CN113679889A

CN113679889A - Acellular matrix composite material and preparation method and application thereof

Info

Publication number: CN113679889A
Application number: CN202110820609.3A
Authority: CN
Inventors: 俞小华; 陈家煜; 姜乃璋; 孙卫; 楼其磊
Original assignee: Hangzhou Xianshi Biotechnology Co ltd
Current assignee: Hangzhou Xianshi Biotechnology Co ltd
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2021-11-23
Anticipated expiration: 2041-07-20
Also published as: CN113679889B

Abstract

The invention discloses an acellular matrix composite material and a preparation method and application thereof, wherein the acellular matrix composite material is prepared by sterilizing, micro-granulating, acellular and the like pretreated materials from vascular tissues and dermal tissues to obtain two different types of acellular matrices, and compounding the vascular acellular matrices and the dermal acellular matrices according to a certain proportion to form the acellular biological matrix material capable of inducing local vascularization of tissues. The invention adopts a decellularization method combining a physical method and a chemical method, effectively removes antigen substances possibly causing human body immune reaction, and can reserve the original basic bracket structure and main biochemical components of extracellular matrix to the maximum extent; and the revascularization promoting factor contained in the vascular acellular matrix is fully utilized to promote early vascularization of the tissue defect, provide rich oxygen and nutrient substances for the surrounding new tissues and contribute to repair and repair of pathological changes and defective tissues.

Description

Acellular matrix composite material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedicine and tissue engineering materials, and particularly relates to a acellular matrix composite material and a preparation method and application thereof.

Background

With the development of tissue engineering and regenerative medicine, people have developed a great deal of research on scaffold materials related to tissue engineering technology, including polymer materials, metal materials, bioceramics, heterogeneous materials and the like, but the materials show certain complications or adverse reactions in clinical application. Therefore, in recent years, a great deal of interest and research has been focused on acellular matrix-derived biomaterials, and research into acellular matrix-derived biomaterials as scaffolds for tissue engineering is being actively conducted.

The tissue is composed of cells and extracellular matrix (ECM), and the purpose of preparing the acellular matrix is to remove cell components with strong immunogenicity in the tissue, such as epidermal cells, hair follicles, sebaceous glands, sweat glands, vascular endothelial cells and fibroblasts in dermis and the like, and to retain cell matrix components with relatively weak immunogenicity and a complete three-dimensional space structure of the dermal tissue. The biological scaffold material composed of ECM is widely applied to the fields of surgical reconstruction and tissue and organ regeneration medicine, such as treatment after skin burn and scald, repair after cartilage injury, hernia repair and the like. The ECM is a secretory product of cells in tissues and organs, has dynamic interaction with the cells, can reflect the change of microenvironment in time, has important effects on cell migration, differentiation and proliferation, has a three-dimensional structure which is very close to the natural environment of in-vivo cell growth, can play a role of a scaffold material, and contains various growth factors which play an important role in promoting tissue repair and reconstruction. The ECM has certain activity after cell removal treatment and is an ideal environment for cell growth, so the ECM can avoid rejection reaction and over-strong immune inflammatory reaction after transplantation without influencing the transplantation effect, and can provide a biological 'template' or a bracket for guiding tissue regeneration, thereby achieving the aim of repairing tissue defects to the maximum extent.

However, the main problem faced by the current tissue engineering scaffold is how to rapidly reconstruct the vascular system of the neogenetic tissue. Since adequate blood supply is the determining factor for ensuring the survival of the new tissue in vivo, the vasculature of the new tissue requires a strict restriction of the tissue size, and if the tissue is too large and the new blood vessels are insufficient, the tissue will not have sufficient access to nutrients and oxygen, which will lead to necrosis of the new tissue before neovascularization. Therefore, how to reconstruct the vascular system of the tissue engineering neogenetic tissue while constructing the tissue engineering neogenetic tissue becomes a critical issue for the transition of the tissue engineering technology from basic research to clinical application.

In conclusion, although the conventional acellular matrix has been widely used in the field of tissue engineering and regenerative medicine due to its components and active ingredients, it cannot form enough vascular tissue for the new tissue during the tissue repair process due to its insufficient vascularization ability, resulting in repair failure or tissue necrosis.

Therefore, there is a need in the art to provide a tissue repair material that facilitates the vascularization process in the tissue regeneration process to overcome the deficiencies of the existing conventional materials in the repair of defective tissue.

Disclosure of Invention

The present invention aims to solve the above technical problems, and provides an acellular matrix composite material, a method for preparing the acellular matrix composite material, and related applications of the acellular matrix composite material in the medical field.

The invention firstly provides an acellular matrix composite material with a vascularization induction function, which is a three-dimensional porous reticular structure formed by compounding a blood vessel acellular matrix and a dermis acellular matrix, wherein the weight ratio of the blood vessel acellular matrix to the dermis acellular matrix is 1: 1-2: 1.

Further, the vascular and dermal acellular matrices are derived from soft tissue of a mammal; preferably, the mammal includes pig, cattle and human.

Research has shown that extracellular matrix components retained after different kinds of heterogeneous or xenogeneic connective tissues are decellularized are not completely the same, and various kinds of small molecular substances play a key role in tissue repair, wherein vascular endothelial cell growth factor (VEGF) plays an important role in vascular system regeneration, and promotes vascular regeneration by promoting mitosis of vascular endothelial cells, increase of vascular permeability, and increasing chemotaxis of monocytes. The vascular tissue contains a large amount of growth factors and signal molecules related to angiogenesis, and the vascular acellular material is used as a key component of the tissue repair material, so that the vascularization process in the tissue regeneration process is facilitated.

According to the invention, the vascular acellular matrix and the dermal acellular matrix in a certain mass ratio are compounded, so that vascularization of the composite material in a tissue repair process can be remarkably promoted, the repair condition of a defective tissue is improved, and the method is an effective way for effectively improving the biological material based on the acellular matrix.

Another object of the present invention is to provide a method for preparing the above acellular matrix composite, the method for preparing the acellular matrix composite comprising the steps of:

the preparation of the vascular tissue acellular matrix material comprises the following steps:

(1) cleaning a blood vessel tissue raw material with deionized water to remove blood and dirt, cutting the whole artery into tissue raw materials with the length, width and height of the required specification and size, uniformly grinding the tissue raw materials into particles with micro particle size by using a colloid mill, and freezing and storing the tissue raw materials at low temperature;

(2) and (3) cell removal: putting the vascular tissue raw material obtained in the step (1) into an enzyme solution for degradation treatment, cleaning, centrifuging, washing to remove the enzyme solution, and putting the vascular tissue raw material into a decellularization solution for overnight treatment;

(3) washing the product obtained in the step (2) by using a washing liquid, and centrifuging to remove a supernatant;

(4) and (3) secondary decellularization: performing secondary treatment on the product obtained in the step (3) by using a cell-free solution, removing the cell-free solution on the surface by using a cleaning solution, finally cleaning by using sterile deionized water, centrifuging to remove supernatant fluid to obtain a blood vessel cell-free matrix, and performing low-temperature freezing storage;

(II) preparing the dermal acellular matrix microparticle material, which comprises the following steps:

(5) collecting dermal tissue raw material, washing blood and dirt on the dermal tissue raw material with deionized water, cutting the dermal tissue raw material into tissue raw material with required size of length, width and height, and freezing and storing at low temperature;

(6) and (3) disinfection and sterilization: slowly unfreezing, placing the tissue raw material in a disinfection solution for disinfection, and fully cleaning the tissue raw material with sterile normal saline after disinfection;

(7) micro-granulation: uniformly grinding the sterilized tissue raw material into particles with tiny particle sizes by using a colloid mill;

(8) and (3) cell removal: soaking the product obtained in the step (7) in a cell removal solution to remove cells, and then cleaning and centrifuging to remove the cell removal solution;

(9) washing the product obtained in the step (8) by using sterile normal saline, centrifuging to remove supernatant fluid to obtain dermal acellular matrix microparticles, and freezing and storing at low temperature;

(III) the preparation of the acellular matrix composite material comprises the following steps:

(10) compounding: unfreezing the vascular acellular matrix and the dermal acellular matrix microparticles, uniformly dispersing the vascular acellular matrix and the dermal acellular matrix microparticles in a solvent according to the mass ratio, and freeze-drying to obtain an acellular matrix composite;

(11) and (3) sterilization: and (3) performing terminal sterilization treatment on the acellular matrix composite.

Further, the cell removal liquid is selected from at least one of TritonX-100, hydroxyethyl piperazine ethanesulfonic acid, polyethylene glycol octyl phenyl ether, sodium deoxycholate, sodium dodecyl aminopropionate or fatty alcohol-polyoxyethylene ether; preferably, the concentration of the cell removal liquid is 0.1% -1%, and the cell removal liquid is vibrated on a shaking table for 4-24 h.

Further, the enzyme solution is at least one of nuclease, pancreatin or neutral protease, the enzyme concentration is 0.5-5%, and the degradation treatment step is shaking for 4-24h on a shaking table.

Further, the cleaning solution used for cleaning is at least one selected from phosphate buffer solution, sterile physiological saline, sterile deionized water or ethylenediamine tetraacetic acid.

Further, the cleaning step is as follows: controlling the mass ratio of the acellular matrix to the cleaning solution to be 1: 0.5-1: 5, shaking the acellular matrix on a shaking table for 0.5-2h, centrifuging the acellular matrix at 3000g for 1-10min to remove supernatant, and repeating the process for 1-5 times.

Further, the disinfectant for disinfection is selected from at least one of sodium hydroxide, alcohol, peracetic acid or hydrogen peroxide.

Further, the step of micro-granulation is: uniformly mixing the tissue raw material and sterile normal saline according to the mass ratio of 1:1-1:5, slowly pouring the mixture into a material bin in a colloid mill, and grinding at 2000-60000rpm for 1-10min to obtain microparticles with the diameter of 1-300 microns.

The mixing of step (10) may be achieved by a shaker, homogenizer or sonicator.

The radiation irradiated in step (11) is selected from one of gamma rays, electron rays or X-rays. The irradiation dose is 5-40 kGy; preferably, the irradiation dose is 20-30 kGy.

The invention also aims to provide application of the acellular matrix composite material, which comprises application of the composite material in preparing a scaffold material for promoting tissue vascularization.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method of the invention adopts a decellularization method combining a physical method and a chemical method, effectively removes antigen substances possibly causing human body immune reaction, and simultaneously furthest retains the original basic scaffold structure and main biochemical components of the extracellular matrix; on the basis, elastin in the vascular matrix is removed in the acellular process, so that the vascular matrix is not beneficial to processing and forming and maintains the mechanical strength of the vascular matrix, the non-vascular tissue matrix and the acellular matrix extracted from blood vessels are compounded, the fiber characteristics of the non-vascular acellular matrix can be reserved to obtain composite materials with different properties, the vascularization promoting factors such as VEGF and the like contained in the vascular acellular matrix are fully utilized, the vascularization at the tissue defect part can be facilitated, the effective vascularization is beneficial to providing rich oxygen and nutrient substances for the peripheral new tissues, and the repair and repair of pathological changes and defective tissues are facilitated.

Drawings

FIG. 1A is a graph showing the HE staining effect of a tissue raw material before decellularization, B is a graph showing the HE staining effect of a matrix after decellularization, C is an enlarged view of the HE staining effect of the tissue raw material before decellularization, and D is an enlarged view of the HE staining effect of the matrix after decellularization;

FIG. 2 is a graph showing the comparison of DNA content before and after decellularization of a tissue starting material;

FIG. 3 is a graph comparing the type I collagen content before and after decellularization of tissue starting material;

FIG. 4 is a graph showing the comparison of VEGF factor content before and after decellularization of the vascular tissue starting material;

FIG. 5A, B is an SEM image of pore sizes in acellular matrix composites; FIG. C, D is an SEM image of a triple helix structure of collagen in an acellular matrix composite;

FIG. 6 is a microscopic photograph of the composite of example 1 and comparative example 1 under a vascular experiment;

FIG. 7 is a graph comparing the results of quantifying capillary length in the angiogenesis test for the composite materials of example 1 and comparative example 1;

fig. 8 is a staining diagram of HE and CD31 tissue sections of blank control group, defect control group and experimental group after animal experiment rabbit endometrium defect implantation material experiment.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Example 1

A preparation method of an acellular matrix composite material comprises the following steps:

taking a blood vessel tissue raw material, washing with deionized water to remove blood and dirt, and cutting the whole artery into tissue raw materials with the length, width and height of the required specification size.

Weighing 500g of tissue raw materials, uniformly mixing the tissue raw materials with sterile normal saline according to the mass ratio of 1:3, pouring the mixture into a colloid mill, uniformly crushing the mixture, and centrifuging the mixture to remove supernatant fluid to obtain a micro-granulated intermediate; placing the micro-granulated intermediate into a pancreatin solution with the mass 3 times that of the intermediate, placing the mixture on a shaking table for oscillation treatment for 8 hours, centrifuging to remove supernatant, adding physiological saline with the mass 3 times that of the intermediate, placing the mixture on the shaking table for oscillation cleaning for 2 hours, centrifuging to remove supernatant, and repeating the cleaning process for 3 times to obtain an enzyme treated and cleaned intermediate; adding a sodium deoxycholate solution with the mass being 4 times that of the enzyme-treated and washed intermediate, placing the intermediate on a shaking table overnight, shaking the intermediate for 18 hours, centrifuging the intermediate to remove supernatant, adding EDTA phosphate buffer solution with the mass being 3 times that of the intermediate, placing the intermediate on the shaking table, shaking the intermediate for 2 hours, centrifuging the intermediate to remove supernatant, and repeatedly washing the intermediate for three times to obtain a cell-free washed intermediate; and adding 3 times of the mass of the sodium deoxycholate solution into the intermediate after the acellular cleaning, placing the intermediate on a shaking table for oscillation treatment for 2 hours, centrifuging to remove supernatant, adding 3 times of the mass of the phosphate buffer solution of EDTA, placing the intermediate on the shaking table for oscillation cleaning for 1 hour, centrifuging to remove the supernatant, and repeating the process for 2 times by using water for injection to obtain the intermediate of the vascular acellular matrix.

And (II) cleaning and cutting the obtained dermal tissue, weighing 500g of the dermal tissue, uniformly mixing the dermal tissue with a peroxyacetic acid solution according to the mass ratio of 1:3, placing the mixture on a shaking table, shaking overnight for 24 hours, and centrifuging a centrifugal bottle after shaking to remove supernatant. Adding sterile physiological saline with the mass of 3 times into the precipitate, placing the precipitate on a shaking table, shaking and cleaning for 10min, centrifuging a centrifugal bottle after shaking is finished, removing supernatant, and repeating the process for 3 times to obtain a sterilized intermediate.

Uniformly mixing the sterilized intermediate and sterile normal saline according to the mass ratio of 1:3, pouring the mixture into a colloid mill for grinding for 3min, collecting the ground micro particles, filling the particles into a centrifugal bottle, and centrifuging to remove supernatant to obtain the micro-granulated intermediate.

Uniformly mixing the micro-granulated intermediate and the sodium deoxycholate solution according to the mass ratio of 1:4, placing the mixture on a shaking table, shaking the mixture overnight for 24 hours, and centrifuging a centrifugal bottle after shaking to remove supernatant. Adding sterile physiological saline 4 times the mass of the mixture, placing the mixture in a shake washing tank for 10min, centrifuging a centrifugal bottle after shaking to remove supernatant, and repeating the process for 10 times to obtain a dermal acellular matrix rear intermediate.

And (III) mixing the intermediate of the vascular acellular matrix with the intermediate of the dermal acellular matrix according to the mass ratio of 1:1, uniformly mixing the mixture with sterile deionized water according to the mass ratio of 1:1, putting the mixture into a mould, freezing the mixture for 24 hours at the temperature of minus 20 ℃ for forming, freezing and drying the mixture for 48 hours at the temperature of minus 50 ℃, putting the mixture into a closed container, and performing irradiation by 20kGy (irradiation dose unit) and then storing the mixture at room temperature.

Characterization example 1

HE staining was performed on the biological tissues before and after decellularization in example 1, and the staining results are shown in FIG. 1, and it is apparent from FIG. 1(A) that a large number of nuclei exist in the cells of the tissues before decellularization, and it is apparent from FIG. 1(B) that the nuclei of the tissues after decellularization are completely removed, demonstrating that the preparation method can efficiently remove the cells and antigens.

Characterization example 2

100mg of the biological Tissue material before and after decellularization of example 1 was analyzed with DNeasy Blood & Tissue Kit (Qiagen) and the change in DNA content before and after decellularization was calculated, and the results are shown in FIG. 2.

As is evident from FIG. 2, the DNA content of the decellularized tissue was much lower than that of the tissue before decellularization, demonstrating that the preparation method can efficiently decellularize.

Characterization example 3

Taking 100mg of the biological tissue material before and after decellularization of example 1, hydrolyzing the material at 110 ℃ by using 6mol/L hydrochloric acid to prepare a test solution, precisely measuring 0.5mL of blank (water), a hydroxyproline control solution and the test solution, respectively adding 1mL of isopropanol and 0.5mL of an oxidant solution, mixing, standing at room temperature for 4min, respectively adding 6.5mL of a color developing agent, mixing, placing each tube in a water bath at 60 ℃ for heating for 15min, cooling, measuring an absorbance value by using a blank control and 560nm, and performing linear regression on the absorbance by using the concentration of the hydroxyproline control solution to obtain a regression equation, thereby calculating the content of hydroxyproline in the test solution, wherein the result is shown in FIG. 3.

The collagen content was calculated as follows:

collagen content (%) ═ hydroxyproline content (%) ÷ 13% × 100%, where 13% is the proportion of hydroxyproline in collagen.

It is apparent from fig. 3 that no significant difference in collagen content occurred before and after decellularization of the biological tissue material.

Characterization example 4

200mg of the biological tissue material before and after decellularization of example 1 was taken, analyzed and processed by using a Porcine (Porcine) vascular endothelial cell growth factor (VEGF) ELISA test kit, and the change of VEGF factor content before and after decellularization was calculated, and the result is shown in FIG. 4.

It is apparent from fig. 4 that there is a significant difference in VEGF content before and after decellularization of the biological tissue material, indicating that the decellularization step will release VEGF contained in the cells of the biological tissue, and although some of the factors will be eliminated during the decellularization step, a large amount of VEGF will be adsorbed on the collagen fibers, and the total VEGF concentration of the material will increase.

Characterization example 5

The lyophilized material of example 1 was photographed by scanning electron microscopy, and the results are shown in fig. 5. It is evident from A, B pieces of low power lens in FIG. 5 that the acellular matrix composite has a three-dimensional porous network structure, and that the collagen in the material retains a complete triple helix structure when C, D pieces of high power lens are used.

Comparative example 1

Examination of mixing ratio:

referring to the method of example 1, tissue materials derived from vascular tissues and dermal tissues after pretreatment were subjected to sterilization, micro granulation, decellularization, and the like to obtain different types of acellular matrices. Mixing the vascular acellular matrix and the dermal acellular matrix according to the proportion of 0:1, 1:2, 2:1 and 1:0 respectively, adding sterile deionized water with equal mass after mixing, stirring and mixing uniformly, putting into a closed container after freeze drying, and obtaining the composite material with the vascular acellular matrix content of 0%, 33%, 67% and 100% after being irradiated by 20kGy (irradiation dose unit) and stored at room temperature.

Experimental example 1

The composite materials of example 1 and comparative example 1 were subjected to hemangiogenesis experiments, 0.5g of each material was weighed, 5mL of the culture medium was added at a ratio of 0.1g/mL, the mixture was extracted at 37. + -. 1 ℃ for 24h in a constant temperature shaking chamber, and the culture medium was taken for hemangiogenesis experiments, the results of which are shown in FIG. 6.

As is apparent from fig. 6, the composite material with the vascular acellular matrix content of 50% and 67% has better vascularization effect compared with other groups, and is proved to have quantitative results on the capillary length as shown in fig. 7, but the two groups of materials with the vascular acellular matrix content of 50% and 67% have no significant difference in value, which indicates that the material has stronger vascularization performance when the vascular acellular matrix content of the composite material is between 50% and 67%.

Experimental example 2

By comparative analysis of the experimental results of experimental example 1, a composite material having a vascular acellular matrix content of 67% was selected as a material for the experimental group of the subsequent animal experiments.

The material was processed to a suitable size and implanted into an in vivo model of endometrial uterine horn injury in rats as an experimental group, rats without any treatment were used as a blank group, and rats without the material after molding were used as a control group. After the end of the three estrus periods, the repair of endometrial lesions was assessed by appropriate histological analysis, the results are shown in fig. 8.

As is apparent from the HE and CD31 tissue sections shown in fig. 8, the endometrium layer of the defect control group is obviously thinner than that of the blank control group, the uterine tissue of the experimental group is obviously thicker than that of the defect control group and is slightly thinner than that of the blank group, and the material has obvious function of promoting tissue regeneration on the defect part of the uterine tissue.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. The acellular matrix composite is characterized by being formed by compounding a blood vessel acellular matrix and a dermis acellular matrix and having a three-dimensional porous reticular structure, wherein the weight ratio of the blood vessel acellular matrix to the dermis acellular matrix is 1: 1-2: 1.

2. The acellular matrix composite according to claim 1, wherein the vascular and dermal acellular matrices are derived from soft mammalian tissue; preferably, the mammal includes pig, cattle and human.

3. A method of preparing the acellular matrix composite material according to claim 1 or 2, comprising the steps of:

preparation of vascular tissue acellular matrix

(1) Pretreatment: taking a blood vessel tissue raw material, cleaning to remove blood and dirt, cutting the whole artery into tissue raw materials with required specification and size, uniformly grinding the tissue raw materials into particles with small particle size, then putting the particles into an enzyme solution for degradation treatment, cleaning and centrifuging to remove the enzyme solution, and putting the particles into a cell removal solution for overnight treatment;

(2) preparation of vascular acellular matrix: cleaning the product obtained in the step (1), centrifuging to remove supernatant, performing secondary treatment on the obtained product by using cell removing liquid, cleaning to remove the cell removing liquid on the surface, centrifuging to remove the supernatant, and obtaining the vascular acellular matrix;

(II) preparation of dermal tissue acellular matrix microparticles

(3) Pretreatment: collecting dermal tissue raw material, cleaning to remove blood and dirt, cutting into tissue raw material with required specification and size, sterilizing and cleaning;

(4) dermal acellular matrix microparticulation: uniformly grinding the sterilized tissue raw materials into particles with small particle sizes, soaking the particles in a cell removal solution to remove cells, and cleaning and centrifuging the cells to remove the cell removal solution; washing the obtained substance with sterile normal saline, centrifuging to remove supernatant to obtain dermal acellular matrix microparticles;

(III) preparation of acellular matrix composite

(5) Compounding: dispersing the vascular acellular matrix and dermal acellular matrix microparticles in a solvent uniformly according to a weight ratio, and freeze-drying to obtain the acellular matrix composite.

4. The preparation method according to claim 3, wherein the cell removal solution is at least one selected from the group consisting of Triton X-100, hydroxyethylpiperazine ethanesulfonic acid, polyethylene glycol octylphenyl ether, sodium deoxycholate, sodium dodecylaminopropionate, and fatty alcohol-polyoxyethylene ether; preferably, the concentration of the cell removal liquid is 0.1% -1%, and the cell removal liquid is vibrated on a shaking table for 4-24 h.

5. The method according to claim 3, wherein the enzyme solution in step (1) is at least one selected from the group consisting of nuclease, pancreatin and neutral protease, the enzyme concentration is 0.5-5%, and the degradation treatment step is performed by shaking on a shaker for 4-24 hours.

6. The method according to claim 3, wherein the washing solution used for washing is at least one selected from the group consisting of phosphate buffer, sterile physiological saline, sterile deionized water, and ethylenediaminetetraacetic acid.

7. The method of claim 6, wherein the cleaning step comprises: controlling the mass ratio of the acellular matrix to the cleaning solution to be 1: 0.5-1: 5, shaking the acellular matrix on a shaking table for 0.5-2h, centrifuging the acellular matrix at 3000g for 1-10min to remove supernatant, and repeating the process for 1-5 times.

8. The method according to claim 3, wherein the disinfectant for disinfection in step (3) is at least one selected from the group consisting of sodium hydroxide, alcohol, peracetic acid, and hydrogen peroxide.

9. The method for preparing according to claim 3, characterized in that said step of microparticulating is: uniformly mixing the tissue raw material and sterile normal saline according to the mass ratio of 1:1-1:5, slowly pouring the mixture into a material bin in a colloid mill, and grinding at 2000-60000rpm for 1-10min to obtain microparticles with the diameter of 1-300 microns.

10. Use of the acellular matrix composite according to any one of claims 1 to 2 or produced by the production process according to any one of claims 3 to 9, characterized in that it comprises the use of this composite for the preparation of a scaffold material for promoting vascularization of tissues.