Low immunogenicity biomaterial containing type II collagen

Abstract

The present invention relates to low immunogenic biomaterials containing collagen type II, which also contain other proteins, glycosaminoglycans, in particular the materials of the invention are derived from aquatic animals, such as connective tissues of aquatic animals, such as cartilage tissues and sheath tissues selected from aquatic animals. The invention also relates to a preparation method of the low-immunogenicity biological material containing type II collagen. The invention further relates to application of the low-immunogenicity biological material containing the type II collagen in repair or prevention of cartilage related to bone joints, nasal cartilage or auricular cartilage defect filling and medical cosmetology.

Description

Technical Field

[0001] The present invention relates to a low-immunogenicity biomaterial containing type II collagen, in particular, a low-immunogenicity biomaterial containing type II collagen suitable for medical use. Specifically, it relates to a low-immunogenicity biomaterial containing type II collagen from an aquatic animal source, and a preparation method thereof. The present invention further relates to the use of the low-immunogenicity biomaterial containing type II collagen in cartilage repair or prevention related to bone joints, filling of nasal cartilage or ear cartilage defects, medical cosmetology, and the like. Background Art

[0002] Many studies have shown that, unlike type I and type III collagen, common type II collagen is usually more immunogenic. Type II collagen is often combined with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (ICFA) to create a mouse / rat model of rheumatoid arthritis, which can induce and activate the cellular and humoral immune response of animals. Type II collagen with the telopeptide removed has the same immune effect, which shows that the immunogenicity of type II collagen is not determined by the telopeptide. The immunodominant epitopes in type II collagen and the higher-order structures they form are considered to be important reasons for triggering immunogens.

[0003] At present, collagen or collagen-based materials are mainly derived from natural tissue preparation technology or genetic engineering recombinant expression technology. Genetic engineering recombinant technology mainly prepares human collagen, and the amino acid sequence is completely consistent with human type II collagen; natural tissue preparation technology mainly processes common mammalian tissues by means of decellularization or extraction. Due to the high homology, the amino acid sequence of the mammalian natural type II collagen usually prepared is also approximately the same as that of human type II collagen. It is known that human type II collagen has immunodominant epitope fragments, and these fragments have the same amino acid sequence as common recombinant or natural type II collagen, which leads to certain immune safety risks in existing type II collagen materials and high application limitations.

[0004] In addition, recombinant collagen with specific epitopes is prepared by recombinant construction. Although this scheme is feasible in theory, it is difficult to implement in actual technology. Moreover, the recombinantly constructed type II collagen without immunogenic epitopes has a single component and lacks biological activity.

[0005] Patents disclosed in the prior art regarding immune epitopes of type II collagen mainly involve immune epitope screening, such as patent CN117736341A, and immune tolerance induction (such as patents CN116831975A, CN116392583A, CN117085118A, and CN117120466A), which focus more on the treatment of rheumatoid arthritis (RA), while the materials mainly involve the preparation and application of type II collagen. Patent CN116981766A discloses interstitial materials for encapsulating cells, preparation methods, and applications thereof. The inventor removes immune components from mammalian cartilage, and the resulting scaffold material is used to encapsulate cells, provide microcarriers for cell culture, and improve cell survival rates. The materials containing type II collagen produced in this patent are mainly targeted at cells of non-immune origin. Since the problem of material immune epitopes is not taken into account, there is a high biosafety risk in medical use. Patent CN117339014A discloses a method for preparing and using particles that promote cartilage regeneration. The inventors added type I collagen, hyaluronic acid and other ingredients to type II collagen solution, and added hydroxyapatite after cross-linking. The material is used to treat temporomandibular joint cartilage-related diseases. This patent does not take into account the immunogenicity of type II collagen, and type I collagen and hydroxyapatite are structurally different from articular cartilage. Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the present invention aims to provide a low immunogenicity biomaterial containing type II collagen. The main component of the material is type II collagen, which is particularly derived from aquatic animals. In particular, it can have an amino acid sequence different from that of human or common mammalian sources at specific sites, and does not show obvious immunogenicity. The low immunogenicity of the type II collagen-containing biomaterial of the present invention has greatly expanded the application of type II collagen-containing materials in the medical field. The material also contains other proteins, glycosaminoglycans and other ingredients, which can provide an ideal physiological environment for the repair and prevention of human articular cartilage damage. Moreover, due to the component composition of the low immunogenicity biomaterial containing type II collagen of the present invention, such as containing components such as various subtypes of collagen, glycosaminoglycans, and inorganic salts, it is surprising that the material of the present invention has an improved stable overall structure, and can induce directional differentiation of chondrocytes and promote the maturation of new cartilage, and can provide an ideal bionic physiological environment for the repair and prevention of articular cartilage damage.

[0007] The type II collagen-containing biomaterial of the present invention comprises, in a dry state of the biomaterial, 75 to 95% by mass of protein relative to the total mass of the biomaterial, wherein the type II collagen content is 40 to 90% by mass, in particular 78 to 92% by mass of protein, preferably 80 to 90% by mass of protein, and further comprises 9 to 20% by mass of glycosaminoglycans.

[0008] The low immunogenicity biomaterial containing type II collagen of the present invention can be in the form of sheets, columns, powders, gels, microspheres, etc.

[0009] According to a specific embodiment of the present invention, the low immunogenicity biomaterial containing type II collagen of the present invention is derived from aquatic animals, in particular from connective tissue, including but not limited to cartilage tissue and sheath tissue.

[0010] According to a specific embodiment of the present invention, the aquatic animals described in the present invention include but are not limited to fish, such as those selected from the order Gadidae, Rays, Chimaeras, Acipenserformes, Mugilius, Lanceriformes, Sepia, Siluriformes, Stingrays, Ictalidae, Siluridae, etc., and invertebrates such as jellyfish, etc. Including but not limited to longtail cod, Dali guitarfish, Xu's guitarfish, spotted guitarfish, Chinese fan ray, He's guitarfish, red stingray, Chinese stingray, sharp-nosed stingray, zebrafish, black-lined chimaera, Zeng's rabbit chimaera, Atlantic chimaera, Joe's chimaera, Russian sturgeon, Dali's sturgeon, Schrencki's sturgeon, Chinese sturgeon, Hibernian sturgeon, moon jellyfish, comb jellyfish, purple-striped sea jellyfish, swimming jellyfish, cycad jellyfish, root jellyfish, flag jellyfish, jellyfish, Japanese Sea squid, Humboldt squid, Schrencki's squid, Du's spear squid, Lei's spear squid, northern squid, heterospined squid, white-spotted squid, tailed spear squid, channel catfish, clouded catfish, longnose catfish, large-mouthed catfish, Basa fish, etc.

[0011] According to a specific embodiment of the present invention, the protein component of the biomaterial is mainly type II collagen, and other protein components also include other subtypes of collagen, such as type IX collagen, type XI collagen, type X collagen, type XII collagen, type VI collagen, type V collagen, type III collagen or type I collagen, or elastin, matrix protein, calmodulin, adiponectin or RING finger protein.

[0012] According to a specific embodiment of the present invention, the biomaterial protein component may also contain 0 to 5% by mass of at least one of the following proteins: elastin, matrix protein, calmodulin, adiponectin and / or RING finger protein, relative to the total mass of the biomaterial.

[0013] According to a specific embodiment of the present invention, relative to the total mass of the biomaterial, the biomaterial includes the following collagen:

[0014] - 40 to 90% by mass of type II collagen, in particular 50 to 85% of type II collagen, even 55 to 85%, especially 60 to 80%,

[0015] - 5% to 30% type IX collagen, in particular 8 to 27%, especially 10% to 25%,

[0016] - 0.5% to 15% type XI collagen, in particular 1 to 12%, especially 2 to 10%,

[0017] - 0% to 1% type X collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0018] - 0% to 1% type XII collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0019] - 0% to 1% type VI collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0020] - 0% to 1% type V collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0021] - 0% to 3% type III collagen, in particular 0.01 to 2%, especially less than 1%,

[0022] - 0% to 10% type I collagen, in particular 0.01 to 6%, especially less than 3%.

[0023] According to a specific embodiment of the present invention, the components in the biological material include glycosaminoglycan, inorganic salts and fat in addition to protein.

[0024] According to a specific embodiment of the present invention, the biomaterial further comprises glycosaminoglycans in a dry state, relative to the total mass of the biomaterial, and in particular, the glycosaminoglycan content is 10 to 20 mass%, preferably 11 to 18 mass%, and especially 12 to 17 mass%.

[0025] According to a specific embodiment of the present invention, the biomaterial further comprises inorganic salts, such as phosphates and carbonates, and in particular, the inorganic salt content is 0.1 to 1.5 mass%, preferably 0.2 to 1.2 mass%, and especially 0.3 to 1.0 mass%.

[0026] According to a specific embodiment of the present invention, the biomaterial may further contain fat, in particular, the fat content is less than 0.8% by mass, preferably 0.1% to 0.5% by mass, and especially 0.2% to 0.4% by mass.

[0027] According to a specific embodiment of the present invention, the type II collagen in the material has a specific amino acid sequence, and the amino acid sequence of the 250-274 position of the triple helix region is GPKGQX1GX2X3GX4X5GFKGEX6GPKGEX7G, wherein: X1 is Q, S, H or E, preferably Q and S; X2 is D, T, I or A, preferably D or I; X3 is P, V or G, preferably P and V; X4 is I, L, V, F or G, preferably I, L and F; X5 is P, K or S, preferably P; X6 is H, A or S, preferably H and A; X7 is H, R, S or A, preferably H and R. In particular, according to the actual test results, the sequence sites of the triple helix region may be offset, preferably not more than 10 sites toward the C-terminus or N-terminus.

[0028] According to a specific embodiment of the present invention, the type II collagen in the material has specific amino acid sequences at other sites, and the amino acid sequence of sites 103-121 in the triple helix region is GAX1GX2KGEX3GSX4GENGX5P, wherein: X1 is A, S, K or D, preferably A and S; X2 is S, A, E, R or G, preferably S, A and E; X3 is A, Q, G or P, preferably A; X4 is S, R, D, A or K, preferably S and R; X5 is A, P, Q, preferably A or P. In particular, according to actual test results, the above triple helix region sequence sites may also be offset, preferably not more than 10 sites offset to the C-terminus or N-terminus.

[0029] According to a specific embodiment of the present invention, in the type II collagen of the biomaterial, the number of GPP sequences is 25 to 38, preferably 30 to 36; the number of GG sequences is 4 to 17, preferably 5 to 16, especially 6 to 14.

[0030] According to a specific embodiment of the present invention, in the type II collagen of the biomaterial, the number of H amino acid residues is 4 to 15, preferably 5 to 13, and especially 6 to 12; or the number of G amino acid residues is 345 to 420, preferably 348 to 410, and especially 350 to 400.

[0031] According to a specific embodiment of the present invention, the DNA content of the biological material does not exceed 50 ng / mg, preferably does not exceed 30 ng / mg, and more preferably does not exceed 20 ng / mg.

[0032] According to a specific embodiment of the present invention, when the biomaterial is solid, it has a porous structure, preferably with multi-level pore sizes.

[0033] According to a specific embodiment of the present invention, when the biomaterial is implanted subcutaneously in mice for 30 days, the IgG and IgM levels are the same as those of the negative control, and the HE staining results are the same as those of the negative control.

[0034] According to a specific embodiment of the present invention, when the biomaterial is injected into the rabbit joint for 7 days and 14 days, the IgG and IgM levels of the synovial fluid in the joint cavity are no different from those of the control.

[0035] The present invention also relates to a composite material comprising the low immunogenic biomaterial containing type II collagen of the present invention. In particular, the composite material of the present invention comprises: the biomaterial containing type II collagen, and at least one selected from the following substances: type I collagen, type III collagen, sodium hyaluronate, methylcellulose, sodium carboxymethylcellulose, chitosan, polylactic acid, hydroxyapatite, sodium alginate, etc.

[0036] The present invention also relates to using the type II collagen-containing biomaterial or the composite material as a surface coating or structural graft of polyethylene terephthalate, polyethylene glycol, polyethylene glycol diacrylate, silicone, polyurethane, polyvinyl alcohol, acrylate, polymethyl methacrylate, or polydioxanone material. Surprisingly, when the type II collagen-containing biomaterial or the composite material is used as a surface coating or structural graft of polyethylene terephthalate, polyethylene glycol, polyethylene glycol diacrylate, silicone, polyurethane, polyvinyl alcohol, acrylate, polymethyl methacrylate, or polydioxanone material, it exhibits improved and more excellent biocompatibility and bioactivity.

[0037] The present invention also relates to a method for preparing the low immunogenicity biomaterial containing type II collagen, comprising the following steps:

[0038] (1) a pretreatment step, including a step of removing impurities and an optional step of removing fishy smell, wherein the step of removing impurities includes a step of removing impurities from aquatic animal tissues; the step of removing fishy smell includes a step of immersing the material in a deodorizing agent and stirring the reaction to remove fishy smell substances;

[0039] (2) Decellularization step: decellularizing the tissue obtained in step (1) by physical and / or chemical methods; preferably, decellularizing by a method that does not cause protein denaturation;

[0040] (3) washing step: rinsing the decellularized matrix obtained in step (2), optionally using purified water for rinsing;

[0041] (4) Optional drying and shaping steps: drying the obtained decellularized matrix, such as freeze-drying, to form a fixed shape, such as a solid or gel according to the final requirements.

[0042] (5) Optional subsequent steps: packaging and sterilizing the obtained materials, such as sterilizing the materials by irradiation or ethylene oxide.

[0043] According to a specific embodiment of the present invention, the deodorization process in step (1) has a reaction time of 12 to 72 hours, preferably 24 to 48 hours, and a reaction temperature of 4 to 15°C.

[0044] According to a specific embodiment of the present invention, the deodorizing agent in step (1) is selected from acids, oxidants, salts, alcohols, phenols, biological enzymes or mixtures thereof.

[0045] According to a specific embodiment of the present invention, the acid in step (1) is selected from one or more of hydrochloric acid, acetic acid, formic acid, sulfuric acid, citric acid, malic acid, phosphoric acid, and lactic acid. In particular, the concentration of the acid is 0.0005 to 0.03 mol / L, especially 0.001 to 0.02 mol / L, or even 0.0015 to 0.01 mol / L.

[0046] According to a specific embodiment of the present invention, the oxidant in step (1) is selected from one or more of hydrogen peroxide, peracetic acid, sodium peroxide, ammonium persulfate, sodium hypochlorite, and potassium bromate. In particular, the concentration of the oxidant is 0.1 to 5% by mass, preferably 0.5 to 4% by mass, and particularly 1 to 3% by mass.

[0047] According to a specific embodiment of the present invention, the salt in step (1) is selected from one or more of sodium chloride, potassium chloride, phosphate, borate, and sodium acetate. In particular, the concentration of the salt substance is 1% to 6% by mass, preferably 1.5 to 4% by mass, and particularly 2 to 3% by mass.

[0048] According to a specific embodiment of the present invention, the alcohol described in step (1) is selected from one or more of ethanol, isopropanol, methanol, pentanediol, and perillyl alcohol. In particular, the concentration of the alcohol is 5 to 40% by mass, preferably 10 to 35% by mass, and in particular 15 to 30% by mass.

[0049] According to a specific embodiment of the present invention, the phenol in step (1) is selected from one or more of ethyl maltol and carvacrol, and in particular, the concentration of the phenol is 0.005 to 0.02% by mass, preferably 0.006 to 0.015% by mass, and in particular 0.008 to 0.012% by mass.

[0050] According to a specific embodiment of the present invention, the biological enzyme described in step (1) is selected from one or more of trypsin, pepsin, chymotrypsin, neutral protease, papain, lipase, aldehyde dehydrogenase, and subtilisin. In particular, the concentration of the biological enzyme is 0.001 to 0.10% by mass, preferably 0.003 to 0.09% by mass, and particularly 0.003 to 0.08% by mass.

[0051] According to a specific embodiment of the present invention, in the preparation method:

[0052] - The decellularization step is carried out by a chemical method; in the chemical method, the reagent used does not contain a protein denaturing component, especially the protein denaturing component is selected from one or more of a surfactant, an acid, an alkali, and a biological enzyme; the treatment time is 0 to 48 hours, especially 0.5 to 24 hours, or even 1 to 12 hours, and the temperature is 2 to 15°C, especially 3 to 12°C, or even 4 to 10°C, wherein, in particular, the surfactant is selected from one or more of sodium dodecyl sulfate, TritonX-100, TritonX-114, Tween 20, Tween 80, thiobetaine 10, and 3-[(3-cholamidopropyl)dimethylaminopropyl]-1-propanesulfonic acid inner salt (CHAPS); in particular, the surfactant concentration used is 0.05% to 1% by mass, especially 0.08% to 0.5% by mass, or even 0.1% to 0.3% by mass; in particular, the acid used is selected from hydrochloric acid, acetic acid, formic acid, , sulfuric acid, citric acid, malic acid, phosphoric acid, lactic acid, in particular, the acid used has a concentration of 0.0005 to 0.03 mol / L, in particular 0.001 to 0.02 mol / L, or even 0.0015 to 0.01 mol / L; in particular, the base is selected from one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, and calcium hydroxide, in particular, the base used has a concentration of 0.005 to 0.3 mol / L, in particular 0.01 to 0.2 mol / L, or even 0.015 to 0.1 mol / L; in particular, the biological enzyme is selected from one or more of trypsin, pepsin, chymotrypsin, neutral protease, papain, lipase, aldehyde dehydrogenase, elastase, and subtilisin, in particular, the biological enzyme used has a concentration of 0.001 mass % to 0.10 mass %, preferably 0.003 mass % to 0.09 mass %, in particular 0.003 mass % to 0.08 mass %; or

[0053] - The decellularization step is performed by a physical method, wherein the physical method is selected from at least one of the following methods (preferably not causing protein denaturation): one or more of high pressure treatment, laser treatment, ultrasonic treatment, ultraviolet irradiation, and freeze-thaw treatment that do not cause protein denaturation, in particular, the treatment time is 0 to 24 h, in particular 0.5 to 15 h, or even 1 to 12 h, in particular, the temperature is 2 to 10° C., in particular 3 to 9° C., or even 4 to 8° C.; or

[0054] - The decellularization step uses both the chemical method and the physical method.

[0055] According to a specific embodiment of the present invention, the cleaning process uses pre-cooled purified water or pre-cooled saline solution for cleaning, and in particular, the saline solution is selected from at least one of phosphate saline solution, borate saline solution, tris(hydroxymethylaminomethane) saline solution, tris(hydroxymethylaminomethane)-ethylenediaminetetraacetic acid saline solution, acetic acid-sodium acetate saline solution, acetic acid-ammonium acetate saline solution, and citric acid-sodium citrate saline solution. In particular, when the cleaning solution is a saline solution, purified water is used for further cleaning after cleaning.

[0056] The present invention also relates to the use of the biomaterial and / or the composite material in preparing products suitable for cartilage repair or prevention related to bone joints, filling of nasal cartilage or ear cartilage defects, soft tissue filling for medical cosmetic purposes, and wound hemostasis. Moreover, the biomaterial and the composite material of the present invention can be applied to soft / hard bone defect filling, biological ink, cell expansion and cultivation, initiator substrate, residual cavity filling, biological glue, etc.

[0057] The present invention relates to the following embodiments:

[0058] Item 1. A biomaterial containing type II collagen, characterized in that, in a dry state, the biomaterial comprises 75% to 95% by mass of protein relative to the total mass of the biomaterial, wherein the content of type II collagen is 40% to 90% by mass, and further comprises 9% to 20% by mass of glycosaminoglycans.

[0059] Item 2. The biomaterial according to Item 1 above, wherein the biomaterial is derived from an aquatic animal, in particular, the biomaterial is derived from the connective tissue of an aquatic animal, and is particularly selected from the cartilage tissue and the sheath tissue of an aquatic animal.

[0060] Item 3. The biological material according to any of the preceding items, wherein the aquatic animal is selected from the group consisting of longtail cod, Da guitarfish, Xu's guitarfish, spotted guitarfish, Chinese fan ray, He's guitarfish, red stingray, Chinese stingray, sharp-nosed stingray, zebrafish, black-lined chimaera, Zeng's rabbit chimaera, Atlantic chimaera, Joe's chimaera, Russian sturgeon, Da's sturgeon, Acipenser schrenckii, Chinese sturgeon, Hibernian sturgeon, moon jellyfish, comb jellyfish, purple-striped sea jellyfish, swimming jellyfish, nematode jellyfish, root jellyfish, flag jellyfish, jellyfish, Japanese Sea squid, Humboldt squid, Siberian squid, Du's spear squid, Lei's spear squid, northern squid, heterospined squid, white-spotted squid, tailed spear squid, channel catfish, clouded catfish, long-snout catfish, large-mouthed catfish, and Basa fish.

[0061] Item 4. The biomaterial according to any one of the preceding items, wherein the biomaterial is in the form of sheets, columns, powders, gels, or microspheres.

[0062] Item 5. The biomaterial according to any one of the preceding items, wherein in a dry state, the biomaterial comprises 78 to 92% by mass of protein, preferably 80 to 90% by mass of protein, relative to the total mass of the biomaterial.

[0063] Item 6. A biomaterial according to any of the preceding items, wherein the protein in the biomaterial also includes at least one selected from the following proteins: type IX collagen, type XI collagen, type X collagen, type XII collagen, type VI collagen, type V collagen, type III collagen and type I collagen.

[0064] Item 7. A biomaterial according to any of the preceding items, wherein the protein in the biomaterial further comprises at least one of the following proteins in an amount of 0 to 5% by mass, relative to the total mass of the biomaterial: elastin, matrix protein, calmodulin, adiponectin and RING finger protein.

[0065] Item 8. The biomaterial according to any one of the preceding items, wherein the biomaterial comprises the following collagen relative to the total mass of the biomaterial:

[0066] - 50% to 85% type II collagen, in particular 55% to 85%, especially 60% to 80%,

[0067] - 5% to 30% type IX collagen, in particular 8 to 27%, especially 10% to 25%,

[0068] - 0.5% to 15% type XI collagen, in particular 1 to 12%, especially 2 to 10%,

[0069] - 0% to 1% type X collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0070] - 0% to 1% type XII collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0071] - 0% to 1% type VI collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0072] - 0% to 1% type V collagen, in particular 0.01 to 0.6%, especially less than 0.3%,

[0073] - 0% to 3% type III collagen, in particular 0.01 to 2%, especially less than 1%,

[0074] - 0% to 10% type I collagen, in particular 0.01 to 6%, especially less than 3%.

[0075] Item 9. The biomaterial according to any of the preceding items, wherein the biomaterial further comprises glycosaminoglycans, in particular, the glycosaminoglycan content is 10 to 20%, preferably 11 to 18%, especially 12 to 17%, relative to the total mass of the biomaterial.

[0076] Item 10. A biomaterial according to any of the preceding items, wherein the biomaterial further comprises inorganic salts, such as phosphates and carbonates, in particular, the inorganic salt content is 0.1 to 1.5%, preferably 0.2 to 1.2%, especially 0.3 to 1.0%, relative to the total mass of the biomaterial.

[0077] Item 11. The biomaterial according to any of the preceding items, wherein the biomaterial further comprises fat, in particular, the fat content is 0 to 5.5%, preferably 0.01 to 2%, especially less than 1%, relative to the total weight of the biomaterial.

[0078] Item 12. A biomaterial according to any of the preceding items, wherein the amino acid sequence of the triple helix region 250-274 positions of type II collagen in the biomaterial is GPKGQX1GX2X3GX4X5GFKGEX6GPKGEX7G, wherein: X1 is Q, S, H or E, preferably Q and S; X2 is D, T, I or A, preferably D or I; X3 is P, V or G, preferably P and V; X4 is I, L, V, F or G, preferably I, L and F; X5 is P, K or S, preferably P; X6 is H, A or S, preferably H and A; X7 is H, R, S or A, preferably H and R.

[0079] Item 13. A biomaterial according to Item 12, wherein the amino acid sequence at positions 250-274 of the triple helical region of type II collagen in the biomaterial is shifted, preferably by no more than 10 positions toward the C-terminus or N-terminus.

[0080] Item 14. A biomaterial according to any of the preceding items, wherein the amino acid sequence of the triple helical region 103-121 positions of type II collagen in the biomaterial is GAX1GX2KGEX3GSX4GENGX5P, wherein: X1 is A, S, K or D, preferably A and S; X2 is S, A, E, R or G, preferably S, A and E; X3 is A, Q, G or P, preferably A; X4 is S, R, D, A or K, preferably S and R; X5 is A, P, Q, preferably A or P.

[0081] Item 15. The biomaterial according to Item 14 above, wherein the amino acid sequence of positions 103-121 of the triple helical region of type II collagen in the biomaterial is shifted, preferably by no more than 10 positions toward the C-terminus or N-terminus.

[0082] Item 16. A biomaterial according to any of the preceding items, wherein the number of GPP sequences in the type II collagen of the biomaterial is 25 to 38, preferably 28 to 37, and especially 30 to 36; or the number of GG sequences is 4 to 17, preferably 5 to 16, and especially 6 to 14.

[0083] Item 17. A biomaterial according to any of the preceding items, wherein the number of H amino acid residues in the type II collagen of the biomaterial is 4 to 15, preferably 5 to 13, and especially 6 to 12; or the number of G amino acid residues is 345 to 420, preferably 348 to 410, and especially 350 to 400.

[0084] Item 18. The biomaterial according to any of the preceding items, wherein the DNA content of the biomaterial does not exceed 50 ng / mg, preferably does not exceed 30 ng / mg, and more preferably does not exceed 20 ng / mg.

[0085] Item 19. The biomaterial according to any one of the preceding items, wherein the biomaterial has a porous structure, preferably having multi-level pore sizes.

[0086] Item 20. A method for preparing a biomaterial according to any one of the preceding items, comprising the following steps:

[0087] (1) a pretreatment step, including a step of removing impurities and an optional step of removing fishy smell, wherein the step of removing impurities includes a step of removing impurities from aquatic animal tissues; the step of removing fishy smell includes a step of immersing the material in a deodorizing agent and stirring the reaction to remove fishy smell substances;

[0088] (2) Decellularization step: decellularizing the tissue obtained in step (1) by physical and / or chemical methods; preferably, decellularizing by a method that does not cause protein denaturation;

[0089] (3) washing step: rinsing the decellularized matrix obtained in step (2), optionally using purified water for rinsing;

[0090] (4) Optional drying and shaping steps: drying the obtained decellularized matrix, such as freeze-drying, to form a fixed shape, such as a solid or gel according to the final requirements;

[0091] (5) Optional subsequent steps: packaging and sterilizing the obtained materials, such as sterilizing the materials by irradiation or ethylene oxide.

[0092] Item 21. The method according to Item 20, wherein the deodorization process described in step (1) has a reaction time of 12 to 72 hours, preferably 24 to 48 hours; and a reaction temperature of 4 to 15°C.

[0093] Item 22. The method according to any one of Items 20-21 above, wherein the deodorizing agent is selected from an acid, an oxidizing agent, a salt, an alcohol, a phenol, a biological enzyme or a mixture thereof.

[0094] Item 23. According to the method of Item 22, the acid is selected from one or more of hydrochloric acid, acetic acid, formic acid, sulfuric acid, citric acid, malic acid, phosphoric acid, and lactic acid, and in particular, the concentration of the acid is 0.0005 to 0.03 mol / L, especially 0.001 to 0.02 mol / L, or even 0.0015 to 0.01 mol / L.

[0095] Item 24. The method according to Item 22, wherein the oxidant is selected from one or more of hydrogen peroxide, peracetic acid, sodium peroxide, ammonium persulfate, sodium hypochlorite, and potassium bromate, and in particular, the concentration of the oxidant is 0.1 to 5% by mass, preferably 0.5 to 4% by mass, and in particular 1 to 3% by mass.

[0096] Item 25. The method according to Item 22, wherein the salt is selected from one or more of sodium chloride, potassium chloride, phosphate, borate, and sodium acetate, and in particular, the concentration of the salt substance is 1 to 6 mass%, preferably 1.5 to 4 mass%, and in particular 2 to 3 mass%.

[0097] Item 26. The method according to Item 22, wherein the alcohol is selected from one or more of ethanol, isopropanol, methanol, pentanediol, and perillyl alcohol, and in particular, the concentration of the alcohol is 5 to 40% by mass, preferably 10 to 35% by mass, and in particular 15 to 30% by mass.

[0098] Item 27. The method according to Item 22, wherein the phenol is selected from one or more of ethyl maltol and carvacrol, and in particular, the concentration of the phenol is 0.005 to 0.02% by mass, preferably 0.006 to 0.015% by mass, and in particular 0.008 to 0.012% by mass.

[0099] Item 28. According to the method of Item 22, the biological enzyme is selected from one or more of trypsin, pepsin, chymotrypsin, neutral protease, papain, lipase, aldehyde dehydrogenase, and subtilisin, and in particular, the concentration of the biological enzyme is 0.001 to 0.10% by mass, preferably 0.003 to 0.09% by mass, and in particular 0.003 to 0.08% by mass.

[0100] Item 29. The method according to any one of Items 20 to 28, wherein:

[0101] - The decellularization step is carried out by a chemical method; in the chemical method, the reagent used does not contain a protein denaturing component, especially the protein denaturing component is selected from one or more of a surfactant, an acid, an alkali, and a biological enzyme; the treatment time is 0 to 48 hours, especially 0.5 to 24 hours, or even 1 to 12 hours, and the temperature is 2 to 15°C, especially 3 to 12°C, or even 4 to 10°C, wherein, in particular, the surfactant is selected from one or more of sodium dodecyl sulfate, TritonX-100, TritonX-114, Tween 20, Tween 80, thiobetaine 10, and 3-[(3-cholamidopropyl)dimethylaminopropyl]-1-propanesulfonic acid inner salt (CHAPS); in particular, the surfactant concentration used is 0.05% to 1% by mass, especially 0.08% to 0.5% by mass, or even 0.1% to 0.3% by mass; in particular, the acid used is selected from hydrochloric acid, acetic acid, formic acid, , sulfuric acid, citric acid, malic acid, phosphoric acid, lactic acid, in particular, the acid used has a concentration of 0.0005 to 0.03 mol / L, in particular 0.001 to 0.02 mol / L, or even 0.0015 to 0.01 mol / L; in particular, the base is selected from one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, and calcium hydroxide, in particular, the base used has a concentration of 0.005 to 0.3 mol / L, in particular 0.01 to 0.2 mol / L, or even 0.015 to 0.1 mol / L; in particular, the biological enzyme is selected from one or more of trypsin, pepsin, chymotrypsin, neutral protease, papain, lipase, aldehyde dehydrogenase, elastase, and subtilisin, in particular, the biological enzyme used has a concentration of 0.001 mass % to 0.10 mass %, preferably 0.003 mass % to 0.09 mass %, in particular 0.003 mass % to 0.08 mass %; or

[0102] - The decellularization step is performed by a physical method, wherein the physical method is selected from at least one of the following methods (preferably not causing protein denaturation): one or more of high pressure treatment, laser treatment, ultrasonic treatment, ultraviolet irradiation, and freeze-thaw treatment that do not cause protein denaturation, in particular, the treatment time is 0 to 24 h, in particular 0.5 to 15 h, or even 1 to 12 h, in particular, the temperature is 2 to 10° C., in particular 3 to 9° C., or even 4 to 8° C.; or

[0103] - The decellularization step uses both the chemical method and the physical method.

[0104] Item 30. A method according to any one of items 20 to 29, wherein the cleaning process uses pre-cooled purified water or pre-cooled saline solution for cleaning, and in particular the saline solution is selected from at least one of phosphate saline solution, borate saline solution, tris(hydroxymethylaminomethane) saline solution, tris(hydroxymethylaminomethane)-ethylenediaminetetraacetic acid saline solution, acetic acid-sodium acetate saline solution, acetic acid-ammonium acetate saline solution, and citric acid-sodium citrate saline solution. In particular, when the cleaning solution is a saline solution, purified water is used for further cleaning after cleaning.

[0105] Item 31. A composite material, comprising a biomaterial according to any one of Items 1 to 19 above or a biomaterial obtained according to the method described in any one of Items 20 to 30 above, optionally, the biomaterial is used as a surface coating or graft selected from at least one of the following substances: polyethylene terephthalate, polyethylene glycol, polyethylene glycol diacrylate, silicone, polyurethane, polyvinyl alcohol, acrylate, polymethyl methacrylate, polydioxanone.

[0106] Item 32. The composite material according to Item 31, further comprising at least one selected from the following substances: type I collagen, type III collagen, sodium hyaluronate, methyl cellulose, sodium carboxymethyl cellulose, chitosan, polylactic acid, hydroxyapatite, sodium alginate.

[0107] Item 33. Use of the biomaterial according to any one of the preceding items 1 to 19, or the biomaterial obtained according to the method described in any one of the preceding items 20 to 30, or the composite material described in any one of the preceding items 31 to 32 in the preparation of products for bone and joint related cartilage repair or prevention, soft / hard bone defect filling such as nasal cartilage or ear cartilage defect filling, soft tissue filling for medical cosmetic purposes, and wound hemostasis.

[0108] Item 34. The application according to Item 33, which is used for bio-ink, cell expansion culture, initiator substrate, residual cavity filling, and bio-glue.

[0109] Item 35. The use according to Item 33, wherein the biomaterial is used as a surface coating or structural graft of polyethylene terephthalate, polyethylene glycol, polyethylene glycol diacrylate, silicone, polyurethane, polyvinyl alcohol, acrylate, polymethyl methacrylate, and polydioxanone materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 It is a total ion current chromatogram of a biological material according to an embodiment of the present invention.

[0111] Figure 2 It is an electron microscope image of a cross section of a biological material according to an embodiment of the present invention. DETAILED DESCRIPTION

[0112] Example 1

[0113] This example examines the protein content, glycosaminoglycan content, fat content, and inorganic salt content of the biomaterial of the present invention.

[0114] Take the cartilage tissue of the northern Pacific squid, remove the fish meat and other impurities, and wash it. Add 0.01 mol / L citric acid and remove the fishy smell at 10°C for 24 hours. Add 0.4% sodium hydroxide and react for 12 hours, then wash with citric acid-sodium citrate solution, and then wash repeatedly with purified water. After drying the material, crush it into powder, and sterilize it with ethylene oxide to obtain a low immunogenic biomaterial containing type II collagen.

[0115] The protein content of the material was determined by referring to the method in Appendix A of the YY / T 1511-2017 standard, the glycosaminoglycan content was determined by referring to the method in the YY / T 1810-2022 standard, the inorganic salt content was determined by referring to the method in 4.7 of the YY / T 1453-2016 standard, and the fat content was determined by referring to the method in 4.11.

[0116] Comparative Example 1.1: Squid type II gelatin was prepared according to the method of Example 1 in patent CN11 1330075A, and the protein content, glycosaminoglycan content, fat content and inorganic salt content of the material were measured.

[0117] Comparative Example 1.2: Porcine cartilage was selected, and acellular cartilage matrix was prepared according to the scheme of Example 1 of patent CN108653814A, and the protein content, glycosaminoglycan content, fat content and inorganic salt content of the material were measured.

[0118] The measurement results are shown in Table 1.

[0119] Table 1 Component detection of different animal-derived biological materials

[0120] Grouping Protein content Glycosaminoglycan content Fat Content Inorganic salt content Example 1 85.2％ 13.9％ 0.2％ 0.7％ Comparative Example 1.1 77.1％ 8.6％ 0.9％ 1.9％ Comparative Example 1.2 82.6％ 5.1％ 1.6％ 3.1％

[0121] From the results, it can be seen that the biological material of the present invention has high protein content and glycosaminoglycan content, and low fat content and inorganic salt content; although the raw material of comparative example 1.1 is also aquatic animal tissue, the protein content and glycosaminoglycan content of the material are low, and the inorganic salt content is high; although the material of comparative example 1.2 has a high protein content, the glycosaminoglycan content is low, and the fat content and inorganic salt content are high.

[0122] Example 2

[0123] This example investigates the protein composition of the biomaterial of the present invention.

[0124] Take the cartilage tissue of the sturgeon, remove impurities such as fish meat, and wash it. Add 0.005 mol / L phosphoric acid and 2% sodium chloride, and remove the fishy smell at 8°C for 48 hours. Add 0.3% concentration of 3-[(3-cholesterol aminopropyl) dimethylamino]-1-propanesulfonic acid (CHAPS) and react for 12 hours, then ultrasonically treat for 2 hours at 8°C to decellularize. Wash with phosphate saline solution, and then wash repeatedly with purified water. After drying the material, make it into a column, and sterilize it with ethylene oxide to obtain a low immunogenic biomaterial containing type II collagen.

[0125] The method of the reference (Jacek R Wisniewski, Alexandre Zougman, NagarjunaNagaraj and Matthias Mann. Nature Methods, 2009, 6: 359-362) is to treat the biological material before testing, and after centrifugal drying, dissolve it in 0.1% formic acid, bottle it and load it for online LC-MS analysis. The analytical column uses a C18 column, the mobile phase A is 0.1% formic acid, and the mobile phase B is 80% acetonitrile and 0.1% formic acid. Gradient chromatography is used, and the concentration of mobile phase B increases from 5% to 38% within 30 minutes. The spray voltage of the mass spectrometer is 1.9kV, and the heating temperature of the ion transfer tube is 320°C. The primary mass spectrometer scan range is 350-1500m / z, and the maximum injection time is 100ms. The mass spectrometer data is processed and searched and analyzed using Peak software, and the protein proportion is calculated based on the protein peak area.

[0126] The measurement results are shown in Table 2.

[0127] Table 2 Protein composition analysis

[0128] Protein information Average molecular weight (peptide chain) Protein Specific Gravity Type II collagen 134881 52.74％ Type IX collagen 27171 26.41％ xI collagen 162542 11.28％ Collagen Type I 129953 6.80％ Type X collagen 73353 0.29％ Type VI collagen 64473 0.21％ Type XII collagen 315245 0.03％ Calmodulin 127430 01％ Adiponectin 23503 0.15％ RING finger protein 81785 0.08％

[0129] From the results, it can be seen that the main protein of the material of the present invention is type II collagen, followed by type IX collagen and type XI collagen, and also contains other subtypes of collagen such as type I collagen, as well as calmodulin, adiponectin and RING finger protein.

[0130] Example 3

[0131] This example examines the amino acid sequence of the biomaterial of the present invention.

[0132] Take the cartilage tissue of Acipenser schrenckii, remove impurities such as fish meat, and wash it. Add 0.001 mol / L hydrochloric acid and 0.003% pepsin to remove the fishy smell at 10°C for 24 hours. Add 0.1% concentration of Triton X-100 and react for 24 hours to decellularize. Wash with tris(hydroxymethylaminomethane)-ethylenediaminetetraacetic acid solution, and then wash repeatedly with purified water. After drying the material, make it into sheets, and after irradiation sterilization, obtain a low immunogenic biomaterial containing type II collagen.

[0133] The sample to be tested was prepared according to the method of Example 2, and the amino acid sequence was analyzed by LC-MS. The chromatographic mobile phase A was an aqueous solution containing 0.1% formic acid, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid. Gradient elution was used, and the mobile phase B increased from 2% to 80% within 70 minutes, and then decreased to 2% after 15 minutes. The spray voltage of the mass spectrometer was 3.5 kV, and the capillary temperature was 300 ° C. The original mass spectrometry data was searched by peaks software to confirm the type II collagen sequence.

[0134] Comparative Example 3.1: The amino acid sequence information of human type II collagen was collected from the NCBI database, and the same statistical analysis was performed as in Example 3.

[0135] The total ion current chromatogram of Example 3 is shown in Figure 1 The statistics of specific sites in the amino acid sequence and the number of related amino acid sequences and amino acid residues are shown in Table 3.

[0136] Table 3 Analysis of amino acid sequences at specific sites and statistics of specific amino acid residues

[0137]

[0138] The amino acid sequences of type II collagen in the material of the present invention at positions 250-274 and 103-121 are different from those of human type II collagen, and the sequences in this region may lead to differences in the local spatial conformation of type II collagen. Compared with the comparative example, the biomaterial of the present invention has fewer GPP sequences, more GG sequences, and more H amino acid residues and G amino acid residues.

[0139] Example 4

[0140] This example investigates the DNA content of the biological material of the present invention.

[0141] The samples of Example 2 of the present invention were taken, and the DNA content of the animal tissue raw materials and the biological materials after process treatment was determined according to the YY / T 1876-2023 standard.

[0142] Comparative Example 4.1: Chicken cartilage was processed according to the method of Example 1, and the DNA content of the animal tissue raw material and the processed biological material was determined.

[0143] The DNA content detection results are shown in Table 4.

[0144] Table 4 DNA content detection

[0145] Grouping Raw material DNA content (ng / mg) DNA content after process treatment (ng / mg) Example 4 79.4±6.2 12.1±1.7 Comparative Example 4.1 384.9±10.3 59.7±4.8

[0146] From the test results, it can be seen that the DNA content of the animal tissue raw materials selected by the present invention is lower, which has obvious advantages over the comparative example. After being processed by the process of the present invention, the DNA content is significantly reduced.

[0147] Example 5

[0148] This example investigates the immunogenic response of the biomaterial of the present invention in animal joints.

[0149] Take the microparticle sample of Example 1 of the present invention, disperse it in physiological saline to make a suspension for use. Establish an osteoarthritis New Zealand rabbit model to evaluate the immunogenicity of the material: The osteoarthritis model was established by the method of rabbit anterior cruciate ligament transection (ACLT) combined with meniscectomy (MTT), and the animals were treated with penicillin for 4 days after surgery; after the model was successfully established, 0.3 mL of the sample suspension was injected into the rabbit joint cavity, and the ELISA kit method was used to detect the immunoglobulin IgG and IgM content of the synovial fluid on the 7th and 14th days. Commercially available sodium hyaluronate was injected into the rabbit knee joint as a control.

[0150] The test results are shown in Table 5.

[0151] Table 5 Immunoglobulin content of samples and control articles at 7 and 14 days after implantation

[0152]

[0153] The test results show that when the material of the present invention is injected into the animal joints for 7 days and 14 days, the immunoglobulin content in the animal synovial fluid is not significantly different from that in the control, indicating that the material of the present invention has good immune safety.

[0154] Example 6

[0155] This example investigates the immunogenic response of the biomaterial of the present invention in animal joints.

[0156] The sample of Example 3 of the present invention was subjected to gold spraying treatment and observed with a scanning electron microscope at a magnification of 540 times.

[0157] The electron microscopy results are shown in Figure 2 As can be seen from the figure, the biomaterial of the present invention has a porous structure with multiple levels of pore sizes.

[0158] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A biomaterial containing type II collagen, characterized in that In a dry state, the biomaterial comprises 75 to 95% by mass of protein relative to the total mass of the biomaterial, wherein the content of type II collagen is 40 to 90% by mass, and further comprises 9 to 20% by mass of glycosaminoglycan.

2. The biomaterial according to claim 1, wherein the biomaterial is derived from an aquatic animal, in particular, the biomaterial is derived from the connective tissue of an aquatic animal, and is particularly selected from the cartilage tissue and thethecal tissue of an aquatic animal.

3. The biomaterial according to any one of the preceding claims, wherein Relative to the total mass of the biomaterial, the biomaterial includes the following collagen: - 50% to 85% type II collagen, in particular 55% to 85%, especially 60% to 80%, - 5% to 30% type IX collagen, in particular 8 to 27%, especially 10% to 25%, - 0.5% to 15% type XI collagen, in particular 1 to 12%, especially 2 to 10%, - 0% to 1% type X collagen, in particular 0.01 to 0.6%, especially less than 0.3%, - 0% to 1% type XII collagen, in particular 0.01 to 0.6%, especially less than 0.3%, - 0% to 1% type VI collagen, in particular 0.01 to 0.6%, especially less than 0.3%, - 0% to 1% type V collagen, in particular 0.01 to 0.6%, especially less than 0.3%, - 0% to 3% type III collagen, in particular 0.01 to 2%, especially less than 1%, - 0% to 10% type I collagen, in particular 0.01 to 6%, especially less than 3%.

4. The biomaterial according to any one of the preceding claims, wherein the amino acid sequence of positions 250-274 of the triple helical region of type II collagen in the biomaterial is GPKGQX1GX2X3GX4X5GFKGEX6GPKGEX7G, wherein: X1 is Q, S, H or E, preferably Q and S; X2 is D, T, I or A, preferably D or I; X3 is P, V or G, preferably P and V; X4 is I, L, V, F or G, preferably I, L and F; X5 is P, K or S, preferably P; X6 is H, A or S, preferably H and A; X7 is H, R, S or A, preferably H and R.

5. The biomaterial according to any one of the preceding claims, wherein the amino acid sequence of the triple helical region 103-121 of type II collagen in the biomaterial is GAX1GX2KGEX3GSX4GENGX5P, wherein: X1 is A, S, K or D, preferably A and S; X2 is S, A, E, R or G, preferably S, A and E; X3 is A, Q, G or P, preferably A; X4 is S, R, D, A or K, preferably S and R; X5 is A, P, Q, preferably A or P.

6. A method for preparing a biomaterial according to any one of the preceding claims, comprising the following steps: (1) a pretreatment step, including a step of removing impurities and an optional step of removing fishy smell, wherein the step of removing impurities includes a step of removing impurities from aquatic animal tissues; the step of removing fishy smell includes a step of immersing the material in a deodorizing agent and stirring the reaction to remove fishy smell substances; (2) Decellularization step: decellularizing the tissue obtained in step (1) by physical and / or chemical methods; preferably, decellularizing by a method that does not cause protein denaturation; (3) Cleaning step: rinsing the decellularized matrix obtained in step (2), optionally using purified water for rinsing; (4) Optional drying and shaping steps: drying the obtained decellularized matrix, such as freeze-drying, to form a fixed shape, such as a solid or gel according to the final requirements; (5) Optional subsequent steps: packaging and sterilizing the obtained materials, such as sterilizing the materials by irradiation or ethylene oxide.

7. The method according to claim 6, wherein: - The decellularization step is carried out by a chemical method; in the chemical method, the reagent used does not contain a protein denaturing component, especially the protein denaturing component is selected from one or more of a surfactant, an acid, an alkali, and a biological enzyme; the treatment time is 0 to 48 hours, especially 0.5 to 24 hours, or even 1 to 12 hours, and the temperature is 2 to 15°C, especially 3 to 12°C, or even 4 to 10°C, wherein, in particular, the surfactant is selected from one or more of sodium dodecyl sulfate, TritonX-100, TritonX-114, Tween 20, Tween 80, thiobetaine 10, and 3-[(3-cholamidopropyl)dimethylaminopropyl]-1-propanesulfonic acid inner salt (CHAPS); in particular, the surfactant concentration used is 0.05% to 1% by mass, especially 0.08% to 0.5% by mass, or even 0.1% to 0.3% by mass; in particular, the acid used is selected from hydrochloric acid, acetic acid, formic acid, , sulfuric acid, citric acid, malic acid, phosphoric acid, lactic acid, in particular, the acid used has a concentration of 0.0005 to 0.03 mol / L, in particular 0.001 to 0.02 mol / L, or even 0.0015 to 0.01 mol / L; in particular, the base is selected from one or more of sodium hydroxide, potassium hydroxide, ammonium hydroxide, and calcium hydroxide, in particular, the base used has a concentration of 0.005 to 0.3 mol / L, in particular 0.01 to 0.2 mol / L, or even 0.015 to 0.1 mol / L; in particular, the biological enzyme is selected from one or more of trypsin, pepsin, chymotrypsin, neutral protease, papain, lipase, aldehyde dehydrogenase, elastase, and subtilisin, in particular, the biological enzyme used has a concentration of 0.001 mass % to 0.10 mass %, preferably 0.003 mass % to 0.09 mass %, in particular 0.003 mass % to 0.08 mass %; or - The decellularization step is performed by a physical method, wherein the physical method is selected from at least one of the following methods (preferably not causing protein denaturation): one or more of high pressure treatment, laser treatment, ultrasonic treatment, ultraviolet irradiation, and freeze-thaw treatment that do not cause protein denaturation, in particular, the treatment time is 0 to 24 h, in particular 0.5 to 15 h, or even 1 to 12 h, in particular, the temperature is 2 to 10° C., in particular 3 to 9° C., or even 4 to 8° C.; or - The decellularization step uses both the chemical method and the physical method.

8. The method according to any one of claims 6 to 7, wherein: The cleaning process uses pre-cooled purified water or pre-cooled saline solution for cleaning, and in particular, the saline solution is selected from at least one of phosphate saline solution, borate saline solution, tris(hydroxymethylaminomethane) saline solution, tris(hydroxymethylaminomethane)-ethylenediaminetetraacetic acid saline solution, acetic acid-sodium acetate saline solution, acetic acid-ammonium acetate saline solution, and citric acid-sodium citrate saline solution. In particular, when the cleaning solution is a saline solution, purified water is used for further cleaning after cleaning.

9. A composite material comprising a biomaterial according to any one of claims 1 to 5 or a biomaterial obtained according to the method of any one of claims 6 to 8, wherein the biomaterial is used as a surface coating or graft of at least one of the following substances: polyethylene terephthalate, polyethylene glycol, polyethylene glycol diacrylate, silicone, polyurethane, polyvinyl alcohol, acrylate, polymethyl methacrylate, polydioxanone.

10. Use of the biomaterial according to any one of the preceding claims 1-5, or the biomaterial obtained according to the method according to any one of the preceding claims 6-8, or the composite material according to claim 9 in the preparation of products for cartilage repair or prevention related to bone joints, filling of soft / hard bone defects such as nasal cartilage or ear cartilage defects, soft tissue filling for medical and cosmetic purposes, and wound hemostasis, such as for bio-ink, cell expansion culture, initiator substrate, residual cavity filling, and bio-glue.

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