Preparation method and application of acellular biological matrix dressing

Abstract

The invention relates to the field of dressings, in particular to a preparation method and application of an acellular biological matrix dressing. The decellularized biological matrix dressing is prepared by the following steps: decellularizing mammalian tissues, sequentially carrying out ion induction and crushing to obtain a crushed product, carrying out surface finishing and vacuum drying on the crushed product, and mixing to obtain the decellularized biological matrix dressing. The obtained acellular biological matrix dressing has a rougher surface and a more uniform roughness degree, so that the stability of the final product acellular biological matrix dressing and the capability of promoting cell adhesion are improved. When in use, the particle can be directly applied to various wounds, the complexity of using the particle in the prior art is reduced, and the particle can be directly covered on full-thickness skin defect wounds (such as deep wet wounds) and superficial wounds (such as dry epidermis injury).

Description

Technical Field

[0001] The present invention relates to the field of dressings, and in particular to a preparation method and application of a decellularized biomatrix dressing. Background Art

[0002] The decellularized matrix obtained after biological tissue is decellularized is widely used in the field of regenerative medicine. ACell has applied for a patent, and its patent content discloses a granular tissue graft with components of different densities. By controlling the density of the second particle to be at least 150% of the density of the first particle, the low-density particles are absorbed first and the high-density particles are absorbed later, thereby prolonging the degradation time of the decellularized matrix. At the same time, the document also records that before the preparation and production of this kind of particle mixture, the site used for repair must be evaluated first, and after obtaining the optimal absorption rate of the particle composition, the specific density value of the first density particle should be calculated, and then the specific density value of the second density is deduced again. Summary of the invention

[0003] In order to reduce the cumbersomeness of the prior art when using this kind of particles, making it easier to use, it is intended to obtain a decellularized biomatrix dressing, which does not need to be used according to the wound to calculate the absorption rate to be proportioned, but has adaptive and self-regulating capabilities, and can be directly applied to various wounds. In order to solve the above problems, the first aspect of the present invention provides a method for preparing a decellularized biomatrix dressing, wherein the decellularized biomatrix dressing is decellularized from mammalian tissue, and then ion-induced and crushed in turn to obtain a crushed product, and the crushed product is surface-finished and vacuum-dried, and then mixed to obtain the decellularized biomatrix dressing.

[0004] As a preferred embodiment, the acellular biomatrix dressing has not been treated with enzymes.

[0005] In this scheme, the acellular biomatrix dressing has not been treated with enzymes, has a three-dimensional helical protein structure, retains the natural fiber network, and can be used to support cell regeneration.

[0006] Preferably, the acellular biomatrix dressing has not been cross-linked.

[0007] As a preferred embodiment, the mammalian tissue is selected from one or two of the small intestinal submucosa, pericardium, bladder basement membrane, peritoneum, and dermis of a mammal.

[0008] Preferably, the mammalian tissue decellularization is specifically as follows: the mammalian tissue is washed in a 0.1%-0.3% peracetic acid aqueous solution for 3-4 hours, and then rinsed with a buffer solution for 3-10 minutes.

[0009] Preferably, the decellularization of mammalian tissue is specifically as follows: after mechanical peeling of the mammalian tissue (mainly referring to the removal of the muscle layer), the mammalian tissue is washed in a 0.1%-0.3% peracetic acid aqueous solution for 3-4 hours, and then rinsed with a buffer solution for 3-10 minutes.

[0010] As a preferred embodiment, ion induction is specifically as follows: soaking the decellularized mammalian tissue in an acid solution for 15-30 minutes, immediately adding an alkaline solution to neutralize to a neutral pH, then standing for 10-15 minutes, taking out the treated mammalian tissue, and obtaining a pre-treated material.

[0011] Preferably, the ion induction is specifically as follows: take a container, add an acid solution into the container, soak the decellularized mammalian tissue in the acid solution for 15-30 minutes, during the soaking, the acid solution completely immerses the decellularized mammalian tissue, after soaking for 15-30 minutes, immediately continue to add alkaline solution to the container to neutralize to a neutral pH, then let it stand for 10-15 minutes, take out the treated mammalian tissue, and obtain a pre-treated material.

[0012] Preferably, the acid solution is an inorganic acid aqueous solution and / or an organic acid aqueous solution.

[0013] Preferably, the acid solution includes at least an aqueous solution of an inorganic acid.

[0014] The pH of the acid solution is 2.7-5.5.

[0015] Preferably, the pH of the acid solution is 4.1-5.5.

[0016] The acid solution is an inorganic acid aqueous solution or a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution.

[0017] The acid solution is a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution.

[0018] When the acid solution is a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution, the volume ratio of the inorganic acid aqueous solution to the organic acid aqueous solution in the acid solution is (8-20):(3-10).

[0019] The inorganic acid aqueous solution is a hydrochloric acid aqueous solution or a phosphoric acid aqueous solution.

[0020] Furthermore, the inorganic acid aqueous solution is a hydrochloric acid aqueous solution.

[0021] Preferably, the organic acid aqueous solution is selected from one of a citric acid aqueous solution, an acetic acid aqueous solution and a tartaric acid aqueous solution.

[0022] Preferably, the organic acid aqueous solution is acetic acid aqueous solution.

[0023] Preferably, when the acid solution is a mixture of an inorganic acid aqueous solution and an organic acid aqueous solution, the acid solution is prepared by mixing 0.025-0.055 g / mL inorganic acid aqueous solution and 0.025-0.055 g / mL organic acid aqueous solution in a volume ratio of (8-20):(3-10), and then diluting with water to adjust the pH to 2.7-5.5.

[0024] In the process of obtaining acellular biological matrix, a single type of acid solution or a single type of alkaline solution is usually used to remove endotoxins from biological tissues, and the use of such a single acid or alkali is usually to wash the biological tissue with an acid or alkaline solution, followed by rinsing with a buffer solution or water. In this preparation scheme, in order to remove cellular endotoxins more quickly, the decellularized mammalian tissue is immersed in an acid solution. In a large number of experiments, it was unexpectedly found that the acid-soaked tissue was not taken out after immersion, but the alkaline solution was continued to be added to the neutral environment, and it was allowed to stand again. After this treatment, it was crushed, and endotoxins could still be effectively removed. At the same time, it was unexpectedly found that the powder dressing obtained by this treatment method (i.e., ion induction) combined with crushing and surface finishing has a fluffy and rough microscopic surface structure, which may be due to the aggregation of a large number of fibers on the particle surface, which will also provide more sites for cell attachment, which is beneficial to tissue repair. It should be emphasized that the step of continuing to add alkaline solution to the neutral environment after acid immersion, and the step of continuing to keep the aforementioned acid-treated material slowly restored to neutral in a neutral environment for a period of time, followed by crushing and surface finishing, may be because the original triple helical structure of the decellularized tissue after acid treatment is affected to a certain extent, and in the change from acidic environment to neutral environment, it is gradually and slowly induced by ions, and through this gradual charge effect, the particle microstructure is affected, and the embedding of salt ions may also affect the subsequent crushing. This phenomenon is particularly obvious when the acid solution is a mixture of inorganic acid aqueous solution and organic acid aqueous solution.

[0025] As a preferred embodiment, the pulverization is pulverization in liquid nitrogen, and the pulverization method during pulverization in liquid nitrogen is selected from one or more of impact pulverization, shear pulverization and compression pulverization.

[0026] Preferably, the decellularized biomatrix dressing is prepared by decellularizing mammalian tissue, sequentially undergoing ion induction, freeze drying and pulverization to obtain a pulverized product, and the pulverized product is surface treated and vacuum dried before mixing to obtain the decellularized biomatrix dressing.

[0027] Preferably, the pulverization method during pulverization in liquid nitrogen is impact pulverization.

[0028] As a preferred embodiment, the pulverized product includes at least pulverized product A, pulverized product B and pulverized product C; During the crushing, the crushing speed is 120-175m / s, and the crushing time is 2-5min, and the crushing product A is obtained; during the crushing, the crushing speed is 70-100m / s, and the crushing time is 1-4min, and the crushing product B is obtained; during the crushing, the crushing speed is 60-80m / s, and the crushing time is 40-80s, and the crushing product C is obtained.

[0029] As a preferred embodiment, the surface preparation is specifically to put the pulverized product into pure water, ultrasonicate for 10-20 minutes, filter, discard the filtrate, and dry the filter residue to obtain a powder dressing.

[0030] Preferably, the surface preparation is specifically to put the pulverized product into pure water, ultrasonicate for 10-20 minutes, filter, discard the filtrate, and vacuum dry the filter residue to obtain the powder dressing.

[0031] As a preferred embodiment, the pulverized product A is surface treated and vacuum dried to obtain powder dressing A; the pulverized product B is surface treated and vacuum dried to obtain powder dressing B; the pulverized product C is surface treated and vacuum dried to obtain powder dressing C.

[0032] As a preferred embodiment, powder dressing A, powder dressing B and powder dressing C are mixed in a mass ratio of (0.9-1.7):1:(5.6-10.8) to obtain a decellularized biomatrix dressing.

[0033] Preferably, powder dressing A, powder dressing B and powder dressing C are mixed in a mass ratio of (0.9-1.7):1:(5.6-9.5).

[0034] Further preferably, powder dressing A, powder dressing B and powder dressing C are mixed in a mass ratio of (0.9-1.7):1:(5.6-9.3).

[0035] Preferably, powder dressing A, powder dressing B and powder dressing C are mixed in a mass ratio of (1-1.7):1:(5.6-9.3).

[0036] The mass ratio of powder dressing A, powder dressing B and powder dressing C is (0.9-1.7): 1: (5.6-10.8), which can not only further promote cell adhesion, but also make the surface of the acellular biomatrix dressing more uniform, which means that the particles have higher adaptive and self-regulating stability, which is helpful for their application and reduces the risk of uncontrollable local application effects on different wound surfaces. It should be noted that in actual experiments, the mass ratio of powder dressing A, powder dressing B and powder dressing C can have a certain error range, and this error range is controlled within (0.9-1.7): 1: (5.6-10.8).

[0037] Generally speaking, for a biomaterial system of the same type including particles of different sizes, large particles always degrade slower than small particles. By utilizing the small particles to degrade first, the active factors are preferentially released to promote healing, and the large particles are degraded later, playing a certain scaffolding role, so that the synergistic system of small and large particles has a certain self-regulating ability. However, it was found in the experiment that this gradient system may accelerate the formation of scars and tissue contracture. The preparation technology overcomes the above problems to a certain extent. The decellularized biomatrix dressing obtained by mixing the powder dressing A, powder dressing B and powder dressing C, which are sequentially ion-induced, crushed, surface-finished and vacuum-dried, in a mass ratio of (0.9-1.7):1:(5.6-10.8), shows a certain effect of inhibiting tissue contraction or hyperplasia, reducing scars and contractures, and making the new tissue have good elasticity in cell experiments. The inventors speculate that such a design may affect the immune response, and the release and regulation of pathway conversion factors during tissue repair are affected. At the same time, the system has a specific multi-level pore structure, forming pore heterogeneity, guiding the adhesion and arrangement of fibroblasts, regulating the interface stress through surface roughness, improving the balance between the shape maintenance and degradation of the new tissue, and achieving the synergistic cooperation of the three particles. The relevant mechanism still needs to be further confirmed. At the same time, the significance and stability of this inhibitory effect also need to be further verified.

[0038] The particle size range of powder dressing A is 10-250 μm; the particle size range of powder dressing B is 200-500 μm; the particle size range of powder dressing C is 750-1200 μm.

[0039] Preferably, the particle size dispersity index (PDI) values ​​of powder dressing A, powder dressing B and powder dressing C are: 1.5<PDI<3.

[0040] Preferably, 1.5<PDI<2.9.

[0041] Preferably, 1.6≤PDI≤2.7.

[0042] The particle size dispersion index (PDI) is used to describe the dispersion of particle size distribution. The calculation formula is PDI = (D90- D10) / D50, where D90 represents the diameter of particles that account for 90% of the diameter, D50 represents the diameter of particles that account for 50% of the diameter, and D10 represents the diameter of particles that account for 10% of the diameter. The smaller the PDI value, the more concentrated the particle size distribution.

[0043] A second aspect of the present invention provides an application of a decellularized biomatrix dressing on a wound surface material, wherein the wound surface material includes a partial-thickness skin wound surface material and a full-thickness skin wound surface material.

[0044] When applied on wound surface materials, the acellular biomatrix dressing can be used alone or in combination with a biological patch.

[0045] Beneficial effects:

[0046] (1) Compared with decellularized matrix materials that have been cross-linked or / and treated with enzymes, decellularized matrix materials that have not been cross-linked or treated with enzymes not only avoid the effects of enzymes and cross-linkers on their own three-dimensional helical protein structure, but also retain the natural fiber network and the bioactive substances (such as growth factors) they contain to a large extent. Their functionality in supporting cell regeneration will not be greatly reduced due to cross-linking or / and enzyme treatment. Moreover, for the preparation process of this technology that requires decellularization and then ion induction and pulverization, the stability of the pulverization can be improved, so that the surface of the pulverized product has a more uniform degree of roughness, thereby significantly improving the product stability of the final product, the decellularized biomatrix dressing. The decellularized biomatrix dressing is not prone to local surface reaction problems during application. Furthermore, it is also helpful to control the quality and performance of the final product in large-scale production, so as to obtain subsequent research on the target product.

[0047] (2) After mammalian tissue is decellularized, it is sequentially subjected to ion induction and pulverization to obtain a pulverized product. After surface finishing and vacuum drying, the pulverized product has a rougher surface, which improves the ability of the final product, the decellularized biomatrix dressing, to promote cell adhesion.

[0048] (3) The acellular biomatrix dressing obtained by the present invention can be directly applied to various wound surfaces when in use, reducing the cumbersomeness of using such particles in the prior art, and can be directly covered on full-thickness skin defect wound surfaces (such as deep moist wounds) and superficial wound surfaces (such as dry epidermal injuries).

[0049] (4) The decellularized biomatrix dressing composed of powder dressing A, powder dressing B and powder dressing C with a mass ratio of (0.9-1.7):1:(5.6-10.8) simulates the dynamic evolution of the natural extracellular matrix through adaptive moisture regulation and gradient degradation characteristics, can match the needs of different wound microenvironments, significantly inhibit tissue contracture during wound healing, and reduce scar hyperplasia caused by abnormal collagen arrangement. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is a scanning electron microscope image of the decellularized biomatrix dressing of Example 1, Figure 1 (a) and (b) are electron microscope images of the same position at different magnifications, where: Figure 1 (a) is the field of view at a magnification of 500 times. Figure 1 (b) is the field of view at a magnification of 1000 times; Figure 2 This is a scanning electron microscope image of the decellularized biomatrix dressing of Comparative Example 4; Figure 3This is a scanning electron microscope image of the decellularized biomatrix dressing of Comparative Example 5. DETAILED DESCRIPTION

[0051] The reagents used in the present invention are all commercially available. The natural tissues in the examples are obtained from fresh tissues of pigs (weighing about 120 kg) raised in enclosures within half an hour after death.

[0052] Example 1

[0053] This example provides a method for preparing a decellularized biomatrix dressing. The decellularized biomatrix dressing is prepared by decellularizing mammalian tissue, sequentially undergoing ion induction and pulverization to obtain a pulverized product, and the pulverized product is surface treated and vacuum dried to obtain a mixed product.

[0054] Specifically: Decellularization of mammalian tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-reared pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0055] Ion induction: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 4.1) into the container, and soak the decellularized mammalian tissue in the acid solution for 25 minutes. During the soaking, the acid solution completely immerses the decellularized mammalian tissue. After soaking for 25 minutes, immediately continue to add an alkaline solution (specifically, a 3wt% sodium hydroxide aqueous solution) into the container to neutralize to a neutral pH, then let it stand for 10 minutes, take out the treated mammalian tissue, and obtain a pre-treated material.

[0056] Crushing: After the pre-treated product was freeze-dried, it was impact crushed in liquid nitrogen to obtain a crushed product. The crushing speed was 130m / s and the crushing time was 4min to obtain crushed product A; the crushing speed was 100m / s and the crushing time was 2min to obtain crushed product B; the crushing speed was 60m / s and the crushing time was 50s to obtain crushed product C.

[0057] Surface preparation: Put the pulverized product A into pure water for ultrasonic treatment for 15 minutes, filter, discard the filtrate, and vacuum dry the residue to obtain powder dressing A; put the pulverized product B into pure water for ultrasonic treatment for 15 minutes, filter, discard the filtrate, and vacuum dry the residue to obtain powder dressing B; put the pulverized product C into pure water for ultrasonic treatment for 15 minutes, filter, discard the filtrate, and vacuum dry the residue to obtain powder dressing C.

[0058] Powder dressing A, powder dressing B and powder dressing C were mixed in a mass ratio of 1:1:7.4 to obtain a decellularized biomatrix dressing.

[0059] Example 2

[0060] This example provides a method for preparing a decellularized biomatrix dressing, which is different from Example 1 in that ion induction: a container is taken, an acid solution is added to the container (the acid solution is a mixture of hydrochloric acid aqueous solution and acetic acid aqueous solution, which is mixed by 0.025 g / mL inorganic acid aqueous solution and 0.037 g / mL organic acid aqueous solution at a volume ratio of 14:6, and then diluted with water to adjust the pH to 5.5), and the decellularized mammalian tissue is immersed in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized mammalian tissue. After immersion for 25 minutes, an alkaline solution (specifically 3wt% sodium hydroxide aqueous solution) is immediately added to the container to neutralize it to a neutral pH, and then it is allowed to stand for 10 minutes, and the treated mammalian tissue is taken out to obtain a pre-treated material.

[0061] After surface preparation and vacuum drying, powder dressing A, powder dressing B and powder dressing C were mixed in a mass ratio of 1.7:1:9.3 to obtain a decellularized biomatrix dressing.

[0062] Example 3

[0063] This example provides a method for preparing a decellularized biomatrix dressing, which is different from Example 2 in that ion induction: a container is taken, an acid solution is added to the container (the acid solution is a mixture of hydrochloric acid aqueous solution and acetic acid aqueous solution, which is mixed by 0.025 g / mL inorganic acid aqueous solution and 0.037 g / mL organic acid aqueous solution at a volume ratio of 14:6, and then diluted with water to adjust the pH to 4.5), and the decellularized mammalian tissue is immersed in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized mammalian tissue. After immersion for 25 minutes, an alkaline solution (specifically 1wt% sodium hydroxide aqueous solution) is immediately added to the container to neutralize it to a neutral pH, and then it is allowed to stand for 15 minutes, and the treated mammalian tissue is taken out to obtain a pre-treated material.

[0064] After surface preparation and vacuum drying, powder dressing A, powder dressing B and powder dressing C are mixed in a mass ratio of 1.4:1:5.6 to obtain a decellularized biomatrix dressing.

[0065] Comparative Example 1 This example provides a method for preparing a decellularized biomatrix dressing, which is different from Example 1 in that the decellularized biomatrix dressing is composed of powder dressing A and powder dressing B. The decellularized biomatrix dressing is derived from mammalian tissue and has not been subjected to enzyme treatment or cross-linking treatment.

[0066] The decellularized biomatrix dressing is prepared by decellularizing mammalian tissue, and then ion-induced and crushed in sequence to obtain a crushed product. The crushed product is surface-treated and vacuum-dried to obtain a mixed product.

[0067] Specifically: Decellularization of mammalian tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-reared pigs (weighing about 120 kg), washed with 0.2% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0068] Ion induction: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 4.3) into the container, and immerse the decellularized mammalian tissue in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized mammalian tissue. After immersion for 25 minutes, immediately continue to add an alkaline solution (specifically, a 2wt% sodium hydroxide aqueous solution) into the container to neutralize to a neutral pH, then let it stand for 10 minutes, take out the treated mammalian tissue, and obtain a pre-treated material.

[0069] Crushing: After the pre-treated product is freeze-dried, it is impact crushed in liquid nitrogen to obtain a crushed product. The crushing speed is 120m / s, and the crushing time is 5min to obtain crushed product A; the crushing speed is 70m / s, and the crushing time is 65s to obtain crushed product B.

[0070] Surface preparation: Put the crushed product A into pure water for ultrasonic treatment for 15 minutes, filter, discard the filtrate, and vacuum dry the residue to obtain powder dressing A; put the crushed product B into pure water for ultrasonic treatment for 15 minutes, filter, discard the filtrate, and vacuum dry the residue to obtain powder dressing B.

[0071] The powder dressing A and the powder dressing B were mixed in a mass ratio of 1:1 to obtain a decellularized biomatrix dressing.

[0072] Comparative Example 2 This example provides a method for preparing a decellular biomatrix dressing. The difference from Example 1 is that in the decellular biomatrix dressing, the mass ratio of powder dressing A, powder dressing B and powder dressing C is 1.7:1:12.3.

[0073] Comparative Example 3 This example provides a method for preparing a decellularized biomatrix dressing, which is different from Example 1 in that the decellularized biomatrix dressing is composed of sieved powder dressing A, sieved powder dressing B, and sieved powder dressing C. The decellularized biomatrix dressing is derived from mammalian tissue and has not been subjected to enzyme treatment or cross-linking treatment.

[0074] The sieved decellularized biomatrix dressing is prepared by decellularizing mammalian tissue, and then ion-induced and crushed in sequence to obtain a crushed product. The crushed product is surface-treated and vacuum-dried, and then sieved and mixed.

[0075] Compared with Example 1, each of the sieved powder dressings A, B and C has a narrower particle size distribution.

[0076] Specifically: Decellularization of mammalian tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-reared pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0077] Ion induction: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 6.3) into the container, and soak the decellularized mammalian tissue in the acid solution for 25 minutes. During the soaking, the acid solution completely immerses the decellularized mammalian tissue. After soaking for 25 minutes, immediately continue to add an alkaline solution (specifically, a 3wt% sodium hydroxide aqueous solution) into the container to neutralize to a neutral pH, then let it stand for 10 minutes, take out the treated mammalian tissue, and obtain a pre-treated material.

[0078] Crushing: After the pre-treated product is freeze-dried, it is impact crushed in liquid nitrogen to obtain a crushed product. The crushing speed is 150m / s, and the crushing time is 130s, and the crushing product A is obtained; the crushing speed is 90m / s, and the crushing time is 2min, and the crushing product B is obtained; the crushing speed is 70m / s, and the crushing time is 40s, and the crushing product C is obtained.

[0079] Surface finishing: The crushed products A, B and C were respectively placed in pure water for ultrasonic treatment for 15 minutes, filtered, the filtrate was discarded, and the filter residue was vacuum dried to obtain powder dressing A, powder dressing B and powder dressing C.

[0080] After powder dressing A passes through a 200-mesh sieve, particles below 200 mesh are collected, and then passed through a 230-mesh sieve again to collect sieved powder dressing A above 230 mesh. Powder dressing B passes through a 40-mesh sieve to collect sieved powder dressing B above 40 mesh. Powder dressing C passes through a 20-mesh sieve to collect sieved powder dressing C below 20 mesh.

[0081] The sieved powder dressing A, the sieved powder dressing B and the sieved powder dressing C were mixed in a mass ratio of 1.3:1:8.2 to obtain a decellularized biomatrix dressing.

[0082] Comparative Example 4 This example provides a method for preparing a decellularized biomatrix dressing. Unlike Example 1, the decellularized biomatrix dressing is derived from mammalian tissue that has been cross-linked and has not been treated with enzymes. The decellularized biomatrix dressing is made by decellularizing mammalian tissue, cross-linking (specifically, immersing in a 0.5% genipin aqueous solution, soaking for 72 hours, and washing with PBS buffer), and then ion-induced and crushed to obtain a crushed product. The crushed product is surface-treated and vacuum-dried to obtain a mixture.

[0083] Crushing: After the pre-treated product is freeze-dried, it is impact crushed in liquid nitrogen to obtain a crushed product. The crushing speed is 170m / s, and the crushing time is 4min, and the crushing product A is obtained; the crushing speed is 100m / s, and the crushing time is 3min, and the crushing product B is obtained; the crushing speed is 80m / s, and the crushing time is 70s, and the crushing product C is obtained.

[0084] Comparative Example 5 This example provides a method for preparing a decellularized biomatrix dressing. The difference from Example 1 is that the decellularized biomatrix dressing is prepared by decellularizing mammalian tissue, and then subjected to salt treatment and pulverization to obtain a pulverized product. The pulverized product is surface treated and vacuum dried to obtain a mixed product.

[0085] Specifically: Decellularization of mammalian tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-reared pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0086] Salt treatment: Take a container, add 25wt% sodium chloride aqueous solution into the container, soak the decellularized mammalian tissue in the sodium chloride aqueous solution for 20 minutes, during the soaking, the sodium chloride aqueous solution completely immerses the decellularized mammalian tissue, after soaking for 20 minutes, take out the treated mammalian tissue to obtain the pre-treated material.

[0087] The operation methods of crushing, surface finishing, vacuum drying and mixing are consistent with those in Example 1.

[0088] Comparative Example 6 This example provides a method for preparing a decellularized biomatrix dressing. The difference from Example 1 is that the decellularized biomatrix dressing is prepared by decellularizing mammalian tissue, acid-treating and pulverizing in sequence to obtain a pulverized product, and the pulverized product is surface-treated and vacuum-dried to obtain a mixed product.

[0089] Specifically: Decellularization of mammalian tissue (specifically bladder basement membrane): The bladder basement membrane was obtained from fresh pig bladders within half an hour after death of closed-reared pigs (weighing about 120 kg), washed with 0.15% peracetic acid aqueous solution for 3 hours, and then rinsed with PBS buffer solution for 10 minutes.

[0090] Acid treatment: Take a container, add an acid solution (specifically, a hydrochloric acid aqueous solution with a pH of 4.5) into the container, and immerse the decellularized mammalian tissue in the acid solution for 25 minutes. During the immersion, the acid solution completely immerses the decellularized mammalian tissue. After immersion for 25 minutes, take out the treated mammalian tissue, rinse it with water, and obtain the pre-treated material.

[0091] Crushing: After the pre-treated product is freeze-dried, it is impact crushed in liquid nitrogen to obtain a crushed product. The crushing speed is 130m / s, and the crushing time is 4min, and the crushing product A is obtained; the crushing speed is 100m / s, and the crushing time is 2min, and the crushing product B is obtained; the crushing speed is 60m / s, and the crushing time is 50s, and the crushing product C is obtained.

[0092] Surface finishing: The crushed products A, B and C were respectively placed in pure water for ultrasonic treatment for 15 minutes, filtered, the filtrate was discarded, and the filter residue was vacuum dried to obtain powder dressing A, powder dressing B and powder dressing C.

[0093] Powder dressing A, powder dressing B and powder dressing C were mixed in a mass ratio of 1.1:1:7.4 to obtain a decellularized biomatrix dressing.

[0094] Test Results 1. Morphology characterization: The natural biological particles obtained in Example 1 and Comparative Examples 4-5 were characterized by SEM scanning electron microscopy. The results are as follows: Figure 1-3 It can be observed that the particles have a fluffy and rough microscopic surface structure; while the natural biological particles of Comparative Examples 4-5 have a relatively smooth and dense surface, and the particle edges are relatively sharp.

[0095] 2. Endotoxin content: The decellularized biomatrix dressings obtained in Examples 1-3 and Comparative Examples 1-6 were respectively extracted with water for 2 h, and the endotoxin content was detected by dynamic photometry. The results are shown in Table 1: Table 1 Example Endotoxin content (EU / g) Example 1 0.68 Example 2 0.73 Example 3 0.79 Comparative Example 1 0.63 Comparative Example 2 0.55 Comparative Example 3 0.83 Comparative Example 4 2.15 Comparative Example 5 4.74 Comparative Example 6 2.77 3. Contact angle test: The contact angles of the acellular biomatrix dressings of Examples 1-3 and Comparative Examples 1-6 were obtained by droplet penetration method. Each sample was measured 10 times and the average value was taken. The smaller the contact angle, the higher the hydrophilicity and the higher the performance of promoting cell adhesion. During the test, it was found that for some samples, the contact angle hysteresis phenomenon was very obvious. Generally speaking, the rougher the microscopic surface of the material, the greater the amplitude of the contact angle hysteresis. The calculation of the contact angle hysteresis is the difference Δθ=θA -θR, where θA is the maximum contact angle (i.e., advancing contact angle), θR is the minimum contact angle (i.e., receding contact angle), the results are shown in Table 2, on this basis, the experiment was repeated, and the contact angles of the powder dressings A, powder dressings B, and powder dressings C obtained in Examples 1-3 and Comparative Examples 4-6 were tested 10 times, respectively. Obvious contact angle hysteresis was also observed in Examples 1-3, while there was basically no contact angle hysteresis or the contact angle hysteresis was not significant in Comparative Examples 4-6. The standard deviation of the 10 contact angle hysteresis Δθ of the powder dressings A, powder dressings B, and powder dressings C was calculated to reflect the discrete degree of the contact angle hysteresis. The results are shown in Table 3: Table 2 Example Contact angle (water, °) Δθ average value (°) Example 1 22.3 5.7 Example 2 25.5 5.4 Example 3 24.6 6.1 Comparative Example 1 41.7 4.9 Comparative Example 2 38.2 5.0 Comparative Example 3 27.4 4.6 Comparative Example 4 44.8 1.6 Comparative Example 5 32.6 1.2 Comparative Example 6 29.5 0.8 As can be seen from Table 2, the contact angle of the natural biological particle combination of Examples 1-3 is small, indicating that it has a high ability to promote cell adhesion. At the same time, the contact angle hysteresis of Examples 1-3 is obvious, which shows that the natural biological particle combination of Examples 1-3 has a relatively rough microscopic surface, which is also consistent with the test results of the SEM scanning electron microscope, and the rough microscopic surface is further conducive to cell adhesion.

[0096] Table 3

[0097] As can be seen from Table 3, in Examples 1-3, the standard deviation of Δθ is significantly smaller than that of Comparative Examples 4-6, which indicates that the contact angle hysteresis of Examples 1-3 is relatively stable, reflecting that each powder dressing in Examples 1-3 has a relatively uniform degree of roughness, while the roughness of each powder dressing in Comparative Examples 4-6 is relatively uneven, which means that the products obtained in Examples 1-3 are more stable.

[0098] 4. Cell experiment: It is known that overexpression or local high concentration of TGF-β1 (transforming growth factor) will stimulate fibroblasts to transform into myofibroblasts that can synthesize and secrete α-SMA (α-smooth muscle actin), leading to scar contracture. After culturing human keloid fibroblasts (purchased from Pronocell, item number CM-H235) to 3-4 generations, 10 μL of cell suspension was aspirated and inoculated into a 24-well (10 wells) plate cover glass. After 24 hours, when the cells were cultured to 80% fusion, the decellularized biomatrix dressing obtained in Example 1 was added to 2 wells, the decellularized biomatrix dressing obtained in Comparative Example 1 was added to 2 wells, the decellularized biomatrix dressing obtained in Comparative Example 2 was added to 2 wells, and the decellularized biomatrix dressing obtained in Comparative Example 3 was added to 2 wells. The aforementioned 8 wells were used as the experimental group, and the remaining 2 wells were not added with any substance as the control group. After 72 hours of continuous culture, the culture medium of the experimental group and the control group was completely aspirated, and the cells were fixed with 40% paraformaldehyde after being rinsed with PBS buffer. Then, 2 ml of BSA blocking solution was added, and the cells were incubated at 37°C in a carbon dioxide atmosphere. Rabbit anti-TGF-β1 and rabbit anti-α-SMA were added, and the cells were kept at 4°C overnight, and then washed with PBST buffer, and DAPI fluorescent dye was added. The nuclei were stained for 4 hours and observed under a fluorescence microscope. The results showed that TGF-β1 and α-SMA in the control group were significantly expressed in the cytoplasm. Compared with the control group, the expression of TGF-β1 and α-SMA in the sample group of comparative example 3 in the experimental group was close to that in the control group; the expression of TGF-β1 and α-SMA in the sample group of Example 1 in the experimental group was significantly reduced, and the expression of TGF-β1 and α-SMA in the sample groups of comparative examples 1 and comparative examples 2 in the experimental group was weakly reduced. This shows that the sample of Example 1 has a significant effect of inhibiting tissue contraction or hyperplasia and reducing scars and contractures.

Claims

1. A method for preparing an acellular biomatrix dressing, characterized in that: The decellularized biomatrix dressing is prepared by decellularizing mammalian tissue, sequentially undergoing ion induction and pulverization to obtain a pulverized product, and the pulverized product is surface treated and vacuum dried before being mixed to obtain the decellularized biomatrix dressing.

2. The method for preparing the acellular biomatrix dressing according to claim 1, characterized in that: The decellularized biomatrix dressing is not treated with enzymes.

3. The method for preparing the acellular biomatrix dressing according to claim 1, characterized in that: The mammalian tissue is selected from one or two of the small intestinal submucosa, pericardium, bladder basement membrane, peritoneum, and dermis of a mammal.

4. The method for preparing the acellular biomatrix dressing according to any one of claims 1 to 3, characterized in that: The ion induction is specifically as follows: soaking the decellularized mammalian tissue in an acid solution for 15-30 minutes, immediately adding an alkaline solution to neutralize to a neutral pH, then standing for 10-15 minutes, taking out the treated mammalian tissue, and obtaining a pre-treated product.

5. The method for preparing the acellular biomatrix dressing according to claim 1, characterized in that: The pulverization is pulverization in liquid nitrogen, and the pulverization method during pulverization in liquid nitrogen is selected from one or more of impact pulverization, shear pulverization and compression pulverization.

6. The method for preparing the acellular biomatrix dressing according to claim 5, characterized in that: The pulverized products at least include pulverized product A, pulverized product B and pulverized product C; During the crushing, the crushing speed is 120-175m / s, and the crushing time is 2-5min, and the crushing product A is obtained; during the crushing, the crushing speed is 70-100m / s, and the crushing time is 1-4min, and the crushing product B is obtained; during the crushing, the crushing speed is 60-80m / s, and the crushing time is 40-80s, and the crushing product C is obtained.

7. The method for preparing the acellular biomatrix dressing according to claim 1, characterized in that: The surface treatment is specifically to put the crushed product into pure water, perform ultrasound for 10-20 minutes, filter, discard the filtrate, and take the filter residue.

8. The method for preparing the acellular biomatrix dressing according to claim 6, characterized in that: The pulverized product A is subjected to surface finishing and vacuum drying to obtain powder dressing A; the pulverized product B is subjected to surface finishing and vacuum drying to obtain powder dressing B; the pulverized product C is subjected to surface finishing and vacuum drying to obtain powder dressing C.

9. The method for preparing the acellular biomatrix dressing according to claim 8, characterized in that: Powder dressing A, powder dressing B and powder dressing C are mixed in a mass ratio of (0.9-1.7):1:(5.6-10.8) to obtain a decellularized biomatrix dressing.

10. An application of the preparation method of the acellular biomatrix dressing according to claim 1 on wound surface materials, characterized in that: The wound surface materials include partial-layer skin wound repair materials and full-layer skin wound repair materials.

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