Aquatic animal-derived acellular matrix injection and preparation method thereof

Abstract

The invention relates to an aquatic animal-derived acellular matrix injection which comprises an acellular matrix, a stabilizer and a pH regulator, and a preparation method of the aquatic animal-derived acellular matrix injection. The invention also relates to an application of the aquatic animal-derived acellular matrix injection in preparation of a product for wound repair, or an application in preparation of a product for surgical operations, or a product for bone defect repair, cartilage defect repair, cornea repair, tissue thickening and residual cavity filling.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to a method for preparing an aquatic animal-derived decellularized matrix injection, which is expected to be applied to orthopedic fields such as osteoarthritis and cartilage damage. Background Art

[0002] There are two common types of joint injuries in clinical practice. One is traumatic acute injury caused by sports that causes pressure or tearing of the articular cartilage, such as athletes tearing the meniscus of the knee. The other more common injury is degeneration (osteoarthritis caused by long-term wear and tear) or long-term inflammation (rheumatoid arthritis, etc.). The most ideal treatment for the above two injuries is to use non-invasive treatment methods in the early stage of injury to relieve joint pain and induce cartilage repair. The current non-invasive treatment methods are mainly to relieve joint pain symptoms, such as intra-articular corticosteroid injection, lubricant injection, joint lavage or arthroscopic synovial cleaning, physical therapy, etc., but they cannot guide cartilage repair and are not long-term treatment options. The ideal non-invasive treatment method should introduce the concept of tissue engineering, meet the bionic design as much as possible, combine the components required for articular cartilage repair and the unique physical and chemical properties of synovial fluid in the joint cavity, simulate the composition and structure of the extracellular matrix of cartilage cells, and provide a microenvironment for the growth and differentiation of chondrocytes and cartilage tissues to spontaneously promote cartilage repair and restore joint function.

[0003] Decellularized cartilage matrix is ​​a decellularized animal cartilage tissue using appropriate methods. While removing immunogenicity, it retains the main components of cartilage matrix, type II collagen and glycosaminoglycans, which is beneficial for providing nutrition for the growth and differentiation of chondrocytes and cartilage tissue and promoting cartilage repair. Compared with decellularized cartilage matrix derived from terrestrial mammals, decellularized cartilage matrix from aquatic animals has become an ideal resource for product development due to its advantages such as low disease infectivity and freedom from religious restrictions. Bioactive materials based on aquatic animal-derived decellularized cartilage matrix have broad application prospects in the repair of cartilage defects caused by osteoarthritis and rheumatoid arthritis.

[0004] At present, there are some patents for the preparation methods and products containing decellularized cartilage matrix, but none of them involve the application of aquatic animal-derived decellularized cartilage matrix in joint cavity injection. Chinese patent CN102188748B discloses a method for preparing a cartilage decellularized matrix membrane, in which the cartilage is taken from the hyaline cartilage of humans, pigs, and sheep. The crushed cartilage tissue is decellularized with Triton and then prepared into a decellularized cartilage matrix slurry, which is air-dried to form a cartilage extracellular matrix membrane. Chinese patent CN113769168A discloses a method for preparing decellularized matrix particles for soft tissue filling and repair, which forms gradient degradation by mixing cross-linked or non-cross-linked decellularized matrix particles with suspending agents to meet the soft tissue repair and reconstruction of different needs. Chinese patent CN111840642B discloses a method for preparing a cartilage decellularized matrix composite scaffold, which completely digests the articular cartilage from rabbits or pigs with an acidic solution containing pepsin and then blends it with natural polymer materials and cell suspension to form a decellularized matrix scaffold for cartilage damage repair.

[0005] Since the decellularized matrix is ​​prepared from animal tissues through a decellularization process, and the content of proline, hydroxyproline and other amino acids in the aquatic animal-derived decellularized matrix is ​​relatively low, the material stability is weaker than that of the terrestrial animal-derived decellularized matrix, which can easily cause the loss of the bionic structure of the decellularized matrix.

[0006] At present, in the art, there is a need for an aquatic animal decellularized matrix injection, in particular, an aquatic animal-derived decellularized cartilage matrix injection, which has an undegraded bionic structure and provides a microenvironment for cell and tissue growth and differentiation. Summary of the invention

[0007] In view of the problems of existing clinical treatment methods and treatment effects, the purpose of the present invention is to provide an aquatic animal-derived decellularized matrix injection and a preparation method thereof, in particular, an aquatic animal-derived decellularized cartilage matrix injection, which especially simulates the unique physicochemical properties of synovial fluid in the joint cavity, provides a microenvironment for the growth and differentiation of chondrocytes and cartilage tissue, so as to spontaneously promote cartilage repair and restore joint function.

[0008] In order to achieve the above technical objectives, the present invention provides an aquatic animal-derived decellularized matrix injection, which has the characteristics of long-term viscoelasticity retention and long-term action, and can be used for large-scale production to meet the medical needs of clinical cartilage repair.

[0009] The inventors of the present invention have discovered that aquatic animal-derived decellularized matrix has a bionic structure similar to that of mammals such as pigs and cows, while maintaining the natural microstructure of the decellularized matrix and expanding its bioavailability. In addition, because it contains more abundant fat and omega-3 unsaturated fatty acids, it has the advantage of effectively reducing inflammatory responses and promoting the formation of new blood vessels in the wound area.

[0010] The inventors of the present invention have found that the aquatic animal-derived decellularized matrix injection provided by the present invention retains the microstructure of the original animal tissue, and is more conducive to inducing rapid cell migration, proliferation and differentiation, promoting cartilage repair, and restoring joint function.

[0011] The aquatic animal-derived decellularized matrix injection provided by the present invention effectively solves the difficulties of the current products on the market, such as short maintenance time and no induction of repair. The lubrication performance of the product can be enhanced by optionally adding a stabilizer. As a joint cavity injection, it has good stability and high viscoelasticity. While supplementing the joint fluid to provide shock-absorbing and lubricating effects, it provides a microenvironment for the growth and differentiation of chondrocytes and cartilage tissue, induces cartilage repair, and restores joint function. In order to obtain the desired functions and technical effects, the aquatic animal-derived decellularized matrix injection provided by the present invention has a low viscosity change rate, a low elastic modulus change rate, and a low hydroxyproline change rate.

[0012] The aquatic animal-derived decellularized matrix injection of the present invention comprises a decellularized matrix, a stabilizer, and a pH adjuster, and has a viscosity at room temperature of 300 to 1800 mPa.s, advantageously 300 to 1500 mPa.s, particularly 300 to 1300 mPa.s, and especially 300 to 1200 mPa.s; an elastic modulus of 20 to 100 Pa, particularly 24 to 68 Pa, particularly 24 to 50 Pa, and especially 25 to 45 Pa.

[0013] According to a specific embodiment of the present invention, the aquatic animal-derived decellular matrix injection comprises the following components: based on the total mass of the aquatic animal-derived decellular matrix injection, the decellular matrix comprises 1% to 3%, particularly 1% to 2%, even 1% to 1.5%, a stabilizer comprises 0.2% to 2%, particularly 0.5% to 1.5%, even 0.8% to 1.2%, an isotonicity regulator comprises 0% to 1%, particularly 0.5% to 0.95%, even 0.7% to 0.9%, a pH regulator comprises 0.1% to 0.6%, particularly 0.1% to 0.5%, even 0.3% to 0.5%. The aquatic animal-derived decellular matrix injection of the present invention can be stored at room temperature for two years or longer.

[0014] In the present invention, "viscosity change rate" refers to the difference between the viscosity of the aquatic animal-derived decellularized matrix injection when stored at a temperature close to human body temperature and the viscosity of the aquatic animal-derived decellularized matrix injection after storage at a temperature close to human body temperature, that is, viscosity change rate = 1-(viscosity after storage at a temperature close to human body temperature, for example, for 6 months) / (viscosity when stored at a temperature close to human body temperature).

[0015] In the present invention, "elastic modulus change rate" refers to the difference between the viscosity of the aquatic animal-derived decellularized matrix injection when stored at a temperature close to human body temperature and the elastic modulus of the aquatic animal-derived decellularized matrix injection after storage at a temperature close to human body temperature, for example, for 6 months, that is, the elastic modulus change rate = 1-(elastic modulus after storage at a temperature close to human body temperature, for example, for 6 months) / (elastic modulus when stored at a temperature close to human body temperature).

[0016] According to the present invention, the aquatic animal-derived decellularized matrix injection of the present invention can maintain a viscosity change rate of less than or equal to 10% and an elastic modulus change rate of less than or equal to 9% under conditions close to human body temperature. In particular, after being stored and maintained at human body temperature for 6 months, the viscosity change is less than 8%, especially less than 6%, or even less than 5%, or the elastic modulus change is less than 8%, especially less than 6%, or even less than 5%.

[0017] According to a specific embodiment of the present invention, the hydroxyproline content in the aquatic animal-derived decellularized matrix of the present invention is 3 wt % to 13 wt %, particularly 5 wt % to 12 wt %, or even 7 wt % to 12 wt %.

[0018] According to the present invention, the hydroxyproline change rate is defined as: hydroxyproline change rate = 1-hydroxyproline content of decellularized matrix / hydroxyproline content of animal tissue before decellularization, as expressed in percentage by weight. According to a specific embodiment of the present invention, the hydroxyproline change rate is less than 3%, particularly 0.1-2%, especially 0.1-1.5%, or even 0.1-1%.

[0019] The aquatic animal-derived decellularized matrix injection can be prepared into a viscous liquid and then filled for injection, or can be freeze-dried into a powder, tablet, granule, pill or sponge form and then hydrated for injection.

[0020] In view of the characteristics of aquatic animal sources, there is no literature in the prior art that discloses that aquatic animal-derived decellularized matrices can simultaneously have a low viscosity change rate, a low elastic modulus change rate, and a low hydroxyproline change rate, that is, it is impossible to simultaneously meet the requirements of good stability and high viscoelasticity of the relevant joint cavity injection fluid, and provide a corresponding structure of a microenvironment with good compatibility and complete growth and differentiation of chondrocytes and cartilage tissue while supplementing the joint fluid to provide shock-absorbing and lubricating effects, thereby inducing cartilage repair and restoring joint function.

[0021] The aquatic animal-derived decellularized matrix of the present invention is preferably selected from aquatic fish, in particular, selected from cartilage of aquatic organisms, wherein the aquatic organism is selected from squid, shark, ray, dragon fish, sturgeon, stingray, mackerel, tuna or saury, longtail cod, blue tip tail hake, Da ploughman, Xu's ploughman, granular ploughman, spotted ploughman, Chinese round fan ray, He's ray, spotted ray, hole ray, red ray, Chinese ray, Gu's ray, sharp-mouthed ray, Siberian sturgeon, Da ploughman, Xu's ploughman, granular ploughman, spotted ploughman, Chinese round fan ray, He's ray, spotted ray, hole ray, red ray, Chinese ray, Gu's ray, sharp-mouthed ray, Siberian sturgeon, Da ploughman, and He's ray. Acipenser 'Chern's sturgeon, naked sturgeon, Acipenser schrenckii, Chinese sturgeon, cone-toothed shark, blacktip shark, spike shark, mackerel shark, epaulette shark, nurse shark, hammerhead shark, whitetip reef shark, Australian tiger shark, catshark, silvertip shark, lemon shark, thresher shark, whale shark, tiger shark, blue shark, moon jellyfish, comb jellyfish, purple-striped sea jellyfish, swimming jellyfish, cycad jellyfish, root-mouthed jellyfish, flag-mouthed jellyfish, jellyfish, Argentine squid, Japan Sea squid, Humboldt squid, Steven's squid, Pacific squid, and tailed squid.

[0022] According to a specific embodiment of the present invention, the aquatic animal-derived decellularized matrix injection solution is added with a stabilizer, including but not limited to natural stabilizers, synthetic stabilizers and other substances that can enhance the stability of the solution. The stabilizer can prevent the aquatic animal-derived decellularized matrix injection solution from precipitation, stratification, flocculation and the like during the storage period, and maintain the uniformity and stability of the aquatic animal-derived decellularized matrix injection solution. Surprisingly, when the aquatic animal-derived decellularized matrix injection solution of the present invention is added with a stabilizer, the product shows significantly enhanced lubricity.

[0023] According to a specific embodiment of the present invention, based on the total mass of the aquatic animal-derived decellularized matrix injection, the stabilizer content is 0.2% to 2%, particularly 0.5% to 1.5%, especially 0.5% to 1.2%, or even 0.8% to 1.2%.

[0024] The stabilizer of the present invention has a purity of more than 95% and is stable at room temperature.

[0025] The natural stabilizer used in the present invention is preferably selected from one or more of gum arabic, gum tragacanth, peach gum, sodium alginate, agar, starch slurry, gelatin, carrageenan, dextrin, alginate, and dextran.

[0026] According to a specific embodiment of the present invention, at least one of the following solutions is preferred:

[0027] - When one or more of gum arabic, gum tragacanth, and peach gum are selected, the mass percentage thereof is preferably 0.2% to 0.27%;

[0028] -When one or more of agar, starch slurry and dextrin are selected, the mass percentage thereof is 0.3% to 0.5%;

[0029] - When one or more of sodium alginate, gelatin and dextran are used, the mass percentage thereof is 0.55% to 1%; or

[0030] -When one or more of carrageenan and alginate are selected, the mass percentage thereof is 0.38% to 0.47%;

[0031] Based on the total mass of aquatic animal-derived decellularized matrix injection solution.

[0032] The synthetic stabilizer used in the present invention is preferably selected from one or more of propylene glycol alginate, methyl cellulose, sodium starch phosphate, sodium carboxymethyl cellulose, carboxymethyl chitin, carboxymethyl chitosan, sodium alginate, casein, sodium polyacrylate, polyethylene oxide, and polyvinyl pyrrolidone.

[0033] According to a specific embodiment of the present invention, at least one of the following schemes is preferred:

[0034] - When one or more of propylene glycol alginate, sodium polyacrylate, polyoxyethylene, and polyvinyl pyrrolidone are selected, the mass percentage thereof is 0.2% to 0.3%;

[0035] - When one or more of methyl cellulose, sodium carboxymethyl cellulose, carboxymethyl chitin and carboxymethyl chitosan are used, the mass percentage thereof is 0.5% to 1%; or

[0036] - When one or more of sodium starch phosphate, sodium alginate, and casein are selected, the mass percentage thereof is 0.3% to 0.45% based on the total mass of the aquatic animal-derived decellularized matrix injection.

[0037] According to the present invention, the change in friction coefficient of the aquatic animal-derived decellularized matrix injection is defined as: change in friction coefficient = (friction coefficient before adding the stabilizer) - (friction coefficient after adding the stabilizer).

[0038] According to a specific embodiment of the present invention, the stabilizer can further enhance the lubrication properties of the aquatic animal-derived decellularized matrix injection, so that the friction coefficient of the aquatic animal-derived decellularized matrix injection changes to a friction coefficient reduction of 25% to 65%, particularly a reduction of 30% to 60%, or even a reduction of 32% to 58%.

[0039] According to a specific embodiment of the present invention, the isotonicity regulator can be selected from one or more of sodium chloride, glucose, glycerol, phosphate or citrate. Based on the total mass of the aquatic animal-derived decellularized matrix injection, the content of the isotonicity regulator is 0.1% to 1%, particularly 0.5% to 0.95%, especially 0.65% to 9%, or even 0.7% to 0.9%.

[0040] According to a specific embodiment of the present invention, the pH regulator is one or more of phosphate buffer solution, citric acid buffer solution, carbonate buffer solution, borate buffer solution, barbital buffer solution, and citrate buffer solution, and the amount of the pH regulator added makes the pH of the composition between 6.0 and 8.0.

[0041] According to a specific embodiment of the present invention, the decellularized matrix has at least one of the following characteristics:

[0042] - DNA removal rate greater than or equal to 97%, in particular greater than 98%, even greater than 99%,

[0043] - a glycosaminoglycan (GAG) content greater than or equal to 15%, in particular greater than 20%, even greater than 25%,

[0044] - a total protein content greater than or equal to 60%, in particular greater than 65%, even greater than 70%,

[0045] - the fibroblast growth factor (FGF) content of the acellular matrix is ​​greater than 30 ng / g, in particular greater than 50 ng / g;

[0046] - the transforming growth factor beta (TGF-β) content of the acellular matrix is ​​greater than 8 ng / g, in particular greater than 10 ng / g;

[0047] The cell proliferation rate of the decellularized matrix is ​​greater than 105%, preferably greater than 110%, or not more than 135%.

[0048] The present invention also relates to a method for preparing the aquatic animal-derived decellularized matrix injection. The method of the present invention can be used to decellularize aquatic animal cartilage while removing immunogenicity and retaining the effective components in the matrix to the maximum extent, while maintaining the natural microstructure of the decellularized matrix and expanding its bioavailability.

[0049] The method for preparing the aquatic animal-derived decellularized matrix injection of the present invention comprises the following steps, wherein the decellularized matrix is ​​prepared by pre-treating and decellularizing tissues of aquatic animals:

[0050] (1) providing a decellularized matrix step, which comprises the following steps:

[0051] (a) Pretreatment step: taking animal tissue, such as cartilage tissue of aquatic animals, and removing the surrounding impurities to remove the foreign tissue;

[0052] (b) decellularization step: decellularizing the tissue obtained in step (a) by physical and / or chemical methods; preferably, decellularizing by a method that does not involve protein denaturation;

[0053] (c) washing step: rinsing the decellularized matrix obtained in step (b), optionally using purified water for rinsing;

[0054] (d) optionally, a drying step: drying the decellularized matrix obtained in step (c), such as freeze-drying, and optionally forming it into a fixed shape;

[0055] (2) Optionally, the aquatic biogenic decellularized matrix and the implantation fluid are micronized to obtain a solution;

[0056] (3) adding a stabilizer, an isotonicity regulator, a pH regulator, and other optional adjuvants to the above-mentioned aquatic biological decellularized matrix solution in sequence, blending them, and obtaining an aquatic animal decellularized matrix injection solution;

[0057] (4) Optional subsequent step: freezing or drying the decellularized matrix solution obtained in step (3) to prepare a powder, tablet, granule, pill or sponge, and optionally packaging and sterilizing the solution.

[0058] According to a specific embodiment of the present invention, the pretreatment step (a) comprises: removing muscle tissue, basement membrane and / or epidermis to obtain tissue material without impurities, for example, obtaining cartilage tissue without impurities. Optionally, then using biological enzymes for enzymolysis at a temperature of 0-15°C for 12-60h, preferably for 13-48h or even 15-36h or especially less than 32h.

[0059] According to a specific embodiment of the present invention, in the pretreatment step (a), the biological enzyme is selected from at least one of elastase, type IV collagenase, papain, trypsin, tyrosinase, neutral protease, pepsin and flavor protease.

[0060] According to a specific embodiment of the present invention, in the pretreatment step (a), the enzymatic hydrolysis system is 0.004-0.006wt% elastase phosphate or pepsin hydrochloric acid solution; the enzymatic hydrolysis time is 12-32h, or even 15-30h; or, the enzymatic hydrolysis system is 0.003-0.005wt% trypsin borate or trypsin phosphate solution; the enzymatic hydrolysis time is 15-30h, or even less than 25h.

[0061] According to a specific embodiment of the present invention, the decellularization step (b) is carried out by a chemical method; the chemical method can be carried out in an alkaline solution, an acidic solution or a salt solution, or an enzyme that does not cause protein denaturation can be used, and the reagents used do not contain protein denaturing components, especially the protein denaturing components are concentrated hydrochloric acid, urea, guanidine hydrochloride, sodium dodecyl sulfate, dithiothreitol, dimethyl sulfoxide, and the alkaline solution used is, for example, selected from one or more of sodium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, and sodium bicarbonate solution; the acidic solution used is, for example, selected from one or more of hydrochloric acid, acetic acid, citric acid, lactic acid, and phosphoric acid solutions containing or not containing enzymes; the enzyme used is, for example, selected from pepsin, papain, trypsin, acid protease or flavor protease; the salt solution used is, for example, selected from one or more of phosphate buffer solution, sodium chloride solution, potassium chloride solution, and calcium chloride solution; or the decellularization treatment is carried out by a physical method, and in the physical method, in particular, a method selected from the following: one or more of high-pressure treatment, laser treatment, high-speed homogenization shearing, and ultraviolet irradiation that do not contain protein denaturing is used.

[0062] According to a specific embodiment of the present invention, the fluid used for implantation in step (2) is selected from one or more of water for injection, physiological saline, phosphate buffer solution, blood, plasma, dextrose, lactated Ringer's solution, carbonate buffer solution, borate buffer solution, and barbiturate buffer solution.

[0063] According to a specific embodiment of the present invention, the solid-liquid mass ratio of the aquatic biogenic decellularized matrix to the fluid used for implantation is 1:2 to 25, preferably 1:3 to 25, especially 1:4 to 20, or even 1:5 to 10.

[0064] According to a specific embodiment of the present invention, the step (2) is carried out under low temperature conditions, particularly at 2 to 10°C, preferably 4 to 8°C.

[0065] According to a specific embodiment of the present invention, the micronization method in step (2) is selected from one or more of ultrafine grinding, ball milling, impact grinding and shear grinding. In particular, the particle size of the microparticles is 15 μm to 1 mm, especially 200 μm to 800 μm.

[0066] According to a specific embodiment of the present invention, the step (3) is carried out under low temperature conditions, particularly at 2 to 10°C, preferably 4 to 8°C.

[0067] According to a specific embodiment of the present invention, the blending is carried out under one or more conditions selected from the group consisting of magnetic stirring, electric stirrer stirring, bubbling or shaking.

[0068] The present invention also relates to the use of the aquatic animal-derived decellularized matrix injection for repairing cartilage defects caused by osteoarthritis, rheumatoid arthritis, etc., and in particular, for preparing products for wound repair, such as for use in products for acute or chronic wound repair, residual cavity filling, and damaged tissue coverage, or for use in preparing for surgical operations, or for use in products such as bone defect repair, cartilage defect repair, corneal repair, tissue thickening, and residual cavity filling.

[0069] The present invention relates to the following embodiments:

[0070] Item 1. An aquatic animal-derived decellularized matrix injection, characterized in that it comprises a decellularized matrix, a stabilizer, and a pH adjuster, and has a viscosity of 300 mPa.s to 1800 mPa.s at room temperature and an elastic modulus of 20 Pa to 100 Pa.

[0071] Item 2. The aquatic animal-derived decellularized matrix injection according to Item 1, characterized in that it is an aquatic animal-derived decellularized cartilage matrix injection.

[0072] Item 3. The aquatic animal-derived decellularized matrix injection according to any of the preceding items is characterized in that the hydroxyproline content in the decellularized matrix is ​​3wt% to 13wt%, particularly 5wt% to 12wt%, or even 7wt%-12wt%, wherein the hydroxyproline change rate between the decellularized matrix and the animal tissue before decellularization is less than 3%, particularly 0.1-2%, especially 0.1-1.5%, or even 0.1-1%.

[0073] Item 4. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that its viscosity at room temperature is 300 to 1800 mPa.s, particularly 300 to 1500 mPa.s, and especially 300 to 1200 mPa.s.

[0074] Item 5. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that the viscosity change rate is less than 10%, particularly less than 8%, particularly less than 6%, or even less than 5% after being stored and maintained at human body temperature for 6 months, or the elastic modulus change rate is less than 9%, particularly less than 8%, particularly less than 6%, or even less than 5%, and in particular, the elastic modulus is 20Pa to 100Pa, particularly 24Pa to 50Pa, particularly 25Pa to 45Pa.

[0075] Item 6. The aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that the aquatic animal-derived decellularized matrix injection is in the form of a viscous liquid or can be made into a powder, tablet, granule, pill or sponge form.

[0076] Item 7. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that, based on the total mass of the aquatic animal-derived decellularized matrix injection, it comprises 1% to 3%, particularly 1% to 2%, or even 1% to 1.5% of the decellularized matrix, 0.2% to 2%, particularly 0.5% to 1.5%, or even 0.8% to 1.2% of the stabilizer, 0% to 1%, particularly 0.5% to 0.95%, or even 0.7% to 0.9% of the isotonicity regulator, and 0.1% to 0.6%, particularly 0.1% to 0.5%, or even 0.3% to 0.5% of the pH regulator.

[0077] Item 8. The aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that the aquatic animal-derived decellularized matrix is ​​derived from aquatic organisms, preferably selected from aquatic fish, and in particular selected from cartilage of aquatic organisms, wherein the aquatic organisms are selected from squid, shark, ray, dragon fish, sturgeon, stingray, mackerel, tuna or saury, longtail cod, blue tip tail hake, Da ploughman, Xu's ploughman, granular ploughman, spotted ploughman, Chinese round fan ray, He's ray, spotted ray, hole ray, red ray, Chinese Stingray, Cuculus stingray, sharp-nose stingray, Siberian sturgeon, Dabry's stingray, naked sturgeon, Acipenser schrenckii, Chinese sturgeon, cone-toothed shark, blacktip shark, spike shark, mackerel shark, epaulette shark, nurse shark, hammerhead shark, whitetip reef shark, Australian tiger shark, catshark, silvertip shark, lemon shark, thresher shark, whale shark, tiger shark, blue shark, moon jellyfish, comb jellyfish, purple-striped sea jellyfish, swimming jellyfish, cycad jellyfish, root-mouthed jellyfish, flag-mouthed jellyfish, jellyfish, Argentine squid, Japan Sea squid, Humboldt squid, Steven's squid, Pacific squid, and tailed squid.

[0078] Item 9. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that the stabilizer is selected from a natural stabilizer and a synthetic stabilizer, in particular, wherein the natural stabilizer is preferably selected from one or more of gum arabic, gum tragacanth, peach gum, sodium alginate, agar, starch slurry, gelatin, carrageenan, dextrin, alginate, and dextran, or wherein the synthetic stabilizer is preferably selected from one or more of propylene glycol alginate, methylcellulose, sodium starch phosphate, sodium carboxymethyl cellulose, carboxymethyl chitin, carboxymethyl chitosan, sodium alginate, casein, sodium polyacrylate, polyethylene oxide, and polyvinyl pyrrolidone.

[0079] Item 10. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that the stabilizer content is 0.2% to 2%, particularly 0.5% to 1.5%, especially 0.5% to 1.2%, or even 0.8% to 1.2%, based on the total mass of the aquatic animal-derived decellularized matrix injection.

[0080] Item 11. The aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that the injection further comprises an isotonicity regulator selected from one or more of sodium chloride, glucose, glycerol, phosphate or citrate.

[0081] Item 12. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that the content of the isotonicity regulator is 0.1% to 1%, particularly 0.5% to 0.95%, especially 0.65% to 9%, or even 0.7% to 0.9%, based on the total mass of the aquatic animal-derived decellularized matrix injection.

[0082] Item 13. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, characterized in that, based on the total mass of the aquatic animal-derived decellularized matrix injection, the pH adjuster is selected from one or more of phosphate buffer solution, citric acid buffer solution, carbonate buffer solution, borate buffer solution, barbital buffer solution, and citrate buffer solution, and the amount of the pH adjuster added makes the pH of the composition between 6.0 and 8.0.

[0083] Item 14. The aquatic animal-derived decellularized matrix injection according to any of the preceding items is characterized in that the friction coefficient of the aquatic animal-derived decellularized matrix injection changes from 25% to 65%, particularly from 30% to 60%, or even from 32% to 58%.

[0084] Item 15. An aquatic animal-derived decellularized matrix injection according to any of the preceding items, wherein the decellularized matrix has at least one of the following characteristics:

[0085] - DNA removal rate greater than or equal to 97%, in particular greater than 98%, even greater than 99%,

[0086] - a glycosaminoglycan (GAG) content greater than or equal to 15%, in particular greater than 20%, even greater than 25%,

[0087] - a total protein content greater than or equal to 60%, in particular greater than 65%, even greater than 70%,

[0088] - the fibroblast growth factor (FGF) content of the acellular matrix is ​​greater than 30 ng / g, in particular greater than 50 ng / g;

[0089] - the transforming growth factor beta (TGF-β) content of the acellular matrix is ​​greater than 8 ng / g, in particular greater than 10 ng / g;

[0090] - the cell proliferation rate of the acellular matrix is ​​greater than 105%, preferably greater than 110%, or not more than 135%; or

[0091] - The pH of the injection solution is 6.0 to 8.0, especially 6.5 to 7.5.

[0092] Item 16. A method for preparing an aquatic animal-derived decellularized matrix injection according to any of the preceding items, wherein the decellularized matrix is ​​prepared from aquatic animal tissues after pretreatment and decellularization, comprising the following steps:

[0093] (1) providing a decellularized matrix step, which comprises the following steps:

[0094] (a) Pretreatment step: taking animal tissue, such as cartilage tissue of aquatic animals, and removing the surrounding impurities to remove the foreign tissue;

[0095] (b) decellularization step: decellularizing the tissue obtained in step (a) by physical and / or chemical methods; preferably, decellularizing by a method that does not involve protein denaturation;

[0096] (c) washing step: rinsing the decellularized matrix obtained in step (b), optionally using purified water for rinsing;

[0097] (d) optionally, a drying step: drying the decellularized matrix obtained in step (c), such as freeze-drying, and optionally forming it into a fixed shape;

[0098] (2) Optionally, the aquatic biogenic decellularized matrix and the implantation fluid are micronized to obtain a solution;

[0099] (3) adding a stabilizer, an isotonicity regulator, a pH regulator or an optional auxiliary agent to the above-mentioned aquatic organism-derived decellularized matrix solution in sequence, blending, and obtaining an aquatic animal-derived decellularized matrix injection solution;

[0100] (4) Optional subsequent step: freezing or drying the decellularized matrix solution obtained in step (3) to prepare a powder, tablet, granule, pill or sponge, and optionally packaging and sterilizing the solution.

[0101] Item 17. The method according to Item 16 above, wherein the pretreatment step (a) comprises: removing muscle tissue, basement membrane and / or epidermis to obtain tissue material without impurities, for example, obtaining cartilage tissue without impurities. Optionally, then using a biological enzyme for enzymatic hydrolysis at a temperature of 0-15°C for 12-60 hours, preferably for 13-48 hours or even 15-36 hours or especially less than 32 hours.

[0102] Item 18. The method according to Item 16 or 17 above, wherein, in the pretreatment step (a), the biological enzyme is selected from at least one of elastase, type IV collagenase, papain, trypsin, tyrosinase, neutral protease, pepsin, and flavor protease.

[0103] Item 19. A method according to any one of Items 16 to 18, wherein in the pretreatment step (a), the enzymatic hydrolysis system is 0.004-0.006 wt% elastase phosphate or pepsin hydrochloric acid solution; the enzymatic hydrolysis time is 12-32 h, or even 15-30 h; or, the enzymatic hydrolysis system is 0.003-0.005 wt% trypsin borate or trypsin phosphate solution; the enzymatic hydrolysis time is 15-30 h, or even less than 25 h.

[0104] Item 20. The method according to any one of items 16 to 19 above, wherein the decellularization treatment step (b) is performed by a chemical method; in the chemical method, the reagents used do not contain protein-denaturing components, especially the protein-denaturing components are concentrated hydrochloric acid, urea, guanidine hydrochloride, sodium dodecyl sulfate, dithiothreitol, dimethyl sulfoxide; and / or the decellularization treatment is performed by a physical method, and in the physical method, in particular, a method selected from the following is used: high-pressure treatment, laser treatment, high-speed homogenization shearing and ultraviolet irradiation that do not contain protein-denaturing components.

[0105] Item 21. The method according to any one of items 18 to 20, wherein the cleaning solution in the cleaning step (c) is selected from one or more of purified water, water for injection, phosphate buffer solution, physiological saline, and edetic acid disodium salt solution.

[0106] Item 22. A method according to any one of items 18 to 21, wherein the fluid used for the implantation in step (2) is selected from one or more of water for injection, physiological saline, phosphate buffer solution, blood, plasma, dextrose, lactated Ringer's solution, carbonate buffer solution, borate buffer solution, and barbiturate buffer solution.

[0107] Item 23. The method according to any one of Items 18 to 22, wherein the solid-liquid mass ratio of the aquatic biological decellularized matrix to the fluid used for implantation is 1:2 to 25, preferably 1:3 to 25, in particular 1:4 to 20, or even 1:5 to 10.

[0108] Item 24. The method according to any one of Items 18 to 23, wherein step (2) is carried out under low temperature conditions, in particular at 2 to 10°C, preferably 4 to 8°C.

[0109] Item 25. The method according to any one of items 18 to 24, wherein the micronization method in step (2) is selected from one or more of ultrafine grinding, ball milling, impact grinding and shear grinding, and in particular, the particle size of the microparticles is 15 μm to 1 mm.

[0110] Item 26. The method according to any one of Items 18 to 25, wherein step (3) is carried out under low temperature conditions, in particular at 2 to 10°C, preferably 4 to 8°C.

[0111] Item 27. The method according to any one of Items 18 to 26, wherein the blending is carried out under one or more conditions selected from the group consisting of magnetic stirring, stirring with an electric stirrer, bubbling or shaking.

[0112] Item 28. Use of the aquatic animal-derived decellularized matrix injection according to any one of the preceding items 1 to 15 and the aquatic animal-derived decellularized matrix injection obtained according to the method described in any one of the preceding items 16 to 27 in the preparation of products for wound repair, such as products for acute or chronic wound repair, residual cavity filling, and damaged tissue coverage, or in the preparation of surgical operations, or in products such as bone defect repair, cartilage defect repair, corneal repair, tissue thickening, and residual cavity filling. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] FIG. 1 is a cross-section of the decellularized matrix of Example 1 before and after shown in Example 7, wherein Figure 1.1 This is a scanning electron micrograph of the matrix before decellularization. Figure 1.2 is a scanning electron micrograph of the decellularized matrix. DETAILED DESCRIPTION

[0114] Various exemplary embodiments of the present invention are now described in detail, which should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention. The following test methods and detection methods, if not otherwise specified, are conventional methods; the reagents and raw materials, if not otherwise specified, are commercially available.

[0115] Embodiment 1:

[0116] The preparation of squid cartilage acellular matrix injection solution comprises the following steps:

[0117] (1) providing a decellularized matrix step, comprising:

[0118] (a) Take the purchased squid cartilage tissue, remove other fat and muscle tissue, wash it, immerse it in 0.005% elastase phosphate solution at 10°C for 15 hours, remove the residual meat tissue with fascia until the complete nasal cartilage part is revealed, and wash it with pre-cooled water;

[0119] (b) Decellularization by chemical method: 500 g of the squid cartilage was added to 5 L of 0.01 M sodium hydroxide solution at a solid-liquid mass ratio of 1:10 at 4°C, and shaken for 120 min; and washed with electric stirring at 4°C for 8 h;

[0120] (c) adding 500 g of the squid cartilage acellular matrix into 10 L of 0.02 M phosphate buffer solution at a solid-liquid mass ratio of 1:20, shaking and washing at 4° C. for 6 h, and changing the solution 2-3 times during the washing process;

[0121] (2) 500 g of the washed squid cartilage acellular matrix was mixed with physiological saline at a solid-liquid mass ratio of 1:5, and ground and homogenized into a suspension at 4°C;

[0122] (3) Add 0.8% carboxymethyl chitosan and 0.01M phosphate buffer solution to the above suspension, stir and homogenize at 4°C to obtain squid cartilage acellular matrix injection.

[0123] Comparative Example 1

[0124] The preparation of squid cartilage acellular matrix injection solution comprises the following steps:

[0125] (1) providing a decellularized matrix step, comprising:

[0126] (a) Take the purchased squid cartilage tissue, remove other fat and muscle tissue, wash it, immerse it in 0.005% elastase phosphate solution at 10°C for 15 hours, remove the residual meat tissue with fascia until the complete nasal cartilage part is revealed, and wash it with pre-cooled water;

[0127] (b) Decellularization by chemical method: 500 g of the squid cartilage was added to 5 L of 0.01 M sodium hydroxide solution at a solid-liquid mass ratio of 1:10 at 4°C, and shaken for 120 min; and washed with electric stirring at 4°C for 8 h;

[0128] (c) adding 500 g of the squid cartilage acellular matrix into 10 L of 0.02 M phosphate buffer solution at a solid-liquid mass ratio of 1:20, shaking and washing at 4° C. for 6 h, and changing the solution 2-3 times during the washing process;

[0129] (2) 500 g of the cleaned squid cartilage was mixed with physiological saline at a solid-liquid mass ratio of 1:5, and the mixture was homogenized into a suspension by grinding at 4°C;

[0130] (3) Add 0.01 M phosphate buffer solution to the above suspension, stir and homogenize at 4° C. to obtain squid cartilage acellular matrix injection solution.

[0131] Example 2

[0132] The preparation of squid cartilage acellular matrix injection solution comprises the following steps:

[0133] (1) providing a decellularized matrix step, comprising:

[0134] (a) Take the purchased squid cartilage tissue, remove other fat and muscle tissue, wash it, immerse it in 0.005% elastase phosphate solution at 10°C for 15 hours, remove the residual meat tissue with fascia until the complete nasal cartilage part is revealed, and wash it with pre-cooled water;

[0135] (b) Decellularization by chemical method: 500 g of the squid cartilage was added to 5 L of 0.05 M sodium hydroxide solution at a solid-liquid mass ratio of 1:10 at 4°C, and shaken for 120 min; and washed by electric stirring at 4°C for 4 h;

[0136] (c) adding 500 g of the squid cartilage acellular matrix into 10 L of 0.02 M phosphate buffer solution at a solid-liquid mass ratio of 1:20, shaking and washing at 4° C. for 6 h, and changing the solution 2-3 times during the washing process;

[0137] (2) 500 g of the cleaned squid cartilage was mixed with physiological saline at a solid-liquid mass ratio of 1:5, and the mixture was homogenized into a suspension by grinding at 4°C;

[0138] (3) Add 1% sodium carboxymethyl cellulose and 0.01 M phosphate buffer solution to the above suspension, stir and homogenize at 4° C. to obtain squid cartilage acellular matrix injection.

[0139] Comparative Example 2

[0140] (1) providing a decellularized matrix step, comprising:

[0141] (a) Take the purchased squid cartilage tissue, remove other fat and muscle tissue, wash it, immerse it in 0.005% elastase phosphate solution at 10°C for 15 hours, remove the residual meat tissue with fascia until the complete nasal cartilage part is revealed, and wash it with pre-cooled water;

[0142] (b) Decellularization by chemical method: 500 g of the squid cartilage was added to 5 L of 0.05 M sodium hydroxide solution at a solid-liquid mass ratio of 1:10 at 4°C, and shaken for 120 min; and washed by electric stirring at 4°C for 4 h;

[0143] (c) adding 500 g of the squid cartilage acellular matrix into 10 L of 0.02 M phosphate buffer solution at a solid-liquid mass ratio of 1:20, shaking and washing at 4° C. for 6 h, and changing the solution 2-3 times during the washing process;

[0144] (2) 500 g of the cleaned squid cartilage was mixed with physiological saline at a solid-liquid mass ratio of 1:5, and the mixture was homogenized into a suspension by grinding at 4°C;

[0145] (3) Add 0.01 M phosphate buffer solution to the above suspension, stir and homogenize at 4° C. to obtain squid cartilage acellular matrix injection solution.

[0146] Example 3

[0147] The preparation of shark cartilage acellular matrix injection solution comprises the following steps:

[0148] (1) providing a decellularized matrix step, comprising:

[0149] (a) Take the purchased shark cartilage tissue, remove other fat and muscle tissue, wash it, immerse it in 0.005% elastase phosphate solution at 10°C for 15 hours, remove the residual meat tissue with fascia until the complete nasal cartilage part is revealed, and wash it with pre-cooled water;

[0150] (b) Decellularization by chemical method: 500 g of the shark cartilage was added to 5 L of 0.01 M sodium hydroxide solution at a solid-liquid mass ratio of 1:10 at 4°C, and shaken for 120 min; and washed under electric stirring at 4°C for 8 h;

[0151] (c) adding 500 g of the shark cartilage acellular matrix into 10 L of 0.02 M phosphate buffer solution at a solid-liquid ratio of 1:20, shaking and washing at 4° C. for 6 h, changing the solution 2-3 times during the washing process;

[0152] (2) 500 g of washed shark cartilage was mixed with physiological saline at a solid-liquid mass ratio of 1:8, and the mixture was homogenized into a suspension by grinding at 4°C;

[0153] (3) Add 1% carboxymethyl chitosan and 0.01 M phosphate buffer solution to the above suspension, stir and homogenize at 4° C. to obtain shark cartilage acellular matrix injection.

[0154] Comparative Example 3

[0155] The preparation of shark cartilage acellular matrix injection solution comprises the following steps:

[0156] (1) providing a decellularized matrix step, comprising:

[0157] (a) Take the purchased shark cartilage tissue, remove other fat and muscle tissue, wash it, immerse it in 0.005% elastase phosphate solution at 10°C for 15 hours, remove the residual meat tissue with fascia until the complete nasal cartilage part is revealed, and wash it with pre-cooled water;

[0158] (b) Decellularization by chemical method: 500 g of the shark cartilage was added to 5 L of 0.01 M sodium hydroxide solution at a solid-liquid mass ratio of 1:10 at 4°C, and shaken for 120 min; and washed under electric stirring at 4°C for 8 h;

[0159] (c) adding 500 g of the shark cartilage acellular matrix into 10 L of 0.02 M phosphate buffer solution at a solid-liquid ratio of 1:20, shaking and washing at 4° C. for 6 h, changing the solution 2-3 times during the washing process;

[0160] (2) 500 g of washed shark cartilage was mixed with physiological saline at a solid-liquid mass ratio of 1:8, and the mixture was homogenized into a suspension by grinding at 4°C;

[0161] (3) Add 0.01 M phosphate buffer solution to the above suspension and stir at 4°C to obtain shark cartilage acellular matrix injection.

[0162] This example illustrates in detail that the intra-articular injection solution prepared by the preparation method of the present invention has the characteristics of high stability and long-term viscoelasticity retention.

[0163] The decellularized matrix injections in Examples 1, 2, and 3 were placed in aging boxes at 25°C and 37°C, respectively, for a 6-month aging test, and samples were taken regularly to determine the viscosity and elastic modulus of the samples (viscosity test method: using a rotational viscometer, according to the third method of the viscosity determination method of Part 4 of the Pharmacopoeia of the People's Republic of China (2020 edition); elastic modulus test method: using a rheometer to record the elastic modulus of the material under specific conditions) to determine the long-term stability of the joint cavity injection samples. The measured results are shown in Table 1. The results show that after aging at 25°C and 37°C for 6 months, the viscosity change rate of all samples was less than 10%, and the elastic modulus change rate was less than 9%.

[0164] Table 1

[0165] Test items Example 1 Example 2 Example 3 Viscosity (initial, mPa.s) 1625 1785 1523 Viscosity (25℃, after aging for 6 months, mPa.s) 1579 1692 1486 Viscosity change rate (25℃, after 6 months of aging) 2.83％ 5.21％ 2.43％ Viscosity (37°C, after aging for 6 months, mPa.s) 1538 1610 1477 Viscosity change rate (37°C, after 6 months of aging) 5.35％ 9.80％ 3.02％ Elastic modulus (initial, Pa) 53 66 59 Elastic modulus (25℃, after aging for 6 months, Pa) 50 63 57 Elastic modulus change rate (25℃, after 6 months of aging) 5.66％ 4.55％ 3.39％ Elastic modulus (37℃, after aging for 6 months, Pa) 49 63 55 Elastic modulus change rate (37℃, after 6 months of aging) 7.55％ 4.55％ 6.78％

[0166] Example 5

[0167] This example illustrates in detail that the intra-articular injection solution prepared by adding a stabilizer in the present invention has the characteristic of low friction coefficient.

[0168] The joint cavity injection solutions prepared in the above Examples 1, 2, 3 and Comparative Examples 1, 2, 3 were subjected to friction coefficient tests (tested using a friction coefficient meter), and the results are shown in Table 1. After adding the stabilizer, the friction coefficients of the prepared joint cavity injection solutions were significantly reduced, indicating that the aquatic animal-derived decellularized matrix injection solution of the present invention can further enhance and improve the lubrication properties of the aquatic animal-derived decellularized cartilage matrix joint cavity injection solution.

[0169] Table 2

[0170]

[0171]

[0172] Example 6: Hydroxyproline content after preparation of acellular matrix

[0173] The aquatic animal tissue decellularized matrix obtained in step (1) of Example 1 was repeatedly rinsed until it was neutral and had an electrical conductivity of less than 10 μS / cm, and then immersed in purified water for 5 h, freeze-dried, and ground into powder using a grinder. Then, the initial hydroxyproline content of the animal tissue was determined according to the method in Appendix B of YY / T 1453-2016, and the hydroxyproline change rate was calculated (hydroxyproline change rate = 1-hydroxyproline content of decellularized matrix / hydroxyproline content of animal tissue before decellularization).

[0174] Comparative Example 6.1 Adult pig skin was selected and acellular matrix was prepared according to the method of Example 1, and the hydroxyproline content was measured. The test results are shown in Table 3 below.

[0175] Table 3

[0176]

[0177] As can be seen from the above table, the change rate of hydroxyproline content in the aquatic animal-derived decellularized matrix of the present invention before and after preparation is small.

[0178] Example 7

[0179] Another squid cartilage tissue was removed from surrounding tissues, washed with purified water, and freeze-dried using the same freeze-drying process as in Example 1. The decellularized matrix in Example 1 and the tissue before animal decellularized matrix were treated with gold spraying and observed using a scanning electron microscope.

[0180] Test results such as Figure 1.1 and Figure 1.2 shown.

[0181] FIG1 is a cross-section of the decellularized matrix of Example 1 of the present invention before and after, showing that the three-dimensional structure of the decellularized matrix before and after remains intact and basically the same, wherein Figure 1.1 It is the three-dimensional structure of the present invention before decellularization. Figure 1.2It is the three-dimensional structure of the decellularized matrix of the present invention.

[0182] It can be seen that the cross-sections of the squid cartilage tissue in the embodiment of the present invention and the squid cartilage tissue acellular matrix of the present invention are both similar multi-layer fiber structures, and the layered fiber structures are complete and basically the same.

[0183] Example 8

[0184] The aquatic animal-derived decellularized cartilage matrix obtained by the decellularization process of the above-mentioned embodiment and comparative example was analyzed. The decellularized matrix of the embodiment was taken, and its DNA residue was determined according to the method of YY / T 1876-2023. The decellularized matrix of the embodiment was taken, and its total protein content was determined according to the method of YY / T 0954-2015. The decellularized matrix of the embodiment was taken, and its glycosaminoglycan (GAG) content was determined according to the method of YYT 1810-2022. The test results are shown in Table 4.

[0185] Table 4

[0186] Test items Example 1 Example 2 Example 3 DNA removal efficiency 99.49％ 98.77％ 99.50％ Total protein content 63.24％ 62.46％ 63.81％ Glycosaminoglycan (GAG) content 15.21％ 15.59％ 17.07％

[0187] The above embodiments are merely exemplary embodiments 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 shall fall within the scope of protection of the present invention.

Claims

1. Aquatic animal-derived decellularized matrix injection, characterized in that: The decellularized matrix comprises a stabilizer, and a pH adjuster, and has a viscosity of 300 mPa.s to 1800 mPa.s at room temperature and an elastic modulus of 20 Pa to 100 Pa.

2. The aquatic animal-derived decellularized matrix injection according to claim 1, characterized in that: It is an aquatic animal-derived decellularized cartilage matrix injection.

3. The aquatic animal-derived decellularized matrix injection according to any one of the preceding claims, characterized in that: The hydroxyproline content in the decellularized matrix is ​​3wt% to 13wt%, particularly 5wt% to 12wt%, or even 7wt%-12wt%, wherein the hydroxyproline change rate between the decellularized matrix and the animal tissue before decellularization is less than 3%, particularly 0.1-2%, especially 0.1-1.5%, or even 0.1-1%.

4. The aquatic animal-derived decellularized matrix injection according to any one of the preceding claims, characterized in that: After storage and maintenance at human body temperature for 6 months, the viscosity change rate is less than 10%, especially less than 8%, especially less than 6%, or even less than 5%, or the elastic modulus change rate is less than 9%, especially less than 8%, especially less than 6%, even less than 5%, especially, the elastic modulus is 20Pa to 100Pa, especially 24Pa to 50Pa, especially 25Pa to 45Pa.

5. The aquatic animal-derived decellularized matrix injection according to any one of the preceding claims, characterized in that: The stabilizer is selected from natural stabilizers and synthetic stabilizers, in particular, the natural stabilizer is preferably selected from one or more of gum arabic, gum tragacanth, peach gum, sodium alginate, agar, starch slurry, gelatin, carrageenan, dextrin, alginate, and dextran, or the synthetic stabilizer is preferably selected from one or more of propylene glycol alginate, methylcellulose, sodium starch phosphate, sodium carboxymethyl cellulose, carboxymethyl chitin, carboxymethyl chitosan, sodium alginate, casein, sodium polyacrylate, polyethylene oxide, and polyvinyl pyrrolidone.

6. The method for preparing the aquatic animal-derived decellularized matrix injection according to any one of the preceding claims, wherein: The decellularized matrix is ​​prepared by pre-treating and decellularizing tissues of aquatic animals, and comprises the following steps: (1) providing a decellularized matrix step, which comprises the following steps: (a) Pretreatment step: taking animal tissue, such as cartilage tissue of aquatic animals, and removing the surrounding impurities to remove the foreign tissue; (b) decellularization step: decellularizing the tissue obtained in step (a) by physical and / or chemical methods; preferably, decellularizing by a method that does not involve protein denaturation; (c) washing step: rinsing the decellularized matrix obtained in step (b), optionally using purified water for rinsing; (d) optionally, a drying step: drying the decellularized matrix obtained in step (c), such as freeze-drying, and optionally forming it into a fixed shape; (2) Optionally, the aquatic biogenic decellularized matrix and the implantation fluid are micronized to obtain a solution; (3) adding a stabilizer, an isotonicity regulator, a pH regulator or an optional auxiliary agent to the above-mentioned aquatic organism-derived decellularized matrix solution in sequence, blending, and obtaining an aquatic animal-derived decellularized matrix injection solution; (4) Optional subsequent step: freezing or drying the decellularized matrix solution obtained in step (3) to prepare a powder, tablet, granule, pill or sponge, and optionally packaging and sterilizing the solution.

7. The method according to claim 6, wherein: The decellularization step (b) is performed by a chemical method; in the chemical method, the reagents used do not contain protein-denaturing components, especially the protein-denaturing components are concentrated hydrochloric acid, urea, guanidine hydrochloride, sodium dodecyl sulfate, dithiothreitol, and dimethyl sulfoxide; and / or the decellularization is performed by a physical method, and in the physical method, a method selected from the following is particularly used: high-pressure treatment, laser treatment, high-speed homogenization shearing, and ultraviolet irradiation that do not contain protein-denaturing components.

8. The method according to any one of claims 6 to 7, wherein: The solid-liquid mass ratio of the aquatic biogenic decellular matrix to the fluid used for implantation is 1:2 to 25, preferably 1:3 to 25, especially 1:4 to 20, even 1:5 to 10.

9. The method according to any one of claims 6 to 8, wherein: The step (2) is carried out under low temperature conditions, particularly at 2 to 10°C, preferably 4 to 8°C.

10. Use of the aquatic animal-derived decellularized matrix injection according to any one of the preceding claims 1-5 and the aquatic animal-derived decellularized matrix injection obtained according to the method described in any one of the preceding claims 6-9 in the preparation of products for wound repair, such as products for acute or chronic wound repair, residual cavity filling, and damaged tissue coverage, or in the preparation of surgical operations, or in products such as bone defect repair, cartilage defect repair, corneal repair, tissue thickening, and residual cavity filling.

Images