Flaky acellular matrix as well as preparation method and application thereof

Abstract

The invention relates to a sheet-shaped acellular matrix and a preparation method and application thereof, the sheet-shaped acellular matrix comprises a sheet layer and a granular layer which are made of an acellular animal membrane extracellular matrix, the granular layer is located on one side of the sheet layer, and the sheet layer and the granular layer are combined together through physical interaction formed after freeze drying. The sheet-shaped acellular matrix takes acellular biological materials derived from human and other animal tissues as raw materials and can be used for promoting skin wound healing, the thin sheet layer provides good barrier protection for the wound, the granular layer is beneficial to maintaining a wet healing environment and is easy to remove after healing, pain of a patient is relieved, and the wound healing effect is improved. And new damage caused by removal is avoided. And meanwhile, the acellular matrix material retains natural extracellular matrix structures and components, so that a favorable microenvironment can be provided for migration, growth, proliferation and differentiation of cells, and wound healing is promoted.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials, and more particularly to a sheet-like decellularized matrix, its preparation method, and its application. Background Technology

[0002] Human skin consists of three layers: the epidermis, dermis, and subcutaneous tissue. Skin is easily injured. Superficial wounds can heal themselves, but when the injury involves the dermis, medical dressings are usually needed to aid healing, often resulting in scar hyperplasia. Medical dressings accelerate wound healing by creating a microenvironment conducive to tissue repair and regeneration, but traditional dressings struggle to address the issue of post-healing scar hyperplasia.

[0003] Biological dressings made from decellularized matrix materials derived from animals have attracted widespread attention in burn treatment. Decellularized matrix materials are natural biomaterials containing a large number of bioactive macromolecules and peptides, possessing excellent physical structures and complex biomolecules, capable of promoting wound healing and skin regeneration. Decellularized biomaterials derived from human and other animal tissues (such as dermis, pericardium, peritoneum, and amnion) can be used to promote skin wound healing.

[0004] Acellular dermal matrix (ADM) can be categorized into allogeneic ADM (human-derived) and xenogeneic ADM (animal-derived) based on the species origin of the dermis. Allogeneic ADM is made from donated homologous human dermis. A meta-analysis showed that it significantly promoted wound healing in diabetic patients and reduced complications. Xenogeneic ADM is primarily derived from pigs and cattle, and its structure is similar to that of allogeneic ADM. Compared to allogeneic ADM, xenogeneic ADM is more cost-effective. For wound repair, bioactive acellular dermal matrix (ADM) containing growth factors (GFs) can provide attachment sites for epidermal cells, promote basement membrane remodeling, and, due to its unique porous structure, promote the migration of endothelial cells and fibroblasts, thereby inducing tissue growth and achieving local reconstruction. Furthermore, the components of the ADM matrix are continuously degraded by collagenase and eventually replaced by newly formed extracellular matrix (ECM) and migrating cells. Studies have evaluated the long-term clinical efficacy of acellular dermal matrix products in burn treatment and post-burn scar repair. After a four-year follow-up, it was found that acellular dermal matrix materials can accelerate wound healing. Compared with the conventional treatment group, the color, texture, thickness, aesthetic changes of the healed skin, and limb function were all significantly improved. Acellular dermal matrix dressings have become one of the most widely used biological dressings. However, the limited supply of donor skin and the risk of disease transmission limit the application of allogeneic ADM, while differences in the molecular structure of the major histocompatibility complex lead to severe inflammatory responses after xenografting of ADM. In addition, the small pore size and low porosity of ADM are not conducive to cell migration and proliferation, which may lead to prolonged healing time, scar formation, and hinder the regeneration of the subcutaneous fat layer.

[0005] Various decellularized biomembranes are also used to treat skin wounds, such as the submucosa of the small intestine, the basement membrane of the bladder, the amnion, and the peritoneum. These decellularized matrix materials mainly contain type I and type III collagen, and also retain abundant growth factors (TGF, FGF, VEGF, and EGF) and fibronectin, which are beneficial for the activation of cell adhesion signaling pathways and the formation of blood vessel morphology. In addition, compared with the dermal matrix, thin-film materials are easier to remove immunogenic substances and reduce inflammatory responses. For example, CN1986001A discloses a biological wound-protecting membrane, which involves degreasing, cross-linking, and removing antigens from the submucosa of the small intestine, and then adding an active modified layer containing fibronectin, laminin, or hydrin to its surface through adsorption and adhesion methods; or mixing a bioadhesive with a broad-spectrum antibacterial drug and applying it to the surface of the submucosa of the small intestine to form an antibacterial sustained-release layer.

[0006] Existing decellularized matrix products for skin defect repair have the following problems:

[0007] (1) Sheet-like decellularized biofilm materials generally show significant degradation after 2 weeks of use, thereby releasing natural components that are beneficial to healing. In the early stage of healing, they mainly play a barrier protection role and cannot actively promote healing.

[0008] (2) Sheet-shaped decellularized biofilm materials have low water absorption capacity and are prone to water loss. Once the wound dries, the dressing will not be able to function.

[0009] In summary, providing a novel sheet-like decellularized matrix has become one of the urgent problems to be solved in this field. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a sheet-like decellularized matrix, its preparation method, and its applications. This sheet-like decellularized matrix comprises a thin sheet layer and a granular layer. The granular layer can absorb a large amount of water, helping to prolong the wound's moisture retention time. During use, the granular layer makes full contact with complex wounds, degrading in the early stages of healing and releasing active substances to actively promote wound healing. The thin sheet layer provides good barrier protection for the wound in the early stages of healing and can also gradually degrade in the middle and later stages of healing, continuing to provide active substances and promote wound healing.

[0011] In a first aspect, the present invention provides a sheet-like decellularized matrix comprising a sheet layer and a granular layer made of decellularized animal membrane extracellular matrix, wherein the granular layer is located on one side of the sheet layer, and the sheet layer and the granular layer are bonded together by physical interaction formed after freeze-drying. The sheet layer is composed of 2-20 layers of decellularized animal membrane extracellular matrix stacked together, and the granular layer is prepared from particles with a particle size of 5-500 μm pulverized from decellularized animal membrane extracellular matrix, and the thickness of the granular layer is 0.1-1 mm.

[0012] In this invention, the granular layer with a specific particle size and thickness can absorb a large amount of water, which helps to prolong the time the wound is moist. When used, the granular layer is in full contact with the complex wound surface and degrades in the early stage of healing, releasing active substances and actively promoting wound healing. The thin sheet layer composed of 2-20 layers of decellularized animal membrane extracellular matrix provides good barrier protection for the wound in the early stage of healing, and can also gradually degrade in the middle and late stages of healing, continuing to provide active substances and promote wound healing.

[0013] The aforementioned 2-20 layers can specifically refer to 2, 3, 5, 10, 15, or 20 layers, etc.

[0014] The particle size of the decellularized animal membrane extracellular matrix particles in the granular layer is 5-500 μm, specifically 5-500 μm, 5-400 μm, 5-300 μm, 5-200 μm, 5-100 μm, 5-50 μm, 50-400 μm, 50-300 μm, 50-200 μm, 50-100 μm, 100-500 μm, 100-400 μm, 100-300 μm, 100-200 μm, etc.

[0015] The specific value of the thickness of the particle layer, which is 0.1-1 mm, can be 0.1 mm, 0.3 mm, 0.5 mm, 0.7 mm, 0.8 mm, 1 mm, etc.

[0016] Preferably, the thin sheet layer has drainage holes inserted through perforations.

[0017] In this invention, the drainage holes are designed to drain wound exudate: some wounds produce exudate, and if too much exudate accumulates on the wound surface and is not drained for a long time, infection can occur; the thin film layer facilitates the exudate drainage by providing drainage holes. The granular layer disperses quickly upon contact with liquid, without affecting exudate drainage, therefore no perforation is required.

[0018] Preferably, the drainage hole exists as a drainage hole or a drainage gap, and more preferably as a drainage gap.

[0019] Preferably, the length of the drainage slit is 4-6 mm (e.g., 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, etc.), and the spacing is 4-6 mm (e.g., 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, etc.).

[0020] Preferably, the drainage hole includes a circular drainage hole or a square drainage hole.

[0021] Preferably, the diameter of the drainage holes is 0.5-2.5 mm (e.g., 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, etc.), and the spacing is 8-12 mm (e.g., 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, etc.).

[0022] In a second aspect, the present invention provides a method for preparing a sheet-like decellularized matrix as described in the first aspect, the method comprising the following steps:

[0023] (1) The biofilm was decellularized, virus-inactivated, defatted, washed and freeze-dried to obtain a decellularized biofilm;

[0024] (2) After wetting the extracellular matrix of decellularized animal membranes, multiple layers are stacked, and then vacuum lamination and freeze-drying are performed to obtain thin sheets;

[0025] The dried decellularized animal membrane extracellular matrix was cut into small pieces and then ground to obtain decellularized animal membrane extracellular matrix particles. The decellularized animal membrane extracellular matrix particles were sieved using a sieve. The decellularized animal membrane extracellular matrix particles that passed through the sieve were mixed with water at a mass-volume ratio of 1 g:(15-30) mL and rehydrated to obtain a paste-like granular layer.

[0026] (3) Wet the sheet layer obtained in step (2) with water, place a frame mold on the wet sheet layer, fill the mold with the paste-like granule layer obtained in step (2) and smooth it, freeze dry, and the granule layer and the sheet layer are combined to obtain sheet-like decellularized matrix.

[0027] The structural diagram of the sheet-like decellularized matrix involved in this invention is shown below. Figure 1 As shown, 1 is a thin sheet layer and 2 is a granular layer.

[0028] The specific values ​​in the above 1 g:(15-30) mL can be 1 g:15 mL, 1 g:17 mL, 1 g:19 mL, 1 g:20 mL, 1 g:21 mL, 1 g:23 mL, 1 g:25 mL, 1 g:27 mL, 1 g:30 mL, etc.

[0029] Preferably, the animal membrane in step (1) is derived from mammals.

[0030] Preferably, the mammal includes any one of pig, cow, sheep or horse.

[0031] Preferably, the mammal is a pig.

[0032] Preferably, the animal membrane includes any one or a combination of at least two of the following: the submucosa of the small intestine, the bladder basement membrane, the peritoneum, the pleura, and the greater omentum.

[0033] Preferably, the rehydration treatment time in step (2) is 4-48 h, specifically 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, etc.

[0034] Preferably, the grinding and pulverizing temperature in step (2) is -196~-10℃, specifically -196℃, -150℃, -100℃, -50℃, -10℃, etc.

[0035] Preferably, the thickness of the frame mold in step (3) is 0.1-1 mm, specifically 0.1 mm, 0.3 mm, 0.5 mm, 0.7 mm, 0.8 mm, 1 mm, etc.

[0036] Thirdly, the present invention provides a composite sheet-like decellularized matrix, wherein the composite sheet-like decellularized matrix comprises the sheet-like decellularized matrix and the microparticle decellularized matrix described in the first aspect.

[0037] The combination of sheet-like decellularized matrix and particulate decellularized matrix can be achieved through contact combination, physical combination, or chemical combination.

[0038] Preferably, the decellularized microparticle matrix is ​​composed of microparticles in a first particle size range, microparticles in a second particle size range, and microparticles in a third particle size range in a mass ratio of (0.2-0.6):1:(1-1.5).

[0039] The particle size of the particles in the first particle size range is 5-50 μm, the particle size of the particles in the second particle size range is 50-150 μm, and the particle size of the particles in the third particle size range is 150-300 μm.

[0040] The particle size range between the first particle size range particles, the second particle size range particles and the third particle size range particles is greater than 50 μm.

[0041] The lower limit of 5-50 μm can be any value between 5 and 50 μm, such as 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 45 μm, etc.; the upper limit can be any value between 5 and 50 μm that is greater than or equal to the lower limit, such as 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, etc.

[0042] The lower limit of 50-150 μm can be any value between 50-150 μm, such as 50 μm, 70 μm, 90 μm, 110 μm, 130 μm, 140 μm, etc.; the upper limit can be any value between 50-150 μm that is greater than or equal to the lower limit, such as 50 μm, 70 μm, 90 μm, 110 μm, 130 μm, 150 μm, etc.

[0043] The lower limit of 150-300 μm can be any value between 150-300 μm, such as 150 μm, 180 μm, 210 μm, 240 μm, 270 μm, 290 μm, etc.; the upper limit can be any value between 150-300 μm that is greater than or equal to the lower limit, such as 150 μm, 180 μm, 210 μm, 240 μm, 270 μm, 300 μm, etc.

[0044] Preferably, the decellularized microparticle matrix is ​​prepared using a method comprising the following steps:

[0045] (1) Animal membrane tissue was decellularized, virus-inactivated, defatted, washed and freeze-dried to obtain decellularized animal membrane extracellular matrix material;

[0046] (2) After cutting the decellularized animal membrane extracellular matrix material into small pieces, grind and pulverize it to obtain microparticle decellularized matrix. The microparticle decellularized matrix is ​​sieved using a sieve to obtain microparticles in the first, second and third particle size ranges. The obtained microparticles in the first, second and third particle size ranges are mixed to obtain microparticle decellularized matrix.

[0047] Preferably, the contact combination is as follows: the microparticle decellularized matrix and the sheet-like decellularized matrix are mixed at a concentration of 1.5-15 mg / cm³. 2 The proportion of contact.

[0048] And / or, the physical combination is performed as follows: the microparticle decellularized matrix is ​​made into a paste with water, coated onto the sheet-like decellularized matrix, and freeze-dried to obtain a physical combination of the microparticle decellularized matrix and the sheet-like decellularized matrix; the dry weight ratio of the microparticle decellularized matrix to the area ratio of the sheet-like decellularized matrix is ​​1.5-15 mg / cm². 2 .

[0049] And / or, the chemical combination is carried out by incubating the physical combination of microparticle decellularized matrix and sheet-like decellularized matrix in an aqueous solution of a crosslinking agent, washing off the crosslinking agent, and freeze-drying to obtain a chemical combination of microparticle decellularized matrix and sheet-like decellularized matrix.

[0050] Preferably, the crosslinking agent comprises any one or a combination of at least two of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDCNHS), dialdehyde, or genipin.

[0051] Fourthly, the present invention provides the application of the sheet-like decellularized matrix as described in the first aspect or the composite sheet-like decellularized matrix as described in the third aspect in the preparation of medical biomaterials that promote wound healing.

[0052] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) The sheet-like decellularized matrix of the present invention combines a thin sheet layer and a granular layer. The granular layer can degrade in the early stage of healing, releasing active substances and actively promoting wound healing; the thin sheet layer provides good barrier protection for the wound in the early stage of healing, and after the granular layer has completed degradation, the thin sheet layer can still continue to contact and react with the tissue, providing active substances and promoting wound healing. The sheet-like decellularized matrix has good mechanical properties, and the thickness of both the thin sheet layer and the granular layer is appropriate, giving it good flexibility and making it easy to adhere to the wound.

[0055] (2) By comprehensively evaluating the degradation time, mechanical properties, particle binding and wound healing of sheet-like decellularized matrix, this invention obtained a sheet layer composed of 2-20 layers of decellularized animal membrane extracellular matrix and a particle layer (0.1-1 mm thick) composed of particles with a particle size of 5-500 μm pulverized from decellularized animal membrane extracellular matrix. The two are combined together by physical interaction formed after freeze-drying, which can exert a greater effect on promoting wound healing.

[0056] (3) The present invention comprises a particle layer with a thickness of 0.1-1 mm composed of particles with a particle size of 5-500 μm. The particle morphology greatly increases the specific surface area of ​​the material, significantly improves water absorption performance, and helps to prolong the time of the moist environment. The moist environment can promote the release of various growth factors; various cells such as neutrophils, macrophages, keratinocytes, and fibroblasts can migrate rapidly; the tissue proteolytic enzymes produced by the wound are conducive to the dissolution of necrotic tissue in the moist environment; no scab formation occurs during use, which can better protect the nerve endings of the wound. At the same time, the specific surface area and contact area of ​​the 5-500 μm particles are relatively reasonable. After being wetted and freeze-dried, more hydrogen bonds are formed between the particles, and they are not easy to fall off. Therefore, a bond of appropriate strength can be formed without the need for additional chemical cross-linking.

[0057] (4) The sheet-like decellularized matrix involved in this invention does not require the introduction of chemical additives during the preparation process, which further improves the biocompatibility and safety of the sheet-like decellularized matrix. Attached Figure Description

[0058] Figure 1 This is a diagram of the sheet-like decellularized matrix structure of the present invention. Detailed Implementation

[0059] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0060] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0061] Definitions:

[0062] Decellularized animal membrane extracellular matrix: refers to the dried membrane-like extracellular matrix material obtained by decellularizing thin animal membranes.

[0063] Decellularized animal membrane extracellular matrix particles: refers to the dried granular material obtained by low-temperature grinding and pulverization of decellularized animal membrane extracellular matrix.

[0064] Granular layer: refers to a layer of material formed by extracellular matrix granules of decellularized animal membrane cells.

[0065] Silicone frame: refers to a hollow rectangular frame formed by cutting a silicone sheet of a certain thickness.

[0066] Stainless steel sheet: refers to a stainless steel sheet with a certain thickness and a smooth and flat surface.

[0067] Low-temperature grinding: refers to grinding at -196~-10℃.

[0068] The steps of decellularization, virus inactivation, defatting, washing, and freeze-drying involved in the following embodiments are as follows:

[0069] (1) Mechanically remove animal membrane tissues, such as the submucosa of the small intestine, the basement membrane of the bladder, and the peritoneum of the bladder;

[0070] (2) First virus inactivation: The animal membrane tissue was shaken for 2 h with a mixed solution of 0.1% peracetic acid and 10% ethanol, and then washed several times with purified water until the pH of the washing solution was about 7.

[0071] (3) Decellularization: The animal membrane tissue was shaken for 2 hours with a mixture of 0.1% sodium dodecyl sulfate and 0.03% trypsin, and then washed several times with purified water until no foam was produced in the washing solution;

[0072] 4) Second virus inactivation: The animal membrane tissue was soaked in a 3% sodium hydroxide aqueous solution for 1 hour, and then washed several times with purified water until the pH of the washing solution was about 7.

[0073] (5) Degreasing: The animal membrane tissue was soaked in a methanol / chloroform mixed solution with a volume ratio of 1.5:1 for 12 h, and then washed several times with purified water until the concentration of chloroform in the washing solution was less than 0.0063%;

[0074] (6) Freeze-drying: Animal membrane tissue was freeze-dried for 24 h to obtain decellularized animal membrane tissue.

[0075] It should be noted that the above-described steps of decellularization, virus inactivation, defatting, washing, and freeze-drying can all be achieved using conventional techniques known in the art. Any such alternative, as long as it aims to achieve the same or similar process objectives, can achieve the technical effects of the present invention.

[0076] Example 1

[0077] This embodiment provides a sheet-like decellularized matrix, the preparation method of which includes the following steps:

[0078] S1: The submucosa of the small intestine and the basement membrane of the bladder of the pig were decellularized, defatted, inactivated by virus, washed, and freeze-dried to obtain the decellularized submucosa of the small intestine and the decellularized basement membrane of the bladder.

[0079] S2: Take the decellularized porcine small intestinal submucosa obtained in S1, moisten it, and stack 10 layers, ensuring that the layers adhere tightly and that no air bubbles are present. The 10 layers of porcine small intestinal submucosa are sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in sequence to obtain a thin sheet. The thin sheet is then cut using a specific die, simultaneously creating a uniformly distributed matrix of slits as drainage gaps (the length of the drainage gaps is 5 mm, and the spacing is 5 mm) to drain exudate during use.

[0080] S3: The decellularized porcine bladder basement membrane obtained in S1 was subjected to low-temperature grinding to obtain decellularized porcine bladder basement membrane particles. Standard sieves with mesh sizes of 100 μm and 250 μm were used to sieve the particles. The 250 μm standard sieve was stacked on top of the 100 μm standard sieve. The particles were poured into the 250 μm standard sieve frame, and the frame was shaken and tapped to obtain particles of 100 μm-250 μm. The particles were rehydrated with purified water at a ratio of 1 g:20 mL, and mixed evenly for 24 h to obtain a paste-like particle layer. The sheet layer obtained in S2 was moistened with purified water. A 0.5 mm thick silica gel frame was placed flat on the sheet layer. The obtained paste-like particle layer was filled into the silica gel frame and smoothed. The mixture was freeze-dried to obtain a sheet-like decellularized matrix composed of the particle layer and the sheet layer. The mixture was then sealed in packaging and sterilized by irradiation.

[0081] Example 2

[0082] This embodiment provides a sheet-like decellularized matrix, the preparation method of which includes the following steps:

[0083] S1: The submucosa of the small intestine and the basement membrane of the bladder of the pig were decellularized, defatted, inactivated by virus, washed, and freeze-dried to obtain the decellularized submucosa of the small intestine and the decellularized basement membrane of the bladder.

[0084] S2: Take the decellularized porcine small intestinal submucosa obtained in S1, moisten it, and stack 10 layers, ensuring that the layers adhere tightly and that no air bubbles are present. The 10 layers of porcine small intestinal submucosa are sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in sequence to obtain a thin sheet. The thin sheet is then cut using a specific die, simultaneously creating a uniformly distributed matrix of slits as drainage gaps (the length of the drainage gaps is 5 mm, and the spacing is 5 mm) to drain exudate during use.

[0085] S3: The decellularized porcine bladder basement membrane obtained in S1 was subjected to low-temperature grinding to obtain decellularized porcine bladder basement membrane particles. Standard sieves with mesh sizes of 5 μm and 300 μm were used to sieve the particles. A 300 μm standard sieve was stacked on top of a 5 μm standard sieve. The particles were then poured into the 300 μm standard sieve frame, and the frame was shaken and tapped to obtain particles ranging from 5 μm to 300 μm. The particles were rehydrated with purified water at a ratio of 1 g:20 mL, and mixed evenly for 24 h to obtain a paste-like particle layer. The sheet layer obtained in S2 was moistened with purified water. A 0.5 mm thick silica gel frame was placed flat on the sheet layer. The obtained paste-like particle layer was filled into the silica gel frame and smoothed. The mixture was then freeze-dried to obtain a sheet-like decellularized matrix composed of the particle layer and the sheet layer. The mixture was then sealed in packaging and sterilized by irradiation.

[0086] Example 3

[0087] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that step S1 involves decellularizing, defatting, virus inactivating, washing, and freeze-drying the porcine small intestinal submucosa to obtain a decellularized porcine small intestinal submucosa, and in step S3, during the preparation of the granular layer, the decellularized porcine bladder basement membrane is adjusted to the decellularized porcine small intestinal submucosa, that is, the granular layer is made using the decellularized porcine small intestinal submucosa. The remaining steps are consistent with Embodiment 1.

[0088] Example 4

[0089] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that, after the decellularized porcine small intestinal submucosa is moistened in step S2, two layers are superimposed, that is, the sheet layer is made of two layers of decellularized porcine small intestinal submucosa. The remaining steps are consistent with Embodiment 1.

[0090] Example 5

[0091] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that, in step S2, the decellularized porcine small intestinal submucosa is soaked and then stacked in 20 layers, that is, the sheet layer is made of 20 layers of decellularized porcine small intestinal submucosa. The remaining steps are consistent with Embodiment 1.

[0092] Example 6

[0093] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that the sieve used to make the particle layer in step S3 has a pore size of 150 μm and 200 μm, resulting in particles of 150 μm-200 μm. The remaining steps are consistent with Embodiment 1.

[0094] Example 7

[0095] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that the sieve used to make the particle layer in step S3 has a pore size of 300 μm and 500 μm, resulting in particles of 300 μm-500 μm. The remaining steps are consistent with Embodiment 1.

[0096] Example 8

[0097] This embodiment provides a sheet-like decellularized matrix, which differs from Example 1 only in that the ratio of particles to purified water is 1 g: 15 mL when making the particle layer in step S3. The remaining steps are the same as in Example 1.

[0098] Example 9

[0099] This embodiment provides a sheet-like decellularized matrix, which differs from Example 1 only in that the ratio of particles to purified water is 1 g: 30 mL when making the particle layer in step S3. The remaining steps are the same as in Example 1.

[0100] Example 10

[0101] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that the rehydration time is 4 hours when making the granular layer in step S3, while the other steps are the same as in Embodiment 1.

[0102] Example 11

[0103] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that the rehydration time in step S3 when making the granular layer is 48 h, while the other steps are the same as in Embodiment 1.

[0104] Example 12

[0105] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that the thickness of the silicone frame is 0.1 mm when the granule layer is made in step S3. The remaining steps are the same as in Embodiment 1.

[0106] Example 13

[0107] This embodiment provides a sheet-like decellularized matrix, which differs from Embodiment 1 only in that the thickness of the silicone frame is 1 mm when the granule layer is made in step S3, while the remaining steps are the same as in Embodiment 1.

[0108] Example 14

[0109] This embodiment provides a composite sheet-like decellularized matrix (a combination of sheet-like decellularized matrix and microparticle decellularized matrix in contact), wherein the preparation method of the sheet-like decellularized matrix is ​​the same as in Embodiment 1; the preparation method of the microparticle decellularized matrix is ​​as follows:

[0110] S1: The submucosa of porcine small intestine was decellularized, virus-inactivated, defatted, washed, and freeze-dried to obtain decellularized porcine small intestinal submucosa.

[0111] S2: The decellularized submucosa of porcine small intestine was cut into small pieces and then ground at low temperature to obtain microparticles of the decellularized matrix. The microparticles were then sieved using sieves with specific pore sizes to obtain microparticles in the first, second, and third particle size ranges, as shown in Table 1. The microparticles in the first, second, and third particle size ranges were mixed in a mass ratio of 0.2:1:1.3 to obtain the microparticle decellularized matrix.

[0112] Table 1

[0113]

[0114] S3: When using, cover the sheet-like decellularized matrix on top of the microparticle decellularized matrix.

[0115] Example 15

[0116] This embodiment provides a composite sheet-like decellularized matrix (a physical combination of sheet-like decellularized matrix and microparticle decellularized matrix), wherein the preparation method of the sheet-like decellularized matrix is ​​the same as in Example 1; the preparation method of the microparticle decellularized matrix is ​​as follows:

[0117] S1: The submucosa of porcine small intestine was decellularized, virus-inactivated, defatted, washed, and freeze-dried to obtain decellularized porcine small intestinal submucosa.

[0118] S2: The decellularized submucosa of porcine small intestine was cut into small pieces and then ground at low temperature to obtain microparticles of decellularized matrix. The microparticles were then sieved using sieves with specific pore sizes to obtain microparticles in the first, second, and third particle size ranges, as shown in Table 2. The microparticles in the first, second, and third particle size ranges were mixed in a mass ratio of 0.4:1:1.3 to obtain the microparticle decellularized matrix.

[0119] Table 2

[0120]

[0121] S3: Take the microparticle decellularized matrix from S2, add an appropriate amount of purified water to make a paste, spread it evenly on one side of the sheet-like decellularized matrix, freeze-dry, and obtain a physical combination of microparticle decellularized matrix and sheet-like decellularized matrix. Seal and package, then sterilize by irradiation.

[0122] Example 16

[0123] This embodiment provides a composite sheet-like decellularized matrix (a chemical combination of sheet-like decellularized matrix and microparticle decellularized matrix). The physical combination of microparticle decellularized matrix and sheet-like decellularized matrix prepared in Example 15 was incubated for 24 h in 50 mL of EDC / NHS solution containing 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 25 mM N-hydroxysuccinimide. Subsequently, the cross-linking agent was removed by washing with purified water, and the mixture was freeze-dried to obtain the chemical combination of microparticle decellularized matrix and sheet-like decellularized matrix. The mixture was then sealed in a package and sterilized by irradiation.

[0124] Comparative Example 1

[0125] This comparative example provides a sheet-like decellularized matrix consisting only of thin lamellar layers, which differs from the sheet-like decellularized matrix described in Example 1 in that it lacks a granular layer. The preparation method includes the following steps:

[0126] S1: The submucosa of the small intestine of pigs is decellularized, defatted, inactivated by virus, washed, and freeze-dried to obtain the decellularized submucosa of the small intestine.

[0127] S2: Take the decellularized porcine small intestinal submucosa obtained in S1, moisten it, and stack 10 layers, ensuring that the layers adhere tightly and that no air bubbles appear. The 10 layers of porcine small intestinal submucosa are sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in sequence to obtain thin sheet layers. The thin sheet layers are then cut, perforated, sealed, packaged, and sterilized by irradiation to obtain sheet-like decellularized matrix consisting only of thin sheet layers.

[0128] Comparative Example 2

[0129] This comparative example provides a sheet-like decellularized matrix with only a granular layer, which differs from the sheet-like decellularized matrix described in Example 1 in that it does not possess a thin sheet layer. The preparation method includes the following steps:

[0130] S1: The porcine bladder basement membrane was decellularized, defatted, inactivated by virus, washed, and freeze-dried to obtain a decellularized porcine bladder basement membrane.

[0131] S2: The decellularized porcine bladder basement membrane obtained in S1 was subjected to low-temperature grinding to obtain decellularized porcine bladder basement membrane particles. Standard sieves with mesh sizes of 100 μm and 250 μm were used to sieve the particles. A 250 μm standard sieve was stacked on top of a 100 μm standard sieve. The particles were poured into the 250 μm standard sieve frame, and the frame was shaken and tapped to obtain particles of 100 μm-250 μm. The particles were rehydrated with purified water at a ratio of 1 g:20 mL, and mixed evenly for 24 h to obtain a paste-like particle layer. A 0.5 mm thick silica gel frame was placed flat on a stainless steel plate, and the obtained paste-like particle layer was filled into the silica gel frame and smoothed. The mixture was then freeze-dried to obtain a particle layer. The product was then sealed in packaging and sterilized by irradiation.

[0132] Comparative Example 3

[0133] This comparative example provides a sheet-like decellularized matrix, the preparation method of which includes the following steps:

[0134] S1: The submucosa of the small intestine and the basement membrane of the bladder of the pig were decellularized, defatted, inactivated by virus, washed, and freeze-dried to obtain the decellularized submucosa of the small intestine and the decellularized basement membrane of the bladder.

[0135] S2: After wetting the submucosa of a single layer of pig small intestine, vacuum laminate and freeze-dry to obtain a thin sheet. The thin sheet is then cut and perforated for later use.

[0136] S3: The decellularized porcine bladder basement membrane obtained in S1 was subjected to low-temperature grinding and pulverization to obtain decellularized porcine bladder basement membrane particles. Standard sieves with mesh sizes of 100 μm and 250 μm were used to sieve the particles. The 250 μm standard sieve was stacked on top of the 100 μm standard sieve. The particles were poured into the 250 μm standard sieve frame, and the frame was shaken and tapped to obtain particles of 100 μm-250 μm. The decellularized porcine bladder basement membrane particles were rehydrated with purified water at a ratio of 1 g:20 mL. The mixture was thoroughly mixed (rehydrated) for 24 h to obtain a paste-like particle layer. The sheet layer obtained in S2 was moistened with purified water. A 0.5 mm thick silica gel frame was placed flat on the sheet layer. The obtained paste-like particle layer was filled into the silica gel frame and smoothed, ensuring the particle layer filled the pores of the sheet layer. The mixture was then freeze-dried to obtain a sheet-like decellularized matrix composed of a particle layer and a sheet layer. Sealed packaging, sterilized by irradiation.

[0137] Comparative Example 4

[0138] This comparative example provides a sheet-like decellularized matrix, which differs from Example 1 only in that, in step S2, the decellularized porcine small intestinal submucosa is soaked and then stacked in 25 layers, that is, the sheet layer is made of 25 layers of decellularized porcine small intestinal submucosa. The remaining steps are consistent with Example 1.

[0139] Comparative Example 5

[0140] This comparative example provides a sheet-like decellularized matrix, which differs from Example 1 only in that the sieve used to make the particle layer in step S3 has a pore size of 500 μm and 600 μm, resulting in particles of 500 μm-600 μm. The remaining steps are consistent with Example 1.

[0141] Comparative Example 6

[0142] This comparative example provides a sheet-like decellularized matrix, which differs from Example 1 only in that the ratio of particles to purified water is 1 g: 10 mL when preparing the particle layer in step S3. The remaining steps are the same as in Example 1.

[0143] Comparative Example 7

[0144] This comparative example provides a sheet-like decellularized matrix, which differs from Example 1 only in that the ratio of particles to purified water in step S3 when preparing the particle layer is 1 g: 35 mL, while the remaining steps are the same as in Example 1.

[0145] Comparative Example 8

[0146] This comparative example provides a sheet-like decellularized matrix, which differs from Example 1 only in that the thickness of the silicone frame is 3 mm when the granule layer is made in step S3, while the other steps are the same as in Example 1.

[0147] Comparative Example 9

[0148] This comparative example provides a sheet-like decellularized matrix, which differs from Example 1 only in that the sieve used to make the particle layer in step S3 has a pore size of 5 μm, resulting in particles smaller than 5 μm. The remaining steps are consistent with Example 1.

[0149] Test Example 1

[0150] In this test, sheet-like decellularized matrix obtained from Examples 1-13 and Comparative Examples 1-9 were used to test their tensile strength.

[0151] Each sheet of decellularized matrix was prepared into a long strip specimen (length 150 mm ± 1 mm, width 10 mm ± 0.5 mm). The specimen was immersed in 0.9% physiological saline at 37℃ ± 1℃ for 3 min, and then excess water was absorbed from the specimen surface. The tensile strength and elongation at break of the specimen were determined using a fabric tensile testing machine according to GB / T3923.1-2013 "Textiles - Tensile Properties of Fabrics - Part 1: Determination of Breaking Strength and Elongation at Break (Strip Method)". The clamp spacing was 75 mm ± 1 mm, and the tensile speed was 100 mm / min. The tensile strength and elongation at break of the specimen were obtained, and the results are shown in Table 3.

[0152] Table 3

[0153]

[0154] Experimental results show that the tensile strength and elongation at break of the sheet-like decellularized matrix are mainly affected by the number of decellularized matrix layers in the sheet; the more decellularized matrix layers, the greater the tensile strength and elongation at break. The granular layer also has a certain influence on the tensile strength and elongation at break of the sheet-like decellularized matrix. Specifically, the tensile strength and elongation at break increase with the increase of the granular layer thickness, the ratio of particle mass to water volume during rehydration, and the rehydration time. This is because as the granular layer thickness, the ratio of particle mass to water volume during rehydration, and the rehydration time increase, the contact between particles increases, forming more hydrogen bonds, thereby improving the relevant mechanical properties. The particle size in the granular layer has almost no effect on the mechanical properties of the sheet-like decellularized matrix.

[0155] Test Example 2

[0156] In this test, sheet-like decellularized matrix obtained from Examples 1-13 and Comparative Examples 1-9 were used to test their bending stiffness.

[0157] Prepare elongated specimens from each sheet of decellularized matrix (length 250 mm ± 1 mm, width 25 mm ± 1 mm). Immerse the specimens in 0.9% physiological saline at 37℃ ± 1℃ for 3 min, then remove and absorb excess water from the specimen surface. Test the bending stiffness of the specimens according to the method specified in YY / T 0472.2. The bending stiffness determines the flexibility of the specimens; the greater the bending stiffness, the worse the flexibility. Record the bending length of the specimens and calculate the bending stiffness using the following formula:

[0158] G = g × c 3 ×10 -3 .

[0159] Wherein, G: bending stiffness per unit width, in mN·cm;

[0160] Where g: mass per unit area, in g / cm³ 2 ;

[0161] Where c: total average bending length, in cm.

[0162] The results are shown in Table 4.

[0163] Table 4

[0164]

[0165] Experimental results show that the softness of sheet-like decellularized matrix is ​​mainly affected by the thickness of the granular layer, and secondly by the number of decellularized matrix layers in the sheet layer. The thicker the granular layer and the more decellularized matrix layers there are, the greater the bending stiffness and the worse the softness. In addition, the particle size of the granular layer and the ratio of particle mass to water volume when the particles are rehydrated also affect the bending stiffness of the sheet-like decellularized matrix material. Larger particle size and a larger ratio of particle mass to water volume result in greater bending stiffness of the sheet-like decellularized matrix material. Softness mainly affects the comfort during use. When there are too many decellularized layers (Comparative Example 4) or the granular layer is too thick (Comparative Example 8), the average bending stiffness of the sheet-like decellularized matrix is ​​above 1.5, indicating poor softness. The sheet-like decellularized matrix material has poor conformability and fit to the human body, which can easily cause discomfort. Furthermore, the sheet layer and granular layer are also prone to displacement.

[0166] Test Example 3

[0167] In this test, the sheet-like decellularized matrix obtained in Examples 1-13 and Comparative Examples 1-9 were used to test their water absorption capacity.

[0168] Each sheet of decellularized matrix was soaked in excess physiological saline at 37°C for 72 h. The mass w0 and w1 of the samples before and after soaking were recorded respectively. The saturated water absorption rate was calculated according to the following formula: Saturated water absorption rate = (w1-w0) / w0×100%.

[0169] The results are shown in Table 5.

[0170] Table 5

[0171]

[0172] Experimental results show that the water absorption capacity of the sheet-like decellularized matrix is ​​mainly related to the properties of the granular layer. Among them, the thickness of the granular layer, the particle size, and the ratio of particle mass to water volume during rehydration have a greater impact. The granular layer thickness and the ratio of particle mass to water volume are positively correlated, while the particle size is negatively correlated.

[0173] Test Example 4

[0174] In this test, the sheet-like decellularized matrix obtained in Examples 1-13 and Comparative Examples 1-9 were used to detect their water loss time.

[0175] The decellularized matrix sheet was soaked in excess physiological saline at 37°C for 72 h. The surface moisture was gently wiped off with absorbent paper, and the sample was weighed and its initial mass (w0) was recorded. The sample was then spread out in a culture dish, with the granular side facing down. Three layers of oily gauze were placed over the sample, with the edges of the gauze extending 5-10 mm beyond the sample below. The gauze was secured with tape and placed in an indoor environment. At specific times, the gauze was removed, the sample was weighed, and its weight (wt) at time t was recorded. This process continued until the difference between two consecutive weighings did not exceed 0.1 g. The time of the last weighing was recorded and considered the time when the decellularized matrix sheet material had completely lost water. The results are shown in Table 6.

[0176] Table 6

[0177]

[0178] Experimental results show that the complete dehydration time of the sheet-like decellularized matrix is ​​mainly related to the number of decellularized matrix layers in the sheet, the thickness of the particle layer, and the ratio of particle mass to water volume when the particles are rehydrated. The more layers, the thicker the layer, and the greater the ratio of particle mass to water volume, the longer the complete dehydration time.

[0179] Test Example 5

[0180] In this test case, the sheet-like decellularized matrix obtained in Examples 1-13 and Comparative Examples 1-9 were used for subcutaneous implantation experiments in rats to detect their degradation ability.

[0181] A skin incision approximately 1 cm long was made at each implantation site on both sides of the rat spine, with a distance of at least 1 cm between each site on one side. Subcutaneous tissue was bluntly dissected down to the superficial fascia and muscle layer, with 3 subcutaneous bursae created on each side of each rat. Each sheet of decellularized matrix sample was immersed in 0.9% physiological saline at 37℃±1℃ for 3 min before implantation. The incisions were closed and the subcutaneous tissue and skin were sutured in layers. The wounds were disinfected with povidone-iodine. On days 5, 7, and 14, the rats were sacrificed, the skin was incised along the midline of the spine, and the subcutaneous tissue was carefully dissected. The experimental material and surrounding tissue were harvested. After sectioning and fixation, HE staining was performed. The implanted sample and its granular and lamellar layers were identified from the HE-stained photographs. The thickness of the granular and lamellar layers was measured, and the residual rate of the sample at each time point was calculated as: residual rate = thickness of granular or lamellar layer at time t / thickness of granular or lamellar layer before implantation × 100%. The results are shown in Tables 7 (granular layer) and 8 (lamellar layer).

[0182] Table 7

[0183]

[0184] Table 8

[0185]

[0186] Experimental results showed that the degradation rate of granular layers was significantly faster than that of lamellar layers. All granular layers were essentially completely degraded within 14 days, while a single layer of decellularized matrix could be completely degraded within 7 days. The degradation time for two or more lamellar layers exceeded 14 days, with the degradation time increasing significantly with the number of layers. The thickness of the granular layer, the particle size, and the ratio of particle mass to water volume during rehydration significantly affected the degradation time, while rehydration time had a relatively smaller impact. Larger particle sizes, thicker granular layers, and higher particle mass to water volume ratios resulted in longer degradation times. Specifically, when the particle size exceeded 500 μm, the granular layer thickness exceeded 3 mm, or the particle mass to water volume ratio was greater than 1 g:15 mL, the degradation time exceeded 14 days, and the longer the rehydration time, the slower the degradation rate.

[0187] Test Example 6

[0188] Burn model wound healing effect test:

[0189] (1) Construction of deep second-degree burn model:

[0190] To create a model of deep second-degree burns in Bama pigs, each defect is a circle with a diameter of 1 cm. The specific method is as follows:

[0191] A deep second-degree burn wound was created on the back of a Bama pig weighing 23-28 kg. An electronic burn instrument was heated to 100°C and a continuous pressure of 1 kg was applied perpendicularly to the pig's back for 30 seconds. Histopathological examination showed necrosis of the entire epidermis, partial necrosis of the dermis, and areas of leukocyte infiltration. The skin appendages in the deep dermis were still alive, achieving a deep second-degree burn.

[0192] (2) Wound treatment:

[0193] (2.1) After wound debridement, the experimental group was treated with sheet-like decellularized matrix or composite sheet-like decellularized matrix provided in Examples 1-16 and Comparative Examples 1-9, respectively.

[0194] (2.1.1) When using sheet-like decellularized matrix alone, soak the sheet-like decellularized matrix in 0.9% physiological saline at 37℃±1℃ for 3 min, while cleaning the wound. Cover the wound with the sheet-like decellularized matrix, with the granular layer facing the wound, and then cover and fix it with Vaseline gauze.

[0195] (2.1.2) When using a composite sheet-like decellularized matrix (a physical or chemical combination of sheet-like decellularized matrix and microparticle decellularized matrix) for treatment, first clean the wound, then hydrate the composite sheet-like decellularized matrix, and then cover it on the wound. Make sure that the microparticle decellularized matrix is ​​in direct contact with the wound and the granular layer of the sheet-like decellularized matrix is ​​in direct contact with the microparticle decellularized matrix. Finally, cover and fix the top with Vaseline gauze.

[0196] (2.1.3) When using composite sheet decellularized matrix (a combination of sheet decellularized matrix and microparticle decellularized matrix) for treatment, first clean the wound, hydrate the sheet decellularized matrix, sprinkle the microparticle decellularized matrix evenly on the wound, and finally cover and fix the wound with Vaseline gauze.

[0197] (2.2) After cleaning the wound, the white group was covered with only Vaseline gauze.

[0198] (3) Calculation of wound healing rate:

[0199] The wound was observed and its area was measured weekly. The wound healing rate was calculated as follows: Wound healing rate = (Initial wound area - Wound area at time t) / Initial wound area × 100%. The results are shown in Table 9.

[0200] Table 9

[0201]

[0202] Experimental results show that the therapeutic effect of sheet-like decellularized matrix is ​​significantly better than that of thin sheet layers or granular layers alone.

[0203] A comparison of the data from Example 1 and Comparative Example 3 shows that when the sheet layer has only one layer of decellularized matrix, both the granular layer and the sheet layer degrade relatively quickly, resulting in a lack of a suitable microenvironment for healing in the later stages of healing. Therefore, the healing speed is significantly lower than that of the sheet layer with multiple layers of decellularized matrix. A comparison of the data from Example 1 and Comparative Example 4 shows that when the number of decellularized matrix layers in the sheet layer exceeds 20, a large amount of undegraded decellularized matrix remains in the sheet layer after wound healing. Furthermore, due to its poor flexibility, premature separation of the sheet layer from the granular layer and displacement of the sheet layer have occurred during treatment. However, data from Example 1 and Examples 4-5 show that a decellularized matrix with 2-10 layers can adhere closely to the wound.

[0204] A comparison of the data from Example 1 and Comparative Example 5 shows that when the maximum particle size of the granular layer exceeds 500 μm, its degradation time exceeds 14 days. Compared with the faster-degrading microparticles, its effect in the early stage of wound healing is weakened, and therefore its healing-promoting effect is also slightly weaker than that of sheet-like decellularized matrix where the above factors are within an appropriate range.

[0205] A comparison of data from Examples 1, 8-9, and Comparative Examples 6-7 shows that sheet-like decellularized matrix with a particle mass-to-water volume ratio of less than 1 g:30 mL dries out approximately 3 days after application. However, sheet-like decellularized matrix with a particle mass-to-water volume ratio greater than 1 g:30 mL and at least two decellularized matrix layers can remain moist for about two weeks (longer than the complete water loss time observed in in vitro experiments, possibly due to the degradation of the lower particle layer into a gel-like substance with stronger water retention). Prematurely dried sheet-like decellularized matrix materials cannot form effective contact with the wound, preventing the release of active ingredients and thus weakening the healing-promoting effect. When the particle mass-to-water volume ratio is outside the range of 1 g:(15~30) mL, the overall healing effect is weakened.

[0206] A comparison of the data from Example 1 and Comparative Example 8 shows that sheet-like decellularized matrix with a granular layer thickness of more than 1 mm also exhibits displacement, which is expected to be related to poor softness. This displacement is not conducive to wound healing.

[0207] A comparison of the data from Example 1 and Comparative Example 9 shows that when the particle size of the particle layer is less than 5 μm, the degradation rate is too fast and it cannot provide a lasting effect. Moreover, since the particle size is close to the nanometer scale, it may cause additional inflammatory reactions.

[0208] Furthermore, a comparison of the data from Examples 1 and 14-16 shows that when sheet-like decellularized matrix and microparticle decellularized matrix are used in combination, the healing speed is further improved, and the synergistic effect of the two can better promote wound healing.

[0209] In summary, this invention provides a sheet-like decellularized matrix. The thin sheet layer provides excellent barrier protection for the wound, while the granular layer degrades rapidly, becoming almost completely degraded in about two weeks. It releases active substances in the early healing stage, actively promoting wound healing. A faster degradation rate is more conducive to promoting early healing, while a slower degradation rate is more conducive to promoting mid-to-late-stage healing. The thin sheet layer degrades more slowly than the granular layer, providing excellent barrier protection in the early healing stage. Significant degradation occurs after two weeks, continuing to provide active substances and promote wound healing. The slower degradation rate is beneficial for providing a longer-lasting barrier protection, thus promoting wound healing. Secondly, the particle morphology of the granular layer in the sheet-like decellularized matrix significantly increases the specific surface area of ​​the material, significantly improving water absorption and extending the duration of the moist environment. Furthermore, the preparation process of the sheet-like decellularized matrix does not introduce any chemical additives, improving the biocompatibility of the material.

[0210] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A sheet-like decellularized matrix, characterized in that, The sheet-like decellularized matrix comprises a sheet layer and a granular layer made of decellularized animal membrane extracellular matrix. The granular layer is located on one side of the sheet layer. The sheet layer and the granular layer are bonded together by physical interaction formed after freeze-drying. The sheet layer is composed of 2-20 layers of decellularized animal membrane extracellular matrix stacked together. The granular layer is prepared from particles with a particle size of 5-500 μm made by pulverizing decellularized animal membrane extracellular matrix. The thickness of the granular layer is 0.1-1 mm.

2. A method for preparing the sheet-like decellularized matrix as described in claim 1, characterized in that, The preparation method includes the following steps: (1) The animal membrane was decellularized, virus-inactivated, defatted, washed and freeze-dried to obtain decellularized animal membrane extracellular matrix; (2) After wetting the extracellular matrix of decellularized animal membranes, multiple layers are stacked, and then vacuum lamination and freeze-drying are performed to obtain thin sheets; The dried decellularized animal membrane extracellular matrix was cut into small pieces and then ground to obtain decellularized animal membrane extracellular matrix particles. The decellularized animal membrane extracellular matrix particles were sieved using a sieve. The decellularized animal membrane extracellular matrix particles that passed through the sieve were mixed with water at a mass-volume ratio of 1 g:(15-30) mL and rehydrated to obtain a paste-like granular layer. (3) Wet the sheet layer obtained in step (2) with water, place a frame mold on the wet sheet layer, fill the mold with the paste-like granule layer obtained in step (2) and smooth it, freeze dry, and the granule layer and the sheet layer are combined to obtain sheet-like decellularized matrix.

3. The method for preparing the sheet-like decellularized matrix according to claim 2, characterized in that, The animal membrane mentioned in step (1) is derived from mammals.

4. The method for preparing the sheet-like decellularized matrix according to claim 2, characterized in that, The rehydration treatment in step (2) takes 4-48 hours.

5. The method for preparing the sheet-like decellularized matrix according to claim 2, characterized in that, The thickness of the frame mold in step (3) is 0.1-1 mm.

6. A composite sheet-like decellularized matrix, characterized in that, The composite sheet-like decellularized matrix includes the sheet-like decellularized matrix and the microparticle decellularized matrix as described in claim 1; The combination of sheet-like decellularized matrix and particulate decellularized matrix can be achieved through contact combination, physical combination, or chemical combination.

7. The composite sheet-like decellularized matrix according to claim 6, characterized in that, The decellularized microparticle matrix is ​​composed of microparticles in a first size range, a second size range, and a third size range in a mass ratio of (0.2-0.6):1:(1-1.5). The particle size of the particles in the first particle size range is 5-50 μm, the particle size of the particles in the second particle size range is 50-150 μm, and the particle size of the particles in the third particle size range is 150-300 μm. The particle size range between the first particle size range particles, the second particle size range particles and the third particle size range particles is greater than 50 μm.

8. The composite sheet-like decellularized matrix according to claim 6, characterized in that, The decellularized microparticle matrix was prepared using a method comprising the following steps: (1) Animal membrane tissue was decellularized, virus-inactivated, defatted, washed and freeze-dried to obtain decellularized animal membrane extracellular matrix material; (2) After cutting the decellularized animal membrane extracellular matrix material into small pieces, grind and pulverize it to obtain microparticle decellularized matrix. The microparticle decellularized matrix is ​​sieved using a sieve to obtain microparticles in the first, second and third particle size ranges. The obtained microparticles in the first, second and third particle size ranges are mixed to obtain microparticle decellularized matrix.

9. The composite sheet-like decellularized matrix according to claim 6, characterized in that, The contact combination method is as follows: the microparticle decellularized matrix and the sheet-like decellularized matrix are mixed at a concentration of 1.5-15 mg / cm³. 2 Proportional contact; And / or, the physical combination is performed as follows: the microparticle decellularized matrix is ​​made into a paste with water, coated onto the sheet-like decellularized matrix, and freeze-dried to obtain a physical combination of the microparticle decellularized matrix and the sheet-like decellularized matrix; the dry weight ratio of the microparticle decellularized matrix to the area ratio of the sheet-like decellularized matrix is ​​1.5-15 mg / cm². 2 ; And / or, the chemical combination is carried out by incubating the physical combination of microparticle decellularized matrix and sheet-like decellularized matrix in an aqueous solution of a crosslinking agent, washing off the crosslinking agent, and freeze-drying to obtain a chemical combination of microparticle decellularized matrix and sheet-like decellularized matrix.

10. The use of the sheet-like decellularized matrix as described in claim 1 or the composite sheet-like decellularized matrix as described in any one of claims 6-9 in the preparation of medical biomaterials that promote wound healing.

Images