Method for preparing a decellularized matrix material

Abstract

The application relates to the technical field of biomedical materials, in particular to a preparation method of a decellularized matrix material. The method comprises the following steps: 1) defatting treatment of mammalian cancellous bone by using a composite defatting solution, wherein the active components in the composite defatting solution comprise 0.5 w / v % to 3 w / v % sodium dodecyl sulfate, 0.5 v / v % to 5 v / v % triton X-100 and 0.2 w / v % to 1 w / v % sodium carbonate; 2) treatment by using a decellularization and deproteinization treatment solution, wherein the decellularization and deproteinization treatment solution comprises the active components in the composite defatting solution, sodium chloride, sodium hydroxide and hydrogen peroxide; 3) virus inactivation treatment by using a virus inactivation treatment solution, wherein the virus inactivation treatment solution comprises sodium hydroxide and hydrogen peroxide; 4) decalcification, cleaning and drying treatment, so as to obtain the decellularized matrix material.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a method for preparing a decellularized matrix material. Background Technology

[0002] Decellularized bone matrix (DBM) is widely used in bone defect repair in fields such as dental implantology, alveolar ridge preservation, maxillofacial surgery, and orthopedics due to its excellent osteoconductivity, osteoinductivity, and biocompatibility. Ideal DBM should completely remove cellular components, antigens, lipids, and pathogens that cause immune rejection, while preserving the three-dimensional structure and bioactive components of natural collagen to the greatest extent possible, and maintaining a suitable porosity to facilitate cell ingrowth and vascularization. However, in clinical practice in oral and maxillofacial surgery, soft tissue defects or poor healing often coexist after tooth extraction or in areas with bone defects. Ideal bone graft materials should not only guide bone regeneration but also promote early healing of the overlying gingiva or soft tissue, reduce postoperative inflammation, atrophy, and scar formation, and provide a favorable soft tissue environment for subsequent implantation or restoration.

[0003] Traditional DBM preparation processes typically involve multiple independent steps, including defatting, decellularization, deproteinization, decalcification, virus / bacterial sterilization, and endotoxin removal. These steps often use single or simple reagents, such as chloroform / methanol defatting, trypsin or Triton X-100 / SDS decellularization, low-pH acid decalcification, ethylene oxide or irradiation sterilization, etc. These methods have several drawbacks: 1) Defatting efficiency is limited, and residual fat may lead to inflammatory reactions; 2) Decellularization and deproteinization processes may excessively damage the natural collagen structure or remove beneficial osteogenic factors; 3) Multi-step independent processing leads to complex processes, long cycles, and a high risk of accumulated chemical reagent residues; 4) Some virus inactivation methods may affect the bioactivity of the material; 5) It is difficult to simultaneously achieve the formation of a bone repair and soft tissue repair environment.

[0004] Therefore, developing an efficient, mild, and integrated processing technology that can ensure the complete removal of immunogenic substances and pathogens while better preserving the beneficial components and structure of the matrix and shortening the production cycle is an important direction for the development of DBM preparation technology. Summary of the Invention

[0005] This invention provides a method for preparing a decellularized matrix material, comprising the following steps:

[0006] 1) The cancellous bone of mammals is degreased using a compound degreasing solution, wherein the active components of the compound degreasing solution include 0.5 w / v % to 3 w / v % sodium dodecyl sulfate, 0.5 v / v % to 5 v / v % Triton X-100 and 0.2 w / v % to 1 w / v % sodium carbonate;

[0007] 2) The treatment is carried out using a decellularized and deproteinized treatment solution, which contains the active components of the composite degreasing solution, as well as sodium chloride, sodium hydroxide and hydrogen peroxide;

[0008] 3) Virus inactivation treatment is performed using a virus-killing solution, which contains sodium hydroxide and hydrogen peroxide;

[0009] 4) Decalcification, washing and drying processes are performed to obtain decellularized matrix material.

[0010] Through the above-mentioned continuous processing steps, the present invention introduces a multi-component synergistic processing system in the processes of degreasing, decellularization, deproteinization, and virus inactivation. This is beneficial to reduce lipid residues, cell residues, DNA residues, and potential pathogen risks while preserving the natural collagen structure and porous scaffold characteristics in the bone matrix, thereby obtaining a decellularized matrix material suitable for tissue repair or bone regeneration.

[0011] In some embodiments, the decellularization and deproteinization treatment solution comprises 0.5 w / v %–1.5 w / v % sodium dodecyl sulfate, 1 v / v %–3 v / v % Triton X-100, 0.2 w / v %–0.5 w / v % sodium carbonate, 1 w / v %–1.5 w / v % sodium chloride, 0.2 mol / L–0.5 mol / L sodium hydroxide, and 3 v / v %–5 v / v % hydrogen peroxide. The decellularization and deproteinization treatment solution further incorporates sodium chloride, sodium hydroxide, and hydrogen peroxide into the active components of the composite degreasing solution, allowing surfactant treatment, osmotic pressure regulation, alkaline degradation, and oxidation treatment to work synergistically within the same treatment system. In some embodiments, the decellularization and deproteinization treatment time can be 6–8 hours. Through the above treatment method, cellular residues, nucleic acid residues, and impurity protein residues in bone tissue can be reduced in a relatively short time, while minimizing excessive damage to the spatial structure of collagen and bone matrix.

[0012] In some embodiments, the virus-killing treatment solution comprises 0.2 mol / L to 0.5 mol / L sodium hydroxide and 4 v / v % to 6 v / v % hydrogen peroxide. Sodium hydroxide can destroy the structure of viruses and other pathogens through an alkaline environment, while hydrogen peroxide can further reduce pathogen activity through oxidation; the combined use of the two is beneficial to improving the efficiency of virus-killing treatment. In some embodiments, the virus-killing treatment time can be 2 to 8 hours. Through the above-mentioned virus-killing steps, the potential pathogen risk in the material can be further reduced on the basis of the already completed composite degreasing and decellularization and deproteinization treatment, thereby improving the safety of the obtained decellularized matrix material when used in biomedical scenarios.

[0013] In some embodiments, the decalcification treatment can be carried out using hydrochloric acid or formic acid. The concentration of the hydrochloric acid or formic acid solution can be 0.5 mol / L to 2.0 mol / L, and the decalcification time can be 1 to 3 hours. Moderate decalcification can reduce the mineral content in bone tissue, improve the subsequent processability and tissue compatibility of the material, and facilitate the exposure of collagen-related structures. In some embodiments, hydrochloric acid decalcification can achieve better decalcification efficiency, while formic acid decalcification can reduce the impact on collagen structure to a certain extent. Those skilled in the art can select appropriate decalcification conditions based on the mineral residue, collagen retention, and mechanical requirements of the target product. After decalcification, residual acid, processing solution, and other soluble impurities can be removed by washing, and a stable decellularized matrix material can be obtained by freeze-drying, vacuum drying, or other suitable drying methods.

[0014] In some embodiments, the mammalian cancellous bone can be bovine or porcine cancellous bone, preferably from bovine or porcine individuals aged 0-6 months. Cancellous bone from younger individuals typically has a more suitable bone matrix structure and processing characteristics, which is beneficial for obtaining decellularized matrix materials with better porosity.

[0015] In some embodiments, a pretreatment step may be included before the composite degreasing treatment. This pretreatment step includes cutting mammalian cancellous bone into bone blocks and washing them until no obvious blood or grease remains. This pretreatment can reduce the impact of blood, free lipids, and surface impurities on subsequent composite degreasing, decellularization, deproteinization, and virus inactivation steps.

[0016] The preparation method of this invention can yield decellularized matrix materials with low lipid residue, low DNA residue, and low potential pathogen risk. In some preferred embodiments, the obtained material can have low fat content, low DNA residue, low endotoxin levels, and preserved collagen and porous bone matrix structures. In some embodiments, the decellularized matrix material can be in the form of blocks, granules, sheets, strips, or other forms suitable for tissue repair. The material can be used for bone defect repair, alveolar ridge preservation, periodontal bone defect repair, bone grafting, soft tissue repair, or other tissue repair-related applications.

[0017] Compared to traditional decellularized matrix material preparation processes, this invention employs a process system that integrates a composite degreasing solution, a composite decellularization and deproteinization treatment solution, and a composite antiviral treatment solution. This system integrates multiple key factors in degreasing, decellularization and deproteinization, and antiviral treatment, thereby improving lipid removal efficiency, cell and DNA removal efficiency, and virus inactivation efficiency while reducing the process complexity and treatment damage risks associated with multiple independent processes. This method effectively balances treatment efficiency, material safety, and matrix structure preservation, thus providing a more efficient, mild, and scalable process for preparing decellularized matrix materials. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The bar chart compares the fat content of the compound degreasing method of this invention with that of single SDS, Triton X-100, sodium carbonate degreasing, and traditional organic solvent degreasing.

[0020] Figure 2 : Schematic diagram of the decrease in virus titer log value during the verification of the virus elimination process of this invention.

[0021] Figure 3 Macroscopic photograph of the decellularized matrix material prepared in the embodiments of the present invention after freeze-drying.

[0022] Figure 4 The scanning electron microscope image (×50) of the product in this embodiment of the invention shows its porous structure.

[0023] Figure 5 Micro-CT analysis results of different materials for the repair of rat skull defects; A is a three-dimensional reconstruction image of Micro-CT, and B is a sagittal tomographic image of Micro-CT.

[0024] Figure 6 Quantitative analysis results of new bone formation at 4 and 8 weeks after implantation of different materials. Detailed Implementation

[0025] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0026] Unless otherwise stated, all terms used in disclosing this invention (including technical and scientific terms) should be understood as having the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention is for the purpose of describing specific embodiments only and is not intended to constitute any limitation on the scope of protection of this invention. Unless the context clearly defines otherwise, the scientific and technical terms used herein, as well as terms and laboratory procedures in related fields such as biomedical materials, tissue engineering, biochemistry, cell biology, virology, polymer and surfactant chemistry, biomaterial processing, and bone repair medicine, are all conventional terms and standard methods well-known and widely used in the art. To facilitate understanding of the technical solutions of this invention, some related terms are further defined and explained below.

[0027] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0028] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0029] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0030] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted.

[0031] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0032] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity greater than or equal to 2.

[0033] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0034] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0035] In this invention, "w / v %" refers to mass-volume percentage concentration, representing the mass of solute in grams per 100 mL of solution; "v / v %" refers to volume-volume percentage concentration, representing the volume of liquid component in milliliters per 100 mL of solution. For solids such as sodium dodecyl sulfate, sodium carbonate, and sodium chloride, or components typically prepared by mass weighing, their percentage concentration can be reasonably understood as w / v % unless otherwise specified in the context. For liquids such as Triton X-100 and hydrogen peroxide, or components typically prepared by volume measurement, their percentage concentration can be reasonably understood as v / v % unless otherwise specified in the context. The above understanding should not change the meaning of the concentration units explicitly stated in the claims.

[0036] In this invention, "decellularized matrix material" refers to a biomedical material obtained by partially or completely processing natural bone tissue through steps such as defatting, decellularization, deproteinization, virus inactivation, decalcification, washing, and drying, retaining at least a portion of the natural bone matrix structure. The decellularized matrix material can be in the form of blocks, sheets, granules, strips, powders, or other forms suitable for implantation, filling, composite processing, or further processing. The degree of decellularization of the decellularized matrix material can be characterized by residual DNA, histological staining, residual cell nuclei, or other commonly used detection methods in the art; its matrix retention can be characterized by hydroxyproline content, collagen staining, scanning electron microscopy, pore structure observation, or other detection methods that can reflect the bone matrix structure and collagen component retention.

[0037] In this invention, "composite degreasing liquid" refers to a liquid treatment system containing two or more effective components for promoting lipid removal. The composite degreasing liquid can be an aqueous solution, or a liquid system with water as the main solvent and further containing buffer components, salts, pH adjusters, or other excipients that do not substantially affect the degreasing effect.

[0038] In this invention, "active component" refers to the component in the corresponding treatment solution that has the main treatment effect on degreasing, decellularization, deproteinization, virus inactivation, or a combination thereof.

[0039] In this invention, "defatting treatment" refers to a process in which bone tissue is brought into contact with a treatment system capable of promoting lipid emulsification, dissolution, saponification, dispersion, or elution, thereby reducing the residual neutral fats, phospholipids, or other lipids in the bone tissue. The defatting treatment can be performed through soaking, agitated soaking, shaking, circulating perfusion, intermittent fluid exchange, or other liquid treatment methods acceptable in the art.

[0040] In this invention, "decellularization and deproteinization treatment solution" refers to a treatment solution used to remove or reduce cellular components, nucleic acid residues, cell membrane components, extraneous proteins, or other biological residues that may trigger an immune response in bone tissue. "Decellularization" refers to the process of removing, lysing, or reducing cells and cell-related components in bone tissue, including reducing cell nuclei, nucleic acids, cell membranes, cytoplasmic components, or cell-related antigenic components. Decellularization does not require the absolute removal of all cell-derived components, but rather refers to reducing cellular residues to a level suitable for the intended use. "Deproteinization" refers to the process of reducing non-target proteins, extraneous proteins, soluble proteins, antigenic proteins, or other protein residues in bone tissue that may affect the biocompatibility of the material. The deproteinization treatment can be performed simultaneously with decellularization treatment, or can partially overlap with defatting, oxidation, or alkaline treatment. The degree of deproteinization can be characterized by total protein residues, specific antigen residues, histological staining, immunostaining, or other commonly used detection methods in the art. Preferably, the deproteinization treatment does not excessively damage the collagen structure in the bone matrix, and collagen retention can be evaluated indirectly or directly by observing hydroxyproline content, collagen staining, or microscopic morphology.

[0041] In this invention, "virus-inactivating treatment solution" refers to a treatment solution used to reduce, inactivate, or remove potential viral activity in bone tissue.

[0042] In this invention, "virus inactivation" or "virus elimination" refers to the process of reducing viral infectivity, replication capacity, or detectable activity. This process may include viral particle structure disruption, viral nucleic acid damage, envelope disruption, capsid protein denaturation, or other mechanisms leading to viral inactivation. Virus inactivation does not require a single mechanism, nor does it require achieving the exact same level of inactivation for all types of viruses. Rather, it refers to reducing the risk of contamination by the target virus, model virus, or potential virus to a level suitable for the intended use of the material under specified process conditions.

[0043] In this invention, "decalcification" refers to the process of reducing the content of calcium salts, hydroxyapatite, or other minerals in bone tissue through acid treatment or other treatment methods that can remove inorganic mineral components. The decalcification can be performed using other commonly used decalcification systems in the art without departing from the technical concept of this invention.

[0044] In this invention, "cleaning" refers to the process of rinsing, soaking, changing the solution, washing, or circulating the bone tissue with water, water for injection, physiological saline, buffer solution, or other liquids suitable for treating biomaterials, in order to reduce residual treatment solution, reaction byproducts, endotoxins, or other soluble impurities. The cleaning can be performed once or multiple times, and the cleaning endpoint can be determined based on pH, conductivity, endotoxin levels, residual reagent detection, or other quality control indicators. In some embodiments, cleaning can be performed until the material is nearly neutral or until no significant treatment solution residue remains.

[0045] In this invention, "drying treatment" refers to the process of reducing the moisture content in materials to facilitate molding, storage, transportation, or subsequent use.

[0046] In this invention, "pretreatment" refers to the preliminary processing or cleaning of mammalian cancellous bone before the compound defatting treatment.

[0047] This invention relates to a method for preparing a decellularized matrix material, comprising the following steps:

[0048] 1) The cancellous bone of mammals is degreased using a compound degreasing solution, wherein the active components of the compound degreasing solution include 0.5 w / v % to 3 w / v % sodium dodecyl sulfate, 0.5 v / v % to 5 v / v % Triton X-100 and 0.2 w / v % to 1 w / v % sodium carbonate;

[0049] 2) The treatment is carried out using a decellularized and deproteinized treatment solution, which contains the active components of the composite degreasing solution, as well as sodium chloride, sodium hydroxide and hydrogen peroxide;

[0050] 3) Virus inactivation treatment is performed using a virus-killing solution, which contains sodium hydroxide and hydrogen peroxide;

[0051] 4) Decalcification, washing and drying processes are performed to obtain decellularized matrix material.

[0052] In this method, each processing step can be designed to remove or reduce lipids, cellular components, extraneous proteins, potential viral activity, and mineral components in bone tissue. This process can reduce immunogenic components and pathogen risk while preserving at least some of the natural bone matrix structure, collagen components, and porous scaffold characteristics. This method is particularly suitable for preparing decellularized matrix materials for bone defect repair, alveolar ridge preservation, periodontal bone defect repair, bone grafting, or other tissue repair applications.

[0053] In this invention, the composite degreasing treatment is carried out using a composite degreasing solution. The active components of the composite degreasing solution include sodium dodecyl sulfate, Triton X-100, and sodium carbonate. Sodium dodecyl sulfate acts as an anionic surfactant to promote lipid emulsification, cell membrane disturbance, and partial protein dissolution; Triton X-100 acts as a nonionic surfactant to promote lipid and membrane structure dispersion; and sodium carbonate provides an alkaline environment and promotes lipid saponification, emulsification, or dissolution. These components can work synergistically in the same treatment system to improve the removal efficiency of lipid components from mammalian cancellous bone. Preferably, the active components in the composite degreasing solution include 1 w / v % to 3 w / v % sodium dodecyl sulfate, 2 v / v % to 5 v / v % Triton X-100 and 0.4 w / v % to 0.9 w / v % sodium carbonate; more preferably, the active components in the composite degreasing solution include 1.5 w / v % to 2.5 w / v % sodium dodecyl sulfate, 3 v / v % to 4 v / v % Triton X-100 and 0.5 w / v % to 0.8 w / v % sodium carbonate.

[0054] In some embodiments, the combined defatting treatment can be performed by soaking, agitated soaking, shaking treatment, circulating perfusion, intermittent fluid exchange, or a combination thereof. The treatment temperature can be room temperature or a temperature range suitable for promoting lipid dissolution without significantly damaging the bone matrix structure. The combined defatting treatment time can be adjusted according to the bone block size, lipid content, treatment solution concentration, and agitation conditions, for example, it can be 4–48 hours, preferably 8–24 hours, and more preferably 10–16 hours. In some embodiments, after the combined defatting treatment, the bone tissue can be washed with water, water for injection, physiological saline, or other cleaning solutions suitable for biomaterial treatment to reduce residual treatment solution and dissolved lipid components on the surface.

[0055] In some embodiments, the bone tissue after composite defatting is further treated with a decellularization and deproteinization solution. This decellularization and deproteinization solution contains the active components of the composite defatting solution, as well as sodium chloride, sodium hydroxide, and hydrogen peroxide. Thus, the decellularization and deproteinization solution can form a composite treatment system comprising surfactants, alkaline salts, inorganic salts, strong alkalis, and oxidants. In this system, the active components of the composite defatting solution can continue to remove lipids and membrane structures; sodium chloride can regulate the osmotic pressure of the system and promote cell lysis or release of cell contents; sodium hydroxide can promote the degradation of cellular components, nucleic acids, and extraneous proteins; and hydrogen peroxide can further destroy cell structures and reduce residual biological components through oxidation. These components can coexist in the same liquid system and work synergistically to form an integrated composite treatment solution.

[0056] In some embodiments, the decellularization and deproteinization treatment solution comprises 0.5 w / v % to 1.5 w / v % sodium dodecyl sulfate, 1 v / v % to 3 v / v % Triton X-100, 0.2 w / v % to 0.5 w / v % sodium carbonate, 1 w / v % to 1.5 w / v % sodium chloride, 0.2 mol / L to 0.5 mol / L sodium hydroxide, and 3 v / v % to 5 v / v % hydrogen peroxide. Preferably, the decellularization and deproteinization treatment solution comprises 0.8 w / v %–1.2 w / v % sodium dodecyl sulfate, 1.5 v / v %–2.5 v / v % Triton X-100, 0.2 w / v %–0.4 w / v % sodium carbonate, 1.1 w / v %–1.3 w / v % sodium chloride, 0.25 mol / L–0.35 mol / L sodium hydroxide, and 3.5 v / v %–4.5 v / v % hydrogen peroxide. The decellularization and deproteinization treatment time can be 4–12 hours, preferably 6–8 hours. A shorter treatment time helps reduce excessive impact on collagen components; a slightly longer treatment time helps reduce DNA and cell residues. In some embodiments, the treatment endpoint can be determined comprehensively based on DNA residue, HE staining, DAPI staining, observation of cell nuclear residue, hydroxyproline content, or other detection indicators. In some implementations, decellularization can be achieved by having less than 20 ng / mg dry weight of residual DNA and / or no obvious nuclear residue observed on histological staining.

[0057] In some implementations, after the decellularization and deproteinization process is completed, the bone tissue can be washed to reduce the residue of the decellularization and deproteinization treatment solution.

[0058] In some embodiments, the bone tissue after decellularization and deproteinization is further treated with a virus-inactivating solution. The virus-inactivating solution contains sodium hydroxide and hydrogen peroxide. Sodium hydroxide provides an alkaline environment and promotes the destruction of viral proteins, envelopes, or nucleic acid structures, while hydrogen peroxide provides an oxidizing environment and promotes the inactivation of viral structures or genetic material. When both are present, a combined alkaline and oxidizing virus-inactivating environment is formed, thereby reducing the potential viral activity in the bone tissue. In some embodiments, the virus-inactivating solution contains 0.2 mol / L to 0.5 mol / L sodium hydroxide and 4 v / v % to 6 v / v % hydrogen peroxide. Preferably, the virus-inactivating solution contains 0.2 mol / L to 0.3 mol / L sodium hydroxide and 4.5 v / v % to 5.5 v / v % hydrogen peroxide. The virus-inactivating treatment time can be 2 to 8 hours, preferably 3 to 6 hours, and more preferably about 4 hours. The effectiveness of virus inactivation treatment can be evaluated through model virus spiking experiments, viral titer reduction values, infectivity detection, cell culture methods, PCR detection, or other virus inactivation verification methods. In some implementations, the effectiveness of virus inactivation can be demonstrated by a viral titer reduction reaching a preset log value.

[0059] In some implementations, after the virus-killing treatment, the bone tissue can be further cleaned to reduce the residue of the virus-killing solution and its reaction.

[0060] In some embodiments, the cleaned bone tissue is further decalcified. Decalcification can be performed using an acidic solution, such as hydrochloric acid or formic acid. In some embodiments, the concentration of the hydrochloric acid or formic acid solution is 0.5 mol / L to 2.0 mol / L, preferably 0.8 mol / L to 1.2 mol / L. The decalcification time can be 1 to 3 hours, preferably 1 to 2 hours. Decalcification reduces the inorganic mineral content in the bone tissue, moderately softening the material and exposing more collagen-related structures. Decalcification conditions can be adjusted according to the target material's hardness, degradation rate, collagen retention, and clinical application scenario.

[0061] In some embodiments, the decalcified bone tissue undergoes a washing process. Washing can be performed using water for injection, water, physiological saline, or other washing solutions suitable for biological materials. Washing can be performed once or multiple times, for example, 2 to 6 times, with each wash lasting 0.5 to 3 hours. In some embodiments, washing can also be part of an endotoxin removal process, reducing endotoxin levels in the material through repeated soaking and solution changes. Endotoxin levels can be evaluated using the Limulus Amebocyte Lysate (LAL) assay or other commonly used detection methods in the art.

[0062] In some embodiments, the decalcified and cleaned bone tissue is dried. The drying process may include freeze-drying, vacuum drying, cryogenic drying, dehydration drying, or other drying methods suitable for biomedical materials. Preferably, the drying process is freeze-drying. The freeze-drying process may include pre-freezing and freeze-drying processes. In some embodiments, pre-freezing may be performed at -20°C to -40°C for 12 to 24 hours, followed by freeze-drying at -40°C to -60°C for 48 to 72 hours. Freeze-drying is beneficial for reducing the moisture content of the material and maintaining its porous structure, but this invention does not preclude the use of other drying methods that can achieve a suitable state for storage, transportation, and use. The drying effect can be characterized by moisture content, appearance, pore structure, mechanical properties, or storage stability.

[0063] In some embodiments, the dried material can be further subjected to shaping processes. These shaping processes may include cutting, trimming, grinding, sieving, pressing, compounding, packaging, or combinations thereof. The resulting decellularized matrix material can be in the form of blocks, flakes, granules, strips, powder, or other forms suitable for the intended use. In some embodiments, the resulting material may have a porous, interconnected structure with pore sizes ranging from 100 to 500 μm, preferably 180 to 400 μm or 200 to 400 μm. The pore structure can be evaluated using scanning electron microscopy, microscopy, pore size analysis, mercury infiltration, or other methods commonly used in the art.

[0064] In some embodiments, the mammalian cancellous bone can be derived from cattle, pigs, sheep, horses, rabbits, or other mammals suitable as bone matrix materials. In a preferred embodiment, the mammalian cancellous bone is cancellous bone from cattle or pigs; in a more preferred embodiment, the mammalian cancellous bone is cancellous bone from cattle or pigs aged 0-6 months. Cancellous bone from young animals typically has a relatively loose trabecular structure and relatively good processability, which facilitates the penetration of the compound defatting solution, decellularization and deproteinization treatment solution, and virus-inactivating treatment solution into the bone tissue, thereby improving the uniformity of subsequent processing. The mammalian cancellous bone can be pretreated before processing, and the pretreatment may include operations such as cutting, trimming, and washing. In some embodiments, the pretreatment includes cutting the mammalian cancellous bone into bone blocks and washing until there is no obvious blood or grease. The bone blocks can be strip-shaped, block-shaped, sheet-shaped, or other forms that facilitate liquid processing, and their size can be adjusted according to the source of raw materials, processing equipment, target product specifications, and the penetration efficiency of the treatment solution.

[0065] In some embodiments, the resulting decellularized matrix material may have low fat residue, low DNA residue, and good collagen retention. Fat content can be determined by Soxhlet extraction or other lipid assay methods; DNA residue can be determined by quantitative real-time PCR, qPCR, or other nucleic acid assay methods; hydroxyproline content can be determined by colorimetric analysis, amino acid analysis, or other collagen-related assay methods. In some embodiments, the resulting decellularized matrix material has a fat content of less than 1.0%, a DNA residue of less than 20 ng / mg dry weight, and / or a hydroxyproline content of more than 11%. In some embodiments, HE staining can show no significant nuclear residue in the treated material, and scanning electron microscopy can show that the material retains a porous and fibrous bone matrix structure.

[0066] In some embodiments, the decellularized matrix material can be further subjected to sterility testing, endotoxin testing, virus inactivation verification, biocompatibility testing, cell adhesion or proliferation testing, in vivo bone repair evaluation, or other quality control and performance evaluation suitable for biomedical materials. The material can be used alone or in combination with other medical materials, such as collagen, gelatin, hyaluronic acid, chitosan, alginate, calcium phosphate, hydroxyapatite, tricalcium β-phosphate, biodegradable polymers, growth factors, drugs, cells, or other components that contribute to tissue repair. Such combinations can be achieved through mixing, coating, impregnation, cross-linking, composite molding, or other conventional methods.

[0067] In the preparation method of this invention, the processes of compound degreasing, decellularization and deproteinization, virus elimination, decalcification, washing, and drying can be appropriately adjusted according to the properties of the raw materials, the size of the bone blocks, the processing equipment, and the specifications of the target product. Without changing the core technical concept of this invention, the processing temperature, processing time, stirring speed, liquid exchange frequency, liquid-to-solid ratio, bone block size, and number of washes in each step can be optimized according to actual production conditions. Preferably, the above adjustments do not substantially alter the combination of active components in the compound degreasing solution, the multi-component composite system in the decellularization and deproteinization treatment solution, or the combination of sodium hydroxide and hydrogen peroxide in the virus elimination treatment solution.

[0068] The decellularized matrix material prepared by the method of this invention can be used to prepare bone repair materials, bone filling materials, alveolar ridge preservation materials, periodontal bone defect repair materials, oral and maxillofacial bone defect repair materials, or orthopedic bone graft materials. Depending on the specific application, the material can be used as a standalone implant material, or as a composite scaffold material, carrier material, or filling material. The morphology, size, degree of decalcification, pore structure, and drying method of the material can be adjusted according to different uses to meet the needs of bone defect filling, cell ingrowth, vascularization, and tissue regeneration.

[0069] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these embodiments are only used to illustrate the technical content of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the specific experimental conditions in the following embodiments are given priority reference to the guidelines provided in this specification, or may be carried out according to generally accepted experimental manuals or conventional experimental conditions, or other experimental methods known in the art, or according to the conditions recommended by the relevant reagent or instrument manufacturers. In specific embodiments, unless otherwise specified, minor deviations within the weighing accuracy range are allowed for the measurement parameters involving raw material components; reasonable deviations due to instrument detection accuracy or operational accuracy are also allowed for parameters such as temperature and time.

[0070] Statistical analysis: All experimental data are expressed as mean ± standard deviation (Mean ± SD), with a sample size of n=6 per group. One-way ANOVA was used for comparisons between groups, and Tukey's HSD test was used for post-hoc multiple comparisons. The comparison objects corresponding to significance markers in each example are as stated in the table footnotes.

[0071] Example 1: Verification of the effect of bovine cancellous bone composite degreasing process

[0072] Several pretreated cancellous bone strips from the same batch of calves were randomly divided into five groups:

[0073] Group 1 (composite degreasing agent of the present invention): Immerse in an aqueous solution containing 1% SDS, 2% Triton X-100 and 0.3% sodium carbonate, and stir and soak for 12 hours.

[0074] Group 2 (single SDS): Immersed in an aqueous solution containing 1% SDS and treated under the same conditions.

[0075] Group 3 (single Triton X-100): Immersed in an aqueous solution containing 2% Triton X-100 and treated under the same conditions.

[0076] Group 4 (single sodium carbonate): Immersed in an aqueous solution containing 0.3% sodium carbonate and treated under the same conditions.

[0077] Group 5 (conventional organic solvents): Immerse in a 2:1 (v / v) mixture of chloroform and methanol at room temperature for 24 hours (refer to CN112807490B).

[0078] After processing, all bone fragments were rinsed with plenty of running water and then freeze-dried. The fat content of the dried bone fragments was determined by Soxhlet extraction, and the results are shown in Table 1.

[0079] Table 1: Comparison of fat content in bone fragments after treatment with different defatting methods (Mean ± SD, n=6)

[0080]

[0081] Note: * P < 0.05, *** P < 0.001, vs. Group 1.

[0082] The statistical results of bone fat content after different defatting methods are as follows: Figure 1 As shown. The results show that the composite degreasing agent of the present invention (group 1) has the best degreasing effect, with significantly lower fat residue than the single-component treatment groups (2,3,4), and is also superior to the traditional organic solvent method (group 5), while avoiding the risk of toxic residue from organic solvents.

[0083] Example 2: Verification of the effect of different compound degreasing agent concentration ratios

[0084] To verify the treatment effect of different concentration ratios of the compound degreasing agent, three groups of compound degreasing agents with different ratios were set up for comparison based on Example 1:

[0085] Group D1: 0.5% SDS, 1% Triton X-100, 0.2% sodium carbonate, soaked for 12 hours.

[0086] Group D2: 1.5% SDS, 3% Triton X-100, 0.5% sodium carbonate, soaked for 12 hours.

[0087] Group D3: 2.5% SDS, 4% Triton X-100, 0.8% sodium carbonate, soaked for 12 hours.

[0088] The fat content was measured after processing, and the results are shown in Table 2.

[0089] Table 2: Comparison of the effects of different compound degreasing agent concentration ratios (Mean ± SD, n=6)

[0090]

[0091] Note: * P < 0.05, vs D2 group; ns, no significant difference.

[0092] The results showed that, within the above test range, all concentration ratios of the compound degreasing agent could control the fat content to below 1.0%, with medium and high concentration ratios showing better results. Considering both the degreasing effect and the compatibility with subsequent processes, the D2 group conditions were preferred for the compound degreasing treatment in subsequent examples.

[0093] Example 3: Verification of the effect of decellularization and deproteinization process

[0094] Bone blocks degreased under the conditions of group D2 in Example 2 were divided into five groups for subsequent treatment: Group A (integrated composite decellularization and deproteinization treatment solution): immersed in a composite aqueous solution containing 1 w / v % SDS, 2 v / v % Triton X-100, 0.3 w / v % sodium carbonate, 1.2 w / v % sodium chloride, 0.3 mol / L NaOH, and 4 v / v % H2O2, and stirred at room temperature for 6 hours. Group B (control group, composite decellularization and deproteinization treatment solution without composite degreasing active components): immersed in a composite aqueous solution containing 1.2 w / v % sodium chloride, 0.3 mol / L NaOH, and 4 v / v % H2O2, and stirred at room temperature for 6 hours. Group C (comparative example, sodium carbonate alone): immersed in a 5 w / v % sodium carbonate aqueous solution, and stirred at room temperature for 15 hours. Group D (comparative example, NaOH alone): immersed in a 0.5 mol / L NaOH aqueous solution, and stirred at room temperature for 6 hours. Group E (Comparative example, single H2O2): Immersed in 4 v / v % H2O2 aqueous solution and stirred at room temperature for 6 hours.

[0095] Table 3: Comparison of the effects of different decellularization and deproteinization methods (Mean ± SD, n=6)

[0096]

[0097] Note: * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Group A; ns, no significant difference.

[0098] The results showed that the integrated composite decellularization and deproteinization treatment solution provided by this invention is superior to the NaCl / NaOH / H2O2 composite treatment solution without composite defatting active components and individual component treatments in reducing DNA residue. It also maintains a high hydroxyproline content while achieving deep decellularization, indicating a good balance between decellularization and deproteinization efficiency and collagen retention. Compared with single NaOH or single H2O2 treatment, the integrated composite system of this invention can more effectively reduce cell nucleus and nucleic acid residue; compared with single sodium carbonate treatment, its DNA residue is further reduced, demonstrating the synergistic treatment effect of surfactants, sodium carbonate, sodium chloride, sodium hydroxide, and hydrogen peroxide in the same treatment system.

[0099] Example 4: Comparison of the effects of different decellularization and deproteinization treatment times

[0100] To verify the effect of decellularization and deproteinization treatment time on product performance, different treatment time groups were set up for comparison based on Example 3:

[0101] Group E1: The conditions of Group A in Example 3 (integrated composite solution) were used for 4 hours.

[0102] Group E2: The conditions of Group A in Example 3 were used for 8 hours.

[0103] Group E3: The conditions of Group A in Example 3 were used for 12 hours.

[0104] After treatment, the residual DNA content and hydroxyproline content were measured, and the results are shown in Table 4.

[0105] Table 4: Comparison of effects of different decellularization and deproteinization treatment times (Mean ± SD, n=6)

[0106]

[0107] Note: * P < 0.05, *** P < 0.001, vs E2 group; ns, no significant difference.

[0108] The results showed that extending the treatment time helped reduce DNA residue, but the hydroxyproline content decreased. Considering both treatment efficiency and collagen retention, the optimal treatment time was 6–8 hours.

[0109] Example 5: Comparative Verification of Different Virus Inactivation Processes

[0110] To verify the advantages of the composite virus-killing process of the present invention, different virus-killing treatments were set up for comparison (in this embodiment, the samples were taken from bone blocks of group E2 in Example 4 after defatting, decellularization, and deproteinization treatment):

[0111] Group F1 (composite solution method of the present invention): Immersed in a composite solution containing 0.25 mol / L NaOH and 5% H2O2, and treated at room temperature for 4 hours.

[0112] Group F2 (stepwise method): First, immerse in 5% H2O2 solution for 2 hours, then rinse and immerse in 0.25 mol / L NaOH solution for 2 hours.

[0113] Group F3 (single NaOH method): Immerse in 0.5 mol / L NaOH solution and treat for 4 hours.

[0114] Group F4 (single H2O2 method): Immerse in 8% H2O2 solution and treat for 4 hours.

[0115] All bone fragments in each group were inoculated with an equal volume of bovine parvovirus (BPV) model virus suspension and then subjected to the above treatment. The residual virus titer was measured after treatment, and the logarithmic decrease in virus titer was calculated. The results are shown in Table 5.

[0116] Table 5: Comparison of the effects of different virus eradication methods (n=6, virus titer reduction is expressed as average value)

[0117]

[0118] Note: *P<0.05, ***P<0.001, vs F1 group.

[0119] The results of viral titer reduction corresponding to different virus inactivation treatments are as follows: Figure 2 As shown in the figure. The results show that the composite solution virus inactivation method (F1) used in this invention has the most significant effect, with a virus inactivation efficiency higher than that of the stepwise method and the single-component treatment method, fully demonstrating the synergistic effect of the composite process.

[0120] Example 6: Comparative verification of different decalcification times (hydrochloric acid)

[0121] Bone fragments from group F1 in Example 5, after defatting, decellularization, and virus inactivation, were decalcified using a 1.0 mol / L HCl solution for different decalcification times.

[0122] Group H1: Decalcification 1.5 hours

[0123] Group H2: Decalcification for 5 hours

[0124] Group H3: Decalcification for 12 hours

[0125] The residual calcium and hydroxyproline content were measured after treatment, and the results are shown in Table 6.

[0126] Table 6: Comparison of the effects of different hydrochloric acid decalcification times (Mean ± SD, n=6)

[0127]

[0128] Note: * P < 0.05, *** P < 0.001, vs H1 group.

[0129] The results showed that a decalcification time of 1.5 hours could moderately remove mineral components while preserving collagen integrity to the greatest extent, resulting in the highest hydroxyproline content. Considering both collagen preservation and decalcification efficiency, the optimal decalcification time was 1.5 hours.

[0130] Example 7: Verification of formic acid decalcification process

[0131] Bone fragments from group F1 in Example 5, after defatting, decellularization, and virus inactivation, were decalcified using a 1.0 mol / L formic acid solution for 1.5 hours. The residual calcium and hydroxyproline content were measured after treatment and compared with those obtained using hydrochloric acid decalcification (1.5 hours). The results are shown in Table 7.

[0132] Table 7: Comparison of the effects of different decalcification acid solutions (Mean ± SD, n=6)

[0133]

[0134] Note: * P < 0.05, vs. hydrochloric acid decalcification group; ns, no significant difference.

[0135] The results showed that formic acid decalcification resulted in a slightly higher calcium residue than hydrochloric acid decalcification within the same time period, but also a higher hydroxyproline content, indicating that formic acid caused less damage to the collagen structure. Both acids can be used in the decalcification step of this invention. Considering the decalcification efficiency, processing cycle, and complete process verification results, hydrochloric acid is the preferred decalcification acid. However, in embodiments where collagen retention or mild decalcification conditions are more important, formic acid can also be used as the decalcification acid.

[0136] Example 8: Preparation process of intact bovine bone and verification of virus inactivation

[0137] The following complete process was used to prepare decellularized matrix materials:

[0138] (1) Pretreatment: Thaw the cancellous bone of young cattle, cut it into strips about 10mm wide, and rinse it with running water.

[0139] (2) Compound degreasing: using the conditions of group D2 in Example 2.

[0140] (3) Decellularization and deproteinization: The conditions of group E2 in Example 4 (integrated composite solution) were used.

[0141] (4) Virus inactivation: Bone blocks were immersed in a composite solution containing 0.25 mol / L NaOH and 5% H2O2 and stirred at room temperature for 4 hours. Bovine parvovirus (BPV) was used as a model virus for spiked inactivation verification. Bone blocks in the experimental group were treated in the above-mentioned virus-inactivating solution containing virus suspension, while bone blocks in the control group were treated in PBS for the same amount of time. The virus titer in the bone blocks and residual solution was measured after treatment. The results showed that the virus inactivation process could reduce the BPV titer by ≥4.8 log, meeting the requirements for virus inactivation.

[0142] (5) Decalcification: Soak in 1.0 mol / L HCl solution for 1.5 hours.

[0143] (6) Cleaning / removing endotoxins: Soak and clean with water for injection 3 times, 1 hour each time.

[0144] (7) Freeze-drying: Pre-freeze at -20℃ for 18 hours, freeze-dry at -50℃ for 60 hours, and cut into 8×8×5 mm³ cubes.

[0145] The final product was characterized as follows: fat content 0.71%, residual DNA 8.5 ng / mg, hydroxyproline content 12.5%, pore size 200 μm–400 μm, endotoxin content <0.25 EU / mL, and sterility test passed. The resulting decellularized matrix material, after lyophilization, exhibited a complete and homogeneous blocky appearance, with the following macroscopic morphology: Figure 3 As shown. Scanning electron microscope morphology as follows. Figure 4 As shown, the material exhibits a rich porous and fibrous network structure.

[0146] Example 9: Validation of the process for preparing intact porcine cancellous bone

[0147] To verify the applicability of the method of this invention to raw materials from different species, a complete process verification was conducted using porcine cancellous bone:

[0148] (1) Pretreatment: Thaw the cancellous bone of piglets, cut it into strips about 10mm wide, and rinse it with running water.

[0149] (2) Compound degreasing: using the conditions of group D2 in Example 2.

[0150] (3) Decellularization and deproteinization: The conditions of group E2 in Example 4 were used.

[0151] (4) Virus inactivation: The bone blocks were immersed in a composite solution containing 0.25 mol / L NaOH and 5% H2O2 and stirred at room temperature for 4 hours. Porcine parvovirus (PPV) was used as a model virus for spiked inactivation verification. The results showed that the virus inactivation process could reduce the PPV titer by ≥4.3 log.

[0152] (5) Decalcification: Soak in 1.0 mol / L HCl solution for 1.5 hours.

[0153] (6) Cleaning / removing endotoxins: Soak and clean with water for injection 3 times, 1 hour each time.

[0154] (7) Freeze-drying: Pre-freeze at -20℃ for 18 hours, freeze-dry at -50℃ for 60 hours.

[0155] The final product was characterized as follows: fat content 0.75%, residual DNA 9.8 ng / mg, hydroxyproline content 12.1%, pore size 180 μm–380 μm, endotoxin content <0.25 EU / mL, and sterility tested satisfactory. The results indicate that the method of this invention also has a good treatment effect on porcine cancellous bone.

[0156] Example 10: Animal in vivo bone repair experiment

[0157] The material prepared in Example 9 was implanted into a rat skull model of critical-sized bone defects (5 mm in diameter, full-thickness defect), with a blank control group and a commercially available DBM product group as controls. Micro-CT and histological analysis at 4 and 8 weeks postoperatively showed that the bone volume fraction (BV / TV), new bone mineral density, and bone healing score of the material group were significantly higher than those of the control group, indicating that it has superior bone induction and bone repair capabilities.

[0158] The effects of different materials on skull defect repair are shown in the figure. Figure 5 In the control group (without implants), no new radiopaque shadows were observed at the bottom and edges of the bone defect at 4 and 8 weeks post-surgery, indicating no significant bone regeneration. In the commercially available DBM group, a small amount of irregular punctate bone formed at the bottom of the defect, and a small amount of irregular new bone appeared at the edge of the defect. The cavity defect was slightly reduced, and most of the defect area was not repaired. In the material group prepared in Example 9, the new bone grew into the scaffold 4 weeks after surgery and continued to grow after 8 weeks, forming a thicker layer of new bone tissue on the bone defect.

[0159] The results of quantitative analysis of new bone formation are shown in Figure 6Four weeks post-implantation, the bone volume fraction (BV / TV) of the control group, the commercially available DBM group, and the material group prepared in Example 9 were (12.8 ± 1.5)%, (20.5 ± 2.3)%, and (30.2 ± 2.8)%, respectively; the bone mineral density (BMD) were (0.40 ± 0.03) g / cm³, (0.58 ± 0.05) g / cm³, and (0.68 ± 0.04) g / cm³, respectively. Eight weeks post-operation, the BV / TV of the control group, the commercially available DBM group, and the material group prepared in Example 9 were (20.5 ± 2.0)%, (35.8 ± 3.2)%, and (48.5 ± 4.0)%, respectively; the BMD were (0.48 ± 0.04) g / cm³, (0.70 ± 0.06) g / cm³, and (0.90 ± 0.04) g / cm³, respectively. 0.07) g / cm³; Compared with the blank control group, the material group prepared in Example 9 showed extremely significant differences in all osteogenic related indicators (BV / TV, BMD, Defect closure, Tb.N, Tb.Th, Tb.Sp, Residual material, BS / BV) at 4 and 8 weeks (P<0.001). Compared with the commercially available DBM group, the BV / TV, BMD, Defect closure, Tb.N, Tb.Th, Tb.Sp, Residual material, and BS / BV of Example 9 showed significant differences at 4 weeks (P<0.01) and 8 weeks (P<0.001), 4 weeks (P<0.01) and 8 weeks (P<0.01), 4 weeks (P<0.05) and 8 weeks (P<0.01), 4 weeks (P<0.05) and 8 weeks (P<0.01), 4 weeks (P<0.05) and 8 weeks (P<0.01), 4 weeks (P<0.05) and 8 weeks (P<0.01), 4 weeks (P<0.05) and 8 weeks (P<0.01), 4 weeks (P<0.05) and 8 weeks (P<0.01), and 4 weeks (P<0.01) and 8 weeks (P<0.01).

[0160] Furthermore, the BV / TV, BMD, Defect closure, Tb.Th, and Tb.N of the newly formed bone in the rat group prepared in Example 9 were significantly higher than those in the other two groups at both 4 and 8 weeks, while Tb.Sp and BS / BV were significantly reduced, and there was less residual material in the scaffold. These results indicate that the material of this invention can more efficiently promote bone defect repair, forming denser and more mature new bone tissue, and exhibits better synergy between material degradation and new bone formation.

[0161] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing a decellularized matrix material, characterized in that, Includes the following steps: 1) The cancellous bone of mammals is degreased using a compound degreasing solution, wherein the active components of the compound degreasing solution include 0.5 w / v % to 3 w / v % sodium dodecyl sulfate, 0.5 v / v % to 5 v / v % Triton X-100 and 0.2 w / v % to 1 w / v % sodium carbonate; 2) Treatment is performed using a decellularization and deproteinization solution, which contains 0.5 w / v %–1.5 w / v % sodium dodecyl sulfate, 1 v / v %–3 v / v % Triton X-100, 0.2 w / v %–0.5 w / v % sodium carbonate, 1 w / v %–1.5 w / v % sodium chloride, 0.2 mol / L–0.5 mol / L sodium hydroxide, and 3 v / v %–5 v / v % hydrogen peroxide; the decellularization and deproteinization treatment time is 6–8 hours. 3) Virus inactivation treatment is performed using a virus-inactivating solution, which contains 0.2 mol / L to 0.5 mol / L sodium hydroxide and 4 v / v % to 6 v / v % hydrogen peroxide; Virus eradication treatment time is 2 to 8 hours; 4) Decalcification, washing and drying processes are performed to obtain decellularized matrix material.

2. The preparation method according to claim 1, characterized in that, Decalcification is performed using hydrochloric acid or formic acid.

3. The preparation method according to claim 2, characterized in that, The concentration of hydrochloric acid or formic acid solution is 0.5 mol / L to 2.0 mol / L.

4. The preparation method according to claim 3, characterized in that, The decalcification time is 1 to 3 hours.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The cancellous bone of the mammals in question is the cancellous bone of cattle or pigs aged 0 to 6 months.

6. The preparation method according to any one of claims 1 to 4, characterized in that, The process includes a pretreatment step prior to step 1), which involves cutting mammalian cancellous bone into bone blocks and washing them until there is no obvious blood or grease.

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