Acellular matrix tubular material and preparation method thereof

Abstract

The invention relates to the technical field of medical materials, in particular to an acellular matrix tubular material and a preparation method thereof. The preparation method comprises the following steps: preparing a thick acellular matrix tissue fiber aggregate; preparing an acellular matrix tissue layer; and preparing the tubular acellular matrix material. And repeatedly pushing and rolling the acellular matrix tissue layer by utilizing a mold through a rolling technology, so that the micro-nano fibers are mutually interwoven, wrapped and tightly bonded together to form the acellular matrix tubular material formed by laminating a single layer of acellular matrix tissue layer or a plurality of layers of single-layer acellular matrix tissue layers. The layers of the prepared acellular tubular scaffold material are stable and tightly bonded, the operation is simple and convenient, the pore diameter and porosity of the tube wall are controllable, a single-cavity, multi-cavity or multi-branch tubular scaffold can be prepared, and the defects that an existing tubular acellular matrix material is compact in structure and needs to be sewn or bonded, and a branch structure is difficult to prepare are overcome.

Description

Technical Field

[0001] The invention relates to the technical field of medical materials, and in particular to a decellularized matrix tubular material and a preparation method thereof. Background Art

[0002] There are many tubular tissues in the human body, such as cardiovascular, peripheral blood vessels, neural ducts, esophagus, intestines, bile ducts, urethra, etc. These tubes play their respective functions. However, some trauma or diseases can cause damage to these tubes and affect their functions. At present, autologous or allogeneic transplantation is often used in clinical practice. However, due to the limited source of transplants, autologous transplantation may cause damage to the donor site function; allogeneic transplantation has immune rejection reactions, and patients need to take immunosuppressive drugs for a long time, which not only increases the economic burden on patients, but also the adverse reactions of immunosuppressive drugs will have a greater impact on the patient's physical health.

[0003] The development of tissue engineering and biomaterials has provided new strategies for repairing defects in human tissues and organs. Scientists have used synthetic polymer materials (such as polycaprolactone, polylactic acid glycolic acid, etc.) or natural materials (such as collagen, gelatin, hyaluronic acid, etc.) to construct a variety of tubular materials for damage repair. However, synthetic polymer materials lack biological activity, and their degradation byproducts are acidic, which can easily cause inflammation and lead to transplantation failure. Scaffolds made of natural polymers usually exhibit poor mechanical properties and cannot simulate the biochemical complexity and ultrastructure of the natural extracellular matrix (ECM), which limits their application. The decellularized matrix material obtained after decellularization of natural tissue has a composition similar to that of natural tissue, so it has good biocompatibility, and can trigger a favorable immune response after implantation into the human body, which is beneficial to tissue repair and remodeling in the human body.

[0004] However, existing tubular materials prepared using decellularized matrices still have the following problems: the decellularized matrices obtained after decellularization of natural tubular tissues (such as decellularized blood vessels, esophagus, etc.) have a dense structure, which restricts cell migration and thus hinders tissue regeneration; slicing natural decellularized tissues or directly using membranous decellularized tissues to prepare tubular materials is cumbersome, has poor controllability, is limited in length, and inevitably requires the use of sutures or adhesives to maintain the tubular structure. In addition, it is difficult to prepare multi-layer tubes, multi-lumen tubes, and tubular materials with multi-branched structures.

[0005] Reference (Alberti KA, Xu Q. Slicing, stacking and rolling: fabrication of nanostructured collagen constructs from tendon sections. Adv HealthcMater. 2013 Jun;2(6):817-21.) discloses a method for preparing a single-cavity and multi-cavity tubular tendon decellularized matrix scaffold, wherein the decellularized tendon tissue is sliced ​​into thin slices and then rolled into a tubular structure, and a multi-cavity tubular scaffold material is further prepared by manual stacking. However, the length of the single-cavity tube prepared by this method is limited, the scaffold tube wall is easily delaminated, is not firm, the pore size is uncontrollable, and the tubes in the multi-cavity tube structure are separated from each other; and it is difficult to prepare a tube scaffold with a branched and "U"-shaped structure by this method.

[0006] Reference (Goldberg LA, Zomer HD, McFetridge C, McFetridge PS. Silicananoparticles enhance interfacial self-adherence of a multi-layeredextracellular matrix scaffold for vascular tissue regeneration. Biotechnol Lett. 2024 Jun;46(3):469-481.) discloses a method for preparing a tubular human decellularized amniotic membrane. The method cuts the human amniotic membrane decellularized tissue into rectangles and then rolls it into a tube. However, the method requires additional adhesive to prevent separation of the layers. In addition, the tubular scaffold prepared by the method has a limited length, a dense structure, and the pore size of the tube wall is difficult to control, and it is difficult to prepare a tubular scaffold with a branched and "U"-shaped structure.

[0007] Patent CN102481390A discloses a vascular graft for treating diseased or damaged blood vessels, which rolls a sheet of acellular tissue matrix into a tube and fixes the edge of the sheet to construct a tubular catheter; Patent CN103977457A discloses a tubular tissue engineering scaffold and a preparation method thereof, which forms a tubular scaffold with multiple layers of thin film by applying a bioadhesive on one or both sides of the film and rolling it around an axis. However, the two preparation methods disclosed above are cumbersome to operate, and the constructed tubular structure requires the use of sutures or adhesives, which has the risk of rupture, and the layers cannot be well fused, making it difficult to prepare multilayer tubes and branched tube structures with different structures. Summary of the invention

[0008] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a method for preparing a decellularized matrix tubular material; the second object of the present invention is to provide a decellularized matrix tubular material.

[0009] In order to achieve the first purpose, the technical solution adopted by the present invention is: A method for preparing a decellularized matrix tubular material comprises the following steps: S100, taking an animal tissue for decellularization, homogenizing the decellularized tissue to obtain a decellularized matrix tissue homogenate containing micro-nano fibers, and centrifuging to obtain a viscous decellularized matrix tissue fiber aggregate; The decellularization method may adopt conventional technical means of the prior art; for example, the decellularization steps are as follows: cut the fresh animal tissue into small pieces, place it in a peracetic acid solution, stir and disinfect it, then wash it with water, then wash it with a sodium dodecyl sulfate (SDS) solution, wash it with a DNA enzyme aqueous solution, and then wash it with water twice, thus completing the decellularization process and obtaining the decellularized animal tissue; S200, spreading the acellular matrix tissue fiber aggregates to obtain a acellular matrix tissue layer; The paving thickness of the acellular matrix tissue fibers can be adjusted according to actual needs, preferably 500um to 5mm. The length and width of the acellular matrix tissue layer are not specifically limited here and can be adjusted according to actual needs. S300, rolling the decellularized matrix tissue layer around an axis and placing it on a mold, and repeatedly rolling the decellularized matrix tissue layer using a rolling technique to further discharge moisture, so that the micro-nano fibers are intertwined, wrapped, and tightly bonded together to form a decellularized matrix tubular material; The rolling technology can be used to push and roll the decellularized micro-nano fiber tissue on the mold in a uniform tubular shape. On the other hand, it can promote the fiber assembly, deposition, wrapping, mutual entanglement and aggregation, and then tightly bond the micro-nano fibers and the layers to each other and seamlessly connect. Compared with the existing decellularized tissue sheet curling around the axis, the repeated rolling and pushing technology can achieve uniform distribution and seamless bonding of decellularized tissue fibers without the use of sutures and adhesives.

[0010] Furthermore, the method further comprises the following steps: S400, pre-freezing the decellularized matrix tubular material and then freeze-drying it, or directly air-drying or air-drying the decellularized matrix tubular material, removing the mold, and obtaining a tubular stent; by combining phase separation technology, directly air-drying or air-drying the cylindrical mold with the decellularized tissue attached, or pre-freezing at low temperature and then freeze-drying it, completely removing moisture and drying it, thereby enhancing the bonding force between fibers, further allowing the fibers to be tightly bonded together, and then removing the mold, thereby preparing a single-lumen tubular stent with different pore sizes and wall thicknesses; The rolling technique is used again to repeatedly roll and further adhere a new layer of decellularized matrix tissue layer on the basis of the original decellularized matrix tissue layer, and then combined with the phase separation technique, directly air-drying, air-drying, or pre-freezing at low temperature and then freeze-drying treatment is performed to make the decellularized tissue fibers tightly bonded without stratification, and then the mold is removed to prepare a single-lumen tubular scaffold with different layer structures; After stacking the molds with the acellular matrix tissue layers attached, they are directly air-dried, air-dried, or pre-frozen at low temperature and then freeze-dried to make the outer layers of the acellular matrix tissue layers tightly bonded, and then the molds are removed to prepare a multi-cavity tubular scaffold; a multi-cavity tubular scaffold material can also be obtained by manually assembling tubular acellular matrix tissues with different inner diameters and numbers; The molds with different diameters attached with the decellularized matrix tissue layer are bonded to each other through the attraction and adhesion of the tissue fibers themselves and fixed into a U-shaped or multi-branched shape, and then air-dried at room temperature, air-dried, or pre-frozen at low temperature and freeze-dried, and then the molds are removed to obtain a tubular stent with branches; Preferably, the pre-freezing temperature is -196°C to -18°C, the freeze-drying temperature is -196°C to -18°C, the pre-freezing time or the freezing time is 6h to 72h, the air-drying time is 30min to 120h, and the air-drying temperature is 0 to 28°C.

[0011] Furthermore, the method further comprises the following steps: S500, mechanically strengthening the tubular stent obtained in S400, the process of this step is as follows: The tubular stent is immersed in a cross-linking agent solution for cross-linking for 1 hour to 24 hours, the cross-linking agent is removed, and the tubular stent is freeze-dried, air-dried or air-dried to obtain a mechanically enhanced tubular stent; The cross-linking solution is water, phosphate buffer or ethanol solution containing a cross-linking agent.

[0012] Furthermore, the cross-linking agent is one or more of glutaraldehyde, genipin, N-hydroxysuccinimide, carbodiimide hydrochloride, transglutaminase and proanthocyanidin.

[0013] Furthermore, the animal tissue in step S100 includes one or more of arteries, veins, heart, liver, kidneys, muscles, skin, fat, meninges, diaphragm, amnion, pericardium, heart valves, small intestinal submucosa, tendons, ligaments, nerves, stomach, trachea, esophagus, intestines, bile duct, urethra, bladder, cornea and placenta.

[0014] Furthermore, in step S100, the homogenization time is 30s to 30min, the centrifugation time is 5min to 30min, and the centrifugation speed is 500rpm to 10000rpm.

[0015] Furthermore, the mold in step S300 is a cylindrical tungsten steel rod, a wooden rod, a silicone rod, a PDMS rod or a polytetrafluoroethylene rod, the diameter of the mold is 10µm to 10cm, the number of push and roll times is 1 to 500 times, and the push and roll time is 10s to 30min.

[0016] In order to achieve the second purpose, the technical solution adopted by the present invention is: A decellularized matrix tubular material, the decellularized matrix tubular material is prepared by any of the above-mentioned methods for preparing decellularized matrix tubular materials, and comprises a lumen and a tube wall, wherein the tube wall encloses the lumen; The tube wall is a single-layer decellularized matrix tissue layer or is formed by stacking several single-layer decellularized matrix tissue layers.

[0017] Furthermore, it also includes active substances, which are arranged on the tube wall, and the active substances include one or more of immune active cells, cytokines, mRNA and small molecule drugs.

[0018] Furthermore, the acellular matrix tubular material is used for the preparation of tissue repair medical devices; The medical device comprises a tubular stent, and the tubular stent is a single-lumen tubular stent, a multi-lumen tubular stent or a branched tubular stent.

[0019] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides a method for preparing a decellular matrix tubular material, which fully retains the biological activity of the decellular matrix material itself, and promotes the interweaving, wrapping, and tight bonding of micro-nano fibers to form a decellular matrix tubular material by repeatedly rolling and rolling using a rolling technique. The rolling technique can be repeatedly rolled, on the one hand, make the decellularized micro-nano fiber tissue on the mold evenly distributed in a tubular shape, and on the other hand, promote the fiber assembly deposition, wrapping, mutual entanglement and aggregation, and then tight bonding, so that the micro-nano fibers and each layer are bonded to each other and seamlessly connected. The method gives full play to the strong self-adhesive force of the decellularized tissue fibers, so that the layers of the prepared decellularized matrix tubular material can be stably and tightly bonded without adhesive bonding and suture stitching, the operation is simple, and the pore size and porosity of the multi-layer tube wall are controllable. If a multi-cavity tubular scaffold is to be prepared, no adhesive is needed between the tube walls, and the walls can be stably combined through the self-adhesive force of the decellularized tissue fibers. The length, diameter, wall thickness, number of layers and pore structure, branch structure and size of the prepared tubular scaffold can be prepared by adjusting the quality of the decellularized tissue fibers, the thickness of the decellularized tissue fiber sheets, the number of push and roll times, the push and roll time, the freezing temperature, the air-drying time, the cross-linking time, etc. to prepare single-layer, multi-layer, multi-cavity and multi-branched decellularized matrix tubular materials with controllable pore size and mechanics. The mechanical strength of the decellularized matrix tubular material can be further enhanced by cross-linking. At the same time, multiple tubular scaffolds can be assembled, which can both enhance the mechanical strength and expand the scope of application.

[0020] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the formation process of the decellularized matrix tubular material provided in Example 1 of the present invention.

[0022] Figure 2 This is a macroscopic image of the decellularized matrix tubular material provided in Example 1 of the present invention, which is made using a tungsten steel rod with a length of 25 cm and a diameter of 5 mm as a mold.

[0023] Figure 3 This is a macroscopic image of a tubular stent with different inner diameters and wall thicknesses provided in Example 1 of the present invention.

[0024] Figure 4 It is a cross-sectional view of the tubular stent after freeze-drying and removal of the mold provided in Example 1 of the present invention and a microscopic view characterized by a scanning electron microscope (SEM).

[0025] Figure 5It is a cross-sectional view of the tubular support provided in Example 1 of the present invention after the mold is removed after air-drying at room temperature and a microscopic image characterized by a scanning electron microscope.

[0026] Figure 6 It is a statistical diagram of the mechanical strength of the tubular stent before and after cross-linking provided in Example 1 of the present invention.

[0027] Figure 7 These are macroscopic and microscopic images of the tubular support provided in Example 2 of the present invention.

[0028] Figure 8 These are stereomicroscope images, bright field images and atomic force microscope images of the homogenate provided in Examples 3 and 4 of the present invention.

[0029] Fig. 9 It is a statistical diagram of the fiber diameter in the homogenate provided in Examples 3 and 4 of the present invention.

[0030] Fig.10 It is a statistical graph of the diameter of collagen fibers in the homogenate provided in Examples 3 and 4 of the present invention.

[0031] Fig.11 This is a macroscopic image of the tubular stent provided in Example 3 of the present invention.

[0032] Fig.12 These are macroscopic and microscopic images of the tubular stent provided in Example 4 of the present invention.

[0033] Fig.13 This is a macroscopic image of the tubular support provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0035] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0036] Example 1 Preparation of a single-cavity porcine muscle acellular matrix tubular scaffold.

[0037] The preparation process is as follows: 1. Decellularization of porcine muscle tissue: Take fresh porcine muscle tissue (1 kg), rinse it with water (2 L), cut it into small pieces with a size of 1 cm × 2 cm × 0.3 cm, soak it in sterile water (2 L) for 30 min to remove blood, disinfect it with 0.1% peracetic acid (2 L) for 30 min, wash it with sterile water (5 L) 3 times, add 1% SDS (3 L) and stir and wash it for 72 h, change the liquid once every 6 hours for the first time, and then change the liquid every 12 hours, then wash it with sterile water (10 L) to remove SDS, then wash it with 500 mL of buffer containing 50 U / mL DNase at 37 ° C for 12 h, and then wash it with sterile water (2 L) to obtain porcine muscle decellularized tissue; 2. Take the pig muscle decellularized tissue (20 g) and put it into a homogenizer, add sterile water (200 mL), homogenize for 10 minutes, and obtain the muscle decellularized matrix tissue homogenate, then centrifuge at room temperature at a speed of 1000 rpm for 5 minutes, remove the supernatant, and obtain a thick decellularized matrix tissue fiber aggregate; 3. Spread the pig muscle acellular tissue fiber aggregates on a flat plate to obtain a sheet of acellular matrix tissue layer with a size of 5 cm×3 cm×0.4 cm; The preparation process from homogenate → fiber aggregate → acellular matrix tissue layer → tubular material, such as Figure 1 As shown; A cylindrical tungsten steel rod with a length of 25 cm and a diameter of 5 mm was used to repeatedly roll the decellularized matrix tissue layer 30 times using a rolling technique to discharge some of the water in the decellularized matrix tissue layer, and to use the attraction between the fibers to promote the assembly and deposition of micro-nano fibers, entanglement and aggregation, and then tightly bond together to form a decellularized matrix tubular material, such as Figure 2 As shown; the diameter and length of the mold can be adjusted according to actual needs to prepare tubular materials with different inner diameters, wall thicknesses and lengths, such as Figure 3 As shown; Fourth, the tungsten steel rod with the acellular matrix tubular material attached was pre-frozen at -80°C and then freeze-dried for 36 hours, or directly air-dried at room temperature for 24 hours, and then the mold was removed to obtain a single-cavity pig muscle acellular matrix tubular scaffold, such as Figure 4 and Figure 5 As shown; The air-dried tubular scaffold obtained above was fully immersed in 0.25% glutaraldehyde solution at 25°C for cross-linking for 2 hours, and then washed with sterile water to remove the cross-linking agent, thereby obtaining a mechanically enhanced tubular scaffold. The mechanical statistics are as follows: Figure 6 As shown, the mechanical strength of the single-lumen acellular matrix tubular material was significantly enhanced after cross-linking.

[0038] In the prior art, due to the use of acellular matrix sheet materials, sutures and adhesives are required to maintain the tubular shape when preparing a tubular structure.

[0039] The tubular stent disclosed in the prior art has a tube wall with uneven thickness, the tube wall layers are easily peeled off, and the pore structure of the tube wall is uncontrollable.

[0040] The tubular stent provided by the present invention can stably maintain the tubular structure without suturing or bonding, and the acellular tissue fibers are evenly distributed on the tube wall, and the pore structure of the tube wall is controllable.

[0041] The tubular stent can be immersed in an active solution according to specific clinical needs. For example, in order to promote tissue repair, the tubular stent can be immersed in a solution containing cells (such as stem cells, immune cells, etc.) or cytokines (such as IL-4, IGF-1, VEGF, etc.) for 12 hours; in order to promote the absorption and action of drugs, such as diclofenac sodium, at the site where the tubular stent is implanted, the tubular stent can be immersed in a solution containing diclofenac sodium for 12 hours, and a biologically active tubular stent loaded with cells or small molecule drugs can be obtained.

[0042] Example 2 Preparation of a double-layer porcine aorta decellularized matrix tubular scaffold.

[0043] 1. Decellularization of porcine aorta: Take fresh porcine aorta tissue (0.5 kg), peel it clean and cut it into small pieces with a size of 1 cm × 0.5 cm × 0.2 cm, soak and wash it with sterile water (1 L) for 20 min, disinfect it with 0.1% peracetic acid (1 L) for 30 min, wash it repeatedly with sterile water (3 L) for 3 times, add 1% SDS (1 L) and stir and wash it for 72 h, change the solution every 6 h, then wash it with sterile water (10 L) to remove SDS, then wash it with 500 mL of buffer containing 50 U / mL DNase at 37 ° C for 12 h, and then wash it with sterile water (5 L) to obtain porcine aorta decellularization tissue; 2. Take the porcine aortic decellularized tissue (15 g) and put it into a homogenizer, add sterile water (300 mL), and homogenize for 15 minutes to obtain the aortic decellularized matrix tissue homogenate, and then centrifuge it at room temperature at a speed of 2000 rpm for 8 minutes, remove the supernatant, and obtain a thick decellularized matrix tissue fiber aggregate; 3. Take a green food-grade dye and drop it into the above-mentioned porcine aortic decellularized tissue fiber aggregate to dye the fiber green, spread the porcine aortic decellularized tissue fiber aggregate on the filter paper to obtain a decellularized matrix tissue layer of 6 cm × 3 cm × 0.2 cm in size, use a cylindrical tungsten steel rod with a diameter of 2 mm to curl the decellularized matrix tissue layer around an axis, use a rolling technique to repeatedly roll it 20 times to discharge the moisture in the decellularized matrix tissue layer, obtain a decellularized matrix tubular material, air-dry at room temperature for 24 hours, and obtain a single-layer porcine aortic decellularized matrix tubular scaffold; Fourth, according to the above three, a 2mm thick, undyed decellularized matrix tissue layer was prepared, and a tungsten steel rod coated with the single-layer porcine aorta decellularized matrix tubular scaffold prepared in the above three was used to roll it around an axis, and the rolling technique was used to repeatedly roll it 30 times, and air-dried at room temperature for 36 hours to obtain a double-layer porcine aorta decellularized matrix tubular scaffold, and its structural morphology is as follows Figure 7 As shown in the figure, it can be seen that the double-layer tubular stent has an inner and outer two-layer dense layered tube wall structure, and the inner layer and the outer layer are tightly bonded without separation; among them, Figure A is a macroscopic image and a local enlarged image of the prepared tubular stent with a double-layer dense structure, and Figure B is a tubular stent with a double-layer dense structure prepared by SEM characterization.

[0044] The double-layered porcine aorta decellularized matrix tubular scaffold was fully immersed in EDC / NHS solution at 4°C for 12 hours for cross-linking, and then washed with sterile water (3L) to remove the cross-linking agent EDC / NHS, thereby obtaining a mechanically enhanced double-layered tubular scaffold. Among them, EDC (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide) is the abbreviation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide; NHS (N-Hydroxy succinimide) is the abbreviation of N-hydroxysuccinimide.

[0045] Example 3 Preparation of a multi-cavity bovine Achilles tendon acellular matrix tubular scaffold.

[0046] The specific preparation process is as follows: Decellularization of bovine Achilles tendon: Take fresh bovine Achilles tendon tissue (1 kg), cut it into small pieces with a size of 1 cm × 0.5 cm × 0.3 cm, then disinfect it with 0.1% peracetic acid (1 L) for 30 min, wash it repeatedly with sterile water (3 L) for 3 times, then add 1% SDS (1 L) and stir and wash for 48 h, change the solution every 12 h, then wash with sterile water (10 L) to remove SDS, then wash with 50 U / mL DNase buffer (500 mL) at 37 ° C for 6 h, and then wash with sterile water (5 L) to obtain decellularized bovine Achilles tendon tissue; 2. Take the decellularized tissue of the bovine Achilles tendon (30 g) and add it to a homogenizer, add sterile water (200 mL), and homogenize for 15 minutes to make the decellularized tissue of the bovine Achilles tendon fibrous, and obtain the Achilles tendon decellularized matrix tissue homogenate, and then centrifuge it at room temperature at a speed of 2000 rpm for 10 minutes, remove the supernatant, and obtain a thick decellularized matrix tissue fiber aggregate; Among them, the fiber morphology in the homogenate is as follows Figure 8 The fiber diameter statistics are shown in Fig. 9As shown, it is around 1.8 μm; the statistics of collagen fiber diameter are as follows Fig.10 As shown, it is around 300nm; 3. The bovine Achilles tendon acellular tissue fiber aggregate was evenly spread on a glass plate and flattened to obtain two pieces of 5cm×4cm×0.4cm sheet-like acellular matrix tissue layers. The two pieces of acellular matrix tissue layers were respectively rolled onto cylindrical tungsten steel rods with diameters of 2mm and 4mm. The rolling technique was used to repeatedly roll the acellular matrix tissue layer 30 times to discharge the water in the acellular matrix tissue layer, and the bovine Achilles tendon acellular matrix tubular material was obtained. Fourth, after stacking cylindrical molds with bovine Achilles tendon decellularized matrix tubular materials, leaving them at room temperature for 48 hours, and removing the molds, a dense multi-lumen tubular scaffold can be prepared; or after leaving silicone tubes of different diameters with bovine Achilles tendon decellularized matrix tubular materials at room temperature for 48 hours, removing the molds, and then stacking them, a dense multi-lumen tubular scaffold can be prepared. There are gaps between the tubular scaffold monomers in the lumen of the tubular scaffold, and its morphological structure is as follows: Fig.11 As shown; Figure A is a multi-lumen tubular stent prepared by first assembling and stacking multiple single-lumen tubular stents and then air-drying them. It can be seen from the figure that the single-lumen tubes can be tightly bonded without adding adhesive; Figure B is a multi-lumen tubular stent prepared by manually stacking the prepared single-lumen tubular stents. It can be seen from the figure that a certain gap is left between the single-lumen tubes of the tubular stent prepared in this way.

[0047] 5. The multi-lumen tubular stent is fully immersed in a 0.5% proanthocyanidin solution, cross-linked at 25° C. for 4 hours, and then washed with sterile water to remove the cross-linking agent, thereby obtaining a mechanically enhanced tubular stent.

[0048] Example 4 Preparation of a double-layered porcine liver decellularized matrix tubular scaffold.

[0049] The specific preparation process is as follows: 1. Decellularization of pig liver: Take fresh pig liver tissue (2.5 kg), wash it with water and cut it into small pieces with a size of 2 cm × 2 cm × 0.3 cm, soak and wash it with sterile water (5 L) for 30 minutes to remove blood, and then wash it with water (3 L) for 10 minutes; disinfect it with 0.1% peracetic acid (5 L) for 30 minutes, wash it repeatedly with sterile water (10 L) for 3 times, add 1% SDS (2 L) and stir and wash it for 48 hours, change the liquid every 6 hours, then wash it with sterile water (15 L) to remove SDS, and then wash it with 800 mL of buffer containing 50 U / mL DNase at 37°C for 6 hours, and then wash it with sterile water (5 L) to obtain decellularized pig liver tissue; 2. Take the pig liver decellularized tissue (15 g) and put it into a homogenizer, add sterile water (300 mL), homogenize for 15 minutes, and obtain the liver decellularized matrix tissue homogenate, then centrifuge at room temperature at 5000 rpm for 5 minutes, remove the supernatant, and obtain a thick decellularized matrix tissue fiber aggregate; Among them, the fiber morphology in the homogenate is as follows Figure 8 The fiber diameter statistics are shown in Fig. 9 As shown, it is around 1.8 μm; the statistics of collagen fiber diameter are as follows Fig.10 As shown, it is around 200nm; 3. Spread the pig liver decellularized tissue fiber aggregates on the filter paper to obtain a sheet of decellularized matrix tissue layer with a size of 2cm×3cm×0.3cm, use a tungsten steel rod with a diameter of 4mm to curl it around the axis, and then use the rolling technology to repeatedly push and roll 25 times to discharge the moisture in the decellularized matrix tissue layer to obtain a decellularized matrix tubular material, place it in a -20℃ refrigerator and freeze it for 12h, and then use a freeze dryer to freeze it for 24h to obtain a decellularized matrix tubular scaffold with a porous wall structure; Fourth, the decellularized matrix tissue layer was prepared according to the above three steps, and a cylindrical tungsten steel rod coated with the single-layer porcine liver decellularized matrix tubular scaffold prepared by the above three steps was used to roll it around an axis, and the rolling technique was used to repeatedly roll it 15 times, and it was dried at room temperature for 36 hours to obtain a double-layer porcine liver decellularized matrix tubular scaffold with a loose inner layer and a dense outer layer. Its morphological structure is as follows Fig.12 As shown in the figure, it can be seen that the inner layer is loose and porous, the outer layer is dense, and the two layers are tightly bonded without delamination; Among them, A is a macroscopic image and a local enlarged image of the double-layer tubular stent. It can be seen from the image that the inner layer of the tubular stent is loose and porous, and the outer layer is dense; Figure B is a characterization of the double-layer tubular stent using SEM.

[0050] 5. The double-layered porcine liver decellularized matrix tubular scaffold was fully immersed in EDC / NHS solution, cross-linked at 4°C for 12 hours, and then washed with sterile water to remove the cross-linking agent to obtain a mechanically enhanced double-layered porcine liver decellularized matrix tubular scaffold.

[0051] Example 5 Preparation of a branched porcine small intestinal submucosa decellularized matrix tubular scaffold.

[0052] 1. Decellularization of porcine small intestinal submucosa: Take porcine small intestinal submucosa tissue (2 kg), rinse it with water (5 L), cut it into small pieces of 2 cm × 1 cm × 0.2 cm, disinfect it with 0.1% peracetic acid (1 L) for 1 hour, wash it repeatedly with sterile water (3 L) for 3 times, add 1% SDS (2 L) and stir and wash it for 72 hours, change the liquid once every 6 hours for the first time, and then change the liquid every 12 hours, then wash it with sterile water (15 L) to remove SDS, then wash it with 50 U / mL DNase buffer (600 mL) at 37°C for 6 hours, and then wash it with sterile water to obtain the decellularized porcine small intestinal submucosa tissue; 2. Take the decellularized tissue (30 g) of the submucosal layer of the small intestine of the pig and add it into a homogenizer. After adding sterile water (300 mL), homogenize for 10 minutes to make the decellularized tissue fibrous, and obtain the decellularized matrix tissue homogenate. Then, centrifuge at room temperature at a speed of 3000 rpm for 10 minutes, remove the supernatant, and obtain a thick decellularized matrix tissue fiber aggregate. 3. Evenly spread the acellular tissue fiber aggregates of the pig small intestinal submucosa on the filter paper and flatten them to obtain three acellular matrix tissue layers with a size of 3 cm×4 cm×0.2 cm; Fourth, the three acellular matrix tissue layers were respectively rolled onto silicone tubes with diameters of 2 mm, 4 mm and 6 mm, and then rolled repeatedly 30 times using a rolling technique to discharge part of the water in the acellular matrix layer, thereby obtaining a tubular acellular matrix material of the submucosal layer of the porcine small intestine; 5. A silicone tube with a diameter of 4 mm wrapped with a decellularized matrix tubular material of the submucosal layer of the porcine small intestine was fixed into a "U" shape. After determining the position of the decellularized matrix tubular materials with different inner diameters connected to the 4 mm decellularized matrix tubular material, the tissue at the connection was removed to form a connection port. The two decellularized matrix tubular materials were then bonded to the corresponding interfaces of the "U"-shaped decellularized matrix tubular material, respectively. After drying at room temperature for 48 hours, the silicone tube mold was removed to obtain an integrated tubular scaffold with a branched structure. The morphological structure of the tubular scaffold is as follows: Fig.13 As shown; 6. The obtained tubular scaffold with branched structure was fully immersed in 50u / g TG enzyme, cross-linked at 25°C for 12h, and then washed with sterile water to remove the cross-linking agent to obtain a mechanically enhanced tubular scaffold.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a decellularized matrix tubular material, characterized in that: The steps include: S100, taking an animal tissue for decellularization, homogenizing the decellularized tissue to obtain a decellularized matrix tissue homogenate containing micro-nano fibers, and centrifuging to obtain a viscous decellularized matrix tissue fiber aggregate; S200, spreading the acellular matrix tissue fiber aggregates to obtain a acellular matrix tissue layer; S300, rolling the decellularized matrix tissue layer around an axis and placing it on a mold, and repeatedly rolling the decellularized matrix tissue layer using a rolling technique to further discharge moisture, so that the micro-nano fibers are intertwined, wrapped, and tightly bonded together to form a decellularized matrix tubular material.

2. The method for preparing the decellularized matrix tubular material according to claim 1, characterized in that: The following steps are also included: S400, pre-freezing the decellularized matrix tubular material and then freeze-drying it, or directly air-drying or air-drying the decellularized matrix tubular material, removing the mold, and obtaining a tubular scaffold.

3. The method for preparing the decellularized matrix tubular material according to claim 2, characterized in that: The following steps are also included: S500, mechanically strengthening the tubular stent obtained in S400, the process of this step is as follows: The tubular stent is immersed in a cross-linking agent solution for cross-linking for 1 hour to 24 hours, the cross-linking agent is removed, and the stent is freeze-dried, air-dried or air-dried to obtain a mechanically enhanced tubular stent.

4. The method for preparing the decellularized matrix tubular material according to claim 3, characterized in that: The cross-linking agent is one or more of glutaraldehyde, genipin, N-hydroxysuccinimide, carbodiimide hydrochloride, glutamine aminotransferase and proanthocyanidin.

5. The method for preparing the decellularized matrix tubular material according to claim 1, characterized in that: The animal tissue in step S100 includes one or more of arteries, veins, heart, liver, kidneys, muscles, skin, fat, meninges, diaphragm, amnion, pericardium, heart valves, small intestinal submucosa, tendons, ligaments, nerves, stomach, trachea, esophagus, intestines, bile duct, urethra, bladder, cornea and placenta.

6. The method for preparing the decellularized matrix tubular material according to claim 1, characterized in that: In step S100, the homogenization time is 30s to 30min, the centrifugation time is 5min to 30min, and the centrifugation speed is 500rpm to 10000rpm.

7. The method for preparing the decellularized matrix tubular material according to claim 1, characterized in that: The mold in step S300 is a cylindrical tungsten steel rod, a wooden rod, a silicone rod, a PDMS rod or a polytetrafluoroethylene rod, the diameter of the mold is 10µm to 10cm, the number of push and roll times is 1 to 500 times, and the push and roll time is 10s to 30min.

8. A decellularized matrix tubular material, characterized in that: The decellularized matrix tubular material is prepared by the preparation method of any one of claims 1 to 7, comprising a lumen and a tube wall, wherein the tube wall encloses the lumen; The tube wall is a single-layer decellularized matrix tissue layer or is formed by stacking several single-layer decellularized matrix tissue layers.

9. The decellularized matrix tubular material according to claim 8, characterized in that: It also includes active substances, which are arranged on the tube wall and include one or more of immune active cells, cytokines, mRNA and small molecule drugs.

10. The decellularized matrix tubular material according to claim 9, characterized in that: The acellular matrix tubular material is used for preparing tissue repair medical devices; The medical device comprises a tubular stent, and the tubular stent is a single-lumen tubular stent, a multi-lumen tubular stent or a branched tubular stent.

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