A decellularized matrix tubular material and preparation method thereof

Abstract

The present invention relates to the field of medical material technology, and in particular to a decellularized matrix tubular material and a preparation method thereof. The preparation method includes the preparation of a dense decellularized matrix tissue fiber aggregate; the preparation of a decellularized matrix tissue layer; and the preparation of a decellularized matrix tubular material. The decellularized matrix tissue layer is repeatedly rolled up using a mold through a rolling technique, so that the micro-nano fibers are interwoven, wrapped, and tightly bonded together to form a decellularized matrix tubular material composed of a single layer or several layers of single-layer decellularized matrix tissue layers. The decellularized tubular scaffold material prepared by the present invention has stable and tight bonding between the layers, is easy to operate, has controllable pore size and porosity of the tube wall, and can be prepared into a single-cavity, multi-cavity or multi-branched tubular scaffold, overcoming the shortcomings of existing tubular decellularized matrix materials, such as dense structure, the need for suturing or bonding, and difficulty in preparing branched structures.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to an acellular 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 tubes, esophagus, intestines, bile ducts, urethra, etc. These tubes perform their respective functions. However, some trauma or diseases can cause damage to these tubes and affect their functions. Currently, 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 financial burden on patients, but also the adverse reactions of immunosuppressive drugs will have a significant impact on the patient's physical health.

[0003] Advances in tissue engineering and biomaterials have provided new strategies for repairing tissue and organ defects. Scientists have constructed a variety of tubular structures for repair using synthetic polymers (such as polycaprolactone and poly(lactic-co-glycolic acid)) or natural materials (such as collagen, gelatin, and hyaluronic acid). However, synthetic polymers lack bioactivity, and their degradation byproducts are acidic, which can easily trigger inflammation and lead to transplant failure. Scaffolds made from natural polymers typically exhibit poor mechanical properties and fail to mimic the biochemical complexity and ultrastructure of the natural extracellular matrix (ECM), limiting their application. Decellularized matrices, obtained by decellularizing natural tissue, have a composition similar to that of native tissue, resulting in excellent biocompatibility. Upon implantation, these materials can trigger a favorable immune response, thus benefiting tissue repair and remodeling.

[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 single-lumen and multi-lumen tubular tendon acellular matrix scaffolds. This method involves slicing acellular tendon tissue into thin slices and rolling them into a tubular structure. Furthermore, the multi-lumen tubular scaffold material is prepared by further manually stacking. However, the single-lumen tubes prepared by this method are limited in length, the scaffold wall is easily delaminated and weak, the pore size is uncontrollable, and the tubes in the multi-lumen tubular structure are separated from each other. Furthermore, this method is difficult to prepare tubular scaffolds with branching or "U"-shaped structures.

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

[0007] Patent CN102481390A discloses a vascular graft for treating diseased or damaged blood vessels. This graft is made by rolling a sheet of acellular tissue matrix into a tube and fixing the edges of the sheet to form a tubular conduit. Patent CN103977457A discloses a tubular tissue engineering scaffold and its preparation method. This tissue engineering scaffold is formed by applying a bioadhesive to one or both sides of the film and rolling it around an axis to form a tubular scaffold with multiple layers of thin film. However, the two disclosed preparation methods are cumbersome to operate. The constructed tubular structure requires the use of sutures or adhesives, which poses a risk of rupture. The layers cannot be well integrated, 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:

[0010] A method for preparing a cell-free matrix tubular material comprises the following steps:

[0011] S100, performing decellularization on an animal tissue, homogenizing the decellularized tissue to obtain a decellularized matrix tissue homogenate containing micro-nanofibers, and centrifuging to obtain a viscous decellularized matrix tissue fiber aggregate;

[0012] The decellularization method can adopt conventional technical means in the prior art; for example, the decellularization steps are as follows: fresh animal tissue is cut into small pieces, placed in a peracetic acid solution, stirred and disinfected, and then washed with water, and then washed with a sodium dodecyl sulfate (SDS) solution, washed with a DNase-containing aqueous solution, and then washed with water twice, thereby completing the decellularization process and obtaining the decellularized animal tissue;

[0013] S200, spreading the acellular matrix tissue fiber aggregates to obtain an acellular matrix tissue layer;

[0014] The thickness of the acellular matrix tissue fibers can be adjusted according to actual needs, preferably 500 μm to 5 mm. The length and width of the acellular matrix tissue layer are not specifically limited here and can be adjusted according to actual needs.

[0015] 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 drain water, so that the micro-nanofibers are interwoven, wrapped, and tightly bonded together to form a decellularized matrix tubular material;

[0016] The repeated rolling technique not only uniformly distributes the acellular micro-nanofibers on the mold into a tubular shape, but also promotes fiber assembly, deposition, wrapping, and intertwining, resulting in tight bonding and seamless adhesion between the micro-nanofibers and the layers. Compared to existing methods of rolling acellular tissue sheets around an axis, this repeated rolling technique achieves uniform distribution and seamless bonding of the acellular fibers without the need for sutures or adhesives.

[0017] Furthermore, the method further comprises the following steps:

[0018] 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, and removing the mold to obtain a tubular scaffold; combining a phase separation technique, directly air-drying or air-drying the cylindrical mold with the decellularized tissue attached, or pre-freezing it at a low temperature and then freeze-drying it, completely removing moisture and drying it, thereby enhancing the bonding strength between the fibers and further tightly bonding the fibers together. The mold is then removed to prepare single-lumen tubular scaffolds with different pore sizes and wall thicknesses.

[0019] The rolling technique is then used to repeatedly roll the material, further adhering a new layer of decellularized matrix tissue to the original layer. The material is then combined with phase separation technology to directly air dry, air dry, or freeze dry after low-temperature pre-freezing, so that the decellularized tissue fibers are tightly bonded and delaminated. The mold is then removed to prepare a single-lumen tubular scaffold with different layer structures.

[0020] After stacking the molds with the acellular matrix tissue layers attached, they are directly air-dried, air-dried, or pre-frozen at low temperatures and then freeze-dried to tightly bond the outer layers of the acellular matrix tissue layers. The molds are then removed to prepare a multi-lumen tubular scaffold. Multi-lumen tubular scaffold materials can also be obtained by manually assembling tubular acellular matrix tissues of different inner diameters and numbers.

[0021] Molds of different diameters with acellular matrix tissue layers adhered thereto are bonded to each other through the attraction and adhesion of tissue fibers themselves and then fixed into a U-shaped or multi-branched shape. The molds are then air-dried or air-dried at room temperature or pre-frozen at low temperature and then freeze-dried, and then the molds are removed to produce a branched tubular scaffold.

[0022] 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.

[0023] Furthermore, the method further comprises the following steps:

[0024] S500: Mechanically strengthen the tubular stent obtained in S400. The process of this step is as follows:

[0025] The tubular scaffold is immersed in a crosslinking agent solution for crosslinking for 1 hour to 24 hours, the crosslinking agent is removed, and the scaffold is freeze-dried, air-dried, or air-dried to obtain a mechanically reinforced tubular scaffold;

[0026] The cross-linking solution is water, phosphate buffer or ethanol solution containing a cross-linking agent.

[0027] Furthermore, the cross-linking agent is one or more of glutaraldehyde, genipin, N-hydroxysuccinimide, carbodiimide hydrochloride, transglutaminase and proanthocyanidin.

[0028] 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.

[0029] Furthermore, in step S100 , the homogenization time is 30 s to 30 min, the centrifugation time is 5 min to 30 min, and the centrifugal speed is 500 rpm to 10000 rpm.

[0030] Furthermore, the mold in step S300 is a cylindrical tungsten steel rod, wooden rod, silicone rod, PDMS rod or 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.

[0031] In order to achieve the second purpose, the technical solution adopted by the present invention is:

[0032] A decellularized matrix tubular material, prepared by any of the above methods for preparing decellularized matrix tubular materials, comprising a lumen and a tube wall, wherein the tube wall encloses the lumen;

[0033] The tube wall is a single layer of acellular matrix tissue layer or is formed by stacking several single layers of acellular matrix tissue layers.

[0034] Furthermore, it also includes active substances, which are arranged on the tube wall. The active substances include one or more of immune active cells, cytokines, mRNA and small molecule drugs.

[0035] Furthermore, the acellular matrix tubular material is used for the preparation of tissue repair medical devices;

[0036] The medical device comprises a tubular stent, which is a single-lumen tubular stent, a multi-lumen tubular stent or a branched tubular stent.

[0037] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0038] The present invention provides a method for preparing a tubular acellular matrix material. While fully preserving the bioactivity of the acellular matrix material itself, the method utilizes a rolling technique to repeatedly roll the material, promoting the interweaving, entangling, and tight bonding of micro-nano fibers to form the tubular acellular matrix material. This rolling technique not only uniformly distributes the acellular micro-nano fibers on the mold into a tubular shape, but also promotes fiber assembly, deposition, entangling, and intertwining, leading to tight bonding and seamless adhesion between the micro-nano fibers and between the layers. This 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 the need for 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 they can be stably bonded 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-rolling times, the push-rolling 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 not only enhances the mechanical strength but also expands the scope of application.

[0039] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a schematic diagram of the formation process of the acellular matrix tubular material provided in Example 1 of the present invention.

[0041] Figure 2 This is a macroscopic image of the acellular 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.

[0042] 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.

[0043] Figure 4 This 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).

[0044] Figure 5This is a cross-sectional view and a microscopic image characterized by a scanning electron microscope of the tubular support provided in Example 1 of the present invention after the mold is removed after air-drying at room temperature.

[0045] Figure 6 This 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.

[0046] Figure 7 These are macroscopic and microscopic images of the tubular stent provided in Example 2 of the present invention.

[0047] Figure 8 These are stereomicroscope images, bright field images, and atomic force microscope images of the homogenates provided in Examples 3 and 4 of the present invention.

[0048] Figure 9 This is a statistical diagram of fiber diameters in the homogenate provided in Examples 3 and 4 of the present invention.

[0049] Figure 10 This is a statistical graph of the diameter of collagen fibers in the homogenate provided in Examples 3 and 4 of the present invention.

[0050] Figure 11 This is a macroscopic view of the tubular stent provided in Example 3 of the present invention.

[0051] Figure 12 These are macroscopic and microscopic images of the tubular stent provided in Example 4 of the present invention.

[0052] Figure 13 This is a macroscopic view of the tubular stent provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0053] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0054] 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.

[0055] Example 1 Preparation of a single-cavity porcine muscle acellular matrix tubular scaffold.

[0056] The preparation process is as follows:

[0057] 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) three times, and then add 1% SDS (3 L) and stir and wash it for 72 h. Change the solution once every 6 hours for the first time, and then change the solution 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;

[0058] 2. Take 20 g of porcine muscle decellularized tissue and place it in a homogenizer. Add sterile water (200 mL) and homogenize for 10 minutes to obtain a muscle acellular matrix tissue homogenate. Then, centrifuge at room temperature at 1000 rpm for 5 minutes, remove the supernatant, and obtain a thick acellular matrix tissue fiber aggregate.

[0059] 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;

[0060] The preparation process from homogenate → fiber aggregate → acellular matrix tissue layer → tubular material, such as Figure 1 As shown;

[0061] 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 promote the assembly and deposition of micro-nano fibers by utilizing the attraction between the fibers, so that the fibers entangled and aggregated with each other, and then bonded tightly together to form a decellularized matrix tubular material. 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;

[0062] Fourth, the tungsten steel rod with the acellular matrix tubular material attached was pre-frozen at -80 ° C and 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. Figure 4 and Figure 5 As shown;

[0063] The air-dried tubular scaffold was fully immersed in 0.25% glutaraldehyde solution at 25°C for 2 hours for cross-linking, and then washed with sterile water to remove the cross-linking agent. A mechanically enhanced tubular scaffold was obtained, and its mechanical statistics are shown as follows: Figure 6As shown, the mechanical strength of the single-lumen acellular matrix tubular material was significantly enhanced after cross-linking.

[0064] 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.

[0065] The tubular stent disclosed in the prior art has walls of varying thickness, is easily peeled off from one layer to another, and has an uncontrollable pore structure.

[0066] 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.

[0067] The tubular stent can be immersed in an active solution according to specific clinical needs. For example, 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. To promote the absorption and action of drugs, such as diclofenac sodium, at the site of tubular stent implantation, the tubular stent can be immersed in a solution containing diclofenac sodium for 12 hours, thereby obtaining a bioactive tubular stent loaded with cells or small molecule drugs.

[0068] Example 2 Preparation of a double-layer porcine aorta decellularized matrix tubular scaffold.

[0069] 1. Decellularization of porcine aorta: Fresh porcine aorta tissue (0.5 kg) was obtained, cleaned and cut into small pieces with a size of 1 cm × 0.5 cm × 0.2 cm. The pieces were soaked and washed with sterile water (1 L) for 20 min, disinfected with 0.1% peracetic acid (1 L) for 30 min, washed repeatedly with sterile water (3 L) for 3 times, and then added with 1% SDS (1 L) and stirred and washed for 72 h. The solution was changed every 6 h, and then washed with sterile water (10 L) to remove SDS. The pieces were then washed with 500 mL of buffer containing 50 U / mL DNase at 37°C for 12 h, and then washed with sterile water (5 L) to obtain porcine aorta decellularized tissue.

[0070] Second, 15 g of porcine aortic decellularized tissue was placed in a homogenizer, sterile water (300 mL) was added, and homogenization was performed for 15 minutes to obtain aortic decellularized matrix tissue homogenate. The homogenate was then centrifuged at room temperature at 2000 rpm for 8 minutes, and the supernatant was removed to obtain a viscous acellular matrix tissue fiber aggregate.

[0071] 3. A green food-grade dye was dripped into the above-mentioned porcine aortic decellularized tissue fiber aggregate to dye the fibers green. The porcine aortic decellularized tissue fiber aggregate was flattened on filter paper to obtain a 6 cm × 3 cm × 0.2 cm decellularized matrix tissue layer. A cylindrical tungsten steel rod with a diameter of 2 mm was used to curl the decellularized matrix tissue layer around an axis. The rolling technique was used to repeatedly roll the decellularized matrix tissue layer 20 times to expel the moisture in the decellularized matrix tissue layer to obtain a decellularized matrix tubular material. The decellularized matrix tubular material was air-dried at room temperature for 24 h to obtain a single-layer porcine aortic decellularized matrix tubular scaffold.

[0072] Fourth, according to the above three, a 2mm thick, undyed acellular matrix tissue layer was prepared. A tungsten steel rod coated with the single-layer porcine aorta acellular matrix tubular scaffold prepared in the above three was used to roll it around an axis. The tungsten steel rod was rolled repeatedly 30 times using a rolling technique and air-dried at room temperature for 36 hours to obtain a double-layer porcine aorta acellular matrix tubular scaffold. Its structural morphology is as follows: Figure 7 As shown in the figure, it can be seen that the double-layer tubular stent has two layers of 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.

[0073] The double-layer 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 (3 L) to remove the cross-linking agent EDC / NHS, thereby obtaining a mechanically enhanced double-layer tubular scaffold.

[0074] 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.

[0075] Example 3 Preparation of a multi-cavity bovine Achilles tendon acellular matrix tubular scaffold.

[0076] The specific preparation process is as follows:

[0077] Decellularization of bovine Achilles tendon: Fresh bovine Achilles tendon tissue (1 kg) was cut into small pieces with a size of 1 cm × 0.5 cm × 0.3 cm. The pieces were then disinfected with 0.1% peracetic acid (1 L) for 30 min and washed three times with sterile water (3 L). 1% SDS (1 L) was then added and stirred for 48 h, with the solution changed every 12 h. The pieces were then washed with sterile water (10 L) to remove SDS. The pieces were then washed with 500 mL of 50 U / mL DNase buffer at 37°C for 6 h and then rinsed again with sterile water (5 L) to obtain decellularized bovine Achilles tendon tissue.

[0078] 2. Take bovine Achilles tendon decellularized tissue (30 g) and add it to a homogenizer. Add sterile water (200 mL) and homogenize for 15 minutes to make the bovine Achilles tendon decellularized tissue fibrous. Obtain Achilles tendon decellularized matrix tissue homogenate. Then, centrifuge at room temperature at 2000 rpm for 10 minutes. Remove the supernatant and obtain a viscous acellular matrix tissue fiber aggregate.

[0079] Among them, the fiber morphology in the homogenate is as follows Figure 8 The fiber diameter statistics are shown in Figure 9 As shown, it is around 1.8 μm; the statistics of collagen fiber diameter are as follows Figure 10 As shown, it is around 300nm;

[0080] 3. The bovine Achilles tendon acellular tissue fiber aggregate was evenly spread on a glass plate and flattened to obtain two sheets of acellular matrix tissue layers, each measuring 5 cm × 4 cm × 0.4 cm. The two acellular matrix tissue layers were respectively rolled onto cylindrical tungsten steel rods with diameters of 2 mm and 4 mm. The rolling technique was used to repeatedly roll the acellular matrix tissue layer 30 times to expel the moisture in the acellular matrix tissue layer, thereby obtaining a bovine Achilles tendon acellular matrix tubular material.

[0081] 4. 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 tubular scaffold lumen, and its morphological structure is as follows: Figure 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 adhesives; 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.

[0082] 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.

[0083] Example 4 Preparation of a double-layered porcine liver acellular matrix tubular scaffold.

[0084] The specific preparation process is as follows:

[0085] 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, and then add 1% SDS (2 L) and stir and wash it for 48 hours. Change the solution 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;

[0086] 2. Take the pig liver decellularized tissue (15g) and put it into a homogenizer. Add sterile water (300mL) and homogenize for 15 minutes to obtain the liver decellularized matrix tissue homogenate. Then, centrifuge it at room temperature at 5000rpm for 5 minutes, remove the supernatant, and obtain a thick decellularized matrix tissue fiber aggregate.

[0087] Among them, the fiber morphology in the homogenate is as follows Figure 8 The fiber diameter statistics are shown in Figure 9 As shown, it is around 1.8 μm; the statistics of collagen fiber diameter are as follows Figure 10 As shown, it is around 200nm;

[0088] Third, porcine liver decellularized tissue fiber aggregates were flattened on filter paper to obtain a sheet of decellularized matrix tissue layer with a size of 2 cm × 3 cm × 0.3 cm. A tungsten steel rod with a diameter of 4 mm was used to curl it around an axis. Then, a rolling technique was used to repeatedly push and roll it 25 times to expel the moisture in the decellularized matrix tissue layer to obtain a decellularized matrix tubular material. The material was placed in a -20°C refrigerator and frozen for 12 hours. It was then freeze-dried in a freeze dryer for 24 hours to obtain a decellularized matrix tubular scaffold with a porous wall structure.

[0089] Fourth, the acellular matrix tissue layer was prepared according to the above three steps. A cylindrical tungsten steel rod coated with a single-layer pig liver acellular matrix tubular scaffold prepared above was rolled around the axis. The cylindrical tungsten steel rod was rolled repeatedly 15 times using a rolling technique and dried at room temperature for 36 hours to obtain a double-layer pig liver acellular matrix tubular scaffold with a loose inner layer and a dense outer layer. The morphological structure is as follows: Figure 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;

[0090] Among them, A is a macroscopic image and a local magnified image of the double-layer tubular scaffold. It can be seen from the image that the inner layer of the tubular scaffold is loose and porous, while the outer layer is dense; Figure B is the characterization of the double-layer tubular scaffold using SEM.

[0091] 5. The double-layer 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-layer porcine liver decellularized matrix tubular scaffold.

[0092] Example 5 Preparation of a branched porcine small intestinal submucosa acellular matrix tubular scaffold.

[0093] 1. Decellularization of porcine small intestinal submucosa: 2 kg of porcine small intestinal submucosa tissue was obtained, rinsed with water (5 L), and cut into small pieces of 2 cm × 1 cm × 0.2 cm. The pieces were disinfected with 0.1% peracetic acid (1 L) for 1 h, washed three times with sterile water (3 L), and then added with 1% SDS (2 L) and stirred and washed for 72 h. The solution was changed once every 6 h for the first time, and then every 12 h. The pieces were then washed with sterile water (15 L) to remove SDS, and then washed with 50 U / mL DNase buffer (600 mL) at 37 ° C for 6 h. The pieces were then washed with sterile water to obtain decellularized porcine small intestinal submucosa tissue.

[0094] Second, 30 g of porcine small intestinal submucosa decellularized tissue was placed in a homogenizer. After adding sterile water (300 mL), the mixture was homogenized for 10 minutes to make the decellularized tissue fibrous, thereby obtaining a cell-free matrix tissue homogenate. The mixture was then centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant was removed to obtain a viscous cell-free matrix tissue fiber aggregate.

[0095] 3. Spread the acellular tissue fiber aggregates of the porcine small intestinal submucosa evenly on filter paper and flatten them to obtain three acellular matrix tissue layers with a size of 3 cm × 4 cm × 0.2 cm;

[0096] Fourth, the three acellular matrix tissue layers were respectively rolled onto silicone tubes with diameters of 2 mm, 4 mm, and 6 mm. The tubes were then rolled repeatedly 30 times using a rolling technique to expel some of the water in the acellular matrix layers, thereby obtaining a tubular acellular matrix material of the porcine small intestinal submucosa.

[0097] 5. A 4mm diameter silicone tube wrapped with a porcine small intestinal submucosa decellularized matrix tubular material was fixed into a "U" shape. After determining the position of the decellularized matrix tubular materials with different inner diameters connected to the 4mm 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. 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: Figure 13 As shown;

[0098] 6. The obtained tubular scaffold with a branched structure was fully immersed in 50 u / g TG enzyme, cross-linked at 25° C. for 12 h, and then washed with sterile water to remove the cross-linking agent to obtain a mechanically enhanced tubular scaffold.

[0099] 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 various embodiments of the present invention.

Claims

1. A method for preparing a cell-free matrix tubular material, characterized in that: The steps include: S100, washing the animal tissue with a sodium dodecyl sulfate solution and an aqueous solution containing DNA enzyme to complete decellularization, homogenizing the decellularized tissue to obtain a decellularized matrix tissue homogenate containing micro-nanofibers, and centrifuging to obtain a viscous decellularized matrix tissue fiber aggregate; The homogenization time is 30 seconds to 30 minutes, the centrifugation time is 5 minutes to 30 minutes, and the centrifugal speed is 500 rpm to 10,000 rpm. S200, spreading the acellular matrix tissue fiber aggregates to obtain an 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 drain water, so that the micro-nanofibers are interwoven, wrapped, and tightly bonded together to form a decellularized matrix tubular material; The mold 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.

2. The method for preparing the acellular matrix tubular material according to claim 1, wherein: 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 acellular matrix tubular material according to claim 2, wherein: The following steps are also included: S500, preparing a mechanically enhanced tubular stent, which includes the following steps: S501, immersing the tubular stent in a cross-linking agent solution for cross-linking for 1 hour to 24 hours, removing the cross-linking agent, and freeze-drying, air-drying or air-drying to obtain a mechanically reinforced tubular stent.

4. The method for preparing the acellular matrix tubular material according to claim 3, wherein: The cross-linking agent in step S501 is one or more of glutaraldehyde, genipin, N-hydroxysuccinimide, carbodiimide hydrochloride, transglutaminase and proanthocyanidin.

5. The method for preparing the acellular matrix tubular material according to claim 1, wherein: 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. A decellularized matrix tubular material, characterized in that: The acellular matrix tubular material is prepared by the method for preparing acellular matrix tubular material according to any one of claims 1 to 5, and comprises a lumen and a tube wall, wherein the tube wall encloses the lumen; The tube wall is a single layer of acellular matrix tissue layer or is formed by stacking several single layers of acellular matrix tissue layers.

7. The acellular matrix tubular material according to claim 6, wherein: It also includes active substances, which are arranged on the tube wall. The active substances include one or more of immune active cells, cytokines, mRNA and small molecule drugs.

8. The acellular matrix tubular material according to claim 7, wherein: The acellular matrix tubular material is used for preparing tissue repair medical devices; The medical device comprises a tubular stent, which is a single-lumen tubular stent, a multi-lumen tubular stent or a branched tubular stent.

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