Aquatic animal-derived acellular matrix hydrogel and preparation method thereof

Abstract

The invention relates to an aquatic animal-derived acellular matrix hydrogel, which comprises an acellular matrix and a non-biological auxiliary agent, and a preparation method of the aquatic animal-derived acellular matrix hydrogel. The invention further relates to application of the aquatic animal-derived acellular matrix hydrogel in preparation of a product for wound repair or application of the aquatic animal-derived acellular matrix hydrogel in preparation of a product for surgical operation.

Description

Technical Field

[0001] The present invention relates to aquatic animal-derived decellularized matrix hydrogel and a preparation method thereof, and also relates to the use of aquatic animal-derived decellularized matrix hydrogel in preparing products for wound repair or in preparing products for surgical operations. Background Art

[0002] Cartilage damage, especially articular cartilage damage, is relatively common in clinical practice. It may be caused by trauma or joint inflammation, and is often accompanied by joint pain and further degeneration of cartilage. Joint replacement surgery is ultimately required to relieve pain and restore motor function. Therefore, articular cartilage defects need to be treated promptly and effectively. At present, hydrogel materials are considered to be the most ideal scaffold materials for cartilage repair because of their suitable mechanical strength, high water content, and strong plasticity. Among them, in situ injected hydrogels are in a flowing state before injection, and after injection into the body, they form in situ gels through a certain mechanism, which can realize the transition from sol state to gel state at the injection site. Therefore, they can be implanted through minimally invasive injection molding without the need for open surgery, which has the advantages of alleviating patient pain and filling irregular cartilage defects.

[0003] From the source of materials for preparing hydrogels, synthetic materials (polycaprolactone, polyethylene glycol, polylactic acid, etc.) have poor biocompatibility, and natural materials (sodium alginate, chitosan, etc.) have poor mechanical properties. Decellularized cartilage matrix derived from cartilage can provide a physical and chemical microenvironment for the survival and differentiation of chondrocytes, and can induce cartilage tissue regeneration. Therefore, it is particularly suitable as a scaffold material for cartilage repair to improve the molding and repair ability of cartilage. At present, there are inventions that use mammalian cartilage to prepare cartilage decellularized matrix materials for cartilage defect repair. For example, patent CN 115475279 discloses a photosensitive cartilage decellularized matrix hydrogel material. This photosensitive hydrogel is prepared by grafting photosensitive groups on mammalian cartilage decellularized matrix. It can be gelled after exposure to light and can be used for in situ articular cartilage defect repair; patent CN 111790007 discloses a thermosensitive deer antler cartilage matrix hydrogel material. This thermosensitive hydrogel is also prepared using mammalian cartilage. However, mammalian decellularized cartilage matrix has high immunogenicity and disease transmission risk, and some mammals also have religious restrictions, which limits the application of such materials. Compared with mammalian cartilage decellularized matrix, cartilage decellularized matrix derived from aquatic animals has unique advantages such as no immunogenicity risk and no disease transmission. It is also rich in collagen and chondroitin sulfate, and contains some bioactive factors for maintaining the phenotype of cartilage tissue. This characteristic can promote cartilage regeneration and generate stable cartilage tissue. However, aquatic animal cartilage mainly contains type II collagen, and its denaturation temperature (25-30℃ for most fish) is lower than the denaturation temperature of mammalian collagen (39-40℃). Therefore, its biggest disadvantage for application in the human body is that it denatures at human body temperature, has poor stability, and has a serious decrease in mechanical strength. In order to overcome the above disadvantages of aquatic animal cartilage or its derivatives, it is often necessary to modify them. Patent CN 115444983 currently discloses the preparation of photosensitive bio-ink that can be used for tissue repair by compounding modified aquatic animal cartilage decellularized matrix with other proteins. However, the prior art does not disclose the use of a single aquatic animal cartilage decellularized matrix to prepare a material for cartilage repair without doping with other biological materials.

[0004] 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.

[0005] The mechanical properties and thermal stability of the cartilage decellularized matrix material itself are poor, and the simple cartilage decellularized matrix hydrogel is difficult to be directly applied to cartilage repair. In this field, there is a demand to add additives to the cartilage decellularized matrix material system to improve the mechanical properties and thermal stability of the hydrogel material, or to achieve the multifunctionalization of natural biomaterials by modification (modifications such as hydroxylation, carboxylation, amination, etc. can give natural materials degradability, tissue adhesion, etc.), increase the temperature sensitivity, mechanical properties, thermal stability, biocompatibility, etc. of biomaterials.

[0006] There is a need in the art to obtain aquatic animal-derived decellularized matrix hydrogels, particularly aquatic animal-derived decellularized cartilage matrix hydrogels, having improved mechanical properties and improved stability. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides an aquatic animal-derived decellularized matrix hydrogel and a preparation method thereof, in particular an aquatic animal-derived decellularized cartilage matrix hydrogel, which solves the problem that aquatic animal-derived decellularized matrix hydrogel has poor stability and greatly reduced mechanical strength under body temperature and body fluid erosion environments. At the same time, the hydrogel has gel-sol transition properties, thereby providing injectable properties and can be used for in situ injection into gel.

[0008] The inventors of the present invention unexpectedly discovered that the aquatic animal-derived decellularized matrix hydrogel of the present invention can be injected into cartilage defects and solidify in situ within a certain period of time. It has the characteristics of maintaining high mechanical strength at body temperature, and can continue to adhere to the defect after being eroded by human fluid, providing physiological conditions for cartilage repair.

[0009] The present invention relates to providing an aquatic animal-derived decellular matrix hydrogel, in particular an aquatic animal-derived decellular cartilage matrix hydrogel, which comprises a decellular matrix and an auxiliary agent of a non-biological material. Before curing, the viscosity of the aquatic animal-derived decellularized matrix hydrogel of the present invention at room temperature is 8000mPa.s to 20000mPa.s, preferably 9000mPa.s to 19000mPa.s, in particular 9500mPa.s to 16000mPa.s, in particular 10000mPa.s to 15000mPa.s; the elastic modulus is 100Pa to 800Pa, preferably 200Pa to 700Pa, in particular 300Pa to 600Pa, in particular 350Pa to 550Pa. After curing, its elastic modulus at room temperature is 3kPa-45kPa, in particular 5kPa-40kPa, in particular 6kPa-38kPa, or even 8kPa-35kPa.

[0010] 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 12.5 wt %, or even 7 wt % to 12 wt %.

[0011] 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 8%, particularly 0.1-7%, especially 0.3-4%, or even 0.5-1%.

[0012] According to a specific embodiment of the present invention, the auxiliary agent of the non-biological material includes but is not limited to at least one thermosensitive substance, photosensitive substance, chemical bonding agent and the like, which can enhance the stability of the decellularized matrix in an in vivo environment.

[0013] According to a specific embodiment of the present invention, the thermosensitive substance includes a thermosensitive compound and an excipient, wherein the thermosensitive compound is one or more of poly (N-isopropylacrylamide), polyvinyl pyrrolidone, poloxamer (F127), and polyethylene glycol-polylactide-co-glycolide copolymer; the excipient is one or more of poloxamer (F68), carbomer, hyaluronic acid, sodium alginate, carboxymethyl cellulose, and hydroxypropyl methylcellulose. In particular, the excipient is selected from one or more of poloxamer (F68), carbomer, hyaluronic acid, sodium alginate, carboxymethyl cellulose, and hydroxypropyl methylcellulose. In particular, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel, the content of the thermosensitive substance is 12% to 35%, in particular 15% to 30%, especially 18% to 28%, or even 20% to 25%.

[0014] According to a specific embodiment of the present invention, the photosensitive substance is a photosensitizer or a photosensitive substance containing a photosensitive group and a photoinitiator, etc., which can convert the aquatic animal-derived decellularized matrix hydrogel from a liquid state to a solid state under the irradiation of a specific light source with a certain irradiation intensity. For example, the photosensitive substance is selected from one or more of riboflavin and rose bengal, and in particular, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel, the content of the photosensitive substance is 0.02 to 0.5%, in particular 0.03 to 0.4%, especially 0.04 to 0.3%, or even 0.05 to 0.2%.

[0015] According to a specific embodiment of the present invention, the chemical bonding agent is selected from one or more compounds of isobutylene dihydrogen ester, genipin, 1-ethyl-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS), diphenylphosphine, D-ribose, glutaraldehyde, 1,4-butanediol glycidyl ether (BDDE), tannic acid, polyamines, and diamine ethane. In particular, the content of the chemical bonding agent is 0.01 to 10%, particularly 0.02 to 8%, especially 0.03 to 5%, or even 0.04 to 2%, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel.

[0016] According to a specific embodiment of the present invention, 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 longtail cod, blue tip tail hake, Dabry's guitarfish, Xu's guitarfish, granular guitarfish, spotted guitarfish, Chinese round fan ray, He's ray, spotted ray, hole ray, red stingray, Chinese stingray, Gu's stingray, sharp-mouthed stingray, Siberian sturgeon, Dabry's guitarfish, naked Belly 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, cat shark, 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, Northern Pacific squid, and tailed squid.

[0017] According to a specific embodiment of the present invention, the aquatic animal-derived decellularized matrix hydrogel comprises 5% to 20% by mass of the decellularized matrix, particularly 6% to 18% by mass, especially 7% to 16% by mass, and even 8% to 15% by mass, and 0.01% to 50% by mass of an auxiliary agent of a non-biological material, particularly 0.1% to 40% by mass, especially 0.5% to 30% by mass, and even 1% to 20% by mass, based on the total mass of the aquatic animal-derived decellularized matrix hydrogel.

[0018] In the present invention, "elastic modulus change rate" refers to the difference between the elastic modulus of the aquatic animal-derived decellularized matrix hydrogel when stored after curing at a temperature close to human body temperature and the elastic modulus of the aquatic animal-derived decellularized matrix hydrogel after curing at a temperature close to human body temperature, for example, 6 months later, that is, the elastic modulus change rate = 1-(elastic modulus after curing at a temperature close to human body temperature, for example, 6 months later) / (elastic modulus when stored after curing at a temperature close to human body temperature).

[0019] According to a specific embodiment of the present invention, compared with a decellularized matrix hydrogel without added additives whose elastic modulus decreases by more than or equal to 50% within 24 hours under body temperature and body fluid erosion conditions, the elastic modulus change rate of the aquatic animal-derived decellularized matrix hydrogel after solidification and storage at human body temperature for 6 months is less than or equal to 20%, particularly less than 18%, and especially less than 15%.

[0020] According to a specific embodiment of the present invention, the gel curing time of the aquatic animal-derived decellularized matrix hydrogel at the cartilage defect in vivo is 1 to 300 s, particularly 60 to 240 s, especially 70 to 180 s, or even 90 to 120 s.

[0021] According to a specific embodiment of the present invention, the aquatic animal-derived decellularized matrix hydrogel is in a gel form or can be made into a powder, tablet, granule, pill or sponge form.

[0022] According to a specific embodiment of the present invention, the decellularized matrix has at least one of the following characteristics:

[0023] - the total protein content of the decellularized matrix is ​​85-99 wt %, preferably 90-98 wt %, more preferably 92-97 wt %;

[0024] - the content of type II collagen in the decellularized matrix is ​​75-99 wt %, particularly 80-95 wt %, especially 85-90 wt %;

[0025] - the content of type III collagen in the decellularized matrix is ​​0-9 wt %, preferably less than 7 wt %, in particular less than 3 wt %;

[0026] - the content of type VI collagen in the decellularized matrix is ​​0-3.5wt%, preferably 0.1-3wt%;

[0027] - the fibroblast growth factor (FGF) content of the acellular matrix is ​​greater than 30 ng / g, in particular greater than 50 ng / g;

[0028] - 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;

[0029] - the cell proliferation rate of the decellularized matrix is ​​greater than 105%, preferably greater than 110%, or not more than 135%; - the water absorption rate of the decellularized matrix is ​​2-8 times, especially 3-7 times, or even 4-6 times of its own weight;

[0030] - the DNA content of the acellular matrix is ​​less than 50 ng / mg, in particular less than 30 ng / mg, especially less than 20 ng / mg;

[0031] The content of type IV collagen in the decellularized matrix is ​​less than 0.5 wt %, in particular less than 0.2 wt %, and especially less than 0.1 wt %; in particular, the decellularized matrix does not contain type IV collagen.

[0032] According to a specific embodiment of the present invention, the aquatic animal-derived decellularized matrix hydrogel is a hydrogel in a broad sense when prepared in a gel state, and its dosage forms include but are not limited to solid gels and fluid gels (including paste-like gels). The solid gel can be broken up before injection.

[0033] According to a specific embodiment of the present invention, when the aquatic animal-derived decellularized matrix hydrogel is injected using a 20G needle at room temperature, the average pushing force is less than or equal to 10N.

[0034] The present invention also relates to a method for preparing aquatic animal-derived decellularized matrix hydrogel, wherein the decellularized matrix is ​​prepared by pre-treating and decellularizing animal tissue, comprising the following steps:

[0035] (1) providing a decellularized matrix step, which comprises the following steps:

[0036] (a) Pretreatment step: taking animal tissue, such as cartilage tissue of aquatic animals, and removing the surrounding impurities to remove the foreign tissue;

[0037] (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;

[0038] (c) washing step: rinsing the decellularized matrix obtained in step (b), optionally using purified water for rinsing;

[0039] (d) drying step: drying the decellularized matrix obtained in step (c), such as freeze-drying, and optionally forming it into a fixed shape;

[0040] (2) Optionally, the aquatic animal-derived decellularized matrix is ​​micronized to obtain a decellularized matrix in a sheet or granular form;

[0041] (3) blending the above-mentioned aquatic animal-derived decellularized matrix and an auxiliary agent to obtain an aquatic animal-derived decellularized matrix hydrogel;

[0042] (4) Optional subsequent step: The decellularized matrix hydrogel obtained in step (3) is optionally packaged and sterilized after being crushed, washed, or freeze-dried.

[0043] According to a specific embodiment of the present invention, the pretreatment step (a) comprises: using biological enzymes for enzymatic hydrolysis at a temperature of 0-15°C for 12-60h, preferably for 12-18h or even 18-21h or especially for 24-48h, and optionally, subsequently removing muscle tissue, basement membrane and / or epidermis to obtain tissue material without impurities, for example, obtaining cartilage tissue without impurities.

[0044] 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, and neutral protease.

[0045] 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 elastase tris(hydroxymethylaminomethane) hydrochloride solution; the enzymatic hydrolysis time is 12-18h, or even 14-17h; or, the enzymatic hydrolysis system is 0.003-0.005wt% trypsin borate or trypsin phosphate solution; the enzymatic hydrolysis time is 15-21h, or even 15-19h.

[0046] According to a specific embodiment of the present invention, 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, dimethyl sulfoxide; and / or the decellularization 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.

[0047] 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.

[0048] According to a specific embodiment of the present invention, in the step (3), the blending is carried out under low temperature conditions, particularly at 2 to 10°C, preferably 4 to 8°C.

[0049] According to a specific embodiment of the present invention, in step (3), when the added non-biological material adjuvant is a temperature-sensitive substance, blending can be performed by adding a flaky or granular decellularized matrix and a temperature-sensitive substance to a fluid for injection (which can be selected from one or more of water for injection, saline, phosphate buffer solution, blood, plasma, dextrose, lactated Ringer's solution, ethanol, propylene glycol, and polyethylene glycol) at low temperature until dissolved and homogenized. The low temperature condition is 2 to 10° C., preferably 4 to 8° C., and the homogenization method includes but is not limited to one or more of magnetic stirring, electric stirrer stirring, bubbling or shaking.

[0050] According to a specific embodiment of the present invention, when the added auxiliary agent of the non-biological material is a photosensitive substance, for example, the photosensitive substance containing a photosensitive group includes one or more photosensitive substances selected from the group consisting of glycidyl methacrylate molecules, methacrylic anhydride molecules, glycidyl acrylate molecules, acrylic anhydride molecules, and acryloyl chloride molecules, and for example, the photoinitiator is a water-soluble initiator such as LAP or I2959. The blending can be performed by first dissolving the sheet-like or granular decellularized matrix in a fluid for injection (which can be selected from one or more of water for injection, physiological saline, phosphate buffer solution, blood, plasma, dextrose, lactated Ringer's solution, ethanol, propylene glycol, and polyethylene glycol), precooling at 0-4°C for 24 hours, then adding one or more photosensitive substances to the precooled decellularized matrix solution, and then slowly adding 3-6M NaOH solution to adjust the pH of the solution and keep it in an alkaline environment, reacting at 0-4°C for 24-48 hours, and then dialyzing the fluid used in the reaction system for 3-5 days. Then, a photoinitiator is mixed into the system and then the mixture is blended homogeneously to obtain the aquatic animal-derived decellularized matrix hydrogel.

[0051] According to a specific embodiment of the present invention, when the photosensitive substance is riboflavin or a photosensitive substance containing a photosensitive group, the specific light source preferably used is ultraviolet light, for example, with a wavelength of 365-370 nm and an irradiation intensity of 3-160 mW / cm 2 When the photosensitive substance is rose bengal, the specific light source preferably used is green light with a wavelength of 532-535nm and an irradiation intensity of 0.1-50mW / cm 2 , the illumination time is 1-30min.

[0052] According to a specific embodiment of the present invention, when the added auxiliary agent of the non-biological material is a chemical bonding agent:

[0053] - When the chemical bonding agent is isocyanate, the mixing may be performed by homogenizing the sheet-like or granular decellularized matrix and isocyanate for 3 to 9 hours;

[0054] - When the chemical bonding agent is genipin, the mixing may be performed by homogenizing the sheet-like or granular decellularized matrix and genipin for 24 to 96 hours;

[0055] When the chemical bonding agent is EDC / NHS, the mixing may be performed by mixing the sheet-like or granular decellularized matrix and EDC / NHS at 4 to 25° C. for 12 to 24 hours.

[0056] According to a specific embodiment of the present invention, when the added non-biological material auxiliary agent is a chemical bonding agent, a solid aquatic animal-derived decellularized matrix hydrogel is obtained after blending, and the solid aquatic animal-derived decellularized matrix hydrogel is optionally crushed, washed, or freeze-dried to obtain a powdered aquatic animal-derived decellularized matrix hydrogel.

[0057] According to a specific embodiment of the present invention, when the auxiliary agent of the non-biological material is a temperature-sensitive substance or a photosensitive substance, the aquatic animal-derived decellularized matrix hydrogel is a fluid gel, which increases its mechanical strength under the action of body temperature and solidifies within a certain period of time when injected into the cartilage defect in the body.

[0058] According to a specific embodiment of the present invention, when the auxiliary agent of the non-biological material is a chemical bonding agent, the aquatic animal-derived decellularized matrix hydrogel can be a powder, which is hydrated to obtain a fluid gel, which is injected into the cartilage defect in the body and solidifies within a certain period of time.

[0059] The present invention also relates to the use of the aquatic animal-derived decellularized matrix hydrogel for repairing cartilage defects caused by osteoarthritis and rheumatoid arthritis, etc., in particular, in the preparation of products for wound repair, or in the preparation of scaffold materials for cartilage repair and regeneration, such as for use in products for acute or chronic wound repair, residual cavity filling, and damaged tissue coverage, or in the preparation of products for surgical operations such as knee microfracture, or for use in products such as bone defect repair, cartilage defect repair, corneal repair, tissue thickening, and residual cavity filling. In particular, it can be used for small defect areas, such as 2-8 cm 2 Application of the product for repair of cartilage defects in the area.

[0060] The present invention relates to the following embodiments:

[0061] Item 1. A decellularized matrix hydrogel of aquatic animal origin, comprising a decellularized matrix and an auxiliary agent of a non-biological material, characterized in that before curing, the hydrogel has a viscosity of 8000 mPa.s to 20000 mPa.s at room temperature and an elastic modulus of 100 Pa to 800 Pa. After curing, the hydrogel has an elastic modulus of 2 kPa to 50 kPa.

[0062] Item 2. The aquatic animal-derived decellularized matrix hydrogel according to Item 1 is characterized in that the hydroxyproline content in the decellularized matrix is ​​3wt% to 13wt%, in particular 5wt% to 12.5wt%, or even 7wt%-12wt%, wherein the hydroxyproline change rate between the decellularized matrix and the animal tissue before decellularization is less than 8%, in particular 0.1-7%, especially 0.3-4%, or even 0.5-1%.

[0063] Item 3. The aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the auxiliary agent of the non-biological material is selected from a thermosensitive substance, a photosensitive substance, a chemical bonding agent or a mixture thereof.

[0064] Item 4. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the thermosensitive substance comprises a thermosensitive compound and an excipient, wherein the thermosensitive compound is one or more of poly (N-isopropylacrylamide), polyvinyl pyrrolidone, poloxamer (F127), and polyethylene glycol-polylactide-co-glycolide copolymer; the excipient is one or more of poloxamer (F68), carbomer, hyaluronic acid, sodium alginate, carboxymethyl cellulose, and hydroxypropyl methylcellulose, in particular, wherein the excipient is selected from one or more of poloxamer (F68), carbomer, hyaluronic acid, sodium alginate, carboxymethyl cellulose, and hydroxypropyl methylcellulose, in particular, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel, the content of the thermosensitive substance is 12% to 35%, in particular 15% to 30%, especially 18% to 28%, or even 20% to 25%.

[0065] Item 5. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the photosensitivity substance is selected from one or more of riboflavin and rose bengal, and in particular, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel, the content of the photosensitivity substance is 0.02 to 0.5%, in particular 0.03 to 0.4%, especially 0.04 to 0.3%, or even 0.05 to 0.2%.

[0066] Item 6. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the chemical bonding agent is selected from one or more compounds of isocyanate, genipin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS), diphenylphosphine, D-ribose, glutaraldehyde, 1,4-butanediol glycidyl ether (BDDE), tannic acid, polyamines, and diamineethane. In particular, the content of the chemical bonding agent is 0.01 to 10%, in particular 0.02 to 8%, especially 0.03 to 5%, or even 0.04 to 2%, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel.

[0067] Item 7. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the aquatic animal-derived decellularized matrix is ​​derived from an aquatic organism, preferably selected from aquatic fish, and in particular selected from the cartilage of an aquatic organism, wherein the aquatic organism is selected from the group consisting of longtail cod, blue tip tail hake, Da guitarfish, Xu's guitarfish, granular guitarfish, spotted guitarfish, Chinese round fan ray, He's ray, spotted ray, hole ray, red stingray, Chinese stingray, Gu's stingray, sharp-mouthed stingray, Siberian sturgeon , Acipenser dabryan, 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, cat shark, silvertip shark, lemon shark, thresher shark, whale shark, tiger shark, blue shark, moon jellyfish, comb jellyfish, purple-striped sea jellyfish, swimming jellyfish, cypress jellyfish, root-mouthed jellyfish, flag-mouthed jellyfish, jellyfish, Argentine squid, Japan Sea squid, Humboldt squid, Steven's squid, Northern Pacific squid, and tail-tailed squid.

[0068] Item 8. An aquatic animal-derived decellular matrix hydrogel according to any of the preceding items, wherein the aquatic animal-derived decellular matrix hydrogel comprises 5% to 20% by mass of the decellularized matrix, particularly 6% to 18% by mass, especially 7% to 16% by mass, and even 8% to 15% by mass, and 0.01% to 50% by mass of an auxiliary agent, particularly 0.1% to 40% by mass, especially 0.5% to 30% by mass, and even 1% to 20% by mass, based on the total mass of the aquatic animal-derived decellularized matrix hydrogel.

[0069] Item 9. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, characterized in that, before solidification, its viscosity at room temperature is 9000mPa.s to 19000mPa.s, particularly 9500mPa.s to 16000mPa.s, especially 10000mPa.s to 15000mPa.s, or its elastic modulus is 100Pa to 800Pa, preferably 200Pa to 700Pa, particularly 300Pa to 600Pa, especially 350Pa to 550Pa.

[0070] Item 10. The aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, characterized in that, after curing, its elastic modulus at room temperature is 3kPa-45kPa, particularly 5kPa-40kPa, especially 6kPa-38kPa, or even 8kPa-35kPa.

[0071] Item 11. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, characterized in that, after curing, the rate of change in elastic modulus after being stored at human body temperature for 6 months is less than or equal to 20%, particularly less than or equal to 18%, and especially less than or equal to 15%.

[0072] Item 12. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the gel curing time of the aquatic animal-derived decellularized matrix hydrogel at the cartilage defect in vivo is 1 to 300 s, particularly 60 to 240 s, especially 70 to 180 s, or even 90 to 120 s.

[0073] Item 13. The aquatic animal-derived decellularized matrix hydrogel according to any one of the preceding items, wherein the aquatic animal-derived decellularized matrix hydrogel is in a gel-like state or can be made into a powder, tablet, granule, pill or sponge form.

[0074] Item 14. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the decellularized matrix has at least one of the following characteristics:

[0075] - the total protein content of the decellularized matrix is ​​85-99 wt %, preferably 90-98 wt %, more preferably 92-97 wt %;

[0076] - the content of type II collagen in the decellularized matrix is ​​75-99 wt %, particularly 80-95 wt %, especially 85-90 wt %;

[0077] - the content of type III collagen in the decellularized matrix is ​​0-9 wt %, preferably less than 7 wt %, in particular less than 3 wt %;

[0078] - the content of type VI collagen in the decellularized matrix is ​​0-3.5wt%, preferably 0.1-3wt%;

[0079] - the fibroblast growth factor (FGF) content of the acellular matrix is ​​greater than 30 ng / g, in particular greater than 50 ng / g;

[0080] - 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;

[0081] - the cell proliferation rate of the decellularized matrix is ​​greater than 105%, preferably greater than 110%, or not more than 135%; - the water absorption rate of the decellularized matrix is ​​2-8 times, especially 3-7 times, or even 4-6 times of its own weight;

[0082] - the DNA content of the acellular matrix is ​​less than 50 ng / mg, in particular less than 30 ng / mg, especially less than 20 ng / mg;

[0083] The content of type IV collagen in the decellularized matrix is ​​less than 0.5 wt %, in particular less than 0.2 wt %, and especially less than 0.1 wt %; in particular, the decellularized matrix does not contain type IV collagen.

[0084] Item 15. A method for preparing an aquatic animal-derived decellularized matrix hydrogel according to any of the preceding items, wherein the decellularized matrix is ​​prepared by pre-treating and decellularizing animal tissue, comprising the following steps:

[0085] (1) providing a decellularized matrix step, which comprises the following steps:

[0086] (a) Pretreatment step: taking animal tissue, such as cartilage tissue of aquatic animals, and removing the surrounding impurities to remove the foreign tissue;

[0087] (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;

[0088] (c) washing step: rinsing the decellularized matrix obtained in step (b), optionally using purified water for rinsing;

[0089] (d) drying step: drying the decellularized matrix obtained in step (c), such as freeze-drying, and optionally forming it into a fixed shape;

[0090] (2) Optionally, the aquatic animal-derived decellularized matrix is ​​micronized to obtain a decellularized matrix in a sheet or granular form;

[0091] (3) blending the above-mentioned aquatic animal-derived decellularized matrix and a non-biological material additive to obtain an aquatic animal-derived decellularized matrix hydrogel;

[0092] (4) Optional subsequent step: packaging and sterilizing the decellularized matrix hydrogel obtained in step (3).

[0093] Item 16. According to the method described in Item 15 above, the pretreatment step (a) comprises: using biological enzymes for enzymatic hydrolysis at a temperature of 0-15°C for 12-60 hours, preferably for 12-18 hours or even 18-21 hours or especially for 24-48 hours, and optionally, subsequently removing muscle tissue, basement membrane and / or epidermis to obtain tissue material without impurities, for example, obtaining cartilage tissue without impurities.

[0094] Item 17. The method according to any one of Items 15-16 above, wherein in the pretreatment step (a), the biological enzyme is selected from at least one of elastase, type IV collagenase, papain, trypsin, tyrosinase, and neutral protease.

[0095] Item 18. A method according to any one of Items 15 to 17, wherein, in the pretreatment step (a), the enzymatic hydrolysis system is 0.004-0.006 wt% of elastase phosphate or elastase tris(hydroxymethyl)aminomethane hydrochloride) solution; the enzymatic hydrolysis time is 12-18 h, or even 14-17 h; or, the enzymatic hydrolysis system is 0.003-0.005 wt% of trypsin borate or trypsin phosphate solution; the enzymatic hydrolysis time is 15-21 h, or even 15-19 h.

[0096] Item 19. The method according to any one of Items 15 to 18 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 among the physical methods, 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.

[0097] Item 20. The method according to any one of items 15 to 19, wherein the micronization method in step (2) is selected from one or more of ultrafine grinding, ball milling, impact grinding and shear grinding.

[0098] Item 21. The method according to any one of Items 15 to 20, wherein in step (3), blending is carried out under low temperature conditions, in particular at 2 to 10°C, preferably 4 to 8°C.

[0099] Item 22. The method according to any one of items 15 to 21, wherein in step (3), 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.

[0100] Item 23. Use of the aquatic animal-derived decellularized matrix hydrogel according to any one of the preceding items 1 to 14 and the aquatic animal-derived decellularized matrix hydrogel obtained according to the method described in any one of the preceding items 15 to 22 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

[0101] Figure 1 A photograph of the aquatic animal-derived cartilage decellularized hydrogel shown in Example 5.1 of the present invention is shown.

[0102] Figure 2The test results of the stability of the hydrogel shown in Example 5.2 of the present invention in an in vivo environment are shown.

[0103] Figure 3 The elastic modulus test results of the hydrogel shown in Example 5.3 of the present invention before and after curing are shown.

[0104] Figure 4 The aging test results of the hydrogel shown in Example 5.4 of the present invention before and after curing are shown.

[0105] Figure 5.1 and Figure 5.2 The scanning electron microscope images of the decellularized matrix shown in Example 5.5 of the present invention before and after decellularization are shown. DETAILED DESCRIPTION

[0106] Example 1

[0107] The method for preparing the aquatic animal-derived cartilage decellularized matrix in the embodiment of the present invention is as follows:

[0108] (1) Take the purchased longtail cod and keep the nasal cartilage, remove the fish meat, fish scales, epidermis and other impurities, wash it, immerse it in 10°C 0.005% elastase phosphate solution for 15 hours, remove the residual meat tissue with fascia until the complete nasal cartilage is exposed, and wash it with pre-cooled water.

[0109] (2) Decellularization by chemical method: at 4°C, the cartilage was immersed in sodium hydroxide solution at a ratio of 1:20 (g / mL, aquatic animal-derived cartilage obtained in step (1) / sodium hydroxide solution) and shaken for 120 min; at 4°C, the alkaline-washed cartilage was immersed in phosphate buffer at a ratio of 1:10 (g / mL, cartilage / phosphate buffer) and shaken for 120 min.

[0110] (3) At 4°C, the phosphate buffer solution obtained by washing the surface of the cartilage with sterile water for injection was immersed in water at a ratio of 1:10 (g / mL, cartilage / sterile water for injection) and shaken for 180 min. The solution was changed 2-3 times during the cleaning process.

[0111] (4) The cartilage is freeze-dried at -50°C to -40°C in a vacuum to obtain a decellularized matrix of aquatic animal-derived cartilage.

[0112] Example 2

[0113] Example 2.1

[0114] Take 2g of the aquatic animal cartilage decellularized matrix prepared in Example 1 and add it to 20mL of water for injection and stir until the system is uniform without obvious agglomeration. The pH value is 6.8 after testing. 8mg of EDC (final concentration of 2mM) is quickly added, and then 12mgNHS is added to the reaction system (final concentration of 5mM) and fully mixed, and 25°C is blended for 12h. After the blending is completed, the solid aquatic animal decellularized cartilage matrix hydrogel is lyophilized after being crushed and washed with deionized water to obtain a powder state aquatic animal decellularized cartilage matrix hydrogel, which is stored in a 4°C environment. When in use, the powder state aquatic animal decellularized cartilage matrix hydrogel needs to be redissolved in water for injection, and then can be injected into the cartilage defect site, and standing for 2min can complete solidification.

[0115] Example 2.2

[0116] Take 2g of the aquatic animal cartilage decellularized matrix prepared in Example 1 and add it to 20mL of water for injection and stir until the system is uniform and there is no obvious agglomeration. The pH value is tested to be 6.8, add 0.2g of isocyanate (final concentration is 1% w / v), blend at 25°C for 4h, and after blending, centrifuge the reaction product at 12000rpm for 5min, then wash it twice with deionized water and saline, disperse it with deionized water, and freeze-dry it to obtain a powdered aquatic animal decellularized cartilage matrix hydrogel, which is stored in a 4°C environment. When in use, the powdered aquatic animal decellularized cartilage matrix hydrogel needs to be redissolved in water for injection, and then injected into the cartilage defect site, and solidified after standing for 2min.

[0117] Example 3

[0118] Example 3.1

[0119] Take 2g of the aquatic animal cartilage decellularized matrix prepared in Example 1 and add it to 20mL of water for injection and stir until the system is uniform and there is no obvious agglomeration, and precool it at 0°C for 24h. After precooling, add 0.1g of glycidyl methacrylate to the slurry solution, stir evenly, and then slowly add 1M NaOH solution to adjust the pH of the solution to 9.0, and then blend at 4°C for 24h. After blending, dialyze the system with deionized water for 3 days to obtain the modified aquatic animal decellularized cartilage matrix liquid, and then mix in 0.1g of water-soluble photoinitiator LAP. After blending homogenously, the aquatic animal decellularized cartilage matrix hydrogel can be obtained. When used, it can be directly injected into the treatment site. After the injection is completed, the decellularized cartilage matrix can be solidified by irradiation with a specific light source. The irradiation conditions are: use ultraviolet light with a wavelength of 365nm and an irradiation intensity of 10mW / cm 2 , light exposure for 2 minutes.

[0120] Example 3.2

[0121] Take 2g of the aquatic animal cartilage decellularized matrix prepared in Example 1 and add it to 20mL of water for injection and stir until the system is uniform and there is no obvious agglomeration. Add 0.05g of riboflavin under the condition of complete light avoidance, stir thoroughly to make the cartilage decellularized matrix solution and riboflavin mix evenly, and store them in a 4°C environment under the condition of complete light avoidance. When using, just inject the mixed solution into the treatment area, and then use ultraviolet light with a wavelength of 365nm and an irradiation intensity of 50mW / cm 2 , light for 2 minutes, and it can be cured.

[0122] Example 4

[0123] Take 2g of the aquatic animal cartilage decellularized matrix prepared in Example 1 and add it to 20mL of water for injection and stir until the system is uniform without obvious lumps, directly add 4g of solid powdered poloxamer (F127), stir it thoroughly in an ice water bath until it dissolves to obtain a thermosensitive sol with a certain viscosity, and store it in an environment of 4°C after mixing. When used, the sol is directly injected into the treatment site, and it can undergo sol-gel conversion to form a solid gel under the action of human body temperature after contacting the human body.

[0124] Example 5: Three-dimensional structure, stability and elastic modulus of the hydrogel of the test example

[0125] Example 5.1

[0126] Figure 1 is a photograph of a hydrogel according to an embodiment of the present invention. Figure 1 (a) is a photograph of taking 2 g of aquatic animal cartilage decellularized matrix prepared in Example 1 and directly adding it into 20 mL of water for injection and stirring until the system is uniform without obvious lumps, and then standing for 2 minutes and then inverting it. Figure 1 (b) is a photograph of taking 2 g of the aquatic animal-derived decellularized cartilage matrix hydrogel powder prepared in Example 2.1 and adding it to 20 mL of water for injection and stirring until the system is uniform without obvious lumps at room temperature, and then standing for 2 minutes to solidify, and then inverting it. Figure 1 (c) At room temperature, 1 mL of aquatic animal-derived decellularized cartilage matrix hydrogel prepared in Example 3.1 was taken and irradiated at a wavelength of 365 nm and an irradiation intensity of 10 mW / cm 2 Photo after UV light curing for 2 minutes. Figure 1 (d) is a photograph of 1 mL of aquatic animal-derived decellularized cartilage matrix hydrogel prepared in Example 4 after being placed at 37°C for 2 minutes for curing. Figure 1As shown, the aquatic animal-derived cartilage decellularized matrix is ​​in a flowable state after being directly rehydrated, while the aquatic animal-derived cartilage matrix hydrogel prepared in Examples 2, 3, and 4 of the present invention is in a solid state, indicating that the aquatic animal-derived cartilage matrix hydrogel of the present invention has stronger mechanical properties.

[0127] Example 5.2

[0128] Figure 2 This is the test result of the stability of the hydrogel of the embodiment of the present invention in the in vivo environment.

[0129] The hydrogel prepared in the embodiment of the present invention was adhered to the bottom of the vial, and the vial was filled with physiological saline, and the vial was inverted and placed in an environment of 37°C for 24 hours to test the stability of the aquatic animal-derived decellularized cartilage matrix hydrogel under the environment of body temperature and liquid erosion. Figure 2 (a) is a photograph of the hydrogel of the aquatic animal cartilage decellularized matrix prepared in Example 1 after direct rehydration in a simulated in vivo environment before and after incubation for 24 hours. Figure 2 (b) is a photograph of the aquatic animal cartilage decellularized matrix hydrogel prepared in Example 3 before and after curing in a simulated in vivo environment for 24 hours. Figure 2 (c) is a photograph of the hydrogel of the aquatic animal cartilage decellularized matrix prepared in Example 1 after direct rehydration and shaking and falling off after incubation for 24 hours. Figure 2 As shown, the aquatic animal source cartilage decellularized matrix hydrogel prepared in Example 1 after direct rehydration absorbs water and swells after incubation for 24 hours in a simulated in vivo environment, and falls off after shaking, and the adhesion performance is greatly reduced; the aquatic animal source cartilage decellularized matrix hydrogel prepared in Example 3 has no obvious change after incubation for 24 hours in a simulated in vivo environment, and still adheres to the bottom of the vial, indicating that the aquatic animal source cartilage decellularized matrix hydrogel of the present invention is more stable in the in vivo environment.

[0130] Example 5.3

[0131] Figure 3 1 is the elastic modulus test result of the hydrogel before and after curing according to the embodiment of the present invention.

[0132] The hydrogels prepared in Examples 2.1, 3.1 and 4 of the present invention were tested for elastic modulus using a rheometer. Figure 3 As shown, the elastic modulus of the aquatic animal cartilage decellularized matrix hydrogel before curing is about 500Pa, and after curing, the elastic modulus increases to 3kPa-45kPa. This shows that the mechanical strength of the aquatic animal decellularized cartilage matrix hydrogel of the present invention increases significantly after curing, and has stronger adhesion performance.

[0133] Example 5.4

[0134] Figure 4These are the test results of the aging experiment of the hydrogel before and after curing according to the embodiment of the present invention.

[0135] The samples of aquatic animal-derived decellularized cartilage matrix hydrogel prepared in Implementation 2.1 before and after curing were placed in an aging chamber at a temperature of 37°C for a period of 6 months, and the elastic modulus of the samples was measured regularly to determine the long-term stability of the hydrogel samples at body temperature. Figure 4 As shown, the results show that after aging at 37°C for 6 months, the change rate of the elastic modulus of the uncured sample is greater than 50%, and the change rate of the elastic modulus of the cured sample is less than 20%, and the elastic modulus is still at a relatively high level, indicating that the stability of the aquatic animal-derived decellularized cartilage matrix hydrogel at body temperature is greatly increased after curing, thereby improving the shortcoming of poor stability of the aquatic animal-derived decellularized cartilage matrix in the in vivo environment due to containing a large amount of type II collagen.

[0136] Example 5.5

[0137] Figure 5.1 and Figure 5.2 is a cross section of the decellularized matrix before and after the embodiment 1 of the present invention, wherein Figure 5.1 This is a scanning electron micrograph of the matrix before decellularization. Figure 5.2 is a scanning electron micrograph of the decellularized matrix.

[0138] The nasal cartilage tissue of the longtail cod was removed from the surrounding tissue, washed with purified water, and freeze-dried by the same freeze-drying process as in Example 1. The decellularized matrix and animal nasal cartilage tissue in Example 1 were treated with gold spraying and observed with a scanning electron microscope.

[0139] Test results such as Figure 5.1 and Figure 5.2 As shown, the cross-sections of the nasal cartilage tissue of the long-tailed cod and the decellularized matrix of the nasal cartilage tissue of the long-tailed cod of the present invention are both similar multi-layered fiber structures, and the layered fiber structures are complete and basically the same.

[0140] Example 6: Hydroxyproline content after preparation of acellular matrix

[0141] The decellularized matrix was prepared as follows:

[0142] The decellularized matrix of Example 1 was repeatedly rinsed until it became neutral and had an electrical conductivity of less than 10 μS / cm, and then immersed in purified water for 5 hours, and then freeze-dried and ground into powder using a grinder.

[0143] The aquatic animal tissue decellularized matrix prepared in Example 1 was used to determine the hydroxyproline content according to the method in Appendix B of YY / T 1453-2016.

[0144] 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 1.

[0145] Table 1 Hydroxyproline content of decellularized matrix

[0146]

[0147] As can be seen from the above table, the aquatic animal of the present invention has a moderate hydroxyproline content and can play a scaffolding role in the early stage, the change rate of hydroxyproline content before and after preparation is small, and the three-dimensional microporous structure remains relatively intact before and after preparation.

[0148] 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. A decellularized matrix hydrogel derived from aquatic animals, comprising a decellularized matrix and an auxiliary agent of a non-biological material, characterized in that: Before curing, its viscosity at room temperature is 8000mPa.s to 20000mPa.s, and its elastic modulus is 100Pa to 800Pa. After curing, its elastic modulus is 2kPa-50kPa.

2. The aquatic animal-derived decellularized matrix hydrogel according to claim 1, characterized in that: The hydroxyproline content in the decellularized matrix is ​​3wt% to 13wt%, particularly 5wt% to 12.5wt%, or even 7wt%-12wt%, wherein the hydroxyproline change rate between the decellularized matrix and the animal tissue before decellularization is less than 8%, particularly 0.1-7%, especially 0.3-4%, or even 0.5-1%.

3. The aquatic animal-derived decellularized matrix hydrogel according to any one of the preceding claims, wherein the auxiliary agent of the non-biological material is selected from a thermosensitive substance, a photosensitive substance, a chemical bonding agent or a mixture thereof.

4. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding claims, wherein the thermosensitive substance comprises a thermosensitive compound and an excipient, wherein the thermosensitive compound is one or more of poly (N-isopropylacrylamide), polyvinyl pyrrolidone, poloxamer (F127), and polyethylene glycol-polylactide-co-glycolide copolymer; the excipient is one or more of poloxamer (F68), carbomer, hyaluronic acid, sodium alginate, carboxymethyl cellulose, and hydroxypropyl methylcellulose, in particular, wherein the excipient is selected from one or more of poloxamer (F68), carbomer, hyaluronic acid, sodium alginate, carboxymethyl cellulose, and hydroxypropyl methylcellulose, in particular, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel, the content of the thermosensitive substance is 12% to 35%, in particular 15% to 30%, especially 18% to 28%, or even 20% to 25%.

5. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding claims, wherein the photosensitive substance is selected from one or more of riboflavin and rose bengal, and in particular, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel, the content of the photosensitive substance is 0.02 to 0.5%, particularly 0.03 to 0.4%, especially 0.04 to 0.3%, or even 0.05 to 0.2%.

6. An aquatic animal-derived decellularized matrix hydrogel according to any of the preceding claims, wherein the chemical bonding agent is selected from one or more compounds of isocyanate, genipin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS), diphenylphosphine, D-ribose, glutaraldehyde, 1,4-butanediol glycidyl ether (BDDE), tannic acid, polyamines, and diamineethane. In particular, the content of the chemical bonding agent is 0.01 to 10%, particularly 0.02 to 8%, especially 0.03 to 5%, or even 0.04 to 2%, relative to the total mass of the aquatic animal-derived decellularized matrix hydrogel.

7. An aquatic animal-derived decellular matrix hydrogel according to any of the preceding claims, wherein the aquatic animal-derived decellular matrix hydrogel comprises 5% to 20% by mass of the decellularized matrix, particularly 6% to 18% by mass, especially 7% to 16% by mass, even 8% to 15% by mass, and 0.01% to 50% by mass of an auxiliary agent, particularly 0.1% to 40% by mass, especially 0.5% to 30% by mass, even 1% to 20% by mass, based on the total mass of the aquatic animal-derived decellularized matrix hydrogel.

8. A decellularized matrix hydrogel of aquatic animal origin according to any one of the preceding claims, characterized in that: After being cured, the elastic modulus change rate after being stored at human body temperature for example for 6 months is less than or equal to 20%, particularly less than or equal to 18%, and especially less than or equal to 15%.

9. The method for preparing the aquatic animal-derived decellularized matrix hydrogel according to any one of claims 1 to 8, wherein: The decellularized matrix is ​​prepared by pre-treating and decellularizing animal tissues, 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) 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 animal-derived decellularized matrix is ​​micronized to obtain a decellularized matrix in a sheet or granular form; (3) blending the above-mentioned aquatic animal-derived decellularized matrix and a non-biological material additive to obtain an aquatic animal-derived decellularized matrix hydrogel; (4) Optional subsequent step: packaging and sterilizing the decellularized matrix hydrogel obtained in step (3).

10. Use of the aquatic animal-derived decellularized matrix hydrogel according to any one of the preceding claims 1 to 8 and the aquatic animal-derived decellularized matrix hydrogel obtained according to the method described in the preceding claim 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.

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