Stiffness-adjustable acellular matrix hydrogel as well as preparation method and application thereof

Abstract

The invention discloses stiffness-adjustable acellular matrix hydrogel as well as a preparation method and application thereof. According to the invention, various tissue acellular matrixes and methylcellulose are mixed according to different proportions to prepare the hydrogel with adjustable rigidity so as to meet the needs of defect reconstruction of different tissues, and the prepared hydrogel is not limited by the rigidity of initial source tissues, and can be applied to regeneration and repair of other tissues by adjusting the rigidity; the prepared hydrogel does not need exogenous addition of stem cells and growth factors, has thermosensitivity, can realize injectability and plasticity of the prepared material, and is applied to repair and regeneration of tissues with irregular shapes and edges.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a rigidity-adjustable acellular matrix hydrogel and a preparation method and application thereof. Background Art

[0002] Decellularized Matrix (DCM) is a biomaterial that retains the extracellular matrix (ECM) by removing cellular components. DCM is a natural polymer biomaterial that removes the contents of cells from tissues or organs through physical, chemical and enzymatic means, retaining only components such as the cytoskeleton structure and the extracellular matrix, in order to promote cell adhesion, proliferation and differentiation and create a good microenvironment for their growth. In recent years, due to its good cell compatibility, biodegradability and ability to induce tissue regeneration, decellularized matrix hydrogels have attracted much attention in the field of tissue repair and regenerative medicine.

[0003] During the preparation of decellularized matrix, cells and related antigen components are removed, while the integrity and structure of the extracellular matrix are retained. Such as collagen and basement membrane proteins. The complete extracellular matrix structure provides a scaffold structure for cells, which plays an important role in maintaining the integrity, mechanical properties and morphological stability of tissues. In addition, the extracellular matrix is ​​rich in many bioactive components, such as growth factors, intercellular signaling molecules and microvesicles, which induce the migration of host mesenchymal stem cells, and can induce cell proliferation and differentiation, which is conducive to tissue regeneration and repair. Decellularized matrix hydrogels have received widespread attention due to their biocompatibility and plasticity, but the preparation process often requires the addition of exogenous substances such as pepsin, which to some extent affects clinical applications.

[0004] Biomaterials with different tissue stiffness can regulate the differentiation direction of stem cells. When the stiffness is small, it can induce adipogenic differentiation of stem cells, while when the stiffness is large, it can induce osteogenic differentiation of cells. External signals are transmitted into the intracellular mechanical transduction pathway through receptors on the cell surface, regulating target genes and affecting cell proliferation, migration, differentiation and other functions. The stiffness of decellularized matrix plays a vital role in inducing cell behavior, and it is particularly significant in regulating cell migration, proliferation and even the directional differentiation of stem cells. At the same time, studies have found that compared with hydrogels with high stiffness, hydrogels with low stiffness have a stronger ability to stimulate endothelial cells to form blood vessels and synthesize vascular networks. Hydrogels are highly dynamic due to their biological properties (such as inducing cell infiltration and self-degradation). Their adjustable stiffness characteristics will affect cell adhesion, cell tension, etc.; ultimately, they can promote the differentiation of stem cells in different directions.

[0005] Methylcellulose is a cellulose derivative that can be dissolved in water. It is often used to prepare various hydrogel materials and improve the adhesion of objects. Methylcellulose hydrogel is widely used in the medical field, mainly in wound dressings, drug sustained-release systems and tissue engineering. Methylcellulose hydrogel has the following characteristics and applications: High water absorption: Methylcellulose hydrogel can quickly absorb and retain a large amount of water in water to form a hydrogel state. This enables it to effectively maintain a moist environment in wound dressings and promote wound healing. Air permeability and permeability: Methylcellulose hydrogel has good air permeability and permeability, which allows air and water to exchange freely, helping to maintain ventilation and excretion of the wound. Biocompatibility: Methylcellulose hydrogel has good biocompatibility, has no toxic side effects on human tissues, and can promote tissue regeneration and repair. Drug sustained release: Methylcellulose hydrogel can be used to prepare drug sustained-release systems. By adding drugs to hydrogels, controlled release of drugs can be achieved and the duration of drug action in the body can be prolonged. In general, methylcellulose hydrogel is a multifunctional material with broad application prospects in the medical field. It can be used in wound dressings, drug sustained-release systems, tissue engineering, etc., providing new possibilities for disease treatment and tissue repair.

[0006] At present, the modification and improvement of decellularized matrices have promoted the development of tissue engineering biomaterials. However, some tissues are limited in material acquisition and have few sources. In general, after a large number of chemical and biological treatments, the stiffness of the tissue decreases. After transplantation, they often cannot regenerate in the direction of the target tissue. Therefore, it is very critical to prepare decellularized matrix hydrogels with adjustable stiffness to provide appropriate biophysical / chemical cues for host stem cells, promote migration and differentiation, and induce the repair and regeneration of target tissues.

[0007] Disadvantages of existing technology:

[0008] 1. After tissue decellularization, chemical and biological treatments lead to the loss of tissue rigidity, making it difficult to recruit and differentiate stem cells after transplantation;

[0009] 2. It is difficult to achieve regeneration of large-volume tissue defects. Existing decellularized materials have insufficient biological activity and often require exogenous addition of stem cells and growth factors to assist tissue repair.

[0010] 3. Fixed shape. For example, in the case of acellular dermis, the shape of commercially available products is fixed. However, the defect site often has irregular edges and varying depths. Existing materials are difficult to perfectly fit the tissue edges, which not only easily leads to high costs and waste of materials, but may also lead to adverse consequences such as ineffective transplantation.

[0011] 4. The tissue source is fixed and can often only be used to construct tissue defects at the source tissue, which may lead to problems such as insufficient materials and difficult preparation.

[0012] Therefore, it is necessary to prepare a decellularized matrix hydrogel with adjustable stiffness to meet the needs of defect reconstruction in different tissues or at different locations. Summary of the invention

[0013] The purpose of the first aspect of the present invention is to provide a decellularized matrix hydrogel with controllable stiffness.

[0014] The second aspect of the present invention aims to provide a method for preparing the above-mentioned decellularized matrix hydrogel with adjustable stiffness.

[0015] The third aspect of the present invention aims to provide the application of the above-mentioned decellularized matrix hydrogel with adjustable stiffness.

[0016] The fourth aspect of the present invention aims to provide a product comprising the above-mentioned decellularized matrix hydrogel with adjustable stiffness.

[0017] The technical solution adopted by the present invention is:

[0018] In a first aspect of the present invention, a stiffness-adjustable decellular matrix hydrogel is provided, wherein raw materials for preparing the decellular matrix hydrogel include decellular matrix and methylcellulose.

[0019] Preferably, the mass volume ratio of the methylcellulose to the decellularized matrix is ​​0.1-0.2 g / ml; in the present invention, the amount of methylcellulose is limited by the volume of the decellularized matrix. When the volume is constant, the amount of methylcellulose used is constant regardless of the weight.

[0020] Preferably, within this range, the stiffness of the decellularized matrix hydrogel is regulated by adjusting the amount of methylcellulose or the decellularized matrix used.

[0021] Preferably, the stiffness-adjustable decellular matrix hydrogel also includes water.

[0022] Preferably, the decellularized matrix is ​​decellularized matrix powder.

[0023] Preferably, the final concentration of the methylcellulose is 0.05-0.1 g / ml.

[0024] The second aspect of the present invention provides a method for preparing the above-mentioned stiffness-adjustable decellular matrix hydrogel, comprising mixing the decellularized matrix, methylcellulose and deionized water and then allowing the mixture to stand to obtain the stiffness-adjustable decellular matrix hydrogel.

[0025] Preferably, the standing time is 12 to 20 hours.

[0026] Preferably, the standing temperature is 2-6°C.

[0027] Preferably, the method for preparing the decellularized matrix comprises: placing the tissue in a freezing buffer and subjecting it to repeated freeze-thaw treatments, and then subjecting the tissue to 2 to 4 digestion enzyme solution treatments and 1 to 3 polar extractions to obtain the decellularized matrix.

[0028] Preferably, the tissue includes but is not limited to adipose tissue, bone, cartilage, skin, heart, kidney and the like.

[0029] Preferably, the specific operation of the digestive enzyme solution treatment and polar extraction is: placing the tissue treated with the decellularized solution in a first digestive enzyme solution for incubation for 14 to 20 hours, extracting it once in a polar solvent after the incubation is completed, and incubating it in the first enzyme digestion solution for 5 to 7 hours after rinsing; then placing the tissue in a second digestive enzyme solution for incubation for 14 to 20 hours, and performing a second extraction in a polar solvent after the incubation is completed to obtain a decellularized matrix.

[0030] Preferably, the first digestive enzyme comprises 0.2-0.3% trypsin (w / v) and 0.05-0.15% EDTA (w / v).

[0031] Preferably, the second digestive enzyme comprises 50-60 mM Na 2 HPO 4 , 15~20mM KH 2 PO 4 , 4.5~5.5mMMgSO 4 / 7H 2 0, 13000~17000U / ml DNA type II, 80~120μg / ml RNase type IIIA and lipase type VI-S (from porcine pancreas) 1800~2200U / ml.

[0032] Preferably, the polar solvent comprises isopropanol, n-propanol or ethanol.

[0033] Preferably, the extraction time is 40 to 55 hours.

[0034] Preferably, the secondary extraction time is 6 to 10 hours.

[0035] Preferably, the method for preparing the decellularized matrix further comprises the following steps: freeze-drying the cytoplasmic matrix and then grinding it to prepare decellularized matrix powder.

[0036] Preferably, the grinding conditions are -60 to 70°C, 65 to 75 Hz, 8 to 12 cycles, each operation for 25 to 35 seconds, and an interval of 8 to 12 seconds.

[0037] The third aspect of the present invention provides the use of the stiffness-adjustable acellular matrix hydrogel described in the first aspect of the present invention in drug delivery, wound dressing, and tissue engineering.

[0038] The fourth aspect of the present invention provides a product, wherein the product comprises the stiffness-adjustable decellular matrix hydrogel described in the first aspect of the present invention.

[0039] Preferably, the products include repair materials, tissue engineering materials, drug delivery carriers, etc.

[0040] The beneficial effects of the present invention are:

[0041] In view of the problem that the rigidity of decellularized matrix is ​​low and cannot be adjusted, the present invention provides a decellularized matrix hydrogel with adjustable rigidity and a preparation method thereof; specifically, various tissue decellularized matrices are mixed with methylcellulose in different proportions to prepare a hydrogel with adjustable rigidity, so as to meet the needs of reconstruction of different tissue defects; the prepared hydrogel is not limited by the rigidity of the initial source tissue, and can be applied to the regeneration and repair of other tissues by adjusting the rigidity; the prepared hydrogel does not require exogenous addition of stem cells and growth factors; the prepared hydrogel is temperature-sensitive, and the injectability and plasticity of the prepared material can be achieved, so it can be applied to the repair and regeneration of tissues with irregular shapes and edges.

[0042] The prepared decellularized matrix hydrogel is thermosensitive and can become a flowable liquid in a low-temperature environment. It can be injected through a syringe and has the advantage of minimally invasive clinical applications. It is suitable for repairing wounds with irregular edges, varying depths, and deep soft tissue defects. It can be shaped by applying external force and its shape is fixed after the body temperature rises. It is plastic. By using methylcellulose, a method for universally regulating the stiffness of decellularized matrices is proposed, which is applicable to decellularized matrices of all tissue sources. It can even be used to reconstruct tissues from sources different from the initial material by changing the stiffness of the decellularized matrix.

[0043] The present invention also improves the preparation process of the decellularized matrix of each tissue. The decellularized matrix is ​​connected to cytokines and exosomes through peptide bonds, integrin receptor-ligands, electrostatic force interactions, etc., and can play the function of recruiting stem cells and regulating inflammatory responses after transplantation without the need for exogenous addition of stem cells and growth factors. The prepared decellularized matrix can be prepared into powder by freeze-drying and ultra-low temperature grinding technology, and a decellularized matrix hydrogel with adjustable stiffness can be prepared by adjusting the ratio with methylcellulose powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The diagram of the preparation of the decellularized matrix prepared in the embodiment of the present invention is shown.

[0045] Figure 2 The diagram of the preparation of the lyophilized powder of the decellularized matrix prepared in the embodiment of the present invention is shown.

[0046] Figure 3Flow chart of preparing a hydrogel with adjustable stiffness by mixing the decellularized matrix powder prepared in an embodiment of the present invention and methylcellulose powder.

[0047] Figure 4 Thermosensitivity of the decellularized matrix hydrogel prepared in the embodiments of the present invention.

[0048] Figure 5 Front and side views of decellularized matrix hydrogels mixed with different MC contents (low, medium, and high concentrations).

[0049] Figure 6 Stiffness of decellularized matrix hydrogels mixed with different MC contents (low, medium, and high concentrations).

[0050] Figure 7 Rheological property test of decellularized matrix hydrogels mixed with different contents (low, medium and high concentrations) of MC - storage modulus.

[0051] Figure 8 Scanning electron microscopy structure of decellularized matrix hydrogels mixed with different contents (low, medium and high concentrations) of MC.

[0052] Fig. 9 Porosity of decellularized matrix hydrogels mixed with different MC contents (low, medium, and high concentrations). DETAILED DESCRIPTION

[0053] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0054] Decellularized Matrix (DCM): A natural biological material that removes cellular components from tissues / organs through physical, chemical, and enzymatic methods, retaining the extracellular matrix (ECM). Currently, a variety of tissues / organs can be used to prepare decellularized matrices, such as the heart, kidney, pancreas, gastrointestinal tract, fat, and bone.

[0055] Methylcellulose (MC) is a water-soluble cellulose derivative with excellent properties such as high water absorption, air permeability and permeability, and biocompatibility. It is often used to prepare various hydrogel materials and improve the adhesion of objects. It is widely used in wound dressings, drug sustained-release systems, tissue engineering, etc.

[0056] Stiffness: refers to the ability of a material / structure to resist elastic deformation when subjected to force. It is an indicator of the ease of elastic deformation of a material / structure. In the field of medical biology, stiffness is involved in regulating cell proliferation, migration, and differentiation. For example, low stiffness can induce adipogenic differentiation of stem cells, while high stiffness can induce osteogenic differentiation of cells.

[0057] The present invention provides a method for preparing a hydrogel with adjustable stiffness. The system has two main components: ① various types of decellularized matrices: used to provide the bioactive components required for the repair and reconstruction of different tissue defects, without the need for exogenous addition of stem cells, exosomes, growth factors and other components, and can provide extracellular matrix-related biological clues and inductions that effectively promote tissue regeneration; ② methylcellulose: by regulating the concentration of methylcellulose in the system, hydrogels with different stiffness can be prepared to meet the needs of tissue repair and reconstruction with different stiffness. At the same time, the prepared hydrogel is not limited to the stiffness of the initial source tissue, and can be applied to the regeneration and repair of other tissues by adjusting the stiffness.

[0058] Example 1 Preparation of decellularized matrix

[0059] The preparation of the decellularized hydrogel system of the present invention comprises the following steps:

[0060] 1. The decellularized matrix preparation process can be summarized as a 5-day process of washing, extraction, mechanical disruption, and enzyme digestion to prepare adipose decellularized matrix:

[0061] (1) Obtaining adipose tissue.

[0062] (2) During preparation, the tissue was first subjected to repeated freezing and thawing (-80°C to 37°C) three times, wherein the freezing buffer used consisted of a hypotonic tris buffer (pH 8.0) containing 10 mM Tris Base and 5 mM ethylenediaminetetraacetic acid (EDTA).

[0063] (3) The tissue was transferred to an enzyme digestion solution consisting of 0.25% trypsin (w / v) / 0.1% EDTA (w / v) (Gibco, Burlington, Canada) and incubated overnight for about 16 hours. After incubation, the tissue was extracted with a polar solvent in 99.9% isopropanol (v / v) at room temperature for 48 hours to remove lipids; the treated tissue was then rinsed in a rinse buffer (containing 8 g / L NaCl, 200 mg / L KCl, 1 g / L Na 2 HPO 4 and 200mg / L KH 2 PO 4The cells were then rinsed three times (30 min / time) in PBS (pH = 8.0) and digested again by incubation in an enzyme digestion solution consisting of 0.25% trypsin (w / v) / 0.1% EDTA (w / v) (Gibco, Burlington, Canada) for 6 h.

[0064] (4) After digestion was complete, the samples were washed three more times in the wash buffer and transferred to a 55 mM Na 2 HPO 4 , 17mM KH 2 PO 4 、4.9mM MgS0 4 / 7H 2 0, 15000U / ml DNA type II (from bovine pancreas), 100μg / ml RNase type IIIA (from bovine pancreas) and 2000U / ml lipase type VI-S (from porcine pancreas), and treated overnight for 16 hours.

[0065] (5) After 16 h, the matrix was rinsed three times (30 min / time) in the rinse buffer and subjected to a final 8-hour room temperature polar solvent extraction in 99.9% isopropanol (v / v). At the end of the treatment, the tissue was rinsed three times (30 min / time) in the rinse buffer, rinsed three times (30 min / time) in 70% ethanol (v / v), and stored in sterile PBS supplemented with 1% ABAM (v / v).

[0066] 2. Preparation process of decellularized matrix powder

[0067] Decellularized matrix preparation flow chart reference Figure 1 ;

[0068] (1) The stored decellularized matrix was centrifuged at 4000 rpm for 5 min, and the supernatant was removed and dispensed into containers with a large bottom area such as culture dishes. The dispensed decellularized matrix was placed in a -80 degree refrigerator for 4 h to 8 h to completely cool the decellularized matrix.

[0069] (2) Using a vacuum freeze dryer to remove water from the decellularized matrix to prepare a dry tissue.

[0070] Freeze-drying parameters: -180°C, 12-24h.

[0071] (3) The prepared dry decellularized matrix is ​​then transferred to a cryogenic tissue grinder, and prepared into a dry decellularized matrix freeze-dried powder by grinding, and stored at low temperature ( Figure 2 ).

[0072] Grinding parameters: -65 °C, 65-75 Hz, 8-12 cycles, 30 seconds each, 10 seconds interval.

[0073] Example 2 Preparation of decellularized matrix hydrogel

[0074] Decellularized matrix and methylcellulose mixing process, reference Figure 3 :

[0075] (1) The small particle mixing method is used: first weigh out the required mass of methylcellulose, mix the methylcellulose with the dry decellularized matrix powder, and stir to mix the two evenly. The less methylcellulose is added, the lower the stiffness of the final decellularized matrix hydrogel; conversely, the higher the stiffness.

[0076] (2) After mixing the two, add deionized water (DEPC H 2 O), and after being mixed evenly, placed in a refrigerator at 4°C overnight (16 h) to prepare acellular matrix hydrogel.

[0077] (3) Can be sealed and stored at 4°C until use.

[0078] in:

[0079] The formula of Low MC is: 0.5ml of decellularized matrix powder + 0.05g of methylcellulose, mixed with deionized water to make 1ml, where the concentration of methylcellulose is 0.05g / ml;

[0080] Middle MC: 0.5ml of decellularized matrix powder + 0.075g of methylcellulose, mixed with deionized water to make 1ml; the concentration of methylcellulose is 0.075g / ml.

[0081] High MC: 0.5 ml of decellularized matrix powder + 0.10 g of methylcellulose, mixed with deionized water to make 1 ml, where the concentration of methylcellulose is 0.10 g / ml.

[0082] Example 3 Performance testing of decellularized matrix hydrogel

[0083] (1) Temperature sensitivity: The prepared hydrogel material was tested for temperature sensitivity. The results are as follows: Figure 4 As shown, the results showed that the hydrogel prepared in the Middle MC group was in a flowable liquid state at 4°C and could be solidified into a gel state after being placed at 37°C for 60 minutes, showing good temperature sensitivity. The hydrogels prepared in the Low MC and High MC groups also had similar effects.

[0084] (2) Overall: The prepared hydrogel material with gradient stiffness (the methylcellulose content is called gradient increase) was observed, and the results are as follows Figure 5 , Figure 5It showed that there was no obvious difference in appearance among the three groups of decellularized matrix gels, and all three groups of decellularized matrix gels exhibited good plasticity.

[0085] (3) Stiffness: Stiffness analysis: The stiffness of normal adipose tissue of SD rats was measured using a small load biomaterial kinetic performance test system (Bose AT3220, Bose, American). The electromagnetic brake of the device allows an axial motion range of 6 mm, and the load force sensor range is 0-225 N. The measurement steps are as follows: 1) Use a clamp to clamp the sample and apply a preload of 25-30 N to remove the slack in the tissue and induce the tissue to stretch completely; 2) Select a small part of the adipose tissue for preliminary measurement to determine the loading range for formal loading. 3) Perform routine loading. Referring to the loading range of the preliminary measurement, set the routine loading to 8 mm / s. When the applied load reaches the preset size, the sample is expected to rupture. Record the highest force and displacement reached by the sample before rupture, and output the tissue stiffness.

[0086] The results are as follows Figure 6 As shown, the results showed that the higher the MC concentration, the higher the stiffness of the decellularized matrix gel.

[0087] (4) Viscoelasticity: At 25 ± 0.2 °C, a rheometer was used to perform a frequency sweep from 0.01 Hz to 100 Hz, and the logarithm of G' (storage modulus) and G" (loss modulus) versus the logarithmic coordinates of frequency f were plotted. The results are shown in Figure 7 As shown in the figure, by comparing the rheological properties of three groups of decellularized matrix gels with different methyl cellulose contents, it was found that they all met the hydrogel characteristics of G'>G".

[0088] (5) Electron microscopy and porosity: The tissues were fixed with 2% glutaraldehyde (w / v) and 1% ammonium tetroxide (w / v) for 1 h, dehydrated with acetone, sputtered with gold using a MED010 encoder, and examined using a S-3000N scanning electron microscope (SEM) (Hitachi, Tokyo, Japan). Figure 8-9 As shown, the results showed that the higher the methylcellulose content, the smaller the porosity of the decellularized matrix hydrogel.

[0089] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A stiffness-adjustable decellular matrix hydrogel, wherein the raw materials of the stiffness-adjustable decellular matrix hydrogel include decellularized matrix and methylcellulose.

2. The stiffness-adjustable acellular matrix hydrogel according to claim 1, It is characterized in that The mass volume ratio of the methylcellulose and the decellularized matrix is ​​0.1 to 0.2 g / ml; Preferably, the raw materials of the stiffness-adjustable decellular matrix hydrogel also include deionized water.

3. A method for preparing the decellularized matrix hydrogel according to claim 1 or 2, comprising mixing the decellularized matrix, methylcellulose and water and then allowing the mixture to stand to obtain the decellularized matrix hydrogel.

4. The preparation method according to claim 3, It is characterized in that The standing time is 12 to 20 hours.

5. The preparation method according to claim 3, It is characterized in that The method for preparing the decellularized matrix comprises digesting the tissue after repeated freezing and thawing, and then extracting the decellularized matrix with a polar solvent; Preferably, the specific operation of the preparation method of the decellularized matrix is: placing the repeatedly frozen and thawed tissue in a first digestive enzyme solution for incubation for 14 to 20 hours, performing an extraction in a polar solvent after the incubation is completed, and incubating in the first enzyme digestion solution for 5 to 7 hours after rinsing; then placing the tissue in a second digestive enzyme solution for incubation for 14 to 20 hours, performing a second extraction in a polar solvent after the incubation is completed, and centrifuging to remove the supernatant to obtain the decellularized matrix.

6. The preparation method according to claim 5, It is characterized in that The first digestive enzyme comprises 0.2-0.3% trypsin (w / v) / 0.05-0.15% EDTA (w / v); Preferably, the second digestive enzyme comprises 50-60 mM Na 2 HPO 4 ,15~20mM KH 2 PO 4 ,4.5~5.5mMMgS0 4 / 7H 2 0, 13000-17000U DNA type II, 80-120μg / ml RNase type IIIA and 1800-2200U / ml lipase type VI-S.

7. The method according to claim 5, It is characterized in that The polar solvent includes isopropanol, n-propanol or ethanol; Preferably, the time of the primary extraction is 40 to 55 hours; Preferably, the secondary extraction time is 6 to 10 hours; Preferably, the method for preparing the decellularized matrix further comprises the following steps: drying the cytoplasmic matrix and crushing it to prepare decellularized matrix powder.

8. The method according to claim 3, It is characterized in that The tissues include, but are not limited to, adipose tissue, bone, cartilage, skin, heart, and kidney.

9. Use of the acellular matrix hydrogel according to claim 1 or 2 in the preparation of skin dressings, repair materials, and tissue engineering materials.

10. A product comprising the decellularized matrix hydrogel according to claim 1 or 2.

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