Particle acellular matrix as well as preparation method and application thereof

Abstract

The invention relates to a particle acellular matrix and a preparation method and application thereof.The particle acellular matrix comprises acellular matrix particles with gradient particle sizes and can be used for promoting skin wound healing, and the particle acellular matrix makes full contact with a complex wound surface and is degraded in a staged mode; a favorable microenvironment can be continuously provided for migration, growth, proliferation and differentiation of cells, so that wound healing is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical biomaterials, in particular to a microparticle acellular matrix and a preparation method and application thereof. BACKGROUND

[0002] Human skin is composed of three layers: epidermis, dermis, and subcutaneous tissue. Skin is prone to injury. Superficial wounds can repair themselves, but when the damage involves the dermis, medical dressings are usually needed to help wound healing, which is often accompanied by scar hyperplasia after healing. Medical dressings can accelerate wound healing by creating a microenvironment conducive to tissue repair and regeneration, but traditional dressings cannot solve the problem of scar hyperplasia after healing.

[0003] Biological dressings using animal-derived acellular matrix materials have received extensive attention in burn treatment. Acellular matrix materials are natural biomaterials that contain a large number of bioactive macromolecules and peptides, have excellent physical structure and complex biomolecules, and can promote wound healing and skin regeneration. Acellular biological materials derived from human and other animal tissues (such as dermis, pericardium, peritoneum, and amniotic membrane) can be used to promote skin wound healing.

[0004] Acellular dermal matrix (ADM) can be divided into allogeneic ADM (human origin) and xenogeneic ADM (animal origin) according to the species source of the dermis. Allogeneic ADM is made from donated homologous human dermis. A meta-analysis showed that it significantly promotes the healing of wounds in diabetic patients and reduces complications. Xenogeneic ADM is mainly derived from pigs and cows, and its structure is similar to that of allogeneic ADM. Compared with allogeneic ADM, xenogeneic ADM is more cost-effective. For wound repair, bioactive acellular dermal matrix (ADM) containing growth factors (GFs) can provide attachment points for epidermal cells, promote the restructuring of the basement membrane, and due to its unique pore structure, can promote the migration of endothelial cells and fibroblasts, thereby inducing tissue growth and achieving local reconstruction. In addition, the ADM matrix components are continuously degraded by collagenase and eventually replaced by newly formed extracellular matrix (ECM) and migrating cells. Studies have evaluated the long-term clinical effects of acellular dermal matrix products in burn treatment and scar repair after burns. After years of follow-up, it was found that acellular dermal matrix materials can accelerate wound healing, and compared with the conventional treatment group, the color, texture, thickness, aesthetic changes of healed skin, and limb function were significantly improved. Acellular dermal matrix dressing has 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 the differences in the molecular structure of the major histocompatibility complex particles lead to severe inflammatory reactions after xenogeneic ADM transplantation. 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 acellular biomembranes are also used for the treatment of skin wounds, such as small intestinal submucosa, bladder basement membrane, amniotic membrane, peritoneum, etc. Such acellular 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 to the activation of cell adhesion signaling pathway and the formation of blood vessel morphology. In addition, compared with dermal matrix, thin film materials are easier to remove immunogenic substances and reduce inflammatory reactions. For example, CN1986001A discloses a biological wound care membrane. After the small intestinal submucosa is subjected to defatting, cross-linking fixation and antigen removal treatment, an active modification layer containing fibronectin, laminin or vitronectin is added on the surface thereof by adsorption, adhesion and other methods; or a broad-spectrum antibacterial drug is mixed with a biological adhesive to form an antibacterial slow-release layer on the surface of the small intestinal submucosa.

[0006] The existing acellular matrix for wound treatment has the following problems:

[0007] (1) The sheet-shaped acellular matrix may not be fully in contact with the wound when used on a complex wound, thereby affecting the healing effect;

[0008] (2) The degradation rate of the sheet-shaped acellular matrix is often significantly slower than the healing rate of most wounds. In the early stage of wound healing, the active ingredients in the acellular matrix that are beneficial to promoting wound healing cannot be effectively released, to some extent hindering the play of the promoting healing function of such biological materials;

[0009] (3) The particle size of the marketed particulate product is usually 500-1000 μm, and the particle size distribution is random, so the reaction in the early stage of wound healing is relatively small, and the degradation in the body is difficult to accurately control, and the product cannot fully play a role in different stages of wound healing.

[0010] In view of the above, it has become one of the urgent problems in the field to provide a new type of particulate acellular matrix. SUMMARY

[0011] To solve the above technical problems, the present application provides a particulate acellular matrix and a preparation method and application thereof. The particulate acellular matrix is composed of acellular biomaterial microparticles with gradient particle size and can be used to promote skin wound healing. The particulate acellular matrix of the present application is in full contact with the complex wound, and microparticles with different particle size ranges can participate in the reaction with cells and tissues in different stages of wound healing, and can continuously provide a favorable microenvironment for cell migration, growth, proliferation and differentiation, thereby promoting wound healing.

[0012] In a first aspect, the present application provides a particulate acellular matrix, which comprises acellular matrix particles of three or more gradient particle sizes, the acellular matrix particles of the three or more gradient particle sizes comprising at least first particle size particles, second particle size particles and third particle size particles.

[0013] The first particle size range particles have a particle size range of 5-50 μm, the second particle size range particles have a particle size range of 50-150 μm, and the third particle size range particles have a particle size range of 150-300 μm.

[0014] The particle size span between the first particle size range particles, the second particle size range particles and the third particle size range particles is 50 μm or more; and the particle size span in each of the first particle size range particles, the second particle size range particles and the third particle size range particles is independently 50 μm or less.

[0015] The mass ratio of the first particle size range particles, the second particle size range particles and the third particle size range particles is (0.2-0.6):1:(1-1.5).

[0016] The first particle size range particles have an in vivo degradation time of 3-5 days, i.e. they are substantially completely degraded in the inflammatory phase of wound healing (1-3 days after injury) and release bioactive substances sufficiently; and the particle size range is in the range in which the particles can be directly phagocytosed by immune cells such as macrophages, and the phagocytosis of the particles can moderately enhance the secretion of various cytokines by the cells such as macrophages, which is conducive to the recruitment of cells.

[0017] The second particle size range particles have an in vivo degradation time of 6-9 days, i.e. they are substantially completely degraded in the early, middle and late stages of wound healing (4-14 days after injury), and are converted from a particulate form to a gel form during the degradation period, which is conducive to the migration of keratinocytes and fibroblasts, so that epithelialization not only starts from the edges of the wound but also starts from the middle part of the wound, reducing the tension of the wound edges and alleviating scars; and the particles sufficiently release bioactive substances and accelerate blood vessel formation and collagen deposition; and the particle size and the in vivo degradation time in this range show a positive correlation.

[0018] The third particle size range particles have an in vivo degradation time of 10-14 days, i.e. they are substantially completely degraded in the middle and late stages of the proliferation phase of wound healing, which is conducive to the sustained maintenance of an environment conducive to cell migration, blood vessel formation and collagen deposition in the middle and late stages of the proliferation phase, accelerating wound healing; and the particle size and the in vivo degradation time in this range show a positive correlation.

[0019] The span of particle size between the first particle size range microparticles, the second particle size range microparticles and the third particle size range microparticles is greater than 50 microns, and the span of particle size in each of the first particle size range microparticles, the second particle size range microparticles and the third particle size range microparticles is less than 50 microns, which is beneficial to the complete degradation of microparticles of different particle sizes at different times, avoids the similar particle size leading to the similar degradation time and unable to fully play a role in different stages.

[0020] The microparticle decellularized matrix exists in the form of microparticles, retains the microstructure and ultrastructure characteristics of the parent extracellular matrix, and at the same time, the surface area for interaction with host cells is greatly increased, which can fully contact the wound in different stages of wound healing and effectively release active ingredients beneficial to promoting wound healing.

[0021] The present application creatively finds that the first particle size range microparticles, the second particle size range microparticles and the third particle size range microparticles of specific particle size are combined in a mass ratio of (0.2-0.6):1:(1-1.5), and each particle size microparticle degrades in stages, which can provide a favorable microenvironment for cell migration, growth, proliferation and differentiation, and can realize rapid healing of the wound.

[0022] Preferably, the microparticle decellularized matrix comprises 3-4 gradient particle size decellularized matrix microparticles.

[0023] Preferably, the gradient particle size decellularized matrix microparticles comprise first particle size range microparticles, second particle size range microparticles, third particle size range microparticles and fourth particle size range microparticles, and the particle size range of the fourth particle size range microparticles is 300-500 microns.

[0024] The mass ratio of the first particle size range microparticles, the second particle size range microparticles, the third particle size range microparticles and the fourth particle size range microparticles is (0.2-0.6):1:(1-1.5):(0.5-1.5).

[0025] The in-vivo degradation time of the fourth particle size range microparticles is 15-18 days, that is, it is basically completely degraded in the middle and late stages of the wound healing proliferation period of a deeper wound, which is beneficial to continuously maintaining an environment conducive to cell migration, vascularization and collagen deposition for a longer period of time, and accelerating wound healing; the particle size of the microparticles in the range and the in-vivo degradation time show a positive correlation.

[0026] The microparticle acellular matrix is suitable for treating degree II burn and scald, especially deep degree II burn, and the combination of microparticles in the first, second and third particle size ranges is suitable for deep degree II burn with a shallow wound surface, which usually needs about three weeks to completely heal; the combination of microparticles in the first, second, third and fourth particle size ranges is suitable for deep degree II burn with a deep wound surface which needs a longer healing time. The microparticle acellular matrix provided by the application can reasonably select the appropriate gradient particle size of the acellular matrix microparticles for combination according to the degree of the wound surface.

[0027] Meanwhile, by controlling the microparticles in each particle size range in the above range, cells can be better recruited, immune responses can be adjusted, and the overall degradation rate is moderate, which is more conducive to subsequent wound healing.

[0028] The lower limit value of 5-50 μm can be any value less than 50 μm in the range of 5-50 μm, for example, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 45 μm, etc.; the upper limit value can be any value in the range of 5-50 μm and greater than the lower limit value, for example, 6 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, etc.

[0029] The lower limit value of 50-150 μm can be any value less than 150 μm in the range of 50-150 μm, for example, 50 μm, 70 μm, 90 μm, 110 μm, 130 μm, 140 μm, etc.; the upper limit value can be any value in the range of 50-150 μm and greater than the lower limit value, for example, 60 μm, 70 μm, 90 μm, 110 μm, 130 μm, 150 μm, etc.

[0030] The lower limit value of 150-300 μm can be any value less than 300 μm in the range of 150-300 μm, for example, 150 μm, 180 μm, 210 μm, 240 μm, 270 μm, 290 μm, etc.; the upper limit value can be any value in the range of 150-300 μm and greater than the lower limit value, for example, 160 μm, 180 μm, 210 μm, 240 μm, 270 μm, 300 μm, etc.

[0031] The lower limit value of 300-500 μm can be any value less than 500 μm in the range of 300-500 μm, for example, 300 μm, 350 μm, 400 μm, 450 μm, 490 μm, etc.; the upper limit value can be any value in the range of 300-500 μm and greater than or equal to the lower limit value, for example, 400 μm, 350 μm, 400 μm, 450 μm, 500 μm, etc.

[0032] The specific value in the above 50 μm or more can be 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 60 μm, etc.

[0033] The specific value in the above 50 μm or more can be 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 60 μm, etc.

[0034] The specific value in the above 0.2-0.6 can be 0.2, 0.3, 0.4, 0.5, 0.6, etc.

[0035] The specific value in the above 1-1.5 can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.

[0036] The specific value in the above 0.5-1.5 can be 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.

[0037] Preferably, the gradient particle size decellularized matrix microparticles further carry a drug, and the drug comprises a growth factor and / or an antibacterial drug.

[0038] Preferably, the growth factor comprises recombinant human epidermal growth factor and / or recombinant human fibroblast growth factor.

[0039] Preferably, the antibacterial drug comprises any one or a combination of at least two of amoxicillin, ampicillin, tetracycline, doxycycline, gentamicin, ciprofloxacin, moxifloxacin, chloramphenicol or linezolid.

[0040] In a second aspect, the present application provides a preparation method of the microparticle decellularized matrix as described in the first aspect, and the preparation method comprises the following steps:

[0041] (1) The animal membrane tissue is subjected to decellularization, virus inactivation, defatting, washing and freeze-drying treatment to prepare a decellularized animal membrane extracellular matrix material;

[0042] (2) The decellularized animal membrane extracellular matrix material is cut and ground to prepare decellularized matrix microparticles, the decellularized matrix microparticles are screened using a screen to obtain microparticles of different particle size ranges, and the microparticles of different particle size ranges are mixed to prepare the microparticle decellularized matrix.

[0043] Preferably, the source of the animal membrane in step (1) comprises a mammal.

[0044] Preferably, the mammal comprises any one of a pig, a cow, a sheep or a horse, etc.

[0045] Preferably, the mammal is a pig.

[0046] Preferably, the animal membrane comprises any one or a combination of at least two of small intestinal submucosa, bladder basement membrane, peritoneum, pleura or omentum.

[0047] Preferably, the temperature for the grinding and crushing in step (2) is -196℃ to -10℃, for example, -196℃, -150℃, -100℃, -50℃, -10℃, etc.

[0048] Optionally, the decellularized matrix microparticles in step (2) are further loaded with a drug.

[0049] The drug is loaded by mixing and soaking the decellularized matrix microparticles with an aqueous solution containing the drug to obtain decellularized matrix microparticles loaded with the drug.

[0050] In a third aspect, the present application provides a composite decellularized matrix comprising the microparticle decellularized matrix of the first aspect and a sheet decellularized matrix.

[0051] The sheet decellularized matrix has a sheet layer structure or a sheet layer-particle layer composite structure.

[0052] The combination of the microparticle decellularized matrix and the sheet decellularized matrix comprises any one or a combination of at least two of contact combination, physical combination or chemical combination.

[0053] The dry weight of the microparticle decellularized matrix to the area of the sheet decellularized matrix is 1.5-15 mg / cm 2 .

[0054] In the present application, the sheet decellularized matrix can provide barrier protection and biological activity, and is used simultaneously with the microparticle decellularized matrix, which has a significant synergistic effect in promoting wound healing.

[0055] Preferably, the sheet decellularized matrix comprises a sheet layer formed by vacuum lamination of multiple layers of decellularized animal membrane extracellular matrix material.

[0056] The sheet layer is composed of 2-20 layers of decellularized animal membrane extracellular matrix material.

[0057] Preferably, the sheet decellularized matrix further comprises a particle layer made of crushed decellularized animal membrane extracellular matrix material.

[0058] The sheet layer and the particle layer are combined together by physical interaction formed after freeze-drying.

[0059] The particle layer is composed of decellularized animal membrane extracellular matrix particles with a particle size of 5-500 μm.

[0060] The thickness of the particle layer is 0.1-3 mm, and the porosity is 30%-90%.

[0061] In the 2-20 layers, the specific point values can be selected from 2 layers, 5 layers, 8 layers, 11 layers, 14 layers, 17 layers, 20 layers, etc., in the 5-500 μm, the specific point values can be selected from 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, 25 μm, 5 μm, etc., in the 0.1-3 mm, the specific point values can be selected from 0.1 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, etc., in the 30%-90%, the specific point values can be selected from 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc.

[0062] Preferably, the contact combination is achieved by covering the particulate decellularized matrix with the same area of the sheet-shaped decellularized matrix.

[0063] Preferably, the physical combination is achieved by making the particulate decellularized matrix into a paste with water, applying it on the sheet-shaped decellularized matrix, and freeze-drying to obtain a physical combination of the particulate and sheet-shaped decellularized matrix.

[0064] Preferably, the chemical combination is achieved by incubating the physical combination of the particulate and sheet-shaped decellularized matrix in an aqueous solution of a crosslinking agent, washing away the crosslinking agent, and freeze-drying to obtain a chemical combination of the particulate and sheet-shaped decellularized matrix.

[0065] Preferably, the crosslinking agent comprises glutaraldehyde and / or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS).

[0066] The dry weight of the particulate decellularized matrix to the area of the sheet-shaped decellularized matrix is 1.5-15 mg / cm 2 , 1.5 mg / cm 2 , 1.6 mg / cm 2 , 1.7 mg / cm 2 , 1.8 mg / cm 2 , 1.9 mg / cm 2 , 2.0 mg / cm 2 , 3.0 mg / cm 2 , 4.0 mg / cm 2 , 5.0 mg / cm 2 , 6.0 mg / cm 2 , 7.0 mg / cm 2 , 8.0 mg / cm 29.0 mg / cm 2 10.0 mg / cm 2 11.0 mg / cm 2 12.0 mg / cm 2 13.0 mg / cm 2 14.0 mg / cm 2 15.0 mg / cm 2 and the like. By controlling the ratio of the dry weight of the microparticle acellular matrix to the area of the sheet acellular matrix in the above range, the present application can ensure that the composite acellular matrix covers the wound surface well, while controlling the degradation time of the composite acellular matrix in a reasonable range, thereby improving the wound healing effect in many aspects.

[0067] In a fourth aspect, the present application provides the use of the microparticle acellular matrix of the first aspect or the composite acellular matrix of the third aspect in the preparation of a medical biological material for promoting wound healing.

[0068] Compared with the prior art, the present application has the following beneficial effects:

[0069] (1) The microparticle acellular matrix can fully contact with complex wounds, retains the natural extracellular matrix structure and composition, and can provide a favorable microenvironment for cell migration, growth, proliferation and differentiation.

[0070] (2) By adjusting the proportion of microparticles of different particle sizes in the microparticle acellular matrix, the reaction of the microparticles with cells and tissues in the body, including degradation and absorption, can be adjusted. The first particle size range of microparticles is smaller, and phagocytosis and degradation can occur during the inflammatory period of wound healing. The second, third and fourth particle size ranges of microparticles degrade during the proliferation period of wound healing, and the degradation time is equivalent to the period of re-epithelialization of the wound. At the same time, the product dosage can be saved, and the use cost can be reduced.

[0071] (3) The microparticle acellular matrix of the present application has a simple preparation method, does not require complex microparticle processing steps, only needs to control the particle size range of the particle size, and can efficiently promote wound healing by reasonable combination.

[0072] (4) Further, the present application combines the microparticle acellular matrix with the sheet acellular matrix, which can provide high biological activity and barrier protection, and the two have a synergistic effect, and the healing-promoting effect is better than that of each alone. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 To test the healing of each group in the Bama pig deep II degree partial superficial burn model in Example 1. DETAILED DESCRIPTION

[0074] To further clarify the technical means adopted by the present application and its effects, the present application is further described below in conjunction with examples and drawings. It can be understood that the specific implementation described herein is only used to explain the present application, but not to limit the present application.

[0075] Unless otherwise specified in the examples, the techniques or conditions are in accordance with those described in the literature in the art, or in accordance with the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0076] Glossary:

[0077] Decellularized animal membrane extracellular matrix: refers to a dry membrane-shaped extracellular matrix material obtained after decellularization treatment of a thin animal membrane from a mammal.

[0078] Decellularized animal membrane extracellular matrix microparticles: refers to a dry microparticle material obtained by low-temperature grinding and crushing of a decellularized animal membrane extracellular matrix.

[0079] Low-temperature grinding: refers to grinding at -196 to -10°C.

[0080] The steps of decellularization, virus inactivation, defatting, washing, and freeze-drying involved in the following implementation modes are as follows:

[0081] (1) Mechanical peeling of animal membrane tissue, such as pig small intestinal submucosa and pig bladder basement membrane;

[0082] (2) First virus inactivation: the pig bladder basement membrane is shaken with a 0.1% peroxoacetic acid-10% ethanol mixed solution for 2 h, and then washed with purified water multiple times until the pH of the washing solution is about 7;

[0083] (3) Decellularization: the pig bladder basement membrane is shaken with a 0.1% sodium dodecyl sulfate / 0.03% trypsin mixed solution for 2 h, and then washed with purified water multiple times until no foam is generated in the washing solution;

[0084] (4) Second virus inactivation: the pig bladder basement membrane is soaked in a 3% sodium hydroxide aqueous solution for 1 h, and then washed with purified water multiple times until the pH of the washing solution is about 7;

[0085] (5) Defatting: the pig bladder basement membrane is soaked in a 1.5:1 methanol / chloroform mixed solution for 12 h, and then washed with purified water multiple times until the concentration of chloroform in the washing solution is less than 0.0063%;

[0086] (6) Freeze-drying: the pig bladder basement membrane is freeze-dried for 24 h to obtain a decellularized pig bladder basement membrane.

[0087] It should be noted that the above steps of decellularization, virus inactivation, defatting, washing and freeze-drying can be realized by using conventional technical means known in the art. Any such alternative, as long as it is intended to achieve the same or similar process purpose, can achieve the technical effects of the present application.

[0088] Example 1

[0089] This embodiment provides a particulate decellularized matrix, which is prepared as follows:

[0090] (1) The porcine bladder basement membrane is subjected to decellularization, virus inactivation, defatting, washing and freeze-drying according to the above process to obtain a decellularized porcine bladder basement membrane;

[0091] (2) The decellularized porcine bladder basement membrane is cut and ground at low temperature to obtain a particulate decellularized matrix. The particulates are sieved using sieves with specific pore sizes to obtain particulates in a first particle size range, particulates in a second particle size range and particulates in a third particle size range. The pore sizes of the sieves are shown in Table 1. The particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range are mixed in a mass ratio of 0.2:1:1.3.

[0092] Table 1

[0093]

[0094] Example 2

[0095] This embodiment provides a particulate decellularized matrix, which is different from that of Example 1 only in that the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range is adjusted to 0.6:1:1.3.

[0096] Example 3

[0097] This embodiment provides a particulate decellularized matrix, which is different from that of Example 1 only in that the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range is adjusted to 0.4:1:1.

[0098] Example 4

[0099] This embodiment provides a particulate decellularized matrix, which is different from that of Example 1 only in that the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range is adjusted to 0.4:1:1.5.

[0100] Example 5

[0101] This embodiment provides a particulate decellularized matrix, which is prepared as follows:

[0102] (1) The porcine bladder basement membrane is subjected to decellularization, virus inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane;

[0103] (2) The decellularized porcine bladder basement membrane is cut into pieces and then ground at low temperature to obtain a particulate decellularized matrix. The particulates are sieved using sieves with specific pore sizes to obtain particulates in a first particle size range, particulates in a second particle size range and particulates in a third particle size range. The pore sizes of the sieves are shown in Table 2. The particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range are mixed at a mass ratio of 0.4:1:1.3.

[0104] Table 2

[0105]

[0106] Example 6

[0107] This example provides a particulate decellularized matrix, which is different from Example 5 only in that the first particle size range is 5-25 μm, and the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range remains unchanged at 0.4:1:1.3.

[0108] Example 7

[0109] This example provides a particulate decellularized matrix, which is different from Example 5 only in that the first particle size range is 20-40 μm, and the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range remains unchanged at 0.4:1:1.3.

[0110] Example 8

[0111] This example provides a particulate decellularized matrix, which is prepared by the following method:

[0112] (1) The porcine bladder basement membrane is subjected to decellularization, virus inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane;

[0113] (2) The decellularized porcine bladder basement membrane is cut into pieces and then ground at low temperature to obtain a particulate decellularized matrix. The particulates are sieved using sieves with specific pore sizes to obtain particulates in a first particle size range, particulates in a second particle size range and particulates in a third particle size range. The pore sizes of the sieves are shown in Table 3. The particulates in the first particle size range, the particulates in the second particle size range and the particulates in the third particle size range are mixed at a mass ratio of 0.4:1:1.3.

[0114] Table 3

[0115]

[0116] Example 9

[0117] The embodiment provides a particulate acellular matrix, which is different from the embodiment 8 only in that the second particle size range is 100-150 μm, and the mass ratio of the first particle size range, the second particle size range, and the third particle size range remains 0.4:1:1.3.

[0118] Embodiment 10

[0119] The embodiment provides a particulate acellular matrix, which is different from the embodiment 8 only in that the second particle size range is 100-120 μm, and the mass ratio of the first particle size range, the second particle size range, and the third particle size range remains 0.4:1:1.3.

[0120] Embodiment 11

[0121] The embodiment provides a particulate acellular matrix, which is prepared in the following method:

[0122] (1) The porcine bladder basement membrane is subjected to decellularization, virus inactivation, defatting, washing, and freeze-drying to obtain a decellularized porcine bladder basement membrane;

[0123] (2) The decellularized porcine bladder basement membrane is cut and ground at low temperature to obtain a particulate acellular matrix, and the particulate acellular matrix is subjected to screening using a screen with a specific pore size to obtain first particle size range particles, second particle size range particles, and third particle size range particles, and the pore size of the screen is shown in Table 4; and the first particle size range particles, the second particle size range particles, and the third particle size range particles are mixed at a mass ratio of 0.4:1:1.3.

[0124] Table 4

[0125]

[0126] Embodiment 12

[0127] The embodiment provides a particulate acellular matrix, which is different from the embodiment 11 only in that the third particle size range is 200-230 μm, and the mass ratio of the first particle size range, the second particle size range, and the third particle size range remains 0.4:1:1.3.

[0128] Embodiment 13

[0129] The embodiment provides a particulate acellular matrix, which is different from the embodiment 11 only in that the third particle size range is 260-300 μm, and the mass ratio of the first particle size range, the second particle size range, and the third particle size range remains 0.4:1:1.3.

[0130] Embodiment 14

[0131] The present embodiment provides a particulate acellular matrix, which is prepared by the following method:

[0132] (1) The porcine bladder basement membrane is subjected to acellularization, virus inactivation, defatting, washing and freeze-drying to obtain an acellular porcine bladder basement membrane;

[0133] (2) The acellular porcine bladder basement membrane is cut into pieces and then ground at low temperature to obtain a particulate acellular matrix. The particulates are sieved using sieves with specific pore sizes to obtain particulates in a first particle size range, particulates in a second particle size range, particulates in a third particle size range and particulates in a fourth particle size range. The pore sizes of the sieves are shown in Table 5. The particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range and the particulates in the fourth particle size range are mixed at a mass ratio of 0.4:1:1.3:0.5.

[0134] Table 5

[0135]

[0136] Example 15

[0137] The present embodiment provides a particulate acellular matrix, which is different from that of Example 14 only in that the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range and the particulates in the fourth particle size range is adjusted to 0.4:1:1.3:1.

[0138] Example 16

[0139] The present embodiment provides a particulate acellular matrix, which is different from that of Example 14 only in that the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range and the particulates in the fourth particle size range is adjusted to 0.4:1:1.3:1.5.

[0140] Example 17

[0141] The present embodiment provides a particulate acellular matrix, which is different from that of Example 15 only in that the fourth particle size range is 400-430 μm, and the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range and the particulates in the fourth particle size range remains unchanged at 0.4:1:1.3:1.

[0142] Example 18

[0143] The present embodiment provides a particulate acellular matrix, which is different from that of Example 15 only in that the fourth particle size range is 480-500 μm, and the mass ratio of the particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range and the particulates in the fourth particle size range remains unchanged at 0.4:1:1.3:1.

[0144] Example 19

[0145] The present embodiment provides a particulate decellularized matrix, which is prepared as follows:

[0146] (1) The porcine bladder basement membrane is subjected to decellularization, viral inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane;

[0147] (2) The decellularized porcine bladder basement membrane is cut into pieces and then ground at low temperature to obtain a particulate decellularized matrix. The particulates are sieved using sieves with specific pore sizes to obtain particulates in a first particle size range, particulates in a second particle size range, particulates in a third particle size range, and particulates in a fourth particle size range, respectively. The sieve pore sizes are shown in Table 6.

[0148] Table 6

[0149]

[0150] (3) The above particulates are incubated with a 500 μg / mL recombinant human epidermal growth factor solution for 8 h (so that the recombinant human epidermal growth factor solution completely immerses the above particulates), freeze-dried, ground at low temperature, and then sieved using sieve combinations with mesh pore sizes of 230 μm and 200 μm, sieve combinations with mesh pore sizes of 140 μm and 100 μm, and sieve combinations with mesh pore sizes of 45 μm and 5 μm, respectively, to obtain drug-loaded decellularized matrix particulates with particle sizes in the ranges of 5-45 μm, 100-140 μm, and 200-230 μm, respectively. The 5-45 μm drug-loaded decellularized matrix particulates, the 100-140 μm drug-loaded decellularized matrix particulates, and the 200-230 μm drug-loaded decellularized matrix particulates are mixed at a mass ratio of 0.4:1:1.3.

[0151] Example 20

[0152] The present embodiment provides a particulate decellularized matrix, which is prepared as follows:

[0153] (1) The porcine bladder basement membrane is subjected to decellularization, viral inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane;

[0154] (2) The decellularized porcine bladder basement membrane is cut into pieces and then ground at low temperature to obtain a particulate decellularized matrix. The particulates are sieved using sieves with specific pore sizes to obtain particulates in a first particle size range, particulates in a second particle size range, particulates in a third particle size range, and particulates in a fourth particle size range, respectively. The sieve pore sizes are shown in Table 6.

[0155] Table 7

[0156]

[0157] (3) The above microparticles were incubated with 500 μg / mL recombinant human epidermal growth factor solution for 8 h, freeze-dried, cryogenically ground, and sieved with a combination of sieves with mesh sizes of 400 μm and 350 μm, a combination of sieves with mesh sizes of 230 μm and 200 μm, a combination of sieves with mesh sizes of 140 μm and 100 μm, and a combination of sieves with mesh sizes of 45 μm and 5 μm, respectively, to obtain drug-loaded acellular matrix microparticles with particle size ranges of 5-45 μm, 100-140 μm, 200-230 μm, and 350-400 μm, respectively. The 5-45 μm drug-loaded acellular matrix microparticles, the 100-140 μm drug-loaded acellular matrix microparticles, the 200-230 μm drug-loaded acellular matrix microparticles, and the 350-400 μm drug-loaded acellular matrix microparticles were mixed at a mass ratio of 0.4:1:1.3:1.

[0158] Example 21

[0159] This example provides a combination of microparticle acellular matrix and sheet-shaped acellular matrix, which is prepared as follows:

[0160] (1) The porcine bladder basement membrane was subjected to decellularization, viral inactivation, defatting, washing, and freeze-drying to obtain a decellularized porcine bladder basement membrane.

[0161] (2) The decellularized porcine bladder basement membrane was cut and cryogenically ground to obtain microparticle acellular matrix. The microparticles were sieved with sieves with specific mesh sizes to obtain microparticles with a first particle size range, microparticles with a second particle size range, and microparticles with a third particle size range. The mesh sizes of the sieves are shown in Table 8. The microparticles with the first particle size range, the microparticles with the second particle size range, and the microparticles with the third particle size range were mixed at a mass ratio of 0.4:1:1.3 to obtain microparticle acellular matrix.

[0162] Table 8

[0163]

[0164] (3) The decellularized porcine small intestinal submucosa obtained in step (1) was soaked and stacked in 10 layers, with tight adhesion between the layers and no air bubbles. The 10 layers of porcine small intestinal submucosa were sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in sequence to obtain a sheet layer of sheet-shaped acellular matrix.

[0165] (4) The microparticle acellular matrix of step (2) was mixed with an appropriate amount of purified water to form a paste, with a ratio of microparticles to water of 0.1 g:5 mL. The paste was uniformly applied to one side of the sheet layer of sheet-shaped acellular matrix of step (3), with a dry weight ratio of microparticle acellular matrix to the area of sheet-shaped acellular matrix of 1.5 mg / cm 2, and freeze-drying to obtain the physical combination of the particulate acellular matrix and the sheet-like acellular matrix. The sheet-like acellular matrix is cut into appropriate sizes to adapt to the size of the wound surface. For the purpose of facilitating the use, the sheet-like acellular matrix is punched to generate seepage of the drainage wound.

[0166] Example 22

[0167] The present example provides a combined form of the particulate acellular matrix and the sheet-like acellular matrix, which is prepared as follows:

[0168] (1) The porcine bladder basement membrane and the porcine small intestinal submucosa are respectively subjected to decellularization, viral inactivation, defatting, washing, and freeze-drying to obtain the decellularized porcine bladder basement membrane and the decellularized porcine small intestinal submucosa.

[0169] (2) The decellularized porcine bladder basement membrane is cut and then ground at low temperature to obtain the particulate acellular matrix. The particulate acellular matrix is sieved using a sieve with a specific pore size to obtain the first, second, and third particulate acellular matrix with different particle sizes. The pore size of the sieve is shown in Table 9. The first, second, and third particulate acellular matrix are mixed in a mass ratio of 0.4:1:1.3 to obtain the particulate acellular matrix.

[0170] Table 9

[0171]

[0172] (3) The decellularized porcine small intestinal submucosa obtained in step (1) is soaked and then stacked in 10 layers with close adhesion between the layers. The 10 layers of the porcine small intestinal submucosa are sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in sequence to obtain a sheet layer of the sheet-like acellular matrix.

[0173] (4) The particulate acellular matrix of step (2) is mixed with purified water to form a paste, and the ratio of the particulate acellular matrix to water is 0.1 g:5 mL. The paste is uniformly applied to one side of the sheet layer of the sheet-like acellular matrix of step (3), and the dry weight of the particulate acellular matrix to the area of the sheet-like acellular matrix is 1.5 mg / cm 2 , and freeze-drying to obtain the physical combination of the particulate acellular matrix and the sheet-like acellular matrix. The sheet-like acellular matrix is cut into appropriate sizes to adapt to the size of the wound surface. For the purpose of facilitating the use, the sheet-like acellular matrix is punched to generate seepage of the drainage wound.

[0174] (5) Take the particulate acellular matrix and sheet acellular matrix physical combination of step (4), incubate with 50 mL of EDC / NHS solution containing 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 25 mM N-hydroxysuccinimide for 24 h, then wash with purified water to remove the crosslinking agent, freeze-dry, and obtain the particulate acellular matrix and sheet acellular matrix chemical combination.

[0175] Example 23

[0176] This example provides a combination of particulate acellular matrix and sheet acellular matrix, which is prepared as follows:

[0177] (1) The porcine bladder basement membrane and porcine small intestinal submucosa are respectively subjected to decellularization, viral inactivation, defatting, washing, and freeze-drying to obtain decellularized porcine bladder basement membrane and decellularized porcine small intestinal submucosa.

[0178] (2) The decellularized porcine bladder basement membrane is cut and ground at low temperature to obtain particulate acellular matrix. The particulate acellular matrix is sieved using sieves with specific pore sizes to obtain particulate acellular matrix of a first particle size range, particulate acellular matrix of a second particle size range, particulate acellular matrix of a third particle size range, and particulate acellular matrix of a fourth particle size range. The pore sizes of the sieves are shown in Table 10. The particulate acellular matrix of the first particle size range, the second particle size range, the third particle size range, and the fourth particle size range are mixed in a mass ratio of 0.4:1:1.3:1 to obtain particulate acellular matrix.

[0179] Table 10

[0180]

[0181] (3) Take the decellularized porcine small intestinal submucosa obtained in step (1), wet it, and stack 10 layers with tight adhesion between layers without air bubbles. The 10 layers of porcine small intestinal submucosa are sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in turn to obtain a sheet layer of sheet acellular matrix, which is cut and punched.

[0182] (4) Take the particulate acellular matrix of step (2), add an appropriate amount of purified water to make a paste, and evenly apply it to one side of the sheet layer of sheet acellular matrix of step (3). The dry weight of particulate acellular matrix to the area of sheet acellular matrix is 1.5 mg / cm 2 , freeze-dry to obtain a particulate acellular matrix and sheet acellular matrix physical combination.

[0183] Example 24

[0184] This example provides a combination of particulate acellular matrix and sheet acellular matrix, which is prepared as follows:

[0185] (1) The porcine bladder basement membrane and porcine small intestinal submucosa are respectively subjected to decellularization, virus inactivation, defatting, washing and freeze-drying to obtain the decellularized porcine bladder basement membrane and decellularized porcine small intestinal submucosa;

[0186] (2) The decellularized porcine bladder basement membrane is cut and ground at low temperature to obtain the decellularized matrix microparticles. The microparticles are sieved using a specific mesh size to obtain microparticles in a first particle size range, a second particle size range, a third particle size range and a fourth particle size range. The mesh size is shown in Table 11. The microparticles in the first particle size range, the second particle size range, the third particle size range and the fourth particle size range are mixed in a mass ratio of 0.4:1:1.3:1 to obtain the microparticle decellularized matrix.

[0187] Table 11

[0188]

[0189] (3) The decellularized porcine small intestinal submucosa obtained in step (1) is soaked and stacked in 10 layers, with close adhesion between layers and no air bubbles. The 10 layers of porcine small intestinal submucosa are sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying to obtain a sheet layer of sheet-shaped decellularized matrix, which is then cut and punched.

[0190] (4) The microparticle decellularized matrix of step (2) is added with an appropriate amount of purified water to form a paste, which is uniformly applied to one side of the sheet layer of sheet-shaped decellularized matrix of step (3). The dry weight of the microparticle decellularized matrix is 1.5 mg / cm 2 , and the area ratio of the sheet-shaped decellularized matrix is 1.5 mg / cm 2 . Freeze-drying obtains the physical combination of microparticle decellularized matrix and sheet-shaped decellularized matrix.

[0191] (5) The physical combination of microparticle decellularized matrix and sheet-shaped decellularized matrix of step (4) is incubated with 50 mL of EDC / NHS solution containing 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 25 mM N-hydroxysuccinimide for 24 h, then washed with purified water to remove the crosslinking agent, and freeze-dried to obtain the chemical combination of microparticle decellularized matrix and sheet-shaped decellularized matrix.

[0192] Example 25

[0193] This example provides a combined form of microparticle decellularized matrix and sheet-shaped decellularized matrix, which is prepared as follows:

[0194] (1) The porcine bladder basement membrane and porcine small intestinal submucosa are respectively subjected to decellularization, virus inactivation, defatting, washing and freeze-drying to obtain the decellularized porcine bladder basement membrane and decellularized porcine small intestinal submucosa;

[0195] (2) The acellular porcine bladder basement membrane was cut into pieces and ground at low temperature to obtain acellular matrix particles. The particles were sieved using a specific mesh size to obtain particles in a first particle size range, a second particle size range, and a third particle size range. The mesh size is shown in Table 12. The particles in the first particle size range, the second particle size range, and the third particle size range were mixed in a mass ratio of 0.4:1:1.3 to obtain the particulate acellular matrix.

[0196] Table 12

[0197]

[0198] (3) The acellular porcine small intestinal submucosa obtained in step (1) was soaked and stacked in 10 layers, with tight adhesion between layers and no air bubbles. The 10 layers of porcine small intestinal submucosa were sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in turn to obtain a sheet layer of sheet-shaped acellular matrix.

[0199] The acellular porcine small intestinal submucosa obtained in step (1) was ground at low temperature to obtain acellular porcine small intestinal submucosa particles. The particles were sieved using standard sieves with mesh sizes of 250 μm and 100 μm. The 250 μm standard sieve was stacked on the 100 μm standard sieve, and the acellular porcine small intestinal submucosa particles were poured into the sieve frame of the 250 μm standard sieve. The sieve frame was shaken and tapped to obtain particles with a particle size ranging from 100 μm to 250 μm. The acellular porcine small intestinal submucosa particles were mixed with purified water at a ratio of 1:20 g / mL, and the mixture was uniformly mixed for 24 hours to obtain a paste.

[0200] The obtained sheet layer was moistened with purified water, and a 1 mm thick silica gel frame was placed on the sheet layer. The obtained paste was filled into the silica gel frame and smoothed, and freeze-dried to obtain a sheet-shaped acellular matrix with the particle layer and the sheet layer combined together. The sheet-shaped acellular matrix was cut and punched.

[0201] (4) The particulate acellular matrix of step (2) was mixed with purified water to form a paste, which was uniformly applied to one side of the sheet-shaped acellular matrix of step (3). The dry weight of the particulate acellular matrix was 1.5 mg / cm 2 , and the area ratio of the sheet-shaped acellular matrix was 1.5 mg / cm

[0202] Example 26

[0203] This example provides a combination of particulate acellular matrix and sheet-shaped acellular matrix, which is prepared as follows:

[0204] (1) The porcine bladder basement membrane is subjected to decellularization, virus inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane;

[0205] (2) The decellularized porcine bladder basement membrane is cut into pieces and then subjected to low-temperature grinding and pulverization to obtain a particulate decellularized matrix. The particulates are screened using a screen with a specific pore size to obtain particulates in a first particle size range, particulates in a second particle size range, particulates in a third particle size range, and particulates in a fourth particle size range. The pore size of the screen is shown in Table 13. The particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range, and the particulates in the fourth particle size range are mixed in a mass ratio of 0.4:1:1.3:1 to obtain the particulate decellularized matrix.

[0206] Table 13

[0207]

[0208] (3) The decellularized porcine small intestinal submucosa obtained in step (1) is soaked and then stacked in 10 layers with close adhesion between the layers without air bubbles. The 10 layers of the porcine small intestinal submucosa are sandwiched between two stainless steel plates and subjected to vacuum lamination and freeze-drying in sequence to obtain a sheet layer of the sheet-shaped decellularized matrix, which is then cut and punched.

[0209] The decellularized porcine small intestinal submucosa obtained in step (1) is subjected to low-temperature grinding and pulverization to obtain decellularized porcine small intestinal submucosa particles. The decellularized porcine small intestinal submucosa particles are screened using standard screens with mesh hole diameters of 250 μm and 100 μm. The 250 μm standard screen is stacked on the 100 μm standard screen, and the decellularized porcine small intestinal submucosa particles are poured into the sieve frame of the 250 μm standard screen. The sieve frame is shaken and knocked to obtain particles with a particle size ranging from 100 μm to 250 μm. The decellularized porcine small intestinal submucosa particles are mixed with purified water at a ratio of 1:20 g / mL, and the mixture is uniformly mixed for 24 hours to obtain a paste.

[0210] The obtained sheet layer is moistened with purified water, a silica gel frame with a thickness of 1 mm is placed on the sheet layer, the obtained paste is filled into the silica gel frame and smoothed, and freeze-drying is performed to obtain a sheet-shaped decellularized matrix with the particle layer and the sheet layer combined together.

[0211] (4) The particulate decellularized matrix of step (2) is mixed with an appropriate amount of purified water to form a paste, which is uniformly applied to one side of the sheet-shaped decellularized matrix of step (3). The dry weight of the particulate decellularized matrix is 1.5 mg / cm 2 , and the area of the sheet-shaped decellularized matrix is 1 cm

[0212] Example 27

[0213] The present example provides a combination of particulate decellularized matrix and sheet decellularized matrix, taking the particulate decellularized matrix obtained in step (2) of Example 25 and the sheet layer of decellularized matrix obtained in step (3), and using the sheet layer of decellularized matrix to cover the particulate decellularized matrix, the dry weight of the particulate decellularized matrix to the area of the sheet decellularized matrix is 1.5 mg / cm 2 .

[0214] Example 28

[0215] The present example provides a combination of particulate decellularized matrix and sheet decellularized matrix, taking the particulate decellularized matrix obtained in step (2) of Example 26 and the sheet layer of decellularized matrix obtained in step (3), and using the sheet layer of decellularized matrix to cover the particulate decellularized matrix, the dry weight of the particulate decellularized matrix to the area of the sheet decellularized matrix is 1.5 mg / cm 2 .

[0216] Example 29

[0217] The present example provides a combination of particulate decellularized matrix and sheet decellularized matrix, taking the particulate decellularized matrix obtained in step (2) of Example 21 and the sheet layer of decellularized matrix obtained in step (3), and using the sheet layer of decellularized matrix to cover the particulate decellularized matrix, the dry weight of the particulate decellularized matrix to the area of the sheet decellularized matrix is 1.5 mg / cm 2 .

[0218] Example 30

[0219] The present example provides a combination of particulate decellularized matrix and sheet decellularized matrix, taking the particulate decellularized matrix obtained in step (2) of Example 21 and the sheet layer of decellularized matrix obtained in step (3), and using the sheet layer of decellularized matrix to cover the particulate decellularized matrix, the dry weight of the particulate decellularized matrix to the area of the sheet decellularized matrix is 7.5 mg / cm 2 .

[0220] Example 31

[0221] The present example provides a combination of particulate decellularized matrix and sheet decellularized matrix, taking the particulate decellularized matrix obtained in step (2) of Example 21 and the sheet layer of decellularized matrix obtained in step (3), and using the sheet layer of decellularized matrix to cover the particulate decellularized matrix, the dry weight of the particulate decellularized matrix to the area of the sheet decellularized matrix is 15 mg / cm 2 .

[0222] Example 32

[0223] This example provides a combination of particulate acellular matrix and sheet-like acellular matrix, taking the particulate acellular matrix obtained in step (2) of Example 23 and the sheet-like acellular matrix obtained in step (3) of Example 23. In use, the sheet-like acellular matrix is placed on top of the particulate acellular matrix, and the dry weight of the particulate acellular matrix to the area of the sheet-like acellular matrix is 1.5 mg / cm 2 .

[0224] Example 33

[0225] This example provides a combination of particulate acellular matrix and sheet-like acellular matrix, taking the particulate acellular matrix obtained in step (2) of Example 23 and the sheet-like acellular matrix obtained in step (3) of Example 23. In use, the sheet-like acellular matrix is placed on top of the particulate acellular matrix, and the dry weight of the particulate acellular matrix to the area of the sheet-like acellular matrix is 7.5 mg / cm 2 .

[0226] Example 34

[0227] This example provides a combination of particulate acellular matrix and sheet-like acellular matrix, taking the particulate acellular matrix obtained in step (2) of Example 21 and the sheet-like acellular matrix obtained in step (3) of Example 21. In use, the sheet-like acellular matrix is placed on top of the particulate acellular matrix, and the dry weight of the particulate acellular matrix to the area of the sheet-like acellular matrix is 15 mg / cm 2 .

[0228] Comparative Example 1

[0229] This comparative example prepared a particulate acellular matrix, which was prepared as follows:

[0230] (1) Take the pig bladder to obtain the bladder basement membrane, wash it with 0.15% peracetic acid aqueous solution for 3 h, then rinse it with PBS buffer solution for 10 min, and freeze-dry it.

[0231] (2) The freeze-dried pig bladder basement membrane is pulverized in liquid nitrogen to obtain a pulverized product. The pulverization speed is 130 m / s, the pulverization time is 4 min, and the pulverized product A is obtained; the pulverization speed is 100 m / s, the pulverization time is 2 min, and the pulverized product B is obtained; the pulverization speed is 60 m / s, the pulverization time is 50 s, and the pulverized product C is obtained.

[0232] (3) The pulverized product A was put into pure water and ultrasonicated for 15 min, filtered, and the filtrate was discarded. The filter residue was vacuum dried to obtain powder dressing A. The pulverized product B was put into pure water and ultrasonicated for 15 min, filtered, and the filtrate was discarded. The filter residue was vacuum dried to obtain powder dressing B. The pulverized product C was put into pure water and ultrasonicated for 15 min, filtered, and the filtrate was discarded. The filter residue was vacuum dried to obtain powder dressing C. The powder dressing A, the powder dressing B and the powder dressing C were mixed in a mass ratio of 1:1:7.4 to obtain the particulate decellularized matrix decellularized biological matrix.

[0233] Comparative Example 2

[0234] A particulate decellularized matrix was prepared in the following manner:

[0235] (1) The porcine bladder basement membrane was subjected to decellularization, viral inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane.

[0236] (2) The decellularized porcine bladder basement membrane was cut and pulverized in liquid nitrogen to obtain a pulverized product. The pulverization speed was 130 m / s, and the pulverization time was 4 min to obtain a pulverized product A. The pulverization speed was 100 m / s, and the pulverization time was 2 min to obtain a pulverized product B. The pulverization speed was 60 m / s, and the pulverization time was 50 s to obtain a pulverized product C.

[0237] (3) The pulverized product A was put into pure water and ultrasonicated for 15 min, filtered, and the filtrate was discarded. The filter residue was vacuum dried to obtain powder dressing A. The pulverized product B was put into pure water and ultrasonicated for 15 min, filtered, and the filtrate was discarded. The filter residue was vacuum dried to obtain powder dressing B. The pulverized product C was put into pure water and ultrasonicated for 15 min, filtered, and the filtrate was discarded. The filter residue was vacuum dried to obtain powder dressing C. The powder dressing A, the powder dressing B and the powder dressing C were mixed in a mass ratio of 1:1:7.4 to obtain the particulate decellularized matrix decellularized biological matrix dressing.

[0238] Comparative Example 3

[0239] A particulate decellularized matrix was prepared in the following manner:

[0240] (1) The porcine bladder basement membrane was subjected to decellularization, viral inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane.

[0241] (2) The acellular porcine bladder basement membrane was cut into pieces and then ground at low temperature to obtain microparticle acellular matrix. The microparticles were sieved using sieves with specific mesh sizes to obtain microparticles in a first particle size range, microparticles in a second particle size range, and microparticles in a third particle size range. The mesh sizes of the sieves are shown in Table 14. The microparticles in the first particle size range, the microparticles in the second particle size range, and the microparticles in the third particle size range were mixed at a mass ratio of 0:1:1.3.

[0242] Table 14

[0243]

[0244] Comparative Example 4

[0245] This comparative example provides a microparticle acellular matrix, which is different from Comparative Example 3 only in that the mass ratio of the microparticles in the first particle size range, the microparticles in the second particle size range, and the microparticles in the third particle size range is adjusted to 0.4:0:1.3.

[0246] Comparative Example 5

[0247] This comparative example provides a microparticle acellular matrix, which is different from Comparative Example 3 only in that the mass ratio of the microparticles in the first particle size range, the microparticles in the second particle size range, and the microparticles in the third particle size range is adjusted to 0.4:1:0.

[0248] Comparative Example 6

[0249] This comparative example provides a microparticle acellular matrix, which is different from Comparative Example 3 only in that the mass ratio of the microparticles in the first particle size range, the microparticles in the second particle size range, and the microparticles in the third particle size range is adjusted to 1:1:1.3.

[0250] Comparative Example 7

[0251] This comparative example provides a microparticle acellular matrix, which is different from Comparative Example 3 only in that the mass ratio of the microparticles in the first particle size range, the microparticles in the second particle size range, and the microparticles in the third particle size range is adjusted to 0.4:1:2.

[0252] Comparative Example 8

[0253] This example provides a microparticle acellular matrix, which is prepared as follows:

[0254] (1) The porcine bladder basement membrane was subjected to decellularization, viral inactivation, defatting, washing, and freeze-drying to obtain an acellular porcine bladder basement membrane.

[0255] (2) The acellular porcine bladder basement membrane was cut into pieces and then ground at low temperature to obtain a particulate acellular matrix. The particulate acellular matrix was sieved by using a sieve with a specific pore size to obtain particulates in a first particle size range, particulates in a second particle size range, particulates in a third particle size range, and particulates in a fourth particle size range. The pore sizes of the sieves are shown in Table 15. The particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range, and the particulates in the fourth particle size range were mixed at a mass ratio of 0.4:1:1.3:2.

[0256] Table 15

[0257]

[0258] Comparative Example 9

[0259] This comparative example provides a combination of a particulate acellular matrix and a foam dressing. The particulate acellular matrix obtained in step (2) of Example 26 was combined with a commercially available foam dressing. In use, the sheet-shaped foam dressing was placed on top of the particulate acellular matrix.

[0260] Comparative Example 10

[0261] This comparative example provides a combination of a particulate acellular matrix and a foam dressing. The particulate acellular matrix obtained in step (2) of Example 25 was combined with a commercially available foam dressing. In use, the foam dressing was placed on top of the particulate acellular matrix.

[0262] Comparative Example 11

[0263] This comparative example provides a particulate acellular matrix, which was prepared as follows:

[0264] (1) An acellular porcine bladder basement membrane was prepared by decellularization, viral inactivation, defatting, washing, and freeze-drying of a porcine bladder basement membrane;

[0265] (2) The acellular porcine bladder basement membrane was cut into pieces and then ground at low temperature to obtain a particulate acellular matrix. The particulate acellular matrix was sieved by using a sieve with a specific pore size to obtain particulates in a first particle size range, particulates in a second particle size range, particulates in a third particle size range, and particulates in a fourth particle size range. The pore sizes of the sieves are shown in Table 16. The particulates in the first particle size range, the particulates in the second particle size range, the particulates in the third particle size range, and the particulates in the fourth particle size range were mixed at a mass ratio of 0.4:1:1.3:1.

[0266] Table 16

[0267]

[0268] Comparative Example 12

[0269] This comparative example provides a particulate acellular matrix, which was prepared as follows:

[0270] (1) The porcine bladder basement membrane is prepared by decellularization, virus inactivation, defatting, washing and freeze-drying to obtain a decellularized porcine bladder basement membrane;

[0271] (2) The decellularized porcine bladder basement membrane is cut and ground at low temperature to obtain a microparticle decellularized matrix, and a specific pore size screen is used to screen the microparticles to obtain a microparticle decellularized matrix with a particle size range of 150-300 μm.

[0272] Test Example 1

[0273] Partial superficial burn model wound healing effect test:

[0274] (1) Construction of partial superficial deep II degree burn model:

[0275] A partial superficial deep II degree burn model of Bama pigs is prepared, and each defect is a square with a side length of 4 cm. The specific method is as follows:

[0276] A partial superficial deep II degree burn model of Bama pigs is prepared, and each defect is a square with a side length of 4 cm. The specific method is as follows:

[0277] (2) Wound treatment:

[0278] (2.1) The experimental group is treated with the microparticle decellularized matrix provided by Examples 1-13, 19, 21, 22, 25, 27, 29-31 and Comparative Examples 1, 2, 3-7, 10, 11, and 12 or the combination of microparticle decellularized matrix and sheet decellularized matrix after wound debridement.

[0279] (2.1.1) When the microparticle decellularized matrix is used alone for treatment, the wound is first debrided, then the microparticle decellularized matrix is made into a paste with normal saline, evenly applied to the wound, then covered and fixed with Vaseline gauze, and the outermost layer is protected with a sterile dressing. The amount of microparticle decellularized matrix used is 1.5 mg / cm 2 .

[0280] (2.1.2) When the microparticle decellularized matrix and sheet decellularized matrix physical or chemical combination is used for treatment, the wound is first debrided, and the microparticle decellularized matrix and sheet decellularized matrix physical or chemical combination is hydrated, then it is covered on the wound, so that the microparticle decellularized matrix directly contacts the wound, and finally Vaseline gauze is covered and fixed on top, and the outermost layer is protected with a sterile dressing. The sheet decellularized matrix should completely cover the wound and the edge should exceed the wound by 0.1-0.3 mm.

[0281] (2.1.3) When the micro-particle decellularized matrix is used in combination with the sheet-shaped decellularized matrix, first, the wound surface is debrided, and the sheet-shaped decellularized matrix is hydrated, and the micro-particle decellularized matrix is evenly sprinkled on the wound surface, then the hydrated sheet-shaped decellularized matrix is covered, and finally, the vaseline gauze is covered and fixed on top, and the outermost layer is protected by a sterile dressing. Among them, the sheet-shaped decellularized matrix should completely cover the wound surface and the edge should exceed the wound surface by 0.1-0.3 mm.

[0282] (2.1.4) When the micro-particle decellularized matrix is used in combination with the purchased foam dressing for treatment, first, the wound surface is debrided, then the micro-particle decellularized matrix is made into a paste with normal saline, and is evenly applied to the wound surface, and then the foam dressing is covered and fixed on top.

[0283] (2.2) The wound surface of the blank group is only covered and protected with vaseline gauze and sterile dressing after debridement.

[0284] (3) Wound healing rate calculation:

[0285] At the specified time point, the wound surface is photographed, the wound area is measured, and the wound healing rate at different time points is calculated according to the following formula.

[0286] Wound healing rate (%) = (A0-A t ) / A0x100%;

[0287] In the formula, A0 is the initial area of the wound surface, A t is the area of the wound surface at a specific time point d.

[0288] The wound healing rates at different time points are shown in Table 17. The comparison chart of the wound healing of Example 12, Example 21, the group of Comparative Example 1, and the blank group is shown in Figure 1

[0289] Table 17

[0290]

[0291] The above data shows that at different time points, the wound healing rate of the example group is greater than that of the blank group, indicating that the micro-particle decellularized matrix has the function of promoting the healing of deep II degree partial shallow wounds.

[0292] Among them, according to the data comparison of Examples 1-5 and Comparative Examples 3-7, the micro-particles in the appropriate proportion of the first particle size range are beneficial to promote early wound healing, and within a certain proportion range, the more the micro-particles in the first particle size range, the greater the promotion effect on early wound healing, which can also affect the subsequent healing progress; the micro-particles in the appropriate proportion of the second particle size range are beneficial to promote mid-term wound healing; and the micro-particles in the appropriate proportion of the third particle size are beneficial to promote mid-term and late-term wound healing.

[0293] ​From the comparison of the data of Example 12, Example 21-22, 25, 27, it can be seen that, within the appropriate range of the ratio of the particulate decellularized matrix to the sheet decellularized matrix, the combination of the two can produce a synergistic effect, and the healing-promoting effect is better than that of the particulate decellularized matrix alone; the effect of the combination of the sheet decellularized matrix with the particulate decellularized matrix in the granular layer is slightly better than that of the combination of the sheet decellularized matrix with the particulate decellularized matrix in the sheet layer, which is related to the fact that the granular layer increases the water absorption of the sheet decellularized matrix and the granular layer is more easily degraded than the sheet layer. The combination mode of the sheet decellularized matrix and the particulate decellularized matrix has less effect on the healing-promoting effect.

[0294] The proportion of the microparticles with a particle size of 750 μm or more in the microparticles in the Comparative Example 1 group and the Comparative Example 2 group is about 80%, and the proportion of microparticles with a small particle size is very low; the Comparative Example 9 group lacks microparticles in the first particle size range. The above-mentioned comparative example groups degrade more slowly in the early stage of the wound, delaying the effect, so that the wound healing rate at 1 week after the operation is lower than that of each example group, and the overall healing speed is also slower than that of each example group. It shows that the presence of microparticles in the first particle size range is beneficial to the early wound healing.

[0295] The Comparative Example 4 group lacks microparticles in the second particle size range, and the wound healing rate at 1 week after the operation is only slightly lower than that of each example group, but the difference between the wound healing rate and the wound healing rate of each example group gradually increases at 2, 3, and 4 weeks after the operation. It shows that the microparticles in the second particle size range play a role in the middle stage and affect the subsequent wound healing.

[0296] The Comparative Example 5 group lacks microparticles in the third particle size range, and the wound healing rate at 1 week and 2 weeks after the operation is only slightly lower than that of each example group, but the wound healing rate at 3 weeks and 4 weeks after the operation is also larger than that of each example group. It shows that the microparticles in the third particle size range have a promoting effect on the wound healing in the middle and late stages.

[0297] The Comparative Example 6 group has an excessive amount of microparticles in the first particle size range, resulting in a small amount of microparticles in the second particle size range and the third particle size range. The wound healing rate at 1 week after the operation is only slightly lower than that of each example group, but the difference between the wound healing rate and the wound healing rate of each example group gradually increases at 2, 3, and 4 weeks after the operation. It shows that when the proportion of microparticles in the first particle size range is too high, it is still beneficial to the early healing, but it is difficult to maintain the healing-promoting effect in the middle and late stages, and the presence of microparticles in the second particle size range and the third particle size range is beneficial to the wound healing in the middle and late stages

[0298] The comparative example 7 group has an excess of the third particle size range microparticles, resulting in less second particle size range microparticles, and the proportion of the first particle size range microparticles is still within the normal range. The wound healing rate at 1 week after the operation is only slightly lower than that of each example group, but the difference between the wound healing rate and that of each example group gradually increases at 2, 3, and 4 weeks after the operation. This shows that when the proportion of the third particle size range microparticles is too high, it cannot effectively promote the wound healing in the middle stage, and the second particle size range microparticles still need to fully play a role in the middle stage of wound healing.

[0299] The comparative example 10 group uses the microparticle decellularized matrix in combination with the foam dressing. The wound healing rate in each stage of wound healing is higher than that of each example group using the microparticle decellularized matrix alone, but is still lower than that of each example group using the microparticle decellularized matrix in combination with the sheet-shaped decellularized matrix. This shows that the microparticle decellularized matrix has a better healing-promoting effect when used in combination with other functional dressings, and the effect of the microparticle decellularized matrix in combination with the sheet-shaped decellularized matrix is better than that of the microparticle decellularized matrix in combination with other functional dressings.

[0300] The microparticle decellularized matrix of the comparative example 12 group is composed of only a single particle size range microparticle, and does not form a decellularized matrix microparticle with more than three gradient particle sizes, which cannot fully play a role in different stages of wound healing. The healing rate is significantly lower than that of each example group, and the overall healing speed is also slower than that of each example group.

[0301] Test Example 2

[0302] Wound healing effect test of deep partial burn model:

[0303] (1) Construction of deep partial burn model of deep II degree:

[0304] A deep II degree partial burn model of Bama pigs was made, and each defect was a circular shape with a diameter of 1 cm. The specific method is as follows:

[0305] A deep II degree partial burn model of Bama pigs was made, and each defect was a circular shape with a diameter of 1 cm. The specific method is as follows:

[0306] (2) Wound treatment:

[0307] (2.1) The microparticle decellularized matrix or the combination of the microparticle decellularized matrix and the sheet-shaped decellularized matrix provided by the experimental example 1, 14-18, 20, 23, 24, 26, 28, 32-34 and the comparative examples 1, 2, 8, 9, and 11 was used for treatment after the wound debridement of the experimental group.

[0308] (2.2) The blank group was only covered with vaseline gauze and sterile dressing after wound debridement.

[0309] The treatment method, detection index and method were the same as those in Test Example 1. The wound healing rates at different time points are shown in Table 18.

[0310] Table 18

[0311]

[0312] The above data show that the wound healing rates of the example groups are greater than those of the blank group at different time points, indicating that the microparticle acellular matrix has the function of promoting the healing of deep II degree deep wounds.

[0313] From the comparison of the data of Example 1 and Examples 14-18, it can be seen that the fourth particle size range microparticles are beneficial to the late healing of deep II degree deep wounds.

[0314] From the comparison of the data of Example 15 and Examples 23-24, 26, 28, and Comparative Example 10, it can be seen that the combination of microparticle acellular matrix and sheet-shaped acellular matrix produces better healing-promoting effect than the use of microparticle acellular matrix alone and the combination of microparticle acellular matrix and other functional dressings.

[0315] The above data show that microparticles of different particle size ranges can degrade at different stages of wound healing, promote cell migration, proliferation and differentiation, and thus accelerate wound healing.

[0316] The microparticles in the first particle size range have a small particle size and a fast degradation rate, and can be degraded during the inflammation period of wound healing. If the microparticles in the first particle size range are absent, the speed of cell recruitment is not significantly improved, and the promotion effect on wound healing is significantly weakened. If the proportion of the microparticles in the first particle size range is too large, most of the microparticles are degraded in the late stage of wound healing, and cannot continuously play a role, which also weakens the healing promotion effect. If the microparticles in the second particle size range are absent or the proportion of the microparticles in the second particle size range is too low, there are fewer degradation products of the microparticles in the proliferation period of wound healing, and the degradation products of the microparticles also have a certain effect on cell migration. During the absence of the degradation products, the promotion effect on cell migration is also weakened, the re-epithelialization speed is relatively reduced, and the healing promotion effect is weakened. If the proportion of the microparticles in the second particle size range is too high, most of the microparticles are degraded in the late stage of wound healing, and cannot continuously play a role, which weakens the healing promotion effect. If the microparticles in the third particle size range are absent or the proportion of the microparticles in the third particle size range is too low, most of the microparticles are degraded in the late stage of wound healing, and cannot continuously play a role, which weakens the healing promotion effect. If the proportion of the microparticles in the third particle size range is too high, the degradation degree of the microparticles is low in the early stage of wound healing, the promotion effect on cell recruitment, migration, proliferation and differentiation is weakened, and wound healing is not conducive. The microparticles in the fourth particle size range have a long degradation time, and are applied to deep burn wounds. When the particle size of the microparticles is greater than the fourth particle size range, because the volume is large, a foreign body reaction can be caused, inflammation is aggravated, and wound healing is delayed.

[0317] In addition, as can be seen from the comparison of the data of Example 15 and Examples 23-24, 26, 28, and Comparative Example 10, when the microparticle decellularized matrix is combined with the sheet-shaped decellularized matrix, the effect of accelerating healing is obviously better than that of the microparticle decellularized matrix alone, and is also better than that of the microparticle decellularized matrix combined with other types of dressings (such as foam dressings). This is because the sheet-shaped decellularized matrix has high biological activity, can provide barrier protection, and can also maintain a microenvironment conducive to healing, and has a significant synergistic effect with the microparticle decellularized matrix.

[0318] In summary, the present application provides a microparticle decellularized matrix. By adjusting the proportion of microparticles of different particle sizes in the microparticle biological dressing, the degradation and absorption time of the microparticles in the body can be adjusted to match the wound healing time. At the same time, the use of sheet-shaped decellularized matrix can provide good barrier protection for the wound, and the use of microparticle decellularized matrix in combination with the sheet-shaped decellularized matrix can further improve the wound healing effect.

[0319] The applicant declares that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by any person skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

[0320] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details of the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0321] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. A microparticle decellularized matrix, characterized in that, The decellularized microparticle matrix includes decellularized matrix microparticles with three or more gradient particle sizes, and the decellularized matrix microparticles with gradient particle sizes include at least microparticles in a first particle size range, microparticles in a second particle size range, and microparticles in a third particle size range. 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 span between the particles in the first particle size range, the particles in the second particle size range, and the particles in the third particle size range is greater than 50 μm; the particle size span within each of the particles in the first particle size range, the particles in the second particle size range, and the particles in the third particle size range is independently less than 50 μm. The mass ratio of the particles in the first particle size range, the particles in the second particle size range, and the particles in the third particle size range is (0.2-0.6):1:(1-1.5).

2. The microparticle decellularized matrix according to claim 1, characterized in that, The gradient-size decellularized matrix microparticles include microparticles in a first size range, a second size range, a third size range, and a fourth size range, wherein the particle size of the fourth size range microparticles is 300-500 μm. The mass ratio of the particles in the first particle size range, the particles in the second particle size range, the particles in the third particle size range, and the particles in the fourth particle size range is (0.2-0.6):1:(1-1.5):(0.5-1.5).

3. The microparticle decellularized matrix according to claim 1, characterized in that, The gradient-sized decellularized matrix microparticles are also loaded with drugs, which include growth factors and / or antibacterial agents.

4. A method for preparing a microparticle decellularized matrix as described in any one of claims 1-3, characterized in that, The preparation method includes 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 decellularized matrix microparticles. The decellularized matrix microparticles are sieved using a sieve to obtain microparticles of different particle sizes. The microparticles of different particle sizes are mixed to obtain microparticle decellularized matrix.

5. The method for preparing the decellularized microparticle matrix according to claim 4, characterized in that, The source of the animal membrane in step (1) includes mammals; The animal membranes include any one or a combination of at least two of the following: the submucosa of the small intestine, the basement membrane of the bladder, the peritoneum, the pleura, or the greater omentum.

6. The method for preparing the decellularized microparticle matrix according to claim 4, characterized in that, The decellularized matrix microparticles described in step (2) are also loaded with drugs; The drug loading method is as follows: decellularized matrix microparticles are mixed and soaked in an aqueous solution containing the drug to obtain drug-loaded decellularized matrix microparticles.

7. A composite decellularized matrix, characterized in that, The composite decellularized matrix includes the microparticle decellularized matrix and sheet-like decellularized matrix as described in any one of claims 1-3; The sheet-like decellularized matrix has a thin sheet structure or a thin sheet-granular composite structure; The combination of the microparticle decellularized matrix and the sheet-like decellularized matrix includes any one or a combination of at least two of the following: contact combination, physical combination, or chemical combination. The dry weight ratio of the microparticle decellularized matrix to the surface area ratio of the sheet-like decellularized matrix is ​​1.5-15 mg / cm². 2 .

8. The composite decellularized matrix according to claim 7, characterized in that, The sheet-like decellularized matrix comprises a thin sheet layer formed by vacuum lamination of multiple layers of decellularized animal membrane extracellular matrix material; The sheet layer consists of 2-20 layers of decellularized animal membrane extracellular matrix material.

9. The composite decellularized matrix according to claim 8, characterized in that, The sheet-like decellularized matrix also includes a granular layer made from pulverized decellularized animal membrane extracellular matrix material; The sheet layer and the particle layer are bonded together by physical interaction formed after freeze-drying; The granular layer is composed of decellularized animal membrane extracellular matrix particles with a particle size of 5-500 μm; The particle layer has a thickness of 0.1-3 mm and a porosity of 30%-90%.

10. The use of the microparticle decellularized matrix as described in any one of claims 1-3 or the composite decellularized matrix as described in any one of claims 7-9 in the preparation of medical biomaterials that promote wound healing.

Images