CN113583933A

CN113583933A - Powdery thin-cut extracellular matrix microcarrier crushed by vibration thin-cutting and screening rotation and preparation method thereof

Info

Publication number: CN113583933A
Application number: CN202110616152.4A
Authority: CN
Inventors: 黄庆成
Original assignee: Basite Pharmaceutical Technology (changzhou) Co ltd
Current assignee: Basite Pharmaceutical Technology (changzhou) Co ltd
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2021-11-02

Abstract

The invention provides a powdery thin-cut extracellular matrix microcarrier crushed by vibration thin-cutting and screening rotation and a preparation method thereof, the preparation of the powdery thin-cut extracellular matrix microcarrier sequentially comprises the steps of rough skin taking treatment, relaxation of a decompression structure, vibration thin-cutting, section purification, structure purification, rotary screening purification, supercritical carbon dioxide aqueous solution extraction and washing purification and rotary screening crushing treatment to obtain the powdery thin-cut extracellular matrix microcarrier with high uniformity and high purity, and the whole treatment process of the invention uses non-toxic chemical reagents, not only can effectively reduce the immunogenicity of a dermal matrix, but also can fully retain the normal collagen structure of the dermal matrix, has short treatment period, is economic and environment-friendly, realizes a method for preparing the powdery thin-cut extracellular matrix microcarrier with high efficiency, and the obtained powdery thin-cut extracellular matrix microcarrier has better size uniformity, miniaturization and high purity.

Description

Powdery thin-cut extracellular matrix microcarrier crushed by vibration thin-cutting and screening rotation and preparation method thereof

Technical Field

The invention relates to the field of biomedical nano materials, in particular to a powdery thin-cut extracellular matrix microcarrier which is pulverized by vibration thin cutting and screening rotation and a preparation method thereof.

Background

Acellular matrix (ADM) is prepared by removing completely the cell components in the epidermis, subcutaneous tissue and dermis of human cadaver skin or animal skin by physical, chemical and other methods, and only the extracellular matrix containing collagen network in dermis is remained. The acellular dermal matrix is derived from natural skin, and after being treated by a certain physicochemical method, the acellular dermal matrix removes epidermal layers in the skin and cells containing antigen components, only retains collagen components, three-dimensional space structures and basement membrane structures of the dermis, has low immunogenicity and good biocompatibility and mechanical properties, and is an ideal skin repairing and filling material. The tissue repair of acellular matrices is based on the theory of Guided Tissue Regeneration (GTR). When tissue injury occurs, the injured local fibrous connective tissue can be pathologically proliferated to form tissue adhesion, so that local relative anatomical relation is disturbed, and therefore, dysfunction is generated. After the acellular matrix is implanted into the tissue, a layer of physical barrier is formed locally, so that the local tissue adhesion of the wound and the healing process of pathological hyperplastic tissue of the wound can be prevented. The acellular matrix has the local function of separating different tissues through the physical barrier function of the acellular matrix, so that the different tissues respectively complete the healing process, and finally the normal anatomical structure of the local tissues is reconstructed. Acellular matrix has been widely used in clinical practice in otolaryngological department (tympanic membrane repair, correction of nasal deformity, pharyngeal angioplasty), face-to-face cosmetic (lip augmentation, eye cosmetic, facial tissue augmentation), neurosurgery, general surgery, and the like. The acellular matrix has rapid vascularization and higher stability. Collagen fibers in the acellular matrix and matrix components in small blood vessels are helpful for guiding the growth of receptor cells and new blood vessels to form a new extracellular matrix which is used for replacing the implanted acellular matrix; in addition, the implanted acellular matrix may impart toughness, elasticity, water retention, and buffer to mechanical forces to the tissue, and provide a microenvironment for the cells to survive and perform various activities. Based on many characteristics of acellular matrix, acellular matrix can be used in the fields of facial filling and repair in medical cosmetology, biomedicine and tissue engineering.

At present, the preparation methods of the acellular dermal matrix mainly comprise the following methods: 1. dispase II-Triton method: dermis was first treated with Dispase ii and then treated with triton x-100. The method has thorough cell removal, but has great damage to a basement membrane, and is not beneficial to the adhesion and growth of epithelial cells; 2. hypertonic salt-SDS method: the dermis is firstly treated by hypertonic salt, and then cell components in the dermis are thoroughly removed by a film breaking agent sodium dodecyl benzene sulfonate (SDS). The basement membrane of the method is kept intact, but the content of cellulose, elastin and the like in the dermis is higher, and higher immunogenicity is possible; 3. high-permeability salt-sodium hydroxide cavitation method: firstly, hypertonic salt is used for removing epidermis, and then the cells are dehydrated and killed through the water absorption effect of strong alkali ions in sodium hydroxide, so that the cell components in the dermis are completely removed. The collagen fiber structure of the method is complete, the arrangement is normally loose, but the cell removal effect is still to be improved; 4. the repeated freeze thawing method carries out repeated freeze thawing on fresh skin for more than 3 times, and has long operation time, long period and low efficiency. Therefore, the existing preparation techniques for acellular matrixes are various, but the preparation, application and batch production of the acellular matrixes have many defects, such as: the cell components are not completely removed, the antigenicity is high, the histocompatibility is poor, the destructiveness to collagen structures and basement membrane components is large, and the adhesion and the growth of epithelial cells are not facilitated; the preparation process is complex, and the preparation period is too long; high preparation cost and the like. In addition, various heterologous enzymes such as trypsin, pepsin, Dispase II and the like are used in the preparation process of many acellular matrixes, and these heterologous proteins are not easy to remove, and when the acellular matrixes obtained by enzyme treatment are used in human bodies, problems such as immunogenicity and the like are easily caused.

Therefore, aiming at the defects in the existing acellular matrix preparation technology, the invention provides a powdery thin-cut extracellular matrix microcarrier (SCDM) technology which utilizes vibration thin cutting and screening rotary crushing and a preparation method thereof.

Disclosure of Invention

Therefore, the present invention provides a powder-state thin-cut extracellular matrix microcarrier which is pulverized by vibration thin-cutting and screening rotation and has a three-dimensional support suitable for tissue repair growth, and a preparation method thereof.

The technical scheme of the invention is realized as follows:

the preparation method of the powdery thin-cut extracellular matrix microcarrier by utilizing structure segmentation purification sequentially comprises the steps of crude skinning treatment, pressure reduction structure relaxation, vibration thin cutting, section purification, structure purification, rotary screening purification, supercritical carbon dioxide aqueous solution extraction and washing purification and rotary screening crushing treatment to obtain the powdery thin-cut extracellular matrix microcarrier with high uniformity and high purity.

Further, the preparation method is characterized in that: the method comprises the following steps:

step (1): and (3) skin taking and rough treatment: selecting an animal skin layer, unhairing a skin taking area, taking the skin, and disinfecting to obtain a first skin membrane, wherein the first skin membrane does not contain subcutaneous adipose tissues;

step (2): and (3) relaxation of the decompression structure: collapsing the first skin membrane under the action of a pressure lower than normal pressure by adopting a decompression condition to obtain a second skin membrane with a relaxed tissue structure;

and (3): vibrating and thin cutting: carrying out vibration thin cutting on the second skin membrane with a loose tissue structure to obtain a first thin-cut skin membrane with a uniform section;

and (4): section purification: performing section purification on the first thin-cut involucra with uniform sections by adopting sterile water to obtain a second thin-cut involucra;

and (5): structure purification: cutting the second thin-cut film, and performing structure purification reaction for 0.5-24 hours at the reaction temperature of 4-35 ℃ by using structure purification washing liquor, wherein the pH value is controlled to be 7-12, so as to obtain a third thin-cut film;

and (6): and (3) rotary screening and purifying: performing rotary screening purification on the third thin-cut film by adopting rotary screening equipment with a first screen of 10-100 meshes to obtain a first screening thin-cut film retained in the first screen and a first powder thin-cut film outside the first screen;

and (7): and (3) supercritical carbon dioxide aqueous solution extraction, washing and purification: carrying out extraction washing and purification on the first screened thin-cut epithelial membrane and the first powder thin-cut epithelial membrane by adopting an isolated supercritical carbon dioxide aqueous solution at the temperature of 4-35 ℃ to obtain a high-purity first screened thin-cut extracellular mesenchymal epithelial membrane and a high-purity first powder thin-cut extracellular mesenchymal epithelial membrane;

and (8): and (3) rotary screening and crushing treatment: and (3) carrying out rotary screening and rotary crushing on the first powdery thin-cut extracellular matrix epithelial membrane by adopting screening rotary crushing equipment with a second screen to obtain the powdery thin-cut extracellular matrix microcarrier with high uniformity.

Preferably, the sieving rotary pulverizing apparatus is an animal tissue pulverizing and sieving machine including: the crushing module, the crushing and screening cabin, the screening and collecting module and the gas inlet and outlet control valve are arranged on the crushing and screening cabin;

wherein the crushing module comprises a crushing motor, a crushing bearing and a crushing blade;

the crushing and screening cabin comprises an upper crushing and screening cabin cover, a cylindrical crushing and screening cabin body, a lower crushing and screening cabin cover, accommodating spaces and a sealable animal tissue feed port, wherein the accommodating spaces are sequentially formed by combining the upper crushing and screening cabin cover, the cylindrical crushing and screening cabin body and the lower crushing and screening cabin cover;

the screening and collecting module comprises a screen and a screening and collecting disc, wherein the central positions of the screen and the screening and collecting disc are respectively provided with a screen central perforation and a screening and collecting disc central perforation, the screen central perforation and the screening and collecting disc central perforation are used for supporting the bottom of the crushing bearing, and the screening and collecting module is detachably fixed in the crushing and screening cylindrical cabin; in the containing space in the crushing and screening cylindrical cabin, the crushing motor rotates the crushing bearing to drive the crushing blade to crush animal tissues, the crushing bearing is arranged below the crushing blade through the screening and collecting module, and the screen screens the crushed animal tissues and falls into the screening and collecting disc; and the gas inlet and outlet control valve is used for injecting regulating gas to regulate the surface property and temperature of the animal tissue.

Preferably, the conditioning gas is any one selected from the group consisting of liquid nitrogen, dry ice, ozone, and high temperature steam.

Preferably, the rotary screening purification in the step (6) adopts common rotary screening equipment, and also can utilize the Rich mingming rotary screening crushing equipment to replace crushing blades with dispersing rods.

Further, in the step (1), the benzalkonium bromide or peracetic acid with the mass concentration of 0.5-5% is adopted for disinfection.

Further, in the step (2), the first coating is disintegrated under a force of 50 to 500 torr which is lower than the atmospheric pressure for 2 to 8 hours.

Further, the vibration thin cutting is to adjust the skinning thickness parameter to be 0.1-5 mm by using vibration thin cutting equipment with thin cutting thickness parameter control, and to perform thin cutting by using a vibration thin cutting blade module to obtain a first thin cutting skin membrane with a uniform section.

Preferably, the vibration thin cutting comprises a conveying carrying platform and a vibration thin cutting blade module arranged above the conveying carrier, and the second film passes through the thin cutting blade of the vibration thin cutting blade module by the conveying carrying platform to obtain the first thin cutting film.

Further, the sterile water used for section purification is 1-20 times of the weight of the thin-cut involucra.

Further, in the structure purification, the second thin-cut skin membrane is cut into a sheet shape with the size of 1 cm × 1 cm to 10 cm × 10 cm, and the use amount of the structure purification washing liquid is 10 to 50 times of the weight of the second thin-cut skin membrane.

Further, the structural purification washing liquid is one or a combination of more of an alkali reagent aqueous solution, an acid reagent aqueous solution, a supercritical water solution and a surfactant reagent aqueous solution.

More preferably, the alkali reagent aqueous solution is one or more of a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a calcium hydroxide aqueous solution, a sodium sulfide aqueous solution, a calcium thioacetate aqueous solution, an ammonium hydroxide aqueous solution and an ammonia aqueous solution.

The acid reagent aqueous solution is one or a combination of more of acetic acid reagent aqueous solution, hydrochloric acid reagent aqueous solution, fruit acid, hydrogen oxygen acid and hypochlorous acid reagent aqueous solution.

The supercritical water solution is one or a combination of more of a supercritical carbon dioxide pure water solution, a supercritical carbon dioxide/methanol water solution, a supercritical carbon dioxide/ethanol water solution, a supercritical carbon dioxide/propanol water solution and a supercritical carbon dioxide/butanol water solution, and is used at a temperature of 4-35 ℃ and a pressure of 30-300 bar.

The surfactant reagent aqueous solution is an aqueous solution of one or more of octyl phenol polyoxyethylene ether, sorbitan monostearate, polysorbate, poloxamer, nonoxynol, cetyl alcohol, alkyl polyglucoside, lauryl sulfonic acid, dodecyl sulfonic acid, sodium dodecyl sulfonate, dodecyl benzene sulfonic acid, tridecyl benzene sulfonic acid, alkyl phenoxy benzene disulfonic acid, naphthalene sulfonic acid, alkyl naphthalene sulfonic acid and alkenyl naphthalene.

Further, the rotating speed of the rotary screening purification is 1500-5000 r/min.

Further, the first screen is selected from a number 3-9 first mesh having a mesh diameter of 3 mm-9 mm.

Further, the supercritical carbon dioxide aqueous solution is extracted, washed and purified as follows: and placing the first screening thin-cut film and the first powder thin-cut film into a separation cavity, and carrying out extraction washing and purification for 0.5-24 hours.

Further, the rotating speed of the rotating screening and crushing treatment is 1500 to 5000 revolutions per minute.

Further, the second screen is selected from a second mesh ranging from 0.1 to 3, the second mesh having a mesh diameter ranging from 0.1 mm to 3 mm.

Further, the powder-state thin-cut extracellular matrix microcarrier has a particle size distribution in the range of 50-500 microns.

The powdery thin-cut extracellular matrix microcarrier is prepared by the preparation method of the powdery thin-cut extracellular matrix microcarrier by utilizing the structure sectional purification.

Compared with the prior art, the invention has the beneficial effects that: the invention adopts effective combination of decompression relaxation treatment, vibration thin cutting, purification treatment of different sections according to structural characteristics and rotary screening pulverization to prepare the powdery thin-cut extracellular matrix microcarrier, and the powdery thin-cut extracellular matrix microcarrier has good suitability as a three-dimensional support for tissue repair and growth.

Wherein, the effective combination of the decompression relaxation treatment, the vibration thin cutting and the purification treatment of different segments according to the structural characteristics is realized, the membrane structure is fully pulled open by using the decompression relaxation effect, which is not only beneficial to improving the effect of the vibration thin cutting and has more uniform section, but also beneficial to combining the section purification, the structure purification, the rotary screening purification and the supercritical carbon dioxide aqueous solution extraction and washing purification treatment effects of different segments, so that the hydrophobic grease on the surface layer of the membrane structure and the hydrophilic substances in the membrane structure are fully removed and purified, the cells and the antigens are thoroughly removed, heterologous proteins (enzymes) are not introduced, the tissue microstructure, the mechanical property and the immunogenicity are kept, meanwhile, the screening and the crushing are simultaneously carried out by using the screening and rotary crushing steps, the uniformity is further improved, and the synchronous rotary crushing is carried out, so that the screening and crushing efficiency is greatly improved, the method has the advantages that residual cells and impurities are removed more effectively, the size uniformity and the purification efficiency of the prepared powdery thin-cut extracellular matrix microcarrier are improved, the achievement of microminiaturization is facilitated, and the purity of the prepared powdery thin-cut extracellular matrix microcarrier is greatly improved.

Drawings

FIG. 1 is a schematic structural diagram of a vibrating thin-cutting apparatus 50 for thin-cutting extracellular matrix microcarriers in powder form by vibrating thin-cutting and sieving and rotating pulverization according to a second embodiment of the present invention; wherein 103 the second skin, 104 the first thin cut skin, 510 the vibration thin cutting blade module, 515 the thin cutting blade, 530 the transport carrier;

FIG. 2 is a scanning electron microscope of the present invention: (A) example five a first thin cut skin membrane prepared prior to acellular matrix of porcine dermis and (B) a powdered thin cut extracellular matrix microcarrier prepared in example five;

FIG. 3 is a staining pattern of the nuclear Dapi reagent of the present invention: (A) example five a first thin cut skin membrane prepared prior to acellular matrix of porcine dermis and (B) a powdered thin cut extracellular matrix microcarrier prepared in example five;

FIG. 4 is a HE staining pattern of the present invention: (A) example five a first thin cut skin membrane prepared prior to acellular matrix of porcine dermis and (B) a powdered thin cut extracellular matrix microcarrier prepared in example five;

FIG. 5 is a staining chart of the stellerin reagent of the present invention, (A) the first thin-cut skin membrane prepared before the acellular matrix of the dermis of the five pigs in the example and (B) the powdery thin-cut extracellular matrix microcarrier prepared in the example;

FIG. 6 is a staining pattern of glycosaminoglycan, an Ailantain reagent, according to the present invention, (A) a first thin-cut dermal membrane prepared before acellular matrix of dermis of five pigs in example and (B) a powdery thin-cut extracellular matrix microcarrier prepared in example five;

FIG. 7 is a comparison of cytotoxicity experiments of the present invention (A) preparation of a first thin-cut skin membrane before acellular matrix of dermis of five pigs in example and (B) preparation of a powdery thin-cut extracellular matrix microcarrier in example five;

FIG. 8 is a scanning electron microscope image of the cell adhesion behavior of L929 according to the present invention, (A) the first thin-cut skin membrane prepared before the dermal acellular matrix of the fifth example and (B) the powdery thin-cut extracellular matrix microcarrier prepared in the fifth example;

FIG. 9 is a graph showing the comparison of the results of nucleic acid residues in the present invention, (A) the first thin-cut membrane prepared before the acellular matrix of pig dermis in example five and (B) the powdery thin-cut extracellular matrix microcarrier prepared in example five;

FIG. 10 is a graph comparing the results of hemolysis experiments of the present invention (A) the first thin-cut membrane prepared before the acellular matrix of the dermis of the fifth pig example and (B) the powdered thin-cut extracellular matrix microcarrier prepared in the fifth example;

FIG. 11 is a schematic diagram of a powder-state thin-sliced extracellular matrix microcarrier prepared according to the fifth embodiment of the present invention, (A) an optical microscope image and (B) a one hundred-fold SEM image.

FIG. 12 is a schematic diagram of the construction of animal tissue milling screen 10 of the present invention; the crushing module 100, the crushing motor 110, the crushing bearing 120, the crushing blade 130, the crushing and screening cabin 200, the crushing and screening cabin upper cover 210, the crushing and screening cylindrical cabin 220, the crushing and screening cabin lower cover 230, the animal tissue feed inlet 240 capable of being sealed, the containing space 250, the crushing and screening cabin lower cover 260, the crushing and screening cabin upper cover 270, the elastic fixing module 280, the screening and collecting module 300, the screen 310, the screening and collecting tray 330, the control module 400 and the gas inlet and outlet control valve 500.

Fig. 13 is a schematic view showing the structure of the animal tissue pulverizer/sifter 10 according to the present invention in a combined state of (a) a screen and (B) a screen/sifting collection plate.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

First embodiment

The first embodiment of the invention discloses a powdery thin-cut extracellular matrix microcarrier crushed by vibration thin-cutting and screening rotation and a preparation method thereof, wherein the preparation of the powdery thin-cut extracellular matrix microcarrier sequentially comprises the steps of crude skinning treatment, relaxation of a pressure reduction structure, vibration thin-cutting, section purification, structure purification, rotary screening purification, supercritical carbon dioxide aqueous solution extraction and washing purification and rotary screening crushing treatment to obtain the powdery thin-cut extracellular matrix microcarrier with high uniformity and high purity.

Second embodiment

The second embodiment of the present invention discloses a powder-state thin-cut extracellular matrix microcarrier pulverized by vibration thin-cutting and screening rotation, and the preparation method of the powder-state thin-cut extracellular matrix microcarrier comprises the following steps:

step (1): and (3) skin taking and rough treatment: selecting a fresh animal skin layer, unhairing a skin taking area, taking the skin, and sterilizing to obtain a first skin membrane, wherein the first skin membrane does not contain subcutaneous adipose tissues;

step (2): and (3) relaxation of the decompression structure: under the condition of reduced pressure, the first skin membrane is collapsed for 2-8 hours under the action force of 50-500 torr lower than the atmospheric pressure so as to relax the tissue structure, and a second skin membrane with a relaxed tissue structure is obtained;

and (3): vibrating and thin cutting: performing vibration thin cutting on a second skin membrane with a loose tissue structure by using vibration thin cutting equipment with thin cutting thickness parameter control, adjusting the thickness of a skin taking thickness parameter to be 0.1-5 mm, and enabling the obtained first thin cut skin membrane to have a uniform section due to the loose tissue structure, wherein in the vibration thin cutting equipment 50, as shown in fig. 1, the second skin membrane 103 is conveyed by a conveying carrying platform 530 through a thin cutting blade 515 of a vibration thin cutting blade module 510 to obtain a first thin cut skin membrane 104; (ii) a

And (4): section purification: purifying the first thin-cut involucra with uniform sections by adopting sterile water with the weight 1-20 times that of the thin-cut involucra, and removing attachments on the sections to obtain a second thin-cut involucra;

and (5): structure purification: cutting the second thin-cut film into sheets with the size of 1 cm multiplied by 1 cm-10 cm multiplied by 10 cm, performing structure purification reaction for 0.5-24 hours at the reaction temperature of 4-35 ℃ by adopting structure purification washing liquor with the weight 10-50 times that of the second thin-cut film, controlling the pH value between 7-12, and removing stripped substances generated due to loose tissue structure to obtain a third thin-cut film;

wherein, the structure purification washing liquid is one or a combination of more of alkali reagent aqueous solution, acid reagent aqueous solution, supercritical water solution and surfactant reagent aqueous solution;

the alkali reagent aqueous solution is one or a combination of more of a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a calcium hydroxide aqueous solution, a sodium sulfide aqueous solution, a calcium thioacetate aqueous solution, an ammonium hydroxide aqueous solution and an ammonia aqueous solution.

And (6): and (3) rotary screening and purifying: the third thin-cut film is subjected to rotary screening purification by adopting rotary screening equipment with a first screen mesh of 10-100 meshes, and the first screening thin-cut film which is subjected to high-uniformity purification and is retained in the first screen mesh and the first powder thin-cut film outside the first screen mesh can be obtained simultaneously through the screening purification treatment, so that the screening efficiency is improved through rotation;

and (7): and (3) supercritical carbon dioxide aqueous solution extraction, washing and purification: placing the first screened thin-cut epithelial membrane and the first powder thin-cut epithelial membrane into a separation cavity, and performing high-permeability extraction and purification at the temperature of 4-35 ℃ by adopting isolated supercritical carbon dioxide aqueous solution to completely remove residual small molecules to obtain high-purity first screened thin-cut extracellular mesenchymal epithelial membrane and first powder thin-cut extracellular mesenchymal epithelial membrane;

and (8): and (3) rotary screening and crushing treatment: the first powdery thin-cut extracellular interstitial epithelium is subjected to rotary screening and rotary crushing by adopting screening rotary crushing equipment with a second screen, and the powdery thin-cut extracellular interstitial microcarrier with high uniformity is efficiently obtained by the rotary screening and rotary crushing.

The sieving rotary pulverizing apparatus can utilize the animal tissue pulverizing and sieving machine 10 designed by the present invention, as shown in fig. 12, which comprises a pulverizing module, a pulverizing and sieving compartment 200, a sieving and collecting module 300, and a gas inlet and outlet control valve 500, wherein the pulverizing module 100 comprises a pulverizing motor 110, a pulverizing bearing 120, a pulverizing blade 130; the crushing and screening compartment 200 comprises an upper crushing and screening compartment cover 210, a cylindrical crushing and screening compartment body 220, a lower crushing and screening compartment cover 230, and an accommodating space 250 and a sealable animal tissue feed port 240 arranged in the crushing and screening compartment 200, which are sequentially combined; the sieving collection module 300 comprises a sieve 310 and a sieving collection tray 330, wherein the sieve 310 and the sieving collection tray 330 are respectively provided with a sieve center perforation 311 and a sieving collection tray center perforation 331 at the center positions, the sieve center perforation 311 and the sieving collection tray center perforation 331 are used for supporting the bottom of the crushing bearing 120, and the sieving collection module 300 is detachably fixed in the crushing and sieving cylindrical cabin 220; in the accommodating space 250 in the cylindrical cabin 220, the crushing motor 110 of the crushing module 100 rotates the crushing bearing 120 to drive the crushing blade 130, so as to crush the animal tissue, and the crushed animal tissue is arranged below the crushing blade 130 through the screening and collecting module 300, and the screen 310 screens the crushed animal tissue and falls into the screening and collecting tray 330; and the gas inlet and outlet control valve 500 is used to inject a conditioning gas to adjust the surface characteristics and temperature of the animal tissue. The conditioning gas is selected from any one of liquid nitrogen, dry ice, ozone and high temperature steam.

Selectively disassembling and replacing a proper screen 310 and a proper screening and collecting tray 330 to combine the screen and collecting module 300, wherein the center positions of the screen 310 and the screening and collecting tray 330 are respectively provided with a screen center perforation 311 and a screening and collecting tray center perforation 331, and the positions of the screen center perforation 311 and the screening and collecting tray center perforation 331 correspond to each other in the screening and collecting module 300; the screening and collecting module is fixedly arranged in the cylindrical cabin body 220 of the crushing and screening element, and is fixed by the elastic fixing module 28, and the screening and collecting module 300 is fixed, so that the powder bearing 120 of the crushing module 110 is not linked with the movement of the crushing blade 130 when the screening and collecting module 300 is operated by the animal tissue crushing and screening machine 10; the crushing and screening compartment 200 is combined with the crushing and screening compartment upper cover 210, the crushing and screening cylindrical compartment body 220 and the crushing and screening compartment lower cover 230 in sequence to form an accommodating space 250, and is fixed with the crushing and screening compartment upper cover fixing lock 270 by using a crushing and screening compartment lower cover fixing lock 260; further, animal tissues to be treated can be placed into the pulverizing and sieving compartment 200 through the closable animal tissue feed inlet 240; in certain operations, a gas inlet and outlet control valve 500 may be used to inject gas to adjust the sample conditions or temperature, such as liquid nitrogen, dry ice, high temperature steam, etc. The whole system can regulate and control the rotating speed or the temperature and humidity through the control module 400. After crushing, the sample falls into a collection tray and is taken out. The sieving and collecting module 300 can combine multiple layers of screens to obtain particles with different sizes. In addition, the rotary sieving purification in step (6) adopts a common rotary sieving device, and the sieving rotary crushing device 10 designed by the invention can be used to replace the crushing blade 130 with a dispersion rod.

Through the effective combination of vibration thin cutting, rotary screening and crushing and sectional purification according to the structural characteristics, the size uniformity, the miniaturization and the high purity of the first powder thin-cut extracellular matrix microcarrier are improved.

Third embodiment

The third embodiment of the present invention discloses a powder-state thin-cut extracellular matrix microcarrier pulverized by vibration thin-cutting and screening rotation, and the preparation method of the powder-state thin-cut extracellular matrix microcarrier comprises the following steps:

step (1): and (3) skin taking and rough treatment: selecting a fresh pigskin layer, unhairing a skin taking area, taking the pigskin, disinfecting and soaking the pigskin layer for thirty minutes by adopting benzalkonium bromide with the mass concentration of 0.5%, taking the pigskin layer out, washing the pigskin layer by using sterile water, draining the pigskin layer, and wiping the pigskin layer by using a clean towel to obtain a first skin membrane, wherein the first skin membrane does not contain subcutaneous adipose tissues;

step (2): and (3) relaxation of the decompression structure: collapsing the first film for 2 hours under a 50 torr acting force lower than atmospheric pressure using reduced pressure conditions to relax the tissue structure, thereby obtaining a second film having a relaxed tissue structure;

and (3): vibrating and thin cutting: adjusting the thickness parameter of the skin taking thickness to be 0.1 mm by using vibration thin cutting equipment with thin cutting thickness parameter control on the second skin membrane with a loose tissue structure, performing vibration thin cutting by using a vibration thin cutting blade module, and enabling the obtained first thin cutting skin membrane to have a uniform section due to the loose tissue structure;

and (4): section purification: performing section purification on the first thin-cut involucra with a uniform section by adopting sterile water with the weight 1 time that of the thin-cut involucra, and removing attachments on the section to obtain a second thin-cut involucra;

and (5): structure purification: cutting the second thin-cut film into sheets with the size of 1 cm multiplied by 1 cm-10 cm multiplied by 10 cm, performing structure purification reaction for 0.5 hour at the reaction temperature of 4 ℃ by adopting structure purification washing liquor with the weight 10 times that of the second thin-cut film, controlling the pH value between 7 and 12, and removing stripped substances generated by loose tissue structure to obtain a third thin-cut film;

wherein the structural purification washing liquid is an alkali reagent aqueous solution formed by mixing 1% (w/v) ammonium hydroxide aqueous solution and 1% (w/v) potassium hydroxide aqueous solution.

And (6): and (3) rotary screening and purifying: performing rotary screening purification on the third thin-cut film by adopting rotary screening equipment with a first screen mesh of 10 meshes at the rotating speed of 1500 rpm, wherein the first screen mesh is selected from No. 3-9 first mesh holes with the mesh diameter of 3-9 mm; through the screening purification treatment, the first screening thin-cutting leather membrane retained in the first screen and the first powder thin-cutting leather membrane outside the first screen after purification with high uniformity can be obtained at the same time, and the screening efficiency is improved through rotation;

and (7): and (3) supercritical carbon dioxide aqueous solution extraction, washing and purification: placing the first screened thin-cut epithelial membrane and the first powder thin-cut epithelial membrane into a separation cavity, and performing high-permeability extraction and purification for 0.5 hour at the temperature of 4 ℃ by adopting isolated supercritical carbon dioxide aqueous solution to completely remove residual small molecules to obtain high-purity first screened thin-cut extracellular stromal epithelial membrane and first powder thin-cut extracellular stromal epithelial membrane;

and (8): and (3) rotary screening and crushing treatment: the first powdery thin-cut extracellular matrix epithelial membrane is subjected to rotary screening and rotary crushing simultaneously by adopting a screening rotary crushing device with a second screen, namely a cutting type grinder with a vortex separation cavity, wherein the rotating speed is 1500 rpm, as shown in figures 12 and 13, the second screen is selected from No. 0.1-3 second meshes, the second meshes have mesh diameters of 0.1-3 mm, and the powdery thin-cut extracellular matrix microcarrier with high uniformity is efficiently obtained through the rotary screening and rotary crushing, and has the particle size distribution of 300-500 microns.

Fourth embodiment

The fourth embodiment of the present invention discloses a powder-state thin-cut extracellular matrix microcarrier obtained by vibration thin-cutting and screening rotation pulverization, and the preparation method of the powder-state thin-cut extracellular matrix microcarrier comprises the following steps:

step (1): and (3) skin taking and rough treatment: selecting a fresh pigskin layer, unhairing a skin taking area, taking the pigskin, disinfecting and soaking the pigskin layer for thirty minutes by adopting 3% benzalkonium bromide, taking the pigskin layer out, washing the pigskin layer by using sterile water, draining the pigskin layer, and wiping the pigskin layer by using a clean towel to obtain a first skin membrane, wherein the first skin membrane does not contain subcutaneous adipose tissues;

step (2): and (3) relaxation of the decompression structure: collapsing the first membrane for 8 hours under 500 torr acting force below atmospheric pressure to relax the tissue structure to obtain a second membrane with a relaxed tissue structure under reduced pressure;

and (3): vibrating and thin cutting: carrying out vibration thin cutting on the second skin membrane with a loose tissue structure by using vibration thin cutting equipment with thin cutting thickness parameter control, adjusting the thickness of the skin taking thickness parameter to be 3 mm, carrying out vibration thin cutting by using a vibration thin cutting blade module, and enabling the obtained first thin cutting skin membrane to have a uniform section due to the loose tissue structure;

and (4): section purification: performing section purification on the first thin-cut involucra with a uniform section by adopting sterile water with the weight 10 times that of the thin-cut involucra, and removing attachments on the section to obtain a second thin-cut involucra;

and (5): structure purification: cutting the second thin-cut film into sheets with the size of 1 cm multiplied by 1 cm-10 cm multiplied by 10 cm, performing structure purification reaction at the reaction temperature of 25 ℃ for 10 hours by adopting structure purification washing liquor with the weight 30 times that of the second thin-cut film, controlling the pH value between 7 and 12, and removing stripped substances generated by loose tissue structure to obtain a third thin-cut film;

wherein, the structure purification washing liquid is an acid reagent aqueous solution, and the acid reagent aqueous solution is formed by mixing 1% (w/v) acetic acid reagent aqueous solution and 0.5% (w/v) hydrochloric acid reagent aqueous solution;

and (6): and (3) rotary screening and purifying: performing rotary screening purification on the third thin-cut film by adopting rotary screening equipment with a first screen mesh of 50 meshes at the rotating speed of 3000 rpm, wherein the first screen mesh is selected from No. 3-9 first mesh holes with the mesh diameter of 3-9 mm; through the screening purification treatment, the first screening thin-cutting leather membrane retained in the first screen and the first powder thin-cutting leather membrane outside the first screen after purification with high uniformity can be obtained at the same time, and the screening efficiency is improved through rotation;

and (7): and (3) supercritical carbon dioxide aqueous solution extraction, washing and purification: placing the first screened thin-cut epithelial membrane and the first powder thin-cut epithelial membrane into a separation cavity, and performing high-permeability extraction and purification for 10 hours at the temperature of 25 ℃ by adopting isolated supercritical carbon dioxide aqueous solution to completely remove residual small molecules to obtain high-purity first screened thin-cut extracellular stromal epithelial membrane and first powder thin-cut extracellular stromal epithelial membrane;

and (8): and (3) rotary screening and crushing treatment: the first powdery thin-cut extracellular matrix epithelial membrane is subjected to rotary screening and rotary crushing simultaneously by adopting a screening rotary crushing device with a second screen, namely a cutting type grinder with a vortex separation cavity, wherein the rotating speed is 3000 r/min, as shown in figures 12 and 13, the second screen is selected from No. 0.1-3 second meshes, the second meshes have mesh diameters of 0.1-3 mm, and the powdery thin-cut extracellular matrix microcarrier with high uniformity is efficiently obtained through the rotary screening and rotary crushing, and has the particle size distribution of 100-200 microns.

Fifth embodiment

The fifth embodiment of the present invention discloses a powder-state thin-cut extracellular matrix microcarrier ground by vibration thin-cutting and screening rotation, and the preparation method of the powder-state thin-cut extracellular matrix microcarrier comprises the following steps:

step (1): and (3) skin taking and rough treatment: selecting a fresh pigskin layer, unhairing a skin taking area, taking the pigskin, disinfecting and soaking the pigskin layer for thirty minutes by adopting peracetic acid with the mass concentration of 5%, taking the pigskin layer out, washing the pigskin layer by using sterile water, draining the pigskin layer, and wiping the pigskin layer by using a clean towel to obtain a first skin membrane, wherein the first skin membrane does not contain subcutaneous adipose tissues;

step (2): and (3) relaxation of the decompression structure: collapsing the first film for 5 hours under a 200 torr acting force lower than atmospheric pressure under reduced pressure to relax the tissue structure, thereby obtaining a second film having a relaxed tissue structure;

and (3): vibrating and thin cutting: carrying out vibration thin cutting on the second skin membrane with a loose tissue structure by using vibration thin cutting equipment with thin cutting thickness parameter control, adjusting the thickness of the skin taking thickness parameter to be 5 mm, carrying out vibration thin cutting by using a vibration thin cutting blade module, and enabling the obtained first thin cutting skin membrane to have a uniform section due to the loose tissue structure;

and (4): section purification: performing section purification on the first thin-cut involucra with a uniform section by adopting sterile water with the weight 20 times that of the thin-cut involucra, and removing attachments on the section to obtain a second thin-cut involucra;

and (5): structure purification: cutting the second thin-cut film into sheets with the size of 1 cm multiplied by 1 cm-10 cm multiplied by 10 cm, performing structure purification reaction at the reaction temperature of 35 ℃ for 24 hours by adopting structure purification washing liquor with the weight 50 times that of the second thin-cut film, controlling the pH value between 7 and 12, and removing stripped substances generated by loose tissue structure to obtain a third thin-cut film;

wherein, the structure purification washing liquid is supercritical water solution, and the supercritical water solution is ethanol/carbon dioxide supercritical fluid with concentration of 10 (V/V)% and is used under the pressure of 150 bar;

and (6): and (3) rotary screening and purifying: performing rotary screening purification on the third thin-cut film by adopting rotary screening equipment with a first screen mesh of 100 meshes at the rotating speed of 5000 rpm, wherein the first screen mesh is selected from No. 3-9 first mesh holes with the mesh diameter of 3-9 mm; through the screening purification treatment, the first screening thin-cutting leather membrane retained in the first screen and the first powder thin-cutting leather membrane outside the first screen after purification with high uniformity can be obtained at the same time, and the screening efficiency is improved through rotation;

and (7): and (3) supercritical carbon dioxide aqueous solution extraction, washing and purification: placing the first screened thin-cut epithelial membrane and the first powder thin-cut epithelial membrane into a separation cavity, and performing high-permeability extraction and purification for 24 hours at the temperature of 35 ℃ by adopting isolated supercritical carbon dioxide aqueous solution to completely remove residual small molecules so as to obtain high-purity first screened thin-cut extracellular stromal epithelial membrane and first powder thin-cut extracellular stromal epithelial membrane;

and (8): and (3) rotary screening and crushing treatment: the first powdery thin-cut extracellular matrix epithelial membrane is subjected to rotary screening and rotary crushing simultaneously at a rotation speed of 5000 rpm by adopting a screening rotary crushing device with a second screen mesh, namely a cutting type grinder with a vortex separation cavity, as shown in fig. 12 and 13, the second screen mesh is selected from No. 0.1-3 second mesh meshes with mesh diameters of 0.1-3 mm, and the powdery thin-cut extracellular matrix microcarrier with high uniformity is efficiently obtained through the rotary screening and rotary crushing, and has particle size distribution in the range of 50-100 microns.

Sixth embodiment

The decellularization method determines the cell removal efficiency, the preparation methods are different, and the amounts of the retained extracellular matrix and cell determinants (immunogens) are also different, thereby directly influencing the composition of the material, the tissue microstructure, the mechanical properties of the material and the immunogenicity thereof. In order to verify the effectiveness of the decellularization method, the powder-state thin-cut extracellular matrix microcarrier prepared by the method of the fifth embodiment is used for detecting indexes including cell residue, collagen structure, cytotoxicity, cell compatibility, DNA residue, hemolysis and the like, and the detection result is shown in fig. 2-5.

(1) The detection result shows that the cell removing method has the advantage of thorough cell removal. Fig. 2 is a Scanning Electron Microscope (SEM) of the first thin-cut skin membrane prepared before the porcine dermal acellular matrix of example five and a Scanning Electron Microscope (SEM) of the powdery thin-cut extracellular matrix microcarrier prepared in example five, and it can be seen from fig. 2 that the first thin-cut skin membrane before the porcine dermal matrix acellular matrix is dense on the surface [ fig. 2(a) ] and covered with a thicker layer of matrix components, and the structure of the dermal matrix after the acellular matrix is loose [ fig. 2(B) ], and the matrix fibers are distributed in a reticular structure; FIG. 3 shows the nuclear staining (Dapi staining) of the first thin-cut epithelial membrane prepared before the dermal acellular matrix of the fifth pig example (FIG. 3 (A)) and the nuclear staining (Dapi staining) of the powdered thin-cut extracellular matrix microcarrier prepared in the fifth example (FIG. 3 (B)) to detect the residual nucleic acid after the treatment of the present invention. As can be seen from fig. 3(a) and 5(B), the first thin-cut epithelial surface before acellular matrix appeared with specific staining of nucleic acid — bright blue star-like spots, while two blue spots where no specific staining of nucleic acid was seen in the powder-state thin-cut extracellular matrix microcarrier appeared.

(2) The detection result shows that the decellularization method has the advantage that the collagen triple-helix structure is kept intact. As can be seen from the scanning electron microscope image (SEM image) of fig. 2(B), the collagen triple helix structure in the dermal matrix after decellularization was well preserved, showing a substantial helix structure, and the collagen fibers were aggregated into bundles. Fig. 4 is a graph showing HE staining of the first thin-cut skin membrane prepared before decellularized matrix of dermis of pig in example five (fig. 4 (a)) and the HE staining of the powder-state thin-cut extracellular matrix microcarrier prepared in example five (fig. 4 (B)). From fig. 4(a), darker colored substrate staining can be seen, as well as yellow spotted particles, which are fat particles; the staining in a fibrous and dot-like distribution was observed in the HE staining of fig. 4(B), and the structure was the staining of collagen fibers, and the collagen in the dermal matrix was distributed in a network in a horizontal and vertical arrangement, as shown in fig. 4 (B). Fig. 5 is a sirius red staining pattern of collagen in the porcine dermal acellular matrix before and after the first thin cut dermal membrane prepared in the fifth example of the present invention and fig. 5(B) a sirius red staining pattern of the pink thin cut extracellular matrix microcarrier prepared in the fifth example of the present invention, and it can be seen from the results of fig. 4(B) that the collagen of the pink thin cut extracellular matrix microcarrier prepared in the fifth example of the present invention is vertically and horizontally staggered, and well maintains the morphological structure inherent to the collagen in the skin tissue, and no sirius red staining pattern is observed in the first thin cut dermal membrane prepared in the fifth example of the present invention of fig. 5 (a).

(3) The detection result shows that the cell removing method has the advantage of thorough removal of polysaccharide. FIG. 6 shows Eisen blue staining of the first thin-cut epithelial membrane prepared before acellular matrix of pig dermis in example five and Eisen blue staining of the powder thin-cut extracellular matrix microcarrier prepared in example five, and from FIG. 6(A), the blue part is staining of glycosaminoglycan in dermis. As can be seen from FIG. 6(B), glycosaminoglycan in the thin-sliced extracellular matrix microcarrier in powder form prepared by the method of the present invention is substantially removed.

(4) The detection result shows that the cell removing method has the advantage of good cell compatibility detection. FIG. 7 is a cytotoxicity test performed on the thin-cut extracellular matrix microcarrier in powder form prepared in example five. The growth of fibroblasts (L929) in the leaching solution of the material after 24 hours of culture is shown, and the survival rate of the cells in the leaching solution of the material is more than 100, which indicates that the toxicity of the powder-state thin-cut extracellular matrix microcarrier prepared in the fifth example is grade 0. Fig. 8 is a Scanning Electron Microscope (SEM) image of the cell adhesion behavior of the first thin-cut skin membrane material prepared before seeding the L929 cells on the dermal acellular matrix of the fifth pig and the powder-state thin-cut extracellular matrix microcarrier prepared in the fifth example. As can be seen from fig. 8(a), no fine adhesion phenomenon was observed on the material of the first thin-cut epithelial membrane prepared before the dermal acellular matrix of the fifth pig, whereas adhesion and aggregation growth were observed on the thin-cut extracellular matrix microcarrier in powder form prepared in the fifth example, as shown in fig. 8(B), it can be seen from the graph that the cells were closely connected to the thin-cut extracellular matrix microcarrier in powder form prepared in the fifth example through pseudopodia, and showed good cell compatibility.

(5) The detection result shows that the cell removing method has the advantage of low residual quantity of nucleic acid. FIG. 9 is a graph showing the results of comparing the amounts of nucleic acid remaining on the first thin-cut membrane prepared before the acellular matrix of pig dermis in example five and the powder-state thin-cut extracellular matrix microcarrier prepared in example five. As can be seen from the results of the assay in FIG. 9, the amount of nucleic acid remaining in the pig dermis after decellularization was much lower than the maximum concentration required by the standard (50 ng/mg).

(6) The detection result shows that the cell removing method has the advantage of low hemolysis. FIG. 10 is a graph comparing the results of hemolysis experiments performed on the first thin-cut membrane prepared before the dermal acellular matrix of the fifth pig in example and the powder-state thin-cut extracellular matrix microcarrier prepared in example five with rabbit blood. From the results, it can be seen that the hemolysis rate of the thin-cut extracellular matrix microcarrier in powder state prepared in example five is 0.29%, which is far less than the hemolysis rate of-8.19% of the first thin-cut epithelial membrane prepared before the dermal acellular matrix of pig prepared in example five, and is less than the standard requirement that the hemolysis rate of medical devices is less than 5%.

(7) The detection result shows the morphology of the powdery thin-cut extracellular matrix microcarrier obtained by grinding and crushing the acellular method of the invention through a 0.5 mm screen, as shown in fig. 11 (a); FIG. 11(B) is a Scanning Electron Microscope (SEM) image of 100 times of the thin-sliced powdered extracellular matrix microcarrier prepared in example V, which has a distinct scaffold structure.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for preparing a powdery thin-cut extracellular matrix microcarrier by using structure segmentation purification is characterized by comprising the following steps: the preparation of the powdery thin-cut extracellular matrix microcarrier sequentially comprises the steps of rough skin taking treatment, decompression structure relaxation, vibration thin cutting, section purification, structure purification, rotary screening purification, supercritical carbon dioxide aqueous solution extraction and washing purification and rotary screening crushing treatment to obtain the powdery thin-cut extracellular matrix microcarrier with high uniformity and high purity.

2. The method for preparing a thin-sliced extracellular matrix microcarrier using structure-segmentation purification according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

3. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: in the step (1), the disinfection is performed by adopting benzalkonium bromide or peracetic acid with the mass concentration of 0.5-5%.

4. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: in the step (2), the first coating is disintegrated for 2-8 hours under the action of 50-500 torr acting force which is lower than the atmospheric pressure.

5. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the vibration thin cutting is to adjust the thickness parameter of the skinning to be 0.1-5 mm by using vibration thin cutting equipment with thin cutting thickness parameter control, and to perform thin cutting by using a vibration thin cutting blade module to obtain a first thin-cutting skin membrane with a uniform section.

6. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the consumption of sterile water for section purification is 1-20 times of the weight of the thin-cut involucra.

7. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: in the structure purification, the second thin cut film is cut into a sheet shape with the size of 1 cm multiplied by 1 cm to 10 cm multiplied by 10 cm, and the use amount of the structure purification washing liquid is 10 to 50 times of the weight of the second thin cut film.

8. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the structure purification washing liquid is one or a combination of more of an alkali reagent aqueous solution, an acid reagent aqueous solution, a supercritical water solution and a surfactant reagent aqueous solution.

9. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the rotating speed of the rotary screening purification is 1500-5000 r/min.

10. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the first screen is selected from a number 3-9 first mesh having a mesh diameter of 3-9 mm.

11. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the supercritical carbon dioxide aqueous solution is extracted, washed and purified as follows: and placing the first screening thin-cut film and the first powder thin-cut film into a separation cavity, and carrying out extraction washing and purification for 0.5-24 hours.

12. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the rotating speed of the rotating screening and crushing treatment is 1500-5000 revolutions per minute.

13. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the second screen is selected from a second mesh ranging from 0.1 to 3 mesh having a mesh diameter ranging from 0.1 mm to 3 mm.

14. The method for preparing the thin-sliced extracellular matrix microcarrier according to claim 2, which is prepared by using a structure-segmented purification method, wherein the method comprises the following steps: the powdery thin-cut extracellular matrix microcarrier has a particle size distribution in the range of 50-500 microns.

15. The method for preparing the powder-state thin-cut extracellular matrix microcarrier according to any one of claims 1 to 14, wherein the powder-state thin-cut extracellular matrix microcarrier is prepared by the method for preparing the powder-state thin-cut extracellular matrix microcarrier through structure segmentation purification.

16. A sieving rotary pulverizing apparatus used in the method for preparing the thin-cut extracellular matrix microcarrier in a powder state by means of structure segmentation purification according to any one of claims 2 to 14, characterized in that: screening rotary crushing equipment is animal tissue crushing screening machine includes: the device comprises a crushing module, a crushing and screening cabin, a screening and collecting module and a gas inlet and outlet control valve;

wherein the crushing module comprises a crushing motor, a crushing bearing and a crushing blade; the crushing and screening cabin comprises an upper crushing and screening cabin cover, a cylindrical crushing and screening cabin body and a lower crushing and screening cabin cover which are sequentially combined to form an accommodating space and a sealable animal tissue feed inlet arranged on the crushing and screening cabin; the screening and collecting module comprises a screen and a screening and collecting disc, wherein the central positions of the screen and the screening and collecting disc are respectively provided with a screen central perforation and a screening and collecting disc central perforation, the screen central perforation and the screening and collecting disc central perforation are used for supporting the bottom of the crushing bearing, and the screening and collecting module is detachably fixed in the crushing and screening cylindrical cabin; in the containing space in the crushing and screening cylindrical cabin, the crushing motor rotates the crushing bearing to drive the crushing blade to crush the animal tissues, and the crushing motor is arranged below the crushing blade through the screening and collecting module; and the gas inlet and outlet control valve is filled with regulating gas to regulate the surface characteristics and the temperature of the animal tissues.

17. The sieving rotary pulverizing apparatus used in the method for preparing a thin-sliced extracellular matrix microcarrier purified by structural segmentation according to claim 16, wherein: the conditioning gas is any one selected from the group consisting of liquid nitrogen, dry ice, ozone and high temperature steam.

18. A vibration thin-cutting apparatus used in the method for preparing the powder-state thin-cut extracellular matrix microcarrier according to any one of claims 2 to 14, wherein the vibration thin-cutting apparatus comprises: the vibrating thin-cutting equipment comprises a conveying carrying platform and a vibrating thin-cutting blade module arranged above a conveying carrier, wherein the vibrating thin-cutting blade module is provided with a thin-cutting blade.