A decellularized meniscus with high extracellular matrix retention and preparation method thereof

Abstract

The present invention discloses a kind of decellularized meniscus of extracellular matrix high retention and preparation method thereof.The method includes the following steps:(1) biomaterial is subjected to freeze-thaw cycle;(2) the biomaterial after freeze-thaw cycle treatment in step (1) is taken out sequentially through cell elution liquid:A liquid, B liquid, C liquid, D liquid until biomaterial decellularization ends;Wherein when eluting with A liquid, B liquid, C liquid, D liquid, the biomaterial is fixed in container and carried out;Wherein, A liquid is Triton X 100 solution;B liquid, D liquid are PBS;C liquid is the nuclease solution of deoxyribonuclease I and ribonuclease A composition;Cell elution liquid does not contain trypsin;A liquid elution time is 7 days.The decellularization method of the present invention can retain meniscus extracellular matrix components as much as possible while completely removing cell components, provide original ecological microenvironment for cell growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering medical biomaterials, and particularly relates to a decellularized meniscus with high extracellular matrix retention and a preparation method thereof. Background Art

[0002] Meniscal tears are the most common knee injury, leading to joint instability, cartilage degeneration, and even progressive osteoarthritis. Unfortunately, due to the inadequate blood supply to the meniscus, meniscal injuries are difficult to heal without surgical intervention. Clinically, the conventional treatment for meniscal tears is surgical, primarily consisting of arthroscopic suturing, meniscectomy, and allograft meniscal transplantation. Although surgical techniques have advanced over the past few decades, challenges remain in complex meniscal injuries, and the outcomes of both suturing and meniscectomy remain unsatisfactory and uncertain. To circumvent these issues, allograft transplantation has been introduced into clinical practice for patients with extensive meniscal injuries and those in the early or early stages of arthritis. However, the widespread use of commercial meniscal allografts is limited by several obstacles, such as donor shortages, implant size reduction, and immune rejection. Given the poor self-healing capacity of the meniscus, facilitating the reconstruction of extensive meniscal tears remains a significant challenge for sports medicine surgeons.

[0003] Given the rapid onset and growing need for meniscus injuries, coupled with the limited availability and narrow inclusion criteria of meniscus allografts, the development of bioengineering technology has provided us with an ideal scaffold to address this issue. Generally speaking, meniscus engineering scaffolds are made of synthetic or natural materials. When developing new artificial materials to best withstand tensile or compressive loads and absorb shock during joint movement, the real challenge is to mimic the natural structure, biochemistry, and geometric properties of the meniscus. On the other hand, decellularized meniscus obtained from animal sources has been explored, and fortunately, the emergence of meniscus decellularized matrix provides us with a shortcut because of its low immunogenicity, high biocompatibility, good biodegradability, and efficient preservation of natural extracellular matrix. Quality (ECM).

[0004] To date, xenogeneic meniscus tissue obtained by decellularization of meniscus from other species has an anatomical structure and matrix similar to that of human meniscus and is easy to obtain. Therefore, it may become a promising alternative for transplantation. According to previous studies, the entire decellularized meniscus has the advantage of a compatible structure, and the retained extracellular matrix components provide a natural microenvironment for meniscus cells, such as inducing chondrogenic differentiation of mesenchymal stem cells (MSCs). However, because the meniscus is a densely fibrous tissue, the difficulty of decellularization is greatly increased. Although long-term and intense washing can remove cellular components as much as possible, the extracellular matrix components suffer excessive damage. Therefore, a whole-body decellularization method for the meniscus that can completely remove cellular components and retain the extracellular matrix of the meniscus as much as possible needs to be explored. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide an integrally decellularized meniscus with high extracellular matrix retention, so as to solve the problem of integral decellularization of the meniscus in the prior art. The integral meniscus has dense fibrous tissue, making decellularization difficult, and excessive decellularization will cause excessive loss of extracellular matrix.

[0006] One of the technical solutions provided by the present invention is to provide a decellularized meniscus with high extracellular matrix retention, so as to solve the problems of donor shortage, implant shrinkage and immune rejection in existing meniscus transplants.

[0007] The second technical solution provided by the present invention is to provide a method for preparing the acellular meniscus with high extracellular matrix retention.

[0008] The technical problem solved by the present invention is achieved by adopting the following technical solutions.

[0009] A method for preparing acellular meniscus with high extracellular matrix retention, comprising the following steps:

[0010] (1) subjecting the biological material to freeze-thaw cycles;

[0011] Prior to the freeze-thaw cycle, the biological material was washed with phosphate buffered saline;

[0012] The number of freeze-thaw cycles is 3-5 times, wherein the conditions of a single cycle are: freezing in liquid nitrogen for 4-8 minutes and thawing in a water bath at 30-40°C for 8-15 minutes;

[0013] (2) taking out the biomaterial after freeze-thaw cycle treatment in step (1) and sequentially passing through cell elution solutions: solution A, solution B, solution C, and solution D until the biomaterial is decellularized; wherein when eluting with solution A, solution B, solution C, and solution D, the biomaterial is fixed in a container;

[0014] Wherein, the solution A is a Triton X-100 solution; the solutions B and D are PBS; the solution C is a nuclease solution composed of deoxyribonuclease I and ribonuclease A;

[0015] The cell eluate does not contain trypsin;

[0016] When the biomaterial subjected to freeze-thaw cycle treatment is eluted with solution A, the elution time is 7 days.

[0017] Trypsin is a commonly used enzyme for digesting extracellular matrix. Its main function is to decompose the connection between the extracellular matrix and promote the removal of cells. Similarly, acting on the meniscus will also degrade and destroy the extracellular matrix of the meniscus; however, if trypsin is not added, the connection between the cells and the extracellular matrix is tight, and the cell components are difficult to remove effectively. Based on this, the present invention studies the technical problem of how to completely remove the cell components and fully retain the extracellular matrix components under the conditions of completely eliminating the use of trypsin and reducing the cytotoxicity of the scaffold as much as possible.

[0018] As an optional embodiment, in step (1), the phosphate buffer is sterile PBS containing 1% penicillin-streptomycin.

[0019] As an optional embodiment, in step (1), the cleaning frequency is more than 1 time, and each time duration is more than 10 minutes.

[0020] As an optional implementation, in step (1), the number of cleaning times is 3-5 times.

[0021] As an optional embodiment, in step (1), the number of freeze-thaw cycles is 3 times.

[0022] As an optional embodiment, in step (1), in the freeze-thaw cycle, the conditions for a single cycle are: freezing in liquid nitrogen for 4-6 minutes and thawing in a 36-38°C water bath for 8-12 minutes.

[0023] As an optional embodiment, in step (1), in the freeze-thaw cycle, the conditions for a single cycle are: freezing in liquid nitrogen for 5 minutes and thawing in a 37°C water bath for 10 minutes.

[0024] As a preferred embodiment, the solution A is 0.1% Triton X-100 containing 1% penicillin-streptomycin.

[0025] As a preferred embodiment, the elution condition of the solution A is continuous stirring at 80 rpm / min on a shaker at 4°C.

[0026] As a preferred embodiment, the condition for eluting with liquid A is to replace the solution every 24 hours.

[0027] As a preferred embodiment, the solution B is sterile 1% penicillin-streptomycin PBS.

[0028] As a preferred embodiment, the elution condition of the solution B is to wash three times at 4°C with stirring at 150 rpm / min on a shaker, each time for 2 hours.

[0029] As a preferred embodiment, the solution C is a physiological saline solution containing 300 UI / mL DNase (Sigma-Aldrich) and 500 μg / mL ribonuclease A (Sigma-Aldrich).

[0030] As a preferred embodiment, the C solution elution condition is to place the sample in a 37°C constant temperature shaker at 100 rpm / min and incubate for 24 hours.

[0031] As a preferred embodiment, the D solution is sterile 1% penicillin-streptomycin PBS.

[0032] As a preferred embodiment, the D solution elution condition is to use sterile 1% penicillin-streptomycin PBS to wash three times, each time for 6 hours.

[0033] As a preferred embodiment, the D solution elution is performed in a negative pressure suction decellularization device.

[0034] In which, the negative pressure decellularization device may include a first pipe 200, a first container 3, a second pipe 201, a second container 5 and a third pipe 202 that are sealed and connected in sequence, and a third container 8 connected to the third pipe 202; one end of the first pipe 200 is connected to the first opening 300 of the first container 3; one end of the second pipe 201 is connected to the second opening 301 of the first container 3; the other end of the second pipe 201 and one end of the third pipe 202 are respectively connected to the two ends of the second container 5; the second container 5 is used to place the biological material 6, and the biological material 6 cannot enter the second pipe 201 and the third pipe 202, and the solution including cells can enter the second pipe 201 and the third pipe 202; when the position of the third container 8 is higher than the second container 5, and the position of the second container 5 is higher than the first container 3, the decellularized fluid flows from the third container 8 through the second container 5 under the action of negative pressure or gravity, and then reaches the first container 3.

[0035] As an optional embodiment, the biomaterial is bone, dermis, pericardium, cornea, blood vessel, bladder, esophagus, small intestinal mucosal matrix, cartilage, or liver.

[0036] As an optional embodiment, the biomaterial is cartilage.

[0037] As a preferred embodiment, the biomaterial is hyaline cartilage.

[0038] As a preferred embodiment, the biomaterial is fibrocartilage.

[0039] As a preferred embodiment, the biomaterial is a bone / tendon interface complex.

[0040] The present invention also provides a decellularized meniscus with high extracellular matrix retention prepared by the method.

[0041] In certain embodiments of the present invention, the solutions are sterile.

[0042] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0043] (1) The extracellular matrix-highly retained decellularized meniscus described in the present invention utilizes physical methods such as repeated freezing and thawing, a negative pressure decellularization device (see authorized patent: ZL202010103858.6), and optimized decellularization processes to replace the use of some chemical reagents. While completely removing cellular components, it retains the extracellular matrix components of the meniscus as much as possible, providing an original ecological microenvironment for cell growth.

[0044] (2) The extracellular matrix highly retains the decellularized meniscus described in the present invention, completely removes cellular components, and effectively retains the extracellular matrix components: collagen and proteoglycans, providing an original ecological microenvironment that can effectively promote stem cell proliferation and promote stem cell differentiation into cartilage.

[0045] (3) The method for preparing a decellularized meniscus with high extracellular matrix retention described in the present invention uses a repeated freeze-thaw cycle, a negative pressure decellularization device (authorized patent: ZL202010103858.6), and an optimized decellularization process. While completely removing cellular components, it also retains as much extracellular matrix components as possible. The provided native ecological microenvironment can effectively promote stem cell proliferation and promote stem cell cartilage differentiation. The unique repeated freeze-thaw cycle and negative pressure decellularization device processing technology replaces the use of traditional toxic reagents such as SDS, reduces the rinsing time during the decellularization process, and reduces the cytotoxicity of the scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 H&E staining, toluidine blue staining, Sirius red staining, DAPI and Shanghai First-Line Station Synchrotron Radiation Infrared Spectroscopy Microscopy detection of the decellularized meniscus (DMS) with different decellularization treatment time ratios in Example 1 (Figure A), as well as collagen and proteoglycan statistical graphs (Figure B).

[0047] Figure 2 The following are the macroscopic and electron microscopic comparison images of the decellularized meniscus and the non-decellularized meniscus in Example 1.

[0048] Figure 3 This is a statistical graph of CCK-8 detection of human bone marrow mesenchymal stem cells seeded on the decellularized meniscus treated for 7 days in Example 1.

[0049] Figure 4 This is a fluorescence image of the chondrogenic marker SOX9 in which human bone marrow mesenchymal stem cells were seeded on the decellularized meniscus treated for 7 days in Example 1.

[0050] Figure 5 Schematic diagram of the biomaterial decellularization device in Example 1.

[0051] The reference numerals corresponding to the component names in the accompanying drawings are as follows:

[0052] 200. First Pipeline

[0053] 201. Second Pipeline

[0054] 202. The Third Pipeline

[0055] 3. First container

[0056] 300. First Opening

[0057] 301. Second Opening

[0058] 5. Second container

[0059] 6. Biomaterials

[0060] 8. Third container DETAILED DESCRIPTION

[0061] The present invention will be described in detail below with reference to the accompanying drawings.

[0062] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments and drawings.

[0063] In the present invention, the number of freeze-thaw cycles is preferably 3-5 times (the conditions for a single cycle may be: liquid nitrogen freezing for 4-8 minutes, thawing in a 37°C water bath for 8-15 minutes). The data used in the examples are the optimal data found out.

[0064] Example 1

[0065] The preparation method of the decellularized meniscus with high extracellular matrix retention described in this embodiment includes:

[0066] 1. Acquisition of meniscus tissue samples:

[0067] Obtain allogeneic or xenogeneic meniscus (the samples in this example are from adult New Zealand rabbits) and rinse 3-5 times with sterile PBS containing 1% penicillin-streptomycin in a sterile operating cabinet to remove the adherent tissue around the meniscus. The cleaned meniscus tissue sample is stored at -80°C for later use.

[0068] 2. Decellularization of meniscus:

[0069] The meniscus tissue obtained in step 1 was removed and wrapped in sterile tin foil and subjected to three freeze-thaw cycles (freeze in liquid nitrogen for 5 minutes and thaw in a 37°C water bath for 10 minutes). The freeze-thawed specimen was immersed in a sterile 0.1% Triton X-100 solution containing 1% penicillin-streptomycin, sealed, and continuously agitated at 80 rpm / min on a shaker at 4°C for 3, 7, and 14 days, with the solution changed every 24 hours. The Triton X-100 solution was discarded, and the specimen was placed in sterile 1% penicillin-streptomycin in PBS and washed three times for 2 hours each at 4°C on a shaker at 150 rpm / min. The wash solution was discarded, and the specimen was placed in a sterile saline solution containing 300 UI / mL DNase (Sigma-Aldrich) and 500 μg / mL RNase A (Sigma-Aldrich) and incubated at 37°C on a shaker at 100 rpm / min for 24 hours. Take out the sample tissue and place it in the negative pressure decellularization device (see the negative pressure decellularization device structure for details). Figure 5 ) and washed three times with sterile 1% penicillin-streptomycin-containing PBS for 6 hours each time. Decellularized meniscus (DMS) was obtained and stored at -80°C until use.

[0070] like Figure 5 As shown, the decellularization device includes a first pipe 200, a first container 3, a second pipe 201, a second container 5, and a third pipe 202, which are sealed and connected in sequence, and a third container 8 connected to the third pipe 202. One end of the first pipe 200 is connected to the first opening 300 of the first container 3; one end of the second pipe 201 is connected to the second opening 301 of the first container 3; the other end of the second pipe 201 and one end of the third pipe 202 are respectively connected to the two ends of the second container 5. The second container 5 is used to hold biological material 6, which cannot enter the second pipe 201 and the third pipe 202. However, the solution, including cells, can enter the second pipe 201 and the third pipe 202. When the third container 8 is positioned higher than the second container 5, and the second container 5 is positioned higher than the first container 3, the decellularized solution flows from the third container 8 through the second container 5 under the action of negative pressure or gravity, and then reaches the first container 3.

[0071] 3. Characterization of Decellularized Meniscus:

[0072] 3.1 The non-decellularized meniscus and three groups of decellularized meniscus treated at different time periods (3 days, 7 days, and 14 days) were dehydrated, embedded, and sectioned, and then subjected to H&E staining, toluidine blue staining, Sirius red staining, DAPI staining, and Shanghai First-Line Station synchrotron radiation infrared spectroscopy microscopy technology detection.

[0073] A. Place the sample slice (decellularized meniscus) in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and pure water for 5 minutes.

[0074] The method for staining sections with H&E staining is as follows: stain and mount the rehydrated sections in A according to the instructions of the H&E staining kit;

[0075] C. Toluidine blue staining method: stain and mount the rehydrated sections in A according to the instructions of the toluidine blue staining kit;

[0076] D. Sirius red staining method: stain and mount the rehydrated sections in A according to the instructions of the Sirius red staining kit, and photograph them using polarized light;

[0077] E. DAPI: Decellularized scaffolds and non-decellularized scaffolds were embedded in OCT and sliced, the OCT was washed off, DAPI indicator was added, the slices were sealed, and the images were taken under a fluorescence microscope;

[0078] F. The detection method of Shanghai First Line Station Synchrotron Radiation Infrared Spectroscopy Microscope is the same as CN 111084904B.

[0079] like Figure 1 As shown in Table 1, histological staining and statistical analysis showed that the cellular components were completely removed on day 7. Furthermore, under the same conditions of complete cellular removal, the extracellular matrix components (collagen and proteoglycans) retained in the decellularized meniscus on day 7 were significantly higher than those on day 14, and were similar to those on day 3. Therefore, 7 days of decellularization was the optimal time point for this study.

[0080] Table 1

[0081]

[0082] In Table 1: *** means P < 0.001.

[0083] 3.2 The obtained decellularized meniscus (DMS) (decellularized meniscus after 7 days of decellularization) was freeze-dried under sterile conditions and examined by electron microscopy. Figure 2As shown, compared with the non-decellularized meniscus, the decellularized meniscus obtained by this embodiment has a more loose and porous structure, which is more conducive to the growth and proliferation of host cells.

[0084] 3.3 Decellularized meniscus (DMS) (decellularized meniscus treated for 7 days) was placed in a well plate, and human bone marrow mesenchymal stem cells (hBMSC) were cultured at a rate of 2.5 x 10 5 / cm 2 Cells were seeded in well plates, and the group without scaffold served as the control group (CTRL). Complete culture medium (10% fetal bovine serum + 1% double-antibody + α-MEM basal medium) was added. During the culture process, cell viability was detected using the CCK-8 detection kit (CA1210, Solarbio, China) on days 1, 5, 7, 9, and 11. Figure 3 As shown, the cell viabilities in the two groups were comparable, indicating that the scaffolds were non-cytotoxic.

[0085] 3.4 After culturing the cells in 3.3 for 14 days, fix, permeabilize, and block the cells. Incubate with the primary antibody SOX9 (ab185966, Abcam, USA) overnight and add the corresponding secondary antibody. Detect under a fluorescence microscope. Figure 4 As shown in the figure, the number of SOX9-positive cells in the decellularized meniscus group was significantly more than that in the control group, indicating that the decellularized meniscus can effectively promote the differentiation of human bone marrow mesenchymal stem cells (hBMSC) into chondrogenic direction.

[0086] Table 2

[0087] Group SOX9-positive cells CTRL 39％ DMS 68％

[0088] In Table 2: * means P < 0.05.

[0089] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

[0090] Compared with the prior art, the present invention has the following advantages:

[0091] 1. This invention addresses the existing challenges of whole-body meniscus decellularization. The dense fibrous tissue of the whole meniscus makes decellularization difficult, while excessive decellularization can lead to excessive loss of extracellular matrix. This invention utilizes physical methods such as repeated freeze-thaw cycles, a negative pressure decellularization device (authorized patent: ZL202010103858.6), and optimized decellularization processes.

[0092] 2. The technical problem addressed by this invention is to provide a fully decellularized meniscus with a high degree of extracellular matrix retention, addressing the issues of donor shortage, implant shrinkage, and immune rejection associated with existing meniscus transplants. While completely removing cellular components, the extracellular matrix components of the meniscus are preserved to the greatest extent possible, providing a native microenvironment for cell growth.

[0093] 3. The extracellular matrix highly retained decellularized meniscus described in the present invention completely removes cellular components and effectively retains extracellular matrix components: collagen and proteoglycans. The provided original ecological microenvironment can effectively promote stem cell proliferation and promote stem cell differentiation into cartilage.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

1. A method for preparing acellular meniscus with high extracellular matrix retention, characterized in that: It includes the following steps: (1) subjecting the biological material to freeze-thaw cycles; Prior to the freeze-thaw cycle, the biological material was washed with phosphate buffered saline; The number of freeze-thaw cycles is 3-5 times, wherein the conditions of a single cycle are: freezing in liquid nitrogen for 4-8 minutes and thawing in a water bath at 30-40°C for 8-15 minutes; (2) taking out the biomaterial after freeze-thaw cycle treatment in step (1) and sequentially passing through cell elution solutions: solution A, solution B, solution C, and solution D until the biomaterial is completely decellularized; wherein when eluting with solution A, solution B, solution C, and solution D, the biomaterial is fixed in a container; Wherein, the solution A is a 0.1% Triton X-100 solution containing 1% penicillin-streptomycin; the solutions B and D are sterile 1% penicillin-streptomycin PBS; the solution C is a nuclease solution containing 300 UI / mL deoxyribonuclease I and 500 μg / mL ribonuclease A; The conditions for elution with solution A were continuous stirring at 80 rpm / min on a shaker at 4°C; The conditions for the elution with solution B were as follows: washing three times at 4°C on a shaker at 150 rpm / min, each time for 2 hours; The conditions for the elution of liquid C are as follows: incubating in a 37°C constant temperature shaker at 100 rpm / min for 24 hours; The conditions for the D solution elution are to wash with sterile 1% penicillin-streptomycin PBS three times, each time for 6 hours; The cell eluate does not contain trypsin; When the biomaterial subjected to freeze-thaw cycle treatment is eluted with solution A, the elution time is 7 days.

2. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 1, characterized in that: In step (1), the phosphate buffer is sterile PBS containing 1% penicillin-streptomycin; And / or, in step (1), the cleaning is performed at least once, and each cleaning time is at least 10 minutes; And / or, in step (1), the cleaning times are 3-5 times.

3. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 1, wherein: In step (1), the number of freeze-thaw cycles is 3 times; And / or, in step (1), in the freeze-thaw cycle, the conditions of a single cycle are: freezing in liquid nitrogen for 4-6 minutes and thawing in a 36-38°C water bath for 8-12 minutes.

4. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 1, wherein: In step (1), in the freeze-thaw cycle, the conditions for a single cycle are: freezing in liquid nitrogen for 5 minutes and thawing in a 37°C water bath for 10 minutes.

5. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 1, wherein: The condition for the liquid A elution is to replace the solution every 24 hours.

6. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 1, wherein: The solution C is a physiological saline solution containing 300 UI / mL DNase and 500 μg / mL ribonuclease A.

7. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 1, wherein: The D solution elution is performed by placing the cell in a negative pressure suction decellularization device.

8. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 7, characterized in that: The negative pressure decellularization device includes a first pipe 200, a first container 3, a second pipe 201, a second container 5 and a third pipe 202, which are sealed and connected in sequence, and a third container 8 connected to the third pipe 202; one end of the first pipe 200 is connected to the first opening 300 of the first container 3; one end of the second pipe 201 is connected to the second opening 301 of the first container 3; the other end of the second pipe 201 and one end of the third pipe 202 are respectively connected to the two ends of the second container 5; the second container 5 is used to place the biological material 6, and the biological material 6 cannot enter the second pipe 201 and the third pipe 202, and the solution including cells can enter the second pipe 201 and the third pipe 202; when the position of the third container 8 is higher than the second container 5, and the position of the second container 5 is higher than the first container 3, the decellularization fluid flows from the third container 8 through the second container 5 under the action of negative pressure or gravity, and then reaches the first container 3.

9. The method for preparing acellular meniscus with high extracellular matrix retention according to claim 1, wherein: The biological materials are bone, dermis, pericardium, cornea, blood vessel, bladder, esophagus, small intestinal mucosal matrix, cartilage and liver.

10. A decellularized meniscus with high extracellular matrix retention obtained by the method according to any one of claims 1 to 9.

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