Cartilage particles, cartilage scaffolds and preparation methods thereof

Abstract

A cartilage particle, a cartilage scaffold and a preparation method thereof, which relate to the field of cartilage repair materials. The preparation method of the cartilage particle comprises the following steps: providing a cartilage block; coating the surface of the cartilage block with a liquid cryoembedding agent, and then freezing it to obtain a composite material; grinding the composite material to form a primary particle product, mixing the primary particle product with water to dissolve the cryoembedding agent on the primary particle product, and obtaining the cartilage particle. The preparation method can effectively produce cartilage particles, and the cartilage scaffold made of the cartilage particles and collagen is conducive to the adhesion of chondrocytes and improves the mechanical properties.

Description

Technical Field

[0001] The present application relates to the technical field of cartilage repair materials, and in particular to a cartilage particle, a cartilage scaffold, and a preparation method and application thereof. Background Art

[0002] Articular cartilage is a transparent cartilage covering the synovial joints. It not only acts as a fulcrum, but also bears the compression force generated by adjacent joints and muscles. In addition, the shear force generated by joint movement will further wear the joint surface and make it vulnerable to damage. Cartilage tissue damage seriously affects human health. Some common surgical methods for treating cartilage damage include autologous cartilage transplantation and surgical repair. The source of autologous cartilage repair is limited, and surgical repair generally uses osteotomy, which will cause lifelong pain. With the development of tissue engineering technology, new solutions have been provided for cartilage damage repair.

[0003] In the prior art, cartilage repair is performed by constructing bionic scaffold materials. Bionic scaffold materials generally include natural materials and artificial synthetic materials. According to the principle of bionics, in order to obtain a good repair effect, it is necessary to select an engineered tissue scaffold with similar cartilage components. Since xenogeneic cartilage is widely available, xenogeneic cartilage is usually selected as a raw material for processing. The cartilage is made into powder and then compounded with other materials to form a scaffold material.

[0004] However, cartilage is flexible and it is difficult to form cartilage particles by directly grinding cartilage in a ball mill. In addition, the inventors of the present application tried to grind cartilage after freezing it and found that cartilage would regain its elasticity during the ball milling process and it was also difficult to form cartilage particles. Summary of the invention

[0005] The present application provides a cartilage particle, a cartilage scaffold and a preparation method thereof, which can effectively prepare cartilage particles, and the cartilage scaffold prepared by using the cartilage particles and collagen is conducive to the adhesion of chondrocytes and the improvement of mechanical properties.

[0006] This application is implemented as follows:

[0007] In a first aspect, the present application provides a method for preparing cartilage particles, comprising the following steps:

[0008] Provide cartilage blocks;

[0009] Coating the surface of the cartilage block with a liquid freezing embedding agent and then freezing it to obtain a composite material;

[0010] The composite material is ground into a primary particle product, and the primary particle product is mixed with water to dissolve the cryoembedding agent on the primary particle product to obtain cartilage particles.

[0011] In a possible embodiment, the step of coating the surface of the cartilage block with a liquid cryoembedding agent comprises: providing the liquid cryoembedding agent and immersing the cartilage block in the cryoembedding agent.

[0012] In a possible embodiment, the step of coating the surface of the cartilage block with a liquid cryoembedding agent comprises: providing the liquid cryoembedding agent and adhering the cryoembedding agent to the surface of the cartilage block.

[0013] In one possible embodiment, the preparation method includes at least one of the following limitations:

[0014] First limitation: the freezing embedding medium contains polyethylene glycol and polyvinyl alcohol;

[0015] Second limitation: the freezing temperature is -80 to -20°C; optionally, the freezing time is 1 to 24 hours;

[0016] The third limitation: after mixing the initial microparticles with water, the process further includes filtering, washing and drying, wherein the drying step is freeze drying; optionally, the freeze drying temperature is -40 to -20°C.

[0017] In a possible embodiment, the cartilage block provided is a freeze-dried cartilage block.

[0018] In a possible implementation manner, the grinding is performed using a tool having an array of convex points or array of convex strips on the surface.

[0019] Optionally, the tool having the array of convex points or array of convex strips on the surface is sandpaper or a file.

[0020] In a possible embodiment, the method further comprises: performing a decellularization treatment on the cartilage particles obtained after the filtering and washing.

[0021] In a second aspect, the present application provides cartilage particles, which are prepared by the preparation method of cartilage particles in the first aspect, and the cartilage particles are in the form of flakes.

[0022] In a third aspect, the present application provides a method for preparing a cartilage scaffold, comprising the following steps: mixing the cartilage particles described in the second aspect with a collagen solution and injecting the mixture into a mold, and freeze-drying the mixture to obtain a cartilage scaffold.

[0023] In one embodiment, when the cartilage particles are mixed with the collagen solution, chondroitin sulfate is also added for mixing.

[0024] In a fourth aspect, the present application provides a cartilage scaffold, which is prepared by the preparation method of the cartilage scaffold of the third aspect.

[0025] The embodiments of the present application have at least the following beneficial effects:

[0026] The method for preparing cartilage microparticles of the embodiment of the present application is to coat the surface of the cartilage block with a liquid cryoembedded agent, and then freeze it. The cryoembedded agent solidifies and plays a role in supporting the cartilage block tissue, and the obtained composite material has a greater hardness. During the grinding process, since the composite material has a certain hardness and the cryoembedded agent supports the inside of the cartilage block tissue, it can be ground into a preliminary microparticle. Subsequently, the preliminary microparticle is mixed with water to dissolve the cryoembedded agent. After filtering, washing, and drying, the cartilage microparticles are obtained. The method for preparing cartilage microparticles of the present application can effectively prepare flaky cartilage microparticles.

[0027] The preparation method of the cartilage scaffold of the present application is obtained by mixing the cartilage particles of the present application with collagen liquid and injecting them into a mold, and then freeze-drying. Since the cartilage particles are in the form of sheets, the cartilage particles are easy to overlap each other, and are mixed with collagen. After freeze-drying, they form a sponge. The collagen sponge and the flaky cartilage particles are fused and overlapped with each other, forming certain pores inside, and can form a three-dimensional mesh structure. It is used as a cartilage scaffold to facilitate the adhesion of chondrocytes. Moreover, the morphology of the cartilage itself is still retained in the flaky cartilage particles, and the effect of cartilage repair is better. In addition, collagen has certain biological activity, and collagen as a repair material can provide an excellent cell microenvironment, which is suitable for cartilage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a photo of the cartilage block of the embodiment of the present application;

[0030] Figure 2 This is a photo of a composite material obtained after a cartilage block according to an example of the present application is immersed in a freezing embedding medium and then frozen;

[0031] Figure 3 This is an optical microscope image of the cartilage particles of Example 1 of the present application;

[0032] Figure 4 This is an optical microscope image of the cartilage particles of Example 2 of the present application;

[0033] Figure 5 This is a photo of the dried cartilage particles of Example 1 of the present application;

[0034] Figure 6 This is a scanning electron microscope image of the dried cartilage particles of Example 1 of the present application;

[0035] Figure 7 This is a scanning electron microscope image of the dried cartilage particles of Example 2 of the present application. DETAILED DESCRIPTION

[0036] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0037] The following is a detailed description of the cartilage particles and their preparation method and application in the embodiments of the present application:

[0038] In a first aspect, the present application provides a method for preparing cartilage particles, comprising the following steps:

[0039] S1: Provide a cartilage block; wherein the cartilage block can be selected from cartilage of pigs, cattle, sheep, dogs, etc.

[0040] Optionally, the cartilage block provided is a freeze-dried cartilage block, and the hardness of the cartilage block increases after freeze-drying.

[0041] S2: Wrap the surface of the cartilage block with a liquid cryoembedder, and then freeze it to obtain a composite material. The cryoembedder solidifies after freezing, and can play a role in supporting the cartilage block tissue, making the composite material hard. In some embodiments, the cryoembedder contains polyethylene glycol and polyvinyl alcohol. Optionally, when used specifically, a biosharp cryoembedder can be used. It should be noted that the cryoembedder of this application is not limited to a specific manufacturer.

[0042] In other embodiments, the cryoembedding agent contains sucrose, polyvinyl pyrrolidone, gelatin and water. Optionally, the sucrose in the cryoembedding agent is 15-30wt%, the polyvinyl pyrrolidone is 2-3wt%, the gelatin is 5-8wt%, and the balance is water.

[0043] In addition, the freezing temperature is -80 to -20°C, for example, -80°C, -70°C, -60°C, -50°C, -40°C, -30°C or -20°C. Optionally, the freezing time is 1 to 24 hours, for example, any one of 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours and 24 hours or a value between any two of them. Freezing for a longer time under low temperature conditions can ensure that the freezing embedding agent has a better solidification effect, sufficient hardness, can better support the cartilage block tissue, and the hardness of the entire composite material is also better. Exemplarily, the freezing step is performed in a refrigerator.

[0044] In some embodiments, the step of coating the surface of the cartilage block with a liquid cryoembedding agent comprises: providing a liquid cryoembedding agent and immersing the cartilage block in the cryoembedding agent.

[0045] In some other possible embodiments, the step of coating the surface of the cartilage block with a liquid cryoembedding agent includes: providing a liquid cryoembedding agent, and adhering the cryoembedding agent to the surface of the cartilage block, for example, by coating.

[0046] S3: Grinding the composite material into a primary particle product, mixing the primary particle product with water to dissolve the cryoembedding agent on the primary particle product, and obtaining cartilage particles.

[0047] When the cartilage block exists alone, it is elastic and not easy to form microparticles during the grinding process. However, by freezing the cartilage and then ball-milling it, it is found that the cartilage will regain its elasticity during the ball-milling process and it is difficult to form cartilage microparticles. In the technical solution of the present application, during the grinding process, the composite material formed by the cartilage block and the cryoembedding agent is frozen and hardened, so that the composite material has a certain hardness, and the solidified cryoembedding agent supports the internal tissue of the cartilage block to increase the continuity of the tissue, so that it can be effectively ground into a preliminary microparticle product. Subsequently, by mixing the preliminary microparticle product with water, the water can dissolve the cryoembedding agent on the preliminary microparticle product, and after filtering, washing and drying, the cartilage microparticles are obtained.

[0048] In addition, the grinding method is at least one of grinding using a tool having an array of convex points or an array of convex strips on the surface.

[0049] When grinding with a tool having an array of convex points or convex strips on the surface, sandpaper, a file, etc. can be selected. It is understandable that the tool having an array of convex points or convex strips on the surface can also be other tools as long as it can form particles during grinding.

[0050] When sandpaper grinding is adopted, the mesh number of the sandpaper will affect the size of the cartilage particles. Optionally, the mesh number of the sandpaper is 20 to 1000 mesh, for example, any one of 24 mesh, 36 mesh, 40 mesh, 50 mesh, 60 mesh, 80 mesh, 100 mesh, 120 mesh, 150 mesh, 180 mesh, 220 mesh, 280 mesh, 320 mesh, 400 mesh, 500 mesh, 600 mesh, 800 mesh and 1000 mesh. When selecting a file, a suitable file type and specification can be selected as needed.

[0051] Furthermore, after mixing the microparticles with water, the process further includes filtering, washing and drying, wherein the drying step is freeze drying. It should be noted that the drying may also be natural drying, air drying, etc. Optionally, the freeze drying temperature is -40 to -20°C.

[0052] In a second aspect, the present application provides a cartilage particle, which is prepared by the preparation method of the cartilage particle of the present application, and the cartilage particle is in the form of a sheet.

[0053] Among them, the flaky cartilage particles still retain the pores of the cartilage itself, which is more conducive to the adhesion of chondrocytes.

[0054] In a third aspect, the present application provides a method for preparing a cartilage scaffold, comprising the following steps:

[0055] The cartilage particles of the second aspect are mixed with collagen solution and injected into a mold, and then freeze-dried to obtain a cartilage scaffold.

[0056] Since the cartilage particles are in the form of flakes, they can easily overlap with each other and are mixed with collagen. After freeze-drying, they form a sponge. The collagen sponge and the flake cartilage particles fuse and overlap with each other, forming certain pores inside, which can form a three-dimensional network structure. It is used as a cartilage scaffold to facilitate the adhesion of chondrocytes and has better mechanical properties. Moreover, the morphology of the cartilage itself is still retained in the flake cartilage particles, and the effect of cartilage repair is better. In addition, collagen has certain biological activity. As a repair material, collagen can provide an excellent cell microenvironment and is suitable for cartilage repair.

[0057] If cartilage microspheres are mixed with collagen solution and then injected into a mold for freeze drying, the cartilage microspheres are dispersed in the collagen sponge and cannot be effectively overlapped together. The overall mechanical properties of the formed cartilage scaffold are worse than those of the cartilage scaffold of the present application.

[0058] Optionally, when mixing the cartilage particles with the collagen solution, chondroitin sulfate is also added for mixing.

[0059] Among them, chondroitin sulfate exists naturally in connective tissue, and chondroitin sulfate can combine with water molecules in the body to lubricate joints and relieve pain. Chondroitin sulfate is added in the process of preparing cartilage scaffolds, and the obtained cartilage scaffolds can also play a role in lubricating joints when implanted in the body during cartilage repair.

[0060] In a fourth aspect, the present application provides a cartilage scaffold, which is prepared by the preparation method of the cartilage scaffold of the third aspect.

[0061] The cartilage particles, cartilage scaffolds, and preparation methods and applications of the present application are further described in detail below in conjunction with the examples.

[0062] Example 1

[0063] This embodiment provides a method for preparing cartilage particles, which comprises:

[0064] S1: providing cleaned cartilage blocks cut from pig bones;

[0065] S2: Immerse the cartilage block in a cryoembedding agent, and then freeze it in a -80°C refrigerator for 4 hours to obtain a composite material. The cryoembedding agent contains polyethylene glycol and polyvinyl alcohol, and specifically uses a biosharp cryoembedding agent.

[0066] S3: Grind the composite material with a file to form a preliminary microparticle, then mix the preliminary microparticle with water, centrifuge and wash with water, centrifuge and filter again after washing, and finally dry to obtain flaky cartilage microparticles.

[0067] Example 2

[0068] This embodiment provides a method for preparing cartilage particles, which comprises:

[0069] S1: providing cleaned cartilage blocks cut from pig bones;

[0070] S2: Immerse the cartilage block in a cryoembedding agent, and then freeze it in a -20°C refrigerator for 24 hours to obtain a composite material. The cryoembedding agent contains polyethylene glycol and polyvinyl alcohol, and specifically uses a biosharp cryoembedding agent.

[0071] S3: Grind the composite material on 120-mesh sandpaper to form a preliminary microparticle, then mix the preliminary microparticle with water, centrifuge and wash with water, centrifuge again after washing, and finally dry to obtain flaky cartilage microparticles.

[0072] Example 3

[0073] This embodiment provides a method for preparing cartilage particles, which comprises:

[0074] S1: providing freeze-dried cartilage blocks;

[0075] S2: Immerse the cartilage block in a cryoembedding agent, and then freeze it in a -40°C refrigerator for 12 hours to obtain a composite material. The cryoembedding agent contains polyethylene glycol and polyvinyl alcohol, and specifically uses a biosharp cryoembedding agent.

[0076] S3: Grind the composite material on 320-mesh sandpaper to form a preliminary microparticle, then mix the preliminary microparticle with water, centrifuge and wash with water, centrifuge again after washing, and finally dry to obtain flaky cartilage microparticles.

[0077] Example 4

[0078] This embodiment provides a method for preparing cartilage particles, which comprises:

[0079] S1:: Provide cleaned cartilage blocks cut from pig bones;

[0080] S2: Immerse the cartilage block in a cryoembedding agent, and then freeze it in a -60°C refrigerator for 8 hours to obtain a composite material, wherein the cryoembedding agent contains 30wt% sucrose, 2wt% polyvinyl pyrrolidone, 5wt% gelatin, and the balance is water.

[0081] S3: Grind the composite material on 600-mesh sandpaper to form a preliminary microparticle, then mix the preliminary microparticle with water, centrifuge and wash with water, centrifuge again after washing, and finally dry to obtain flaky cartilage microparticles.

[0082] Test example

[0083] (1) Figure 1 As shown, Figure 1 It is a piece of cartilage cut from pig bones; Figure 2 As shown, Figure 2 The composite material is obtained after the cartilage block is immersed in a cryoembedding medium and then frozen.

[0084] (2) After the microparticles of Example 1 and Example 2 were mixed with water and stirred for 5 minutes, the cartilage microparticles were observed under an optical microscope. The results were as follows: Figure 3 and Figure 4 shown.

[0085] (3) The photo of the dried cartilage particles in Example 1 is as follows Figure 5 shown.

[0086] (3) The dried cartilage particles of Example 1 and Example 2 were observed under an electron scanning microscope. The results were as follows: Figure 6 and Figure 7 shown.

[0087] from Figure 3 , Figure 4 , Figure 6 and Figure 7 It can be seen that the cartilage particles in the embodiment of the present application are in the form of flakes, and Figure 3 , Figure 4 It can be seen that the cartilage particles of the present application retain the morphology and structure of the cartilage itself. Figure 6 and Figure 7 It can be seen that the flake-like cartilage particles of the embodiment of the present application are easy to be stacked together, which is conducive to forming a three-dimensional network structure and improving the mechanical properties of the cartilage scaffold.

[0088] The above description is only a specific embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing cartilage particles, characterized in that: The following steps are involved: Provide cartilage blocks; The surface of the cartilage block is coated with a liquid cryoembedding agent, and then frozen to obtain a composite material; the cryoembedding agent contains polyethylene glycol and polyvinyl alcohol; The composite material is formed into a primary particle by grinding using a tool having an array of convex points or array of convex strips on the surface; the primary particle is mixed with water to dissolve the cryoembedding agent on the primary particle to obtain cartilage particles, which are in the form of flakes.

2. The method for preparing cartilage particles according to claim 1, characterized in that: The step of coating the surface of the cartilage block with a liquid cryoembedding agent comprises: providing the liquid cryoembedding agent and immersing the cartilage block in the cryoembedding agent.

3. The method for preparing cartilage particles according to claim 1, characterized in that: The step of coating the surface of the cartilage block with a liquid cryoembedding agent comprises: providing the liquid cryoembedding agent and adhering the cryoembedding agent to the surface of the cartilage block.

4. The method for preparing cartilage particles according to any one of claims 1 to 3, characterized in that: The freezing temperature is -80 to -20°C, and the freezing time is 1 to 24 hours; And / or, after the microparticles are mixed with water, the process further comprises filtering, washing and drying, wherein the drying step is freeze drying, and the freeze drying temperature is -40 to -20°C.

5. The method for preparing cartilage particles according to claim 4, characterized in that: Also includes: The cartilage microparticles obtained after filtering and washing are subjected to a decellularization treatment.

6. A cartilage particle, characterized in that: The cartilage particles are prepared by the method for preparing the cartilage particles according to any one of claims 1 to 5, and the cartilage particles are in the form of flakes.

7. A method for preparing a cartilage scaffold, characterized in that: It includes the following steps: The cartilage particles according to claim 6 are mixed with collagen solution and injected into a mold, and then freeze-dried to obtain a cartilage scaffold.

8. The method for preparing a cartilage scaffold according to claim 7, characterized in that: When the cartilage particles are mixed with the collagen solution, chondroitin sulfate is also added for mixing.

9. A cartilage scaffold, prepared by the preparation method of the cartilage scaffold according to claim 7 or 8.

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