linked immunosorbent assay (ELISA) method with the Zhuocai (brand) Glycosaminoglycan (GAG) ELISA kit (catalog number ZC-39830); 2) Growth factor content: Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of the corresponding growth factors using the Zhuocai (brand) Epidermal Growth Factor (EGF) ELISA kit (catalog number ZC-39463), Fibroblast Growth Factor 2 (FGF2) ELISA kit (catalog number ZC-39480), Transforming Growth Factor β1 (TGF-β1) ELISA kit (catalog number ZC-50021), and Vascular Endothelial Growth Factor (VEGF) ELISA kit (catalog number ZC-39900). Figure 4 ).

[0046] (4) Comparison of decellularized matrix of laryngeal and tracheal cartilage HE staining was performed on the decellularized cartilage matrix of the larynx and trachea prepared according to the method in Example 1. Figure 5 ) and DNA content detection ( Figure 6 To evaluate its decellularization effect and matrix suitability.

[0047] (5) Observation of thermosensitive self-assembly behavior After the decellularized cartilage matrix was incubated at 37°C for 5 minutes, it was inverted and its self-assembly structure and microstructure were observed under a scanning electron microscope (SEM). Figure 7 ).

[0048] 2. Experimental Results 2.1 Evaluation of decellularization effect HE staining revealed significant differences in the decellularization effects among the various embodiments. Figure 2 In Example 1, the decellularized cartilage matrix contained almost no residual cell nuclei, and the extracellular matrix structure remained intact, demonstrating the best decellularization effect. Example 4 showed only scattered residual cell nuclei, with a decellularization effect second only to Example 1. Residual cell nuclei were still observed in Example 3, indicating that its decellularization process was not thorough. In Example 2, the extracellular matrix structure was more severely damaged; although there were fewer residual cell nuclei, the matrix integrity was compromised. Further comparison of the microstructural changes in the key decellularization steps between Example 1 and Example 2 (…) Figure 2 The results showed that the process of Example 1 could better maintain the matrix skeleton while removing cells.

[0049] Quantitative analysis of residual DNA further confirmed the above histological observations. Figure 3Natural cartilage has a high DNA content, and after decellularization, Example 1 showed the lowest DNA residue (0.69~0.78 ng / mg), significantly lower than Example 2 (1.50~2.0 ng / mg). Both met the safety standards for tissue engineering materials. Example 1 demonstrated a significant advantage in DNA removal efficiency. Compared with existing technologies (CN116173303B, CN118995587B, CN114796615B), Example 1 of this invention achieved lower DNA residue (<0.78 ng / mg) while maintaining a relatively large tissue thickness (500-1000 μm). This indicates that the method of this invention can not only effectively remove genetic material and reduce immune risk on a larger scale, but also overcomes the limitations of existing technologies that are generally difficult to apply to thicker cartilage tissues.

[0050] Regarding other immunogenic substances, the residual trends of α-Gal antigen and endotoxin are consistent with those of DNA. Figure 3 Of these, the endotoxin content in Example 1 was extremely low, while the endotoxin levels in natural cartilage were similar to, and significantly higher than, those in Example 2. This indicates that the process in Example 1 can more effectively remove potentially immunogenic substances.

[0051] 2.2 Retention of extracellular matrix components Glycosaminoglycans (GAGs) are important functional components of cartilage matrix. For example... Figure 3 As shown, after decellularization, the GAG ​​retention levels in Examples 1 and 2 were comparable and significantly higher than those in natural cartilage. This indicates that the decellularization method of the present invention can effectively remove cellular components while retaining key bioactive polysaccharides.

[0052] However, different embodiments exhibited selectivity differences in growth factor retention. Figure 4 Overall, Example 1 is more advantageous in retaining TGF-β1 and VEGF.

[0053] 2.3 Applicability of decellularization to different cartilage sources The optimized process of Example 1 was applied to laryngeal cartilage and tracheal cartilage, and the results showed that the process has good universality. Figure 5 HE staining showed that both types of decellularized matrix effectively removed cell nuclei. DNA quantification results ( Figure 6 The results showed that the residual DNA content in the decellularized matrix of the larynx was less than 25 ng / mg, and the residual DNA content in the decellularized matrix of the trachea was less than 50 ng / mg, further confirming the decellularization effect and indicating that the process can be used to prepare cartilage tissue from multiple sources.

[0054] 2.4 Thermosensitive self-assembly characteristics of decellularized matrix The thermosensitivity of the prepared decellularized cartilage matrix was tested, such as... Figure 7 As shown, the matrix rapidly self-assembled after standing at 37°C for 5 minutes and did not flow when inverted. Under a 200x microscope, a uniform, porous network-like gel structure was observed. These results indicate that decellularization did not damage the inherent biophysical properties of the matrix, and the resulting material possesses good temperature-sensitive gelling properties and microstructure, making it suitable for use as an injectable tissue engineering scaffold.

[0055] In summary, this invention provides a decellularized cartilage matrix, its preparation method, and its applications. Addressing the limitations of existing technologies in effectively decellularizing thick cartilage tissue, resulting in high DNA residue and easy loss of extracellular matrix, this invention provides an optimized preparation method. This method can efficiently process cartilage tissue with a thickness of 500-1000 μm. Through a specific combination of decellularization reagents, it removes a large number of cellular components while maximally retaining extracellular matrix components such as glycosaminoglycans. The resulting decellularized cartilage matrix has a DNA residue of <0.78 ng / mg, significantly reduced immunogenicity, and excellent biocompatibility. This invention expands the application of cartilage from various sources, such as knee cartilage, laryngeal cartilage, and tracheal cartilage. The prepared matrix can be used in regenerative medicine fields such as cartilage defect repair and tissue engineering scaffolds.

Claims

1. A method for preparing decellularized cartilage matrix, characterized in that, The preparation method includes the following steps: (1) Clean the cartilage tissue and slice it; (2) The sliced ​​cartilage tissue was subjected to freeze-thaw cycles; (3) The cartilage tissue subjected to freeze-thaw cycles is subjected to multi-stage chemical decellularization treatment, wherein the multi-stage chemical decellularization treatment includes: soaking and washing with at least three decellularization treatment solutions in sequence; (4) The tissue treated in step (3) is washed to obtain decellularized cartilage matrix; The at least three decellularization treatment solutions are selected from at least three of the following: nonionic detergent solutions containing buffer, nuclease treatment solutions, ionic detergent solutions, and ionic detergent solutions containing buffer.

2. The preparation method according to claim 1, characterized in that, In step (3), the decellularization treatment solutions are, in sequence: Triton X-100 solution containing Tris, TNE solution, and SDS solution containing Tris.

3. The preparation method according to claim 2, characterized in that, In step (3), the Triton X-100 solution containing Tris has a Triton X-100 mass-volume concentration of 0.5%~2% and a Tris mass-volume concentration of 0.01%~0.05%; the TNE solution has a TNE mass-volume concentration of 0.1%~0.5%; and the SDS solution containing Tris has an SDS mass-volume concentration of 1%~3% and a Tris mass-volume concentration of 0.01%~0.05%. The different decellularization treatment solutions were used for immersion treatment at temperatures ranging from 4°C to 37°C for 24 to 96 hours.

4. The preparation method according to claim 3, characterized in that, In step (3), the Triton X-100 solution containing Tris has a Triton X-100 mass-volume concentration of 1% and a Tris mass-volume concentration of 0.02%; the TNE solution has a TNE mass-volume concentration of 0.25%; and the SDS solution containing Tris has an SDS mass-volume concentration of 2% and a Tris mass-volume concentration of 0.02%. The Tris-containing Triton X-100 solution was soaked at a temperature of 37°C for 48 hours. The TNE solution was used for immersion treatment at a temperature of 4°C for 24 hours. The Tris-containing SDS solution was soaked at a temperature of 37°C for 96 hours.

5. The preparation method according to claim 1, characterized in that, In step (1), the slicing is the cutting of cartilage tissue into slices with a thickness of 500~1000μm.

6. The preparation method according to claim 1, characterized in that, In step (2), the freeze-thaw cycle treatment includes: freezing the tissue in liquid nitrogen for 1 to 10 minutes, and then thawing it at 30 to 45°C for 20 to 40 minutes; the number of freeze-thaw cycles is 2 to 5.

7. The preparation method according to claim 1, characterized in that, After step (3) is completed, the following steps are also included: soaking the tissue treated in step (3) in water at 2~6℃ for 20~30 hours.

8. The preparation method according to claim 1, characterized in that, The cartilage tissue is derived from hyaline cartilage of the knee joint, laryngeal cartilage, or tracheal cartilage of a pig.

9. The decellularized cartilage matrix prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the decellularized cartilage matrix according to claim 9 in the preparation of tissue-engineered products, characterized in that, The tissue engineering products are selected from scaffolds, hydrogels, bio-inks, filler materials, or patches.

Images