Acellular matrix capable of being quickly gelatinized and application of acellular matrix

Abstract

The invention relates to an acellular matrix capable of being quickly gelatinized and application thereof. The present invention relates to acellular matrices that can be rapidly gelled, in particular acellular matrices (ECM) derived from aquatic animals, where the acellular matrices (ECM) can be gelled in neutral aqueous solutions in vitro. The invention further relates to a preparation method of the acellular matrix capable of being quickly gelatinized, and further relates to application of the acellular matrix or the acellular matrix gel in products for wound repair, medical cosmetology and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, in particular to a decellularized matrix (ECM), especially a decellularized matrix (ECM) derived from aquatic animals, wherein the decellularized matrix (ECM) can be gelled in a neutral aqueous solution in vitro. Background Art

[0002] Decellularized matrix (ECM) refers to the removal of immunogenic cellular components from human or animal tissues to form an animal tissue-derived scaffold that retains only ECM. After being implanted in the body, ECM can be gradually replaced by the patient's own tissue over time, guiding the regeneration of the body's tissues. Natural ECM plays an important role in regulating biophysical stimuli, biochemical and molecular signals, and spatial structures. As a natural degradable biological scaffold material, ECM maintains the natural biological structure and retains most of the biological functions. ECM also has appropriate porosity and adsorption, is suitable for cell adhesion and growth, and has good biocompatibility. Therefore, it also has excellent functional reconstruction effects and is easy to achieve tissue regeneration and reconstruction of tissue biological functions. There are a large number of ECM research reports both at home and abroad, and the results have been transformed into various medical device products and widely used.

[0003] According to existing data, ECM can be mainly divided into solid and gel forms according to its appearance. Solid forms are often made into flakes, granules, columns, powders, etc.

[0004] The common acellular dermal matrices on the market are generally derived from pigs, cattle, sheep and horses, and their production processes and clinical applications have been perfected. However, due to the frequent occurrence of zoonotic diseases such as bovine spongiform encephalopathy (BSE) and highly pathogenic avian influenza (HPAI) in recent years, acellular matrix materials from mammals such as pigs and cattle have become controversial.

[0005] Human allogeneic acellular matrix is ​​also a good in situ tissue repair material, and has shown excellent pro-repair properties in clinical studies. However, up to now, all organ tissues of human origin are still facing a severe shortage of supply. Therefore, the application of this type of allogeneic biomaterial will still be greatly limited by the problem of insufficient donors.

[0006] Aquatic animal-derived decellularized matrices have become one of the hot topics of research at home and abroad in recent years due to their low antigenicity, high biosafety, and rich variety and production. Aquatic animal-derived decellularized matrices have a biomimetic structure similar to that of mammals such as pigs and cattle, and contain more abundant fat and omega-3 unsaturated fatty acids, which can effectively reduce inflammatory responses and promote the formation of new blood vessels in the wound area, and have a significant promoting effect in areas such as the repair of refractory wounds.

[0007] Decellularized matrix is ​​prepared from animal tissues through a decellularization process. Depending on the number and distribution of cells in the matrix, the decellularization process will more or less affect the matrix structure. The content of proline, hydroxyproline and other amino acids in aquatic animal-derived decellularized matrix is ​​low, and the material stability is weaker than that of terrestrial animal-derived decellularized matrix. Inappropriate over-processing conditions can easily cause the loss of the bionic structure of the decellularized matrix.

[0008] Patent CN113769168A discloses a decellularized matrix microparticle product for soft tissue filling and repair and its preparation method, wherein the prepared decellularized matrix is ​​in granular form. The solid decellularized matrix has a microscopic porous structure, which can provide a good environment for cell migration, proliferation and differentiation after implantation; however, if the solid structure is not degraded in time and causes a space-occupying effect in the body, it will also affect tissue healing and regeneration.

[0009] It is known in the art that the gel form of ECM usually has a larger contact specific surface area and a more controllable degradation rate, and has good application prospects. However, due to the stability of the ECM structure, according to the technology known in the art, the gel form usually requires the animal extracellular matrix tissue to be first made into an ECM solid state, then digested and dissolved in an enzyme environment, and the pH is adjusted to neutral. This makes it usually impossible to remove enzyme residues from the prepared gel, which has certain biosafety risks.

[0010] Patent CN105169483A discloses a method for preparing a decellular matrix gel and a decellular matrix gel thereof. The inventor obtains a decellular matrix gel by first preparing a solid decellular matrix, then crushing, enzymatic digestion, and adjusting the pH to neutral. The hydrogel obtained by this method is usually unable to remove enzyme residues, and has certain biosafety risks; even if the residual enzymes are removed by introducing physical methods such as heating, dialysis / ultrafiltration, etc., the original active factors (such as glycosaminoglycans, growth factors, etc.) of the decellular matrix may be lost, reducing the effectiveness of the material. Therefore, it is particularly important to prepare a decellular matrix gel with high biosafety and high bioactivity, and to form a decellular matrix gel in an in vitro non-enzymatic digestion environment while retaining the basic composition and structure of the decellular matrix.

[0011] Patent CN116617462A discloses a neutral environment water-soluble acellular dermal matrix material, its preparation method and application. The inventor digests and dissolves the cellular dermal matrix (ADM) in a hydrochloric acid and enzyme environment, adds salt and adjusts it to neutrality, then dialyzes and freeze-dries it to make a water-soluble acellular dermal matrix material. The neutral water-soluble ADM prepared in this patent is digested in an enzyme environment, and the original active substances of ADM are lost after dialysis. The finished product is actually a fibrous protein solid.

[0012] Patent CN116747353A discloses a photo-crosslinked hydrogel preparation and application based on Wharton's gel decellularized matrix. The inventors digested the decellularized umbilical cord tissue with an enzyme method to obtain a water-soluble decellularized matrix. The water-soluble decellularized matrix obtained in this patent uses an enzyme digestion method, which poses a biosafety risk of enzyme residues.

[0013] The ECM produced by the prior art is either solid or gel-like. The solid form has disadvantages such as poor degradation controllability, and the gel form has problems such as loss of active factors or enzyme residual biological risks, which are subject to certain restrictions in the field of application. Especially in the processing steps, such as the decellularization step or after the decellularization step, if enzyme digestion is used, there will be problems such as enzyme residual biological risks. The decellularized matrix in the prior art has a stable solid structure in water and can only form a suspension. It cannot provide viscoelasticity or gel scaffolds. In some application scenarios, such as residual cavity filling, the material distribution will be uneven due to sedimentation.

[0014] At present, in this field, there is a need for a decellularized matrix material with a solid biomimetic structure. When implanted into the body, it can play the role of a solid scaffold for a certain period of time, inducing cells to migrate to the three-dimensional microporous structure, and then quickly gel, continue to provide a scaffolding effect and gradually release active factors to induce the body's tissue repair and regeneration; if necessary, it can quickly degrade. Summary of the invention

[0015] In view of the deficiencies of the above-mentioned prior art, the present invention aims to provide a rapidly gelling decellularized matrix and its application. The material comprising the decellularized matrix of the present invention is solid with a biomimetic structure. When implanted into the body, it can play the role of a solid scaffold for a certain period of time, inducing cells to migrate into the three-dimensional microporous structure, and then the material quickly gels, continues to provide a scaffolding effect and gradually releases active factors, inducing the body's tissue repair and regeneration; and quickly degrades.

[0016] Moreover, the decellular matrix of the present invention can also be rapidly gelled in a neutral aqueous solution in vitro to form a decellular matrix hydrogel, which can be directly applied to body tissues in the form of injection. In addition, the decellular matrix of the present invention can form a uniform solution of the decellular matrix after being dissolved in water. The decellular matrix of the present invention that can be rapidly gelled and can form a uniform solution after being dissolved in water can be widely used in the fields of inducing hard tissue and soft tissue repair, residual cavity filling, medical cosmetology, etc.

[0017] The applicant has found that the rapidly gelling decellular matrix of the present invention can achieve the above-mentioned needs. The rapidly gelling decellular matrix of the present invention can be suspended in a saline solution (such as physiological saline, phosphate buffer, bicarbonate buffer, tris(hydroxymethyl)aminomethane buffer, etc.) or a polysaccharide solution (such as sodium hyaluronate, chitosan, sodium alginate, sodium carboxymethylcellulose, carboxymethyl chitin, chondroitin sulfate, etc.) in a particulate form, and an implant is made for injection in the fields of medical cosmetology, residual cavity filling, soft / hard tissue defects, etc. The rapid gelling property of the decellularized matrix can provide viscoelastic support for the implant, and can also be induced to repair by releasing active ingredients such as growth factors. The material can also be directly made into a gel in a sterile in vitro environment and then filled into an implant, which can also be used in the fields of medical cosmetology, residual cavity filling, soft / hard tissue defects, etc., and the solute loss caused by filtration sterilization can be avoided. Therefore, the rapidly gelling decellularized matrix of the present invention has the advantages of both solid scaffolds and gels, greatly enriching the scope of application of decellularized matrix materials.

[0018] According to a specific embodiment of the present invention, the decellularized matrix of the present invention is derived from connective tissue, eye tissue, and organ tissue of an animal, specifically including but not limited to skin tissue, bone tissue, corneal tissue, organ tissue, mucosal tissue, or connective tissue of an animal.

[0019] According to a specific embodiment of the present invention, the decellularized matrix of the present invention is derived from aquatic animals, such as skin tissue, cartilage tissue, corneal tissue, organ tissue, mucosal tissue or connective tissue of aquatic animals, preferably selected from skin tissue of aquatic animals.

[0020] According to a specific embodiment of the present invention, the decellularized matrix of the present invention is derived from aquatic animals in cold zones, aquatic animals in temperate zones, aquatic animals in subtropical zones, or aquatic animals in tropical zones. Preferably, the aquatic organisms have a living temperature of 18-30° C. More preferably, the aquatic animals are selected from fish, more preferably temperate, subtropical, or tropical fish.

[0021] According to a specific embodiment of the present invention, the cold zone aquatic animal can also be selected from: polar bears, seals, whales, and Arctic char.

[0022] According to a specific embodiment of the present invention, the temperate aquatic animal is selected from: trout, sea turtle, various freshwater fish such as carp, silver carp and bighead carp.

[0023] According to a specific embodiment of the present invention, the subtropical aquatic animal is selected from the group consisting of: golden pomfret, sea snake, mandarin fish, black porgy, mullet, and black carp.

[0024] According to a specific embodiment of the present invention, the tropical aquatic animal is selected from the group consisting of: mackerel, angel fish, angelfish, clownfish, tilapia, coral reef fish, crocodile, butterfly fish, and guppy.

[0025] According to a specific embodiment of the present invention, the hydroxyproline content in the animal tissue of the present invention is 3 wt% to 11 wt%, in particular 5 wt% to 10.8 wt%, or even 7.5 wt%-10.5 wt%.

[0026] 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 1-6%, or even 1.5-5%.

[0027] According to a specific embodiment of the present invention, the decellularized matrix has a microstructure similar to or substantially identical to that of the original animal tissue.

[0028] According to a specific embodiment of the present invention, the total protein content of the decellularized matrix of the present invention is 85-99 wt %, preferably 90-98 wt %, and more preferably 92-97 wt %.

[0029] According to a specific embodiment of the present invention, the content of type I collagen in the decellularized matrix of the present invention is 75-99wt%, particularly 80-98wt%, and especially 85-97wt%.

[0030] According to a specific embodiment of the present invention, the content of type III collagen in the decellularized matrix of the present invention is 0-9wt%, preferably less than 7wt%, and particularly less than 3wt%.

[0031] According to a specific embodiment of the present invention, the content of type IV collagen in the decellularized matrix of the present invention is less than 0.5 wt %, particularly less than 0.2 wt %, especially less than 0.1 wt %, and preferably substantially free of type IV collagen.

[0032] According to the present invention, "substantially free" means that the content is lower than 0.05 wt%.

[0033] According to a specific embodiment of the present invention, the content of type VI collagen in the decellularized matrix of the present invention is 0-3.5 wt %, preferably 0.1-3 wt %.

[0034] According to a specific embodiment of the present invention, the decellularized matrix of the present invention has an irregular honeycomb porous or lamellar microstructure, and has a water absorption rate of 1-8 times its own weight, particularly 1.5-7 times its own weight, and especially 2-6 times its own weight.

[0035] According to a specific embodiment of the present invention, the DNA content of the decellularized matrix of the present invention is less than 50 ng / mg, particularly less than 30 ng / mg, and especially less than 20 ng / mg.

[0036] According to a specific embodiment of the present invention, the fibroblast growth factor (FGF) content of the decellularized matrix of the present invention is greater than 30 ng / g, in particular greater than 50 ng / g.

[0037] According to a specific embodiment of the present invention, the transforming growth factor β (TGF-β) content of the decellularized matrix of the present invention is greater than 8 ng / g, in particular greater than 10 ng / g.

[0038] According to a specific embodiment of the present invention, the α-gal antigen of the decellularized matrix of the present invention is not detected, wherein the cell proliferation rate of the decellularized matrix is ​​greater than 105%, even greater than 110%, or does not exceed 135%, especially does not exceed 125%, or does not exceed 120%.

[0039] According to a specific embodiment of the present invention, the decellularized matrix of the present invention has an appearance selected from the following: single-layer or multi-layer sheet, tube, mesh, granule or microsphere; or an irregular shape made in combination with auxiliary devices.

[0040] The present invention also relates to a method for preparing the above-mentioned decellularized matrix, wherein the decellularized matrix is ​​prepared by pre-treating and decellularizing animal tissue, and comprises the following steps:

[0041] (1) Pretreatment step: taking animal tissue, such as aquatic animal tissue, and removing the surrounding impurities to remove the foreign tissue;

[0042] (2) decellularization step: decellularizing the tissue obtained in step (1) by physical and / or chemical methods; preferably, decellularizing by a method that does not cause protein denaturation;

[0043] (3) Cleaning step: rinse the decellularized matrix obtained in step (2) and then rinse with purified water;

[0044] (4) Drying step: drying the decellularized matrix obtained in step (3) to form a fixed shape;

[0045] (5) Optionally, the decellularized matrix obtained in step (4) is packaged and sterilized to obtain a finished product.

[0046] According to a specific embodiment of the present invention, the animal tissue is derived from tissues selected from the following: animal skin tissue, cartilage tissue, corneal tissue, organ mucosal tissue or connective tissue, preferably the animal tissue is derived from aquatic animal tissue, in particular selected from aquatic animal skin tissue.

[0047] According to a specific embodiment of the present invention, the pretreatment step 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 dermal tissue without impurities.

[0048] According to a specific embodiment of the present invention, the pretreatment step comprises a step of separating attached muscle tissue, wherein the muscle tissue comprises fascia tissue.

[0049] According to a specific embodiment of the present invention, in the pretreatment step, the biological enzyme is selected from at least one of elastase, type IV collagenase, papain, trypsin, tyrosinase, and neutral protease.

[0050] According to a specific embodiment of the present invention, in the pretreatment step, the concentration of each biological enzyme in the enzymatic hydrolysis system is 0.001-0.01 wt%, preferably 0.004-0.006 wt% or even 0.003-0.005 wt%.

[0051] According to a specific embodiment of the present invention, in the pretreatment step, the solvent of the enzymatic hydrolysis system is selected from at least one of phosphate buffer, borate buffer and tris(hydroxymethyl)aminomethane hydrochloride.

[0052] According to a specific embodiment of the present invention, in the pretreatment step, 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.

[0053] According to a specific embodiment of the present invention, the dermal tissue free of foreign tissue obtained after the pretreatment step has no colored components visible to the naked eye.

[0054] According to a specific embodiment of the present invention, the pretreatment method further comprises, optionally infiltrating the obtained clean tissue, such as dermal tissue, with a protective solution precooled at 2-10°C, and then performing a decellularization treatment; the protective solution is selected from at least one of a phosphate buffer, a sodium chloride solution, and a borate buffer, and the concentration is 0.5-1.5wt%.

[0055] According to a specific embodiment of the present invention, the decellularization step 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; or the decellularization step is performed by a physical method; in the physical method, 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.

[0056] According to a specific embodiment of the present invention, the decellularization treatment further includes a washing step: first, immersing with 15-25°C purified water for 0.5-4h, and then immersing with 4-10°C pre-cooled purified water for 24-72h.

[0057] According to a specific embodiment of the present invention, the aquatic animal-derived decellularized matrix is ​​prepared by pretreatment and decellularization of aquatic animal connective tissue; the pretreatment method comprises: using biological enzymes for enzymatic hydrolysis at a temperature of 10-15°C for 12-60h, or even 15-50h, to remove muscle tissue, basement membrane and epidermis to obtain dermal tissue without impurities.

[0058] According to a specific embodiment of the present invention, the present invention utilizes a one-step decellularization method, combined with the previous processing steps, which can not only greatly improve the effectiveness of clinical safety, but also enable the obtained animal-derived decellularized matrix such as aquatic animals to have a bionic structure similar to or consistent with the original animal tissue.

[0059] According to a specific embodiment of the present invention, in the decellularization step (2), the decellularization treatment is selected from physical methods and / or chemical methods; in the physical method, high-pressure treatment, laser treatment, high-speed homogenization shearing and ultraviolet irradiation that may cause protein denaturation are not included; or, in the chemical method, the reagents used do not contain protein-denaturing components, and the protein-denaturing components are preferably concentrated hydrochloric acid, urea, guanidine hydrochloride, sodium dodecyl sulfate, dithiothreitol, and dimethyl sulfoxide.

[0060] According to a specific embodiment of the present invention, in the washing of the decellularized matrix in the cleaning step (3), the end point of the washing is that the pH of the washing solution is neutral and the conductivity is less than 10 μm / cm; and the purified water immersion time is 4-12 hours.

[0061] According to a specific embodiment of the present invention, the drying method in the drying step (4) can be one of freeze drying and vacuum drying, and the fixed shape is selected from the following appearances: single-layer or multi-layer sheet, tube, mesh, granular or microsphere; or combined with auxiliary equipment to make an irregular shape.

[0062] According to a specific embodiment of the present invention, the sterilization in the subsequent step (5) is selected from at least one of irradiation sterilization or epoxide sterilization such as ethylene oxide sterilization.

[0063] The present invention also relates to a decellularized matrix gel, which is prepared by gelling the decellularized matrix in a neutral aqueous solution in vitro.

[0064] According to a specific embodiment of the present invention, the aqueous solution is selected from one or more of pure water, phosphate buffer, borate buffer, glycine-hydrochloric acid buffer, Tris-hydrochloride buffer, carbonate buffer, barbital buffer, and normal saline, wherein the aqueous solution is particularly a neutral aqueous solution.

[0065] According to a specific embodiment of the present invention, the gelation concentration of the decellularized matrix is ​​less than 30 g / mL.

[0066] The present invention also relates to a method for preparing the aforementioned decellularized matrix gel, comprising the following steps: the decellularized matrix is ​​completely gelled in an aqueous solution in vitro under stirring conditions for 0.5 to 15 days, particularly 2 to 12 days, or even 4 to 10 days. According to a specific embodiment of the present invention, the aqueous solution is selected from one or more of pure water, phosphate buffer, borate buffer, glycine-hydrochloric acid buffer, tris hydrochloride buffer, carbonate buffer, barbital buffer, and physiological saline, wherein the aqueous solution is particularly a neutral aqueous solution.

[0067] According to a specific embodiment of the present invention, the gelation temperature of the decellularized matrix is ​​20-40°C, preferably 25-38°C.

[0068] According to a specific embodiment of the present invention, the decellularized matrix can be made into a gel or solution by controlling the solid content of the decellularized matrix.

[0069] According to a specific embodiment of the present invention, the acellular matrix gels in the Bama pig model with full-thickness cortical defects within 3-7 days after implantation, degrades to be invisible to the naked eye within 7-20 days, and the wound healing rate within 20 days is not less than 80%, preferably not less than 85%.

[0070] According to a specific embodiment of the present invention, the decellularized matrix can be implanted and used again after gelation or degradation.

[0071] According to a specific embodiment of the present invention, the decellularized matrix can be directly used in vitro as a gel or solution.

[0072] According to a specific embodiment of the present invention, the decellularized matrix of the present invention is prepared in a manner that does not cause protein denaturation while retaining its natural biomimetic structure, such as by pretreatment, physical or chemical cell removal and cleaning, drying and molding, and packaging and sterilization.

[0073] According to a specific embodiment of the present invention, the fixed shape of the dried decellularized matrix includes but is not limited to sheet, column, granule, microsphere, special shape, etc., which can be achieved by cutting, crushing, etc.

[0074] According to a specific embodiment of the present invention, sterilization is carried out by one of irradiation or ethylene oxide.

[0075] According to a specific embodiment of the present invention, physical and chemical treatment methods will not significantly affect the bionic structure and active factor components of the decellularized matrix, but will change the physical stability of the overall structure by precisely affecting the covalent cross-links and secondary bonds of some fibrous proteins in the decellularized matrix.

[0076] Specifically, due to the change in the physical stability of the overall structure of the decellularized matrix of the present invention, the material absorbs water and swells in a neutral aqueous solution environment, which accelerates the gradual dissolution of unstable or weakly stable fibrin, affects the overall structure of the decellularized matrix, and ultimately causes the "building collapse" of the decellularized matrix, and the formed "fragments" gradually dissolve in the aqueous solution.

[0077] Furthermore, the macroscopic bionic structure of the decellularized matrix prepared by the present method does not change, and the above-mentioned change in physical stability does not affect the initial bionic scaffold function of the material.

[0078] Furthermore, the decellularized matrix prepared by the method of the present invention can artificially regulate the gelation rate of the material by controlling the physical and chemical treatment conditions.

[0079] According to a specific embodiment of the present invention, the rapid gelation of the decellularized matrix of the present invention is based on its solubility in aqueous solution, and the gelation is based on the high solid concentration of the decellularized matrix in a liquid environment.

[0080] In addition, it can be understood that the body of an animal or human is a moist environment, and the decellularized matrix of this embodiment has a gelation basis; and because the gelation / dissolution of the decellularized matrix requires a certain amount of time, the decellularized matrix can be used as a bionic material to provide a three-dimensional scaffold in the early stage of implantation.

[0081] It can be seen that the decellularized matrix of the present invention has the advantages of solid and gel, which are specifically manifested in structural bionics, increased contact specific surface area, maintained biological activity and controllable degradation rate.

[0082] According to a specific embodiment of the present invention, the decellularized matrix in this embodiment can be dissolved in an in vitro aqueous solution to form a gel or solution for use.

[0083] Due to the characteristics of the decellularized matrix described in the present invention, the decellularized matrix and decellularized matrix gel described in the present invention can be used in wound repair and medical cosmetology products, for example, its application in products for acute or chronic wound repair, residual cavity filling, damaged tissue coverage, and hemostasis, specifically, for surgical operations, or for bone defect repair, cartilage defect repair, corneal repair, tissue thickening, residual cavity filling, hemostasis, and nerve repair.

[0084] Compared with the prior art, the decellularized matrix of the present invention can rapidly gel in a neutral, enzyme-free aqueous solution environment while retaining the microstructure and active factors of the original animal tissue, thereby solving the disadvantages of the degradation rate of solid decellularized matrix materials being difficult to control and poor applicability in irregular residual cavities or wound surfaces, and also solving the biosafety risk of residual enzymes in decellularized matrix gels and the risk of reduced effectiveness such as loss of active factors, which greatly expands the application of decellularized matrix materials. When the decellularized matrix of the present invention is directly implanted in a solid state, the bionic structure of the material can be used to induce rapid cell migration, proliferation and differentiation in the early stage; in the middle stage of implantation, the specific surface area in contact with the body is increased by rapid gelation, while the active factors are gradually released, and the tissue healing and regeneration are accelerated while maintaining a suitable moist environment at the implantation site; in the late stage of implantation, it can be rapidly degraded to avoid the space-occupying effect and delay tissue healing. The decellularized matrix of the present invention can be directly dissolved in a neutral aqueous solution environment, and the operation of making a gel or solution is simple, and it is convenient to use in large-scale production, and can be implanted clinically by minimally invasive injection according to actual use needs. The decellularized matrix of the present invention has high biological safety, and can be customized according to needs and through repeated implantation to be more consistent with the patient's healing period.

[0085] In particular, the present invention relates to the following technical solutions:

[0086] Item 1. A decellularized matrix, characterized in that the hydroxyproline content is 3wt% to 11wt%, particularly 5wt% to 10.8wt%, or even 7.5wt%-10.5wt%, wherein the hydroxyproline change rate between the decellularized matrix and the animal tissue before decellularization is less than 8%, particularly 0.1-7%, especially 1-6%, or even 1.5-5%.

[0087] Item 2. The decellularized matrix according to the preceding item, wherein the decellularized matrix is ​​derived from aquatic animals, such as skin tissue, cartilage tissue, corneal tissue, organ tissue, mucosal tissue or connective tissue of aquatic animals, preferably selected from skin tissue of aquatic animals.

[0088] Item 3. The decellularized matrix according to Item 1 or 2 above, wherein the decellularized matrix is ​​derived from aquatic animals in cold zones, temperate zones, subtropical zones, or tropical zones, and preferably the aquatic organisms live at a temperature of 18-30°C. In particular, the decellularized matrix is ​​derived from aquatic animals in temperate zones, subtropical zones, or tropical zones, and more preferably, the aquatic animals are selected from fish, more preferably temperate, subtropical, or tropical fish.

[0089] Item 4. The decellularized matrix according to any of the preceding items, wherein the total protein content of the decellularized matrix is ​​85-99 wt %, preferably 90-98 wt %, and more preferably 92-97 wt %.

[0090] Item 5. The decellularized matrix according to any of the preceding items, wherein the content of type I collagen in the decellularized matrix is ​​75-99wt%, particularly 80-95wt%, and especially 85-90wt%.

[0091] Item 6. The decellularized matrix according to any of the preceding items, wherein the content of type III collagen in the decellularized matrix is ​​0-9wt%, preferably less than 7wt%, and especially less than 3wt%.

[0092] Item 7. The decellularized matrix according to any of the preceding items, wherein the content of type IV collagen in the decellularized matrix is ​​less than 0.5 wt %, particularly less than 0.2 wt %, and especially less than 0.1 wt %.

[0093] Item 8. The decellularized matrix according to any of the preceding items, wherein the decellularized matrix does not contain type IV collagen.

[0094] Item 9. The decellularized matrix according to any of the preceding items, wherein the content of type VI collagen in the decellularized matrix is ​​0-3.5 wt %, preferably 0.1-3 wt %.

[0095] Item 10. The decellularized matrix according to any of the preceding items, wherein the decellularized matrix has an irregular honeycomb porous or lamellar microstructure, and has a water absorption rate of 2-8 times its own weight, particularly a water absorption rate of 3-7 times its own weight, and especially a water absorption rate of 4-6 times its own weight.

[0096] Item 11. The decellularized matrix according to any of the preceding items, wherein the DNA content of the decellularized matrix is ​​less than 50 ng / mg, particularly less than 30 ng / mg, and especially less than 20 ng / mg.

[0097] Item 12. The decellularized matrix according to any one of the preceding items, wherein the fibroblast growth factor (FGF) content of the decellularized matrix is ​​greater than 30 ng / g, in particular greater than 50 ng / g.

[0098] Item 13. The decellularized matrix according to any one of the preceding items, wherein the transforming growth factor β (TGF-β) content of the decellularized matrix is ​​greater than 8 ng / g, in particular greater than 10 ng / g.

[0099] Item 14. The decellularized matrix according to any of the preceding items, wherein the cell proliferation rate of the decellularized matrix is ​​greater than 105%, preferably greater than 110%, or not more than 135%.

[0100] Item 15. The decellularized matrix according to any of the preceding items, wherein the decellularized matrix has an appearance selected from the following: single-layer or multi-layer sheet, tube, mesh, granule or microsphere; or an irregular shape made in combination with auxiliary equipment.

[0101] Item 16. The method for preparing the decellularized matrix according to any one of Items 1 to 15 above, wherein the decellularized matrix is ​​prepared by pre-treating and decellularizing animal tissue, and comprises the following steps:

[0102] (1) Pretreatment step: taking animal tissue, such as aquatic animal tissue, and removing the surrounding impurities to remove the foreign tissue;

[0103] (2) decellularization step: decellularizing the tissue obtained in step (1) by physical and / or chemical methods; preferably, decellularizing by a method that does not cause protein denaturation;

[0104] (3) washing step: rinsing the decellularized matrix obtained in step (2), optionally using purified water for rinsing;

[0105] (4) Drying step: drying the decellularized matrix obtained in step (3), such as freeze-drying, to form a fixed shape;

[0106] (5) Optionally, in a subsequent step, the decellularized matrix obtained in step (4) is packaged and sterilized.

[0107] Item 17. The method according to Item 16 above, wherein the animal tissue is derived from tissues selected from the following: skin tissue, cartilage tissue, corneal tissue, organ mucosal tissue or connective tissue of an animal, preferably the animal tissue is derived from tissue of an aquatic animal, in particular selected from skin tissue of an aquatic animal.

[0108] Item 18. The method according to any one of Items 16-17 above, wherein the pretreatment step 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 free of impurities, for example, obtaining dermal tissue free of impurities.

[0109] Item 19. The method according to Item 18 above, wherein, in the pretreatment step, the biological enzyme is selected from at least one of elastase, type IV collagenase, papain, trypsin, tyrosinase, and neutral protease.

[0110] Item 20. The method according to any one of Items 18-19 above, wherein, in the pretreatment step, the concentration of each biological enzyme in the enzymatic hydrolysis system is 0.001-0.01wt%, preferably 0.004-0.006wt% or even 0.003-0.005wt%.

[0111] Item 21. The method according to any one of Items 18 to 20 above, wherein, in the pretreatment step, the solvent of the enzymatic hydrolysis system is selected from at least one of phosphate buffer, borate buffer, and tris(hydroxymethyl)aminomethane hydrochloride.

[0112] Item 22. A method according to any one of Items 18 to 21, wherein, in the pretreatment step, the enzymatic hydrolysis system is 0.004-0.006wt% elastase phosphate or elastase tris(hydroxymethyl)aminomethane hydrochloride) solution; the enzymatic hydrolysis time is 12-18h; or, the enzymatic hydrolysis system is 0.003-0.005wt% trypsin borate or trypsin phosphate solution; the enzymatic hydrolysis time is 15-21h.

[0113] Item 23. The method according to any one of Items 16 to 22 above, wherein the dermal tissue free of foreign matter obtained after the pretreatment step has no colored components visible to the naked eye.

[0114] Item 24. The method according to any one of Items 16 to 23 above, wherein the pretreatment method further comprises infiltrating the obtained impurity-free tissue, such as dermal tissue, with a protective solution precooled at 2-10°C, and then performing a decellularization treatment; the protective solution is selected from at least one of phosphate buffer, sodium chloride solution, and borate buffer, and the concentration is 0.5-1.5wt%.

[0115] Item 25. The method according to any one of Items 16 to 24 above, wherein the decellularization step 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; or the decellularization step is performed by a physical method; in the physical method, 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.

[0116] Item 26. The method according to any one of Items 16 to 25 above, wherein the decellularization treatment further includes a washing step: first immersing in 15-25°C purified water for 0.5-4h, and then immersing in 4-10°C pre-cooled purified water for 24-72h.

[0117] Item 27. The method according to any one of Items 16 to 26, wherein the total protein content of the aquatic animal-derived decellularized matrix is ​​85-99 wt %, preferably 90-98 wt %, and more preferably 92-97 wt %;

[0118] and / or, in the fibrous protein of the aquatic animal-derived decellularized matrix, the content of type I collagen is 75-99wt%, particularly 80-95wt%, especially 85-90wt%;

[0119] and / or, the content of type IV collagen is less than 0.5 wt %, in particular less than 0.2 wt %, especially less than 0.1 wt %, preferably substantially free of type IV collagen;

[0120] And / or, the thermal denaturation temperature of the freeze-dried product of the aquatic animal-derived decellularized matrix is ​​35-50° C., preferably 40-48° C.;

[0121] And / or, the residual DNA content of the aquatic animal-derived decellularized matrix is ​​less than 35 ng / mg, preferably less than 20 ng / mg;

[0122] And / or, the aquatic animal-derived decellularized matrix has an irregular honeycomb porous or lamellar microstructure, and has a water absorption rate of 2-8 times its own weight, particularly 3-7 times its own weight, and especially 4-6 times its own weight.

[0123] Item 28. The method according to any one of Items 16 to 27, wherein the hydroxyproline change rate is less than 8%, particularly 0.1-7%, especially 1-6%, or even 1.5-5%.

[0124] Item 29. The method according to any one of Items 16 to 28, wherein the connective tissue of the aquatic animal is the skin tissue of the aquatic animal, preferably the skin tissue of the aquatic fish.

[0125] Item 30. The method according to any one of the preceding items 16 to 29, wherein the aquatic animal-derived decellularized matrix is ​​obtained by pretreatment and decellularization of aquatic animal connective tissue; the pretreatment method comprises: using biological enzymes for enzymatic hydrolysis at a temperature of 10-15°C for 12-60 hours to remove muscle tissue, basement membrane and epidermis to obtain dermal tissue free of foreign tissue.

[0126] Item 31. The method according to any one of items 16 to 30 above, wherein in the decellularization step (2), the decellularization treatment is performed by a physical method and / or a chemical method; the physical method does not include high pressure treatment, laser treatment, high-speed homogenization shearing and ultraviolet irradiation that may cause protein denaturation; or, in the chemical method, the reagents used do not include components that may cause protein denaturation, and the components that may cause protein denaturation are preferably concentrated hydrochloric acid, urea, guanidine hydrochloride, sodium dodecyl sulfate, dithiothreitol, and dimethyl sulfoxide.

[0127] Item 32. A method according to any one of items 16 to 31 above, wherein the end point of the rinsing of the decellularized matrix in the cleaning step (3) is that the pH of the rinsing solution is neutral and the conductivity is less than 10 μm / cm; and the purified water immersion time is 4-12 hours.

[0128] Item 33. The method according to any one of items 16 to 32 above, wherein the fixed shape described in the drying step (4) is selected from the following appearances: single-layer or multi-layer sheet, tube, mesh, granular or microsphere; or an irregular shape made in combination with auxiliary equipment.

[0129] Item 34. The method according to any one of items 16 to 33 above, wherein the sterilization described in the subsequent step (5) is selected from at least one of radiation sterilization or epoxide sterilization such as ethylene oxide sterilization.

[0130] Item 35. A decellularized matrix gel prepared by gelling the decellularized matrix according to any one of Items 1 to 34 in vitro in a neutral aqueous solution.

[0131] Item 36. The decellularized matrix gel according to Item 35 above, wherein the gelation concentration of the decellularized matrix is ​​less than 30 g / mL.

[0132] Item 37. A method for preparing the acellular matrix gel according to Item 35 or 36, comprising the following steps:

[0133] The decellularized matrix completely gels in an aqueous solution in vitro under stirring conditions for 0.5 to 15 days, particularly 2 to 12 days, or even 3 to 10 days.

[0134] Item 38. A method for preparing a decellularized matrix gel according to Item 37 above, wherein the aqueous solution is selected from one or more of pure water, phosphate buffer, borate buffer, glycine-hydrochloric acid buffer, tris(hydroxymethylaminomethane) hydrochloride buffer, carbonate buffer, barbital buffer, and physiological saline, wherein the aqueous solution is particularly a neutral aqueous solution.

[0135] Item 39. The method for preparing the decellularized matrix gel according to any one of Items 37 to 38, wherein the gelation temperature of the decellularized matrix is ​​20-40°C, preferably 25-38°C.

[0136] Item 40. An implant comprising the decellularized matrix according to any one of Items 1 to 15, or the decellularized matrix obtained according to the method for preparing the decellularized matrix according to any one of Items 16 to 34, or the decellularized matrix gel according to any one of Items 35 to 36, or the decellularized matrix gel prepared by the preparation method according to any one of Items 37 to 39.

[0137] Item 41. An implant comprising a decellular matrix obtained according to any one of Items 1 to 15 above, or a decellularized matrix obtained according to the method for preparing a decellularized matrix according to any one of Items 16 to 34, wherein the decellularized matrix is ​​in the form of a microparticle suspension in a saline solution or a polysaccharide solution, wherein the saline solution is such as physiological saline, phosphate buffer, bicarbonate buffer, or tris(hydroxymethyl)aminomethane buffer, and the polysaccharide solution is such as sodium hyaluronate, chitosan, sodium alginate, sodium carboxymethylcellulose, carboxymethyl chitin, or chondroitin sulfate.

[0138] Item 42. Use of the decellularized matrix according to any one of the preceding items 1-15, or the decellularized matrix obtained according to the method for preparing the decellularized matrix according to any one of items 16-34, or the decellularized matrix gel according to any one of items 35-36, or the decellularized matrix gel prepared by the preparation method according to any one of items 37-39, or the implant according to item 40, or the implant according to item 41 in products for wound repair and medical cosmetology.

[0139] Item 43. The use according to Item 42 above, which is used in products for acute or chronic wound repair, residual cavity filling, damaged tissue coverage, and hemostasis.

[0140] Item 44. The use according to Item 42 above, which is used for surgical operations, or for purposes such as bone defect repair, cartilage defect repair, corneal repair, tissue thickening, residual cavity filling, hemostasis, and nerve repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1.1 and Figure 1.2 They are schematic cross-sectional structures of the decellularized matrix prepared in Example 1 of the present invention and the animal original tissue used therein; Figure 1.1 is a schematic diagram of the cross-sectional structure of the decellularized matrix prepared in Example 1 of the present invention, Figure 1.2 It is a schematic diagram of the cross-sectional structure of the animal primary tissue used in Example 1 of the present invention.

[0142] Figure 2 This is the dissolution / gelation condition of the decellularized matrix in Example 2 of the present invention.

[0143] Figure 3 This is the protein composition analysis of the decellularized matrix of Example 6 of the present invention.

[0144] Figure 4 This is the general situation of wound healing after 20 days of implantation of the acellular matrix in Example 7 of the present invention and its control in a full-thickness cortical defect Bama pig model.

[0145] Figure 5 This is the morphology of the acellular matrix of Example 9 of the present invention in the skin defect mouse at different times. DETAILED DESCRIPTION

[0146] In order to better understand the present invention, it is further described below in conjunction with specific embodiments.

[0147] Example 1

[0148] This example examines the microstructural differences between the decellularized matrix of the present invention and its original animal tissue.

[0149] The decellularized matrix was prepared as follows:

[0150] (1) The skin tissue of tilapia was taken, and the fish meat, fish scales, epidermis and other impurities were removed and washed. The skin tissue was immersed in a 0.005% elastase phosphate solution at 10°C for 15 hours, and the residual meat tissue with fascia was removed until a complete dermal structure was revealed. The epidermis and basement membrane layer were removed with a precision cutter until no scale roots and colored tissue were visible to the naked eye, and the skin tissue was washed with pre-cold water.

[0151] (2) Chemical decellularization: at 10°C, the fish skin was immersed in 0.4% sodium hydroxide solution for 2 h and 0.5% Triton X-100 for 24 h to remove residual cells on the fish skin.

[0152] (3) Rinse the fish skin repeatedly until it becomes neutral, and then soak it in purified water for 8 hours.

[0153] (4) Freeze-dry the fish skin and cut it into 5 cm x 5 cm square pieces.

[0154] (5) The fish skin is packaged and irradiated to sterilize the finished product.

[0155] Another tilapia tissue was taken after removing the surrounding tissues, washed with purified water, and freeze-dried using the same freeze-drying process for later use. The decellularized matrix and animal skin tissue were sprayed with gold and observed using a scanning electron microscope.

[0156] Test results such as Figure 1.1 and Figure 1.2 As shown, the cross-sections of the original tilapia skin tissue and the tilapia decellularized matrix of the present invention both have similar multi-layer fiber structures and asymmetric morphologies, and the layered fiber structures are complete and basically consistent.

[0157] Example 2

[0158] This example investigates the in vitro gelation / dissolution time of one of the decellularized matrices prepared in the present invention.

[0159] The decellularized matrix was prepared as follows:

[0160] (1) Take the skin tissue of silver crucian carp, remove the fish meat, scales, epidermis and other impurities and wash it clean.

[0161] (2) Decellularization by chemical method: at 10°C, the fish skin was immersed in 0.05% sodium deoxycholate solution for 3 h and 15% sodium chloride solution for 48 h to remove residual cells on the fish skin.

[0162] (3) Rinse the fish skin repeatedly until the conductivity is less than 10 μS / cm, and then soak it in purified water for 6 h.

[0163] (4) Freeze-dry the fish skin and grind it into powder using a grinder.

[0164] (5) The fish skin powder is packaged and then sterilized with ethylene oxide to obtain a finished product.

[0165] Prepare a 1% concentration of decellularized matrix suspension with purified water, shake continuously, and take samples to observe the dissolution of the decellularized matrix at 12h, 24h, and 72h. Prepare a 10% concentration of decellularized matrix suspension with purified water and saline, shake continuously for 72h, and take samples to observe the gelation of the decellularized matrix.

[0166] Test results such as Figure 2 As shown, the 1% purified water suspension of the decellularized matrix in this embodiment has a clear and transparent solution after 72 hours of dissolution, and the decellularized matrix is ​​completely dissolved; the 10% concentration of the purified water suspension and the physiological saline suspension form a uniform and stable gel after 72 hours of dissolution, in which the physiological saline gel undergoes re-fibrosis to form a hydrogel with a tighter fiber network structure.

[0167] Example 3

[0168] This example investigates the differences in hydroxyproline content after decellularized matrices made from different aquatic animal tissues.

[0169] The decellularized matrix was prepared as follows:

[0170] (1) taking mackerel skin, golden pomfret maw, silver carp and bighead carp skin, salmon skin and cod maw, removing impurities such as fish meat, fish scales and epidermis, and washing them, and splitting the maw into a single layer of membrane;

[0171] (2) Spanish mackerel skin and golden pomfret swim bladder: decellularization was performed using the method of Example 1; silver carp and bighead carp skin: decellularization was performed by freeze-thaw cycles at -18°C to 10°C for 5 times, followed by immersion in 1% peracetic acid for 3 h at 10°C and treatment with 0.01% trypsin solution for 6 h;

[0172] (3) Rinse the fish skin and fish bladder repeatedly until they are neutral and the conductivity is less than 10 μS / cm, and then soak them in purified water for 5 hours.

[0173] (4) Freeze-dry the fish skin and fish maw and grind them into powder using a grinder.

[0174] (5) The fish skin and fish maw powder are packaged and sterilized with ethylene oxide to obtain a finished product.

[0175] The hydroxyproline content of the aquatic animal tissue decellularized matrix prepared above was determined according to the method in Appendix B of YY / T 1453-2016, and the gelation time of the material in an in vitro aqueous solution environment was determined.

[0176] Comparative Example 3.1: According to the preparation method of silver carp and bighead carp skin decellularized matrix in Example 3, salmon skin and cod maw decellularized matrix were prepared, and the hydroxyproline content and gelation time were measured.

[0177] Comparative Example 3.2 Adult pig skin was selected and acellular matrix was prepared according to the method of Example 1, and the hydroxyproline content and gelation time were measured. The test results are shown in Table 1.

[0178] Table 1 Hydroxyproline content of decellularized matrices from different aquatic animal tissues

[0179]

[0180] As can be seen from the above table, the optimal living temperature of cold zone aquatic animals is generally not more than 16°C, the hydroxyproline content of their tissues is relatively low, the gelation time after being made into acellular matrix is ​​too fast, and the ability to serve as a scaffold in the early stage is weak; the hydroxyproline content of pigs exceeds 12%, the stability of the pig skin acellular matrix is ​​high, and it is unable to form a gel in an aqueous solution environment; the temperate, subtropical, and tropical aquatic animals described in Example 3 have a moderate hydroxyproline content, have gelation properties, and can serve as a scaffold in the early stage.

[0181] Example 4

[0182] This example investigates the residual DNA content, hydroxyproline content, α-gal antigen content and growth factor content of the decellularized matrices of various aquatic animal tissues in Example 3 of the present invention.

[0183] Take various fish skin or fish maw decellularized matrices of Example 3, and determine the residual DNA amount thereof according to the method of YY / T 1876-2023; determine the α-gal antigen content thereof according to the method of YY / T 1561-2017; determine the FGF and TGF-β content of the sample according to the determination method of growth factors in a commercially available kit (the kit is the JL42886 kit of Jianglai Biological). Take fish skin or fish maw tissue that has been processed in process step 1 and has not been decellularized, and determine the initial hydroxyproline content of the animal tissue according to the method of Appendix B of YY / T 1453-2016, and calculate the hydroxyproline change rate (hydroxyproline change rate = 1-hydroxyproline content of decellularized matrix / hydroxyproline content of animal tissue before decellularization). The test results are shown in Table 2.

[0184] Comparative Example 4.1 The pig skin of Comparative Example 3.2 was selected and acellular matrix was prepared according to the method of Comparative Example 3.2, and the residual DNA content, hydroxyproline content, α-gal antigen content and growth factor content were measured.

[0185] Table 2 Residual DNA content, hydroxyproline content, α-gal antigen content and growth factor content in the decellularized matrix

[0186]

[0187] From the results in Table 2, it can be seen that the aquatic biological tissue decellularized matrix prepared by the present invention has low residual DNA and no significant difference in α-gal content with negative, indicating that the material has high biological safety; the hydroxyproline change rate of the material is less than 5%, indicating that the influence of the material composition in the preparation process is controllable and the overall structure of the material is well maintained; the higher growth factor content also indicates that the preparation process has no significant impact on the content of beneficial factors. On the contrary, the DNA residue of the prepared porcine skin decellularized matrix is ​​high, and obvious α-gal antigen is measured, indicating that the preparation process of the material fails to completely remove the immunogenic substance; the hydroxyproline content is significantly reduced after the process treatment, indicating that the process causes the loss of protein components in the material.

[0188] Example 5

[0189] This example investigates the cell proliferation rate of the decellularized matrix of the present invention.

[0190] The decellularized matrix derived from the mackerel skin of Example 3 was used as the test sample, and the cell proliferation rates of the decellularized matrix extract solution and its 50% dilution, 1% decellularized matrix solution, and 2% decellularized matrix solution were respectively determined according to the MTT method of GB / T16886.5-2017.

[0191] The test results are shown in Table 3. The results show that the extract and the dissolving solution of the decellularized matrix of the present invention can effectively promote cell proliferation.

[0192] Table 3 Effect of decellularized matrix on cell proliferation

[0193]

[0194] Example 6

[0195] This example investigates the protein composition of the rapidly gelatinized decellularized matrix of the present invention.

[0196] The decellularized matrix derived from tilapia skin in Example 1 was used as the test sample, and the sample was processed according to the scheme of reference (Jacek RWisniewski, Alexandre Zougman, Nagarjuna Nagaraj and Matthias Mann. Nature Methods, 2009, 6: 359-362), and analyzed using LC-MS / MS equipment after centrifugal drying. The analytical column selected a C18 reverse phase chromatographic column, and the gradient was that the mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 30min. The mass spectrometer used a ThermoFisher Q Exactive system combined with a nanoliter spray Nano Flex ion source, the spray voltage was 1.9kV, and the ion transfer tube heating temperature was 275°C. The mass spectrometer scanning mode was in the information-dependent acquisition working mode, the primary mass spectrometer scanning resolution was 70000, the scanning range was 350-2000m / z, and the maximum injection time was 100ms. In each DDA cycle, a maximum of 20 secondary spectra with charges ranging from 2+ to 5+ were collected, and the maximum injection time of secondary mass spectrometry ions was 50ms. The mass spectrometry results were processed and searched using PEAKS Studdio software.

[0197] Protein composition test results Figure 3 As shown, from the results, it can be seen that the fibrous protein in the decellularized matrix is ​​mainly collagen, type I collagen accounts for more than 90%, followed by type VI collagen, a small amount of type V collagen is also detected, and type IV collagen is not detected.

[0198] Example 7

[0199] This embodiment investigates the repair effect of the acellular matrix prepared in Example 1 of the present invention on a full-thickness cortical defect model of Bama pigs.

[0200] The experiment selected female Bama pigs weighing 15-20kg and were housed alone for 1 week to adapt to the environment before surgery. A total of 3 groups were divided: a test group (acellular matrix prepared from Spanish mackerel in Example 3 of the present invention), a blank group (membrane-making only) and a control group (acellular matrix prepared using the method in Example 1 of patent CN116617462A). A wound with a diameter of no more than 4cm was made beside the spine of each Bama pig, the entire cortex was removed, and the materials of the test group and the control group were cut into a size suitable for the wound, and implanted after complete infiltration. All three groups of animals were covered with dressings to avoid infection of the wound during the experiment. The experiment took general photos of the wounds at 0, 2, 6, 12 and 20 days after surgery, and measured and calculated the wound healing rate.

[0201] The general situation of wound healing is as follows Figure 4 The wound healing rate statistics are shown in Table 3. The results show that the decellularized matrix of the present invention gelled 6 days after implantation, degraded 12 days after implantation, and the healing rate exceeded 85% at 20 days after implantation; the wound in the blank group also showed a shrinking trend within 20 days, but the healing rate was less than 60%; the control group degraded slowly, and in the middle and late stages of implantation, a dry scab was formed with animal tissue fluid and blood, causing a space-occupying effect, which affected the wound healing. Since the material degraded 12 days after implantation, the wound healing rate slowed down. It is not difficult to infer that continuing to implant the decellularized matrix after degradation can effectively further shorten the time for complete wound healing.

[0202] Table 4 Wound healing rate of Bama pigs in the experimental group, blank group and control group

[0203] Implantation time 0d 2d 6d 12d 20d Experimental Group 0％ 6.3％ 17.6％ 73.2％ 88.1％ Blank Group 0％ 2.4％ 6.5％ 38.2％ 56.2％ Control group 0％ 3.9％ 6.4％ 29.7％ 37.8％

[0204] Example 8

[0205] This example investigates the skin sensitization and intradermal reaction of the rapidly gelling acellular matrix of the present invention.

[0206] The skin sensitization test and intradermal reaction test of the acellular matrix prepared in Example 3 of the present invention were tested according to the method of GB / T 16886.10. The skin sensitization results are shown in Table 5, and the intradermal reaction results are shown in Table 6.

[0207] Table 5 Skin sensitization test results

[0208]

[0209] Table 6 Intradermal reaction test results

[0210]

[0211]

[0212] As can be seen from the above table, the material obtained by the present invention does not cause skin sensitization and has no irritation.

[0213] Example 9

[0214] This example investigates the morphological changes of the acellular matrix of the present invention at different stages in an animal body.

[0215] Adult db / db mice were selected to create a full-thickness skin defect wound model, and the acellular matrix material from Spanish mackerel according to Example 3 of the present invention was implanted. After bandaging, the changes in the material were regularly observed.

[0216] The test results are as follows Figure 5 As shown. It can be seen that the material structure is intact at the beginning of implantation, and it is flexible and fits tightly to the implantation port after absorbing water; 7 days after implantation, the blood and other tissue fluids of the animal have fully penetrated into the material, but the material morphology remains unchanged, still with the appearance of a sheet-like stent; 14 days after implantation, the material gels and still adheres to the surface of the wound to provide a moisturizing environment, and the wound has healed at this time. This result shows that the decellularized matrix of the present invention can act as a stent in the early stage, and then form an in situ gel to continuously provide a moist environment.

[0217] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited by the embodiments. Any other changes, modifications, combinations, substitutions and simplifications made without departing from the spirit and principles of the present invention shall be considered as equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. Acellular matrix, characterized in that: The hydroxyproline content is 3wt% to 11wt%, particularly 5wt% to 10.8wt%, even 7.5wt%-10.5wt%, wherein the hydroxyproline change rate between the decellularized matrix and the animal tissue before decellularization is less than 8%, particularly 0.1-7%, especially 1-6%, even 1.5-5%.

2. The decellularized matrix according to claim 1, wherein the decellularized matrix is ​​derived from aquatic animals, such as skin tissue, cartilage tissue, corneal tissue, organ tissue, mucosal tissue or connective tissue of aquatic animals, preferably selected from skin tissue of aquatic animals.

3. The decellularized matrix according to claim 1 or 2, wherein the decellularized matrix is ​​derived from aquatic animals in cold zones, temperate zones, subtropical zones, or tropical zones, preferably the aquatic organisms have a living temperature of 18-30° C., in particular the decellularized matrix is ​​derived from aquatic animals in temperate zones, subtropical zones, or tropical zones, more preferably the aquatic animals are selected from fish, more preferably temperate, subtropical, or tropical fish.

4. The decellularized matrix according to any one of claims 1 to 3, wherein the water absorption rate of the decellularized matrix is ​​2 to 8 times of its own weight, in particular, the water absorption rate is 3 to 7 times of its own weight, and even the water absorption rate is 4 to 6 times of its own weight.

5. The decellularized matrix according to any one of the preceding claims 1 to 4, wherein the DNA content of the decellularized matrix is ​​less than 50 ng / mg, in particular less than 30 ng / mg, especially less than 20 ng / mg.

6. The decellularized matrix according to any one of claims 1 to 5, wherein the content of type IV collagen in the decellularized matrix is ​​less than 0.5 wt%, in particular less than 0.2 wt%, especially less than 0.1 wt%; in particular the decellularized matrix does not contain type IV collagen.

7. The decellularized matrix according to any one of claims 1 to 6, wherein the decellularized matrix has at least one of the following characteristics: - the total protein content of the decellularized matrix is ​​85-99 wt %, preferably 90-98 wt %, more preferably 92-97 wt %; - the content of type I collagen in the acellular matrix is ​​75-99 wt %, particularly 80-95 wt %, especially 85-90 wt %; - 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 %; - the content of type VI collagen in the decellularized matrix is ​​0-3.5wt%, preferably 0.1-3wt%; - the fibroblast growth factor (FGF) content of the acellular matrix is ​​greater than 30 ng / g, in particular greater than 50 ng / g; - 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; The cell proliferation rate of the decellularized matrix is ​​greater than 105%, preferably greater than 110%, or not more than 135%.

8. The decellularized matrix according to any one of claims 1 to 7, wherein the decellularized matrix has an appearance selected from the following: single-layer or multi-layer sheet, tube, mesh, granule or microsphere; or an irregular shape made in combination with auxiliary equipment.

9. The method for preparing the decellularized matrix according to any one of claims 1 to 8, wherein: The decellularized matrix is ​​prepared by pre-treating and decellularizing animal tissue, and includes the following steps: (1) Pretreatment step: taking animal tissue, such as aquatic animal tissue, and removing the surrounding impurities to remove the foreign tissue; (2) decellularization step: decellularizing the tissue obtained in step (1) by physical and / or chemical methods; preferably, decellularizing by a method that does not cause protein denaturation; (3) washing step: rinsing the decellularized matrix obtained in step (2), optionally using purified water for rinsing; (4) Drying step: drying the decellularized matrix obtained in step (3), such as freeze-drying, to form a fixed shape; (5) Optional subsequent step: packaging and sterilizing the decellularized matrix obtained in step (4).

10. The method according to claim 9, wherein: The pretreatment step comprises: using biological enzymes to perform enzymatic hydrolysis at a temperature of 0-15°C for 12-60 hours, preferably for 12-18 hours or even for 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 dermal tissue without impurities.

11. The method according to claim 10, wherein: In the pretreatment step, the biological enzyme is selected from at least one of elastase, type IV collagenase, papain, trypsin, tyrosinase, and neutral protease.

12. The method according to any one of the preceding claims 9 to 11, wherein: In the pretreatment step, the enzymatic hydrolysis system is 0.004-0.006wt% elastase phosphate or elastase tris(hydroxymethyl)aminomethane 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.

13. The method according to any one of the preceding claims 9 to 12, wherein: The decellularization step 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; or the decellularization treatment 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.

14. A decellularized matrix gel, which is prepared by gelling the decellularized matrix according to any one of claims 1 to 8 or the decellularized matrix obtained according to the method for preparing the decellularized matrix according to any one of claims 9 to 13 in a neutral aqueous solution in vitro.

15. The decellularized matrix gel according to claim 14, wherein the gelation concentration of the decellularized matrix is ​​less than 30 g / mL.

16. The method for preparing the decellularized matrix gel according to claim 14 or 15, comprising the following steps: The decellularized matrix completely gels in an aqueous solution in vitro under stirring conditions for 0.5 to 15 days, particularly 2 to 12 days, or even 3 to 10 days.

17. The method for preparing the decellularized matrix gel according to claim 16, wherein the aqueous solution is selected from one or more of pure water, phosphate buffer, borate buffer, glycine-hydrochloric acid buffer, Tris-hydrochloride buffer, carbonate buffer, barbiturate buffer, and physiological saline, wherein the aqueous solution is particularly a neutral aqueous solution.

18. The method for preparing the decellularized matrix gel according to any one of claims 16 to 17, wherein the gelation temperature of the decellularized matrix is ​​20-40°C, preferably 25-38°C.

19. An implant comprising a decellular matrix obtained according to any one of claims 1 to 8 or the method for preparing a decellular matrix according to any one of claims 9 to 13, wherein the decellular matrix is ​​in the form of a microparticle suspension in a salt solution or a polysaccharide solution, wherein the salt solution is such as physiological saline, phosphate buffer, bicarbonate buffer, or tris(hydroxymethyl)aminomethane buffer, and the polysaccharide solution is such as sodium hyaluronate, chitosan, sodium alginate, sodium carboxymethylcellulose, carboxymethyl chitin, or chondroitin sulfate.

20. Use of the decellularized matrix according to any one of the preceding claims 1-8, or the decellularized matrix obtained by the preparation method of the decellularized matrix according to any one of claims 9-13, or the decellularized matrix gel according to any one of claims 14-15, or the decellularized matrix gel prepared by the preparation method according to any one of claims 16-18, or the implant according to claim 19 in products for wound repair and medical cosmetology, such as products for acute or chronic wound repair, residual cavity filling, damaged tissue coverage, and hemostasis, or for surgical operations, or for bone defect repair, cartilage defect repair, corneal repair, tissue thickening, residual cavity filling, hemostasis, and nerve repair.

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