Tissue-engineered artificial blood vessel for small-caliber artery regeneration and construction method thereof

Abstract

The construction method comprises the following steps: preparing a PCL near-field direct-writing inner-layer stent and a PGS / PCL hollow pipeline outer-layer stent, nesting the inner-layer stent and the outer-layer stent outside a silica gel tube to obtain a double-layer stent silica gel tube combination body, fixing the double-layer stent silica gel tube combination body in a bioreactor, soaking the double-layer stent silica gel tube combination body in an alcohol bath, and airing the double-layer stent silica gel tube combination body in an ultra-clean workbench; inoculating pig smooth muscle and cartilage cells on the double-layer bracket for culturing; taking out the double-layer stent silica gel tube combination body from the bioreactor in a sterile state, removing the silica gel tube of the double-layer stent silica gel tube combination body, carrying out decellularization treatment, and retaining an extracellular matrix and bioactive substances; obtaining the artificial blood vessel. The caramel sacrifice template is adopted to prepare a hollow pourable pipeline structure, antibiotics, anticoagulant drugs and the like can be loaded, and infection and embolism are effectively avoided; meanwhile, the acellular extracellular matrix is prepared by adopting an acellular technology, immediate implantation can be realized after engineering preparation is completed, the consumed time is short, and the method is suitable for trauma first aid.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a tissue-engineered artificial blood vessel for small-diameter arterial regeneration and its construction method. Background Technology

[0002] Small-diameter arteries refer to arteries in the human body with a diameter of less than 6 millimeters, such as coronary arteries, cerebral arteries, and distal limb arteries. They are important channels for delivering blood to critical organs and are also high-risk sites for vascular diseases. Transplantation is the preferred treatment for small-diameter arterial obstructive diseases. However, the transplant environment for small-diameter arteries differs from that of large arteries such as thoracic and abdominal arteries, making them prone to embolism and resulting in poorer wound healing.

[0003] Currently, ideal small-diameter arterial grafts should meet the following requirements: blood flow patency, tissue integration, and mechanical stability. At present, artificial blood vessels are mainly tissue-engineered vessels. For example, the 3D-printed PCL tubular stent developed by Linkbio has entered the clinical trial stage and shows outstanding performance in blood flow patency, tissue integration, and mechanical stability. However, due to the long autologous implantation time, it cannot meet emergency needs and carries a certain risk of infection. Humacyte has developed a vascular graft with superior anti-infection properties and has been approved by the FDA for clinical use. However, the humanized cells required for its use are not available domestically due to ethical concerns.

[0004] Therefore, it is necessary to propose new measures to overcome the above-mentioned shortcomings. Summary of the Invention

[0005] The purpose of this invention is to provide a tissue-engineered artificial blood vessel for small-diameter arterial regeneration and its construction method, so as to solve the problems of existing artificial blood vessels being unable to regenerate quickly, being prone to embolism, and infection when used for small-diameter arterial transplantation.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration is provided, the method comprising:

[0008] A PCL near-field direct writing inner scaffold and a PGS / PCL hollow tube outer scaffold were fabricated and nested sequentially on a small-diameter silicone tube to obtain a double-layer scaffold-silicone tube assembly.

[0009] The double-layer support silicone tube assembly was fixed inside the bioreactor. After soaking the bioreactor in an alcohol bath, the bioreactor was placed in a clean bench to dry.

[0010] Porcine smooth muscle cells and porcine chondrocytes were inoculated onto a double-layered scaffold, and the culture medium was pumped into a bioreactor for culture.

[0011] Under sterile conditions, the double-layer scaffold silicone tube assembly was removed from the bioreactor, the silicone tubes of the double-layer scaffold silicone tube assembly were removed, and decellularization was performed to retain the extracellular matrix and bioactive substances.

[0012] A tissue-engineered artificial blood vessel for small-diameter arterial regeneration was obtained, whose PGS / PCL hollow conduit outer stent has a drug-loadable hollow perfusion conduit structure.

[0013] Furthermore, the PCL near-field direct writing inner scaffold is a micro / nano fiber network structure obtained by near-field direct writing technology using PCL.

[0014] Furthermore, the outer support layer of the PGS / PCL hollow pipe is prepared, including:

[0015] Sucrose, dextran, fructose, glucose and ultrapure water were mixed and stirred until the color turned slightly yellow. After heating and drying, the mixture was 3D printed to obtain a caramel sacrificial template.

[0016] PCL and PGS were added to a tetrahydrofuran solution to prepare a coating liquid, which was then coated onto the outside of a caramel sacrificial template to obtain a PGS / PCL caramel sacrificial template assembly.

[0017] The PGS / PCL caramel sacrificial template assembly was placed in distilled water to dissolve the flexible sugar sacrificial carrier, thus obtaining the outer support of the PGS / PCL hollow pipe.

[0018] Furthermore, the mixing ratio of sucrose, dextran, fructose, and glucose is 50:8:2:25.

[0019] Furthermore, the caramel sacrificial template is a grid-like circular tube structure, and after being dissolved, the coating on its surface forms the outer support of the PGS / PCL hollow tube.

[0020] Furthermore, the ratio of porcine smooth muscle cells and porcine chondrocytes seeded on the double-layer scaffold was 7:3.

[0021] Furthermore, the double-layer scaffold undergoes decellularization treatment, including:

[0022] SDS solution is injected into the outer support of the PGS / PCL hollow pipe.

[0023] Then wash with PBS and add Triton X-100 solution;

[0024] Continue washing with PBS, add deoxyribonuclease to remove DNA while preserving the main matrix structure.

[0025] On the other hand, a tissue-engineered artificial blood vessel for small-diameter arterial regeneration obtained as described in the method is provided, the tissue-engineered artificial blood vessel comprising a PCL near-field direct-write inner layer stent and a PGS / PCL hollow conduit outer layer stent, and the whole being in the shape of a cylindrical tube;

[0026] The PCL near-field direct writing inner scaffold is a micro / nano fiber network structure;

[0027] The outer support of the PGS / PCL hollow tube has circumferential and longitudinal channels that are interconnected, and the outer support of the PGS / PCL hollow tube is closely attached to the outer support of the PCL near-field direct writing inner support.

[0028] Furthermore, the tissue-engineered artificial blood vessel contains extracellular matrix and bioactive substances of porcine smooth muscle cells and porcine chondrocytes, tightly integrating the PCL near-field direct-write inner scaffold and the PGS / PCL hollow channel outer scaffold into one unit.

[0029] Furthermore, the hollow internal cavity of the outer support of the PGS / PCL hollow pipe serves as a drug loading space.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention provides a tissue-engineered artificial blood vessel for small-diameter arterial regeneration and its construction method. It addresses the cell source issue by using porcine cartilage and smooth muscle cells. Furthermore, a hollow, perfusion-compatible conduit structure is prepared using a caramel sacrificial template, allowing for the loading of multiple drugs such as antibiotics and anticoagulants, effectively preventing infection and embolism in tissue-engineered artificial blood vessels. Simultaneously, a decellularized extracellular matrix is ​​prepared using decellularization technology, enabling immediate implantation after engineered preparation, resulting in a short procedure time and suitability for trauma emergency care. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained from these drawings without creative effort.

[0033] Figure 1 This is a schematic diagram of the tissue-engineered artificial blood vessel construction process provided in an embodiment of the present invention.

[0034] Figure 2 This is a schematic diagram of the assembly of the bioreactor provided in an embodiment of the present invention.

[0035] Figure 3 This is a photograph of the actual bioreactor culture provided in an embodiment of the present invention.

[0036] Figure 4 These are macroscopic images of two artificial blood vessels of different diameters cultured according to embodiments of the present invention. Specifically, a is a macroscopic surface view of a 4mm artificial blood vessel, b is a macroscopic side view of a 4mm artificial blood vessel, c is a macroscopic surface view of a 5mm artificial blood vessel, and d is a macroscopic side view of a 5mm artificial blood vessel.

[0037] Figure 5 These are images showing the shape of the artificial vascular stent before and after decellularization, and the deformation after applying pressure, provided in this embodiment of the invention. In the images, a is the shape of the artificial vascular stent before decellularization, b is the deformation before decellularization under external force, c is the shape of the artificial vascular stent after decellularization, and d is the deformation after decellularization under external force.

[0038] Figure 6 This is a scanning electron microscope image of the surface and cross-section of the artificial blood vessel before decellularization, provided in an embodiment of the present invention.

[0039] Figure 7 This is a scanning electron microscope image of the surface and cross-section of the decellularized artificial blood vessel provided in an embodiment of the present invention.

[0040] Figure 8 This is an image showing the HE and Safranin O staining results of pre-decellularized artificial blood vessel sections provided in an embodiment of the present invention.

[0041] Figure 9 This is a diagram showing the results of safranin O staining of decellularized artificial blood vessel sections provided in an embodiment of the present invention.

[0042] Figure 10 The results are color Doppler ultrasound monitoring results after tissue-engineered artificial blood vessels were implanted into animals, as provided in the embodiments of the present invention. Detailed Implementation

[0043] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0044] In the description of this invention, it should be understood that all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the definitions in this specification shall prevail. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, the reagents used in the embodiments are commercially available products, and the devices used in the embodiments are existing devices. The limitation on the means, reagents, or devices should not be construed as a limitation on the present invention. Means, reagents, or devices of the same type that solve the same technical problem are all within the protection scope of the present invention.

[0045] In the description of this invention, it should be understood that when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0046] It should also be noted that although the order of steps is mentioned in the method description, in some cases, steps may be performed in a different order than that described here, and this should not be interpreted as a restriction on the order of steps.

[0047] This invention provides a tissue-engineered artificial blood vessel that can be used for small-diameter arterial regeneration transplantation. It has a double-layer scaffold structure, with a hollow outer scaffold that can be loaded with anticoagulant and anti-infective drugs. The surface of this tissue-engineered artificial blood vessel is also loaded with extracellular matrix and bioactive substances obtained through decellularization treatment. Therefore, it can meet the multiple requirements of blood flow patency, tissue integration, and mechanical stability required for small-diameter arterial vascular transplants. It can also be rapidly constructed and is suitable for emergency treatment.

[0048] Specifically, the aforementioned tissue-engineered artificial blood vessels are prepared using the following construction method:

[0049] S1: Prepare a PCL near-field direct writing inner scaffold and a PGS / PCL hollow tube outer scaffold, which are then nested sequentially around a small-diameter silicone tube to obtain a double-layer scaffold-silicone tube assembly.

[0050] Near-Field Electrospinning Writing (NFEW) is an advanced micro / nano fabrication technology that combines the principles of electrospinning with high-precision motion control. By overcoming the randomness inherent in traditional electrospinning, it achieves controllable directional deposition of micro / nanofiber fibers. In this invention, the PCL near-field writing inner scaffold utilizes a micro / nanofiber network structure obtained from PCL (polycaprolactone) through near-field writing technology. This network features a fine micromesh, providing an excellent substrate for loading extracellular matrix.

[0051] The specific steps for preparing the outer support of the PGS / PCL hollow pipe are as follows:

[0052] S11: Sucrose, dextran, fructose, glucose, and ultrapure water are mixed and stirred until the color turns slightly yellow. After heating and drying, the mixture is 3D printed to obtain a caramel sacrificial template. The mixing ratio of sucrose, dextran, fructose, and glucose is 50:8:2:25, with an appropriate amount of ultrapure water. For example, 50g of sucrose, 8g of dextran, 2g of fructose, 25g of glucose, and 40ml of ultrapure water can be mixed to prepare the caramel sacrificial template. The flexible sugar mesh planar substrate obtained by this sugar mixture formula has good toughness and plasticity. It is soft and deformable, and can be freely cut into different shapes. It can also be manually extruded, stretched, bent, kneaded, assembled, and glued to obtain the desired three-dimensional structure, and can maintain the desired three-dimensional configuration for a long time.

[0053] S12: PCL and PGS (polyglycerol sebacic acid ester) are added to a tetrahydrofuran solution to prepare a coating liquid, which is then coated on the outside of the caramel sacrificial template to obtain a PGS / PCL caramel sacrificial template assembly.

[0054] S13: The PGS / PCL caramel sacrificial template assembly was placed in distilled water to dissolve the flexible sugar sacrificial carrier, resulting in the outer support of the PGS / PCL hollow tubes. The caramel sacrificial template has a grid-like circular tube structure, and after being dissolved, the coating on its surface forms the outer support of the PGS / PCL hollow tubes.

[0055] S2: Fix the double-layer support silicone tube assembly inside the bioreactor, soak the bioreactor in an alcohol bath, and then place the bioreactor in a clean bench to dry.

[0056] S3: Porcine smooth muscle cells and porcine chondrocytes were inoculated onto a double-layered scaffold, and the culture medium was pumped into a bioreactor for culture. The ratio of porcine smooth muscle cells to porcine chondrocytes inoculated onto the silicone tube conjugate of the double-layered scaffold was 7:3. This combination of cartilage matrix providing strength and smooth muscle matrix providing flexibility ensures that the physical properties and collagen structure of the extracellular matrix cultured in the bioreactor are similar to those of human arteries, resulting in increased matrix secretion and significantly reduced immunogenicity.

[0057] S4: Under aseptic conditions, remove the bilayer scaffold silicone tubing assembly from the bioreactor, remove the silicone tubing from the assembly, and perform decellularization, retaining the extracellular matrix and bioactive substances. The decellularization process of the bilayer scaffold specifically includes:

[0058] S41: Inject SDS solution into the pipe of the outer support of the PGS / PCL hollow pipe;

[0059] S42: Then wash with PBS and add Triton X-100 solution;

[0060] S43: Continue washing with PBS, add deoxyribonuclease to remove DNA while preserving the main matrix structure.

[0061] S5: Obtain a tissue-engineered artificial blood vessel for small-diameter arterial regeneration, wherein the outer stent of the PGS / PCL hollow conduit has a drug-loadable hollow perfusion conduit structure.

[0062] The tissue-engineered artificial blood vessel for small-diameter arterial regeneration obtained by the above method has a special structure, including a PCL near-field direct-writing inner scaffold and a PGS / PCL hollow-channel outer scaffold, with an overall cylindrical shape. The PCL near-field direct-writing inner scaffold has a micro / nano fiber network structure, while the PGS / PCL hollow-channel outer scaffold has circumferential and longitudinal channels that are interconnected, and is tightly attached to the outside of the PCL near-field direct-writing inner scaffold. Simultaneously, the tissue-engineered artificial blood vessel contains extracellular matrix and bioactive substances from porcine smooth muscle cells and porcine chondrocytes, tightly integrating the PCL near-field direct-writing inner scaffold and the PGS / PCL hollow-channel outer scaffold into a single unit.

[0063] The tissue-engineered artificial blood vessel provided by this invention creatively introduces a hollow tube stent structure to construct a perfusionable artificial blood vessel structure. The hollow inner cavity of the outer stent of its PGS / PCL hollow tube serves as a drug-loading space, allowing for the perfusion of various drugs. When anticoagulants are perfused, a local and overall anticoagulant microenvironment is created within the artificial blood vessel, preventing embolism. When antibiotics are perfused, a local and overall anti-infection microenvironment is created within the artificial blood vessel, preventing infection. Of course, other drugs can also be loaded, giving the artificial blood vessel more therapeutic functions.

[0064] The construction method of the present invention will be further described in detail below through specific embodiments.

[0065] Example:

[0066] This embodiment constructs a bioreactor for culturing decellularized extracellular matrix tissue-engineered blood vessels. By culturing porcine-derived cartilage and smooth muscle cells, the physical properties and collagen structure of the extracellular matrix tissue-engineered artificial blood vessel scaffold cultured in the bioreactor are ensured to be similar to those of human arteries. The decellularization technique provides excellent biosafety, preserving the main components and structure of the extracellular matrix. To address the issues of secondary trauma and long-term placement caused by in vivo embedding, this embodiment proposes a novel decellularized extracellular matrix vascular scaffold, which offers simple and efficient in vitro culture conditions. Specifically:

[0067] Step 1: Prepare the outer layer scaffold of PGS / PCL hollow tube using a caramel sacrificial template, and prepare a composite double-layer scaffold by combining it with the inner layer scaffold of PCL near-field direct writing, which will be used as a scaffold material for bioreactor culture.

[0068] Step 2: Assemble and sterilize the various components of the bioreactor for culturing tissue-engineered artificial blood vessels rich in extracellular matrix.

[0069] Step 3: Obtain extracellular matrix artificial blood vessels by inoculating porcine chondrocytes and smooth muscle cells and regularly replacing the culture medium with fresh ones.

[0070] Step 4: Under aseptic conditions, material is harvested from the bioreactor and decellularized to obtain extracellular matrix tissue-engineered artificial blood vessels with low immunoreactivity and high bioactivity.

[0071] Step one is as follows:

[0072] like Figure 1 The material preparation process for a bioreactor includes the following steps:

[0073] 1. Mix 50g sucrose, 8g dextran, 2g fructose, 25g glucose and 40ml ultrapure water at 120℃ and stir until the color is slightly yellow. Place in a 130℃ drying oven for 16 hours and then perform 3D bioprinting to prepare a caramel sacrificial template.

[0074] 2. Prepare a 3% mixed solution of PCL and PGS with tetrahydrofuran solution for coating to prepare the outer support of PGS / PCL hollow pipe.

[0075] A PCL inner layer scaffold was fabricated using near-field direct writing technology. The near-field direct-written PCL scaffold and the PGS / PCL hollow tube scaffold were sequentially nested onto a silicone tube with an outer diameter of 4 mm, forming a structure as shown below. Figure 1 The materials shown can be used for bioreactor cultivation.

[0076] Step two is as follows:

[0077] like Figure 2 This is a schematic diagram of the assembly of a bioreactor, including the following steps:

[0078] (1) Place the double-layer support silicone tube assembly and bioreactor in a 75% alcohol bath for at least 30 minutes, ensuring that the alcohol completely submerges the bioreactor.

[0079] (2) Remove the bioreactor from the ethanol bath, fix the double-layer support silicone tube assembly in the glass tube in the middle of the reactor, and then put the bioreactor back into the ethanol bath for 6 hours.

[0080] (3) Remove the bioreactor from the alcohol bath and drain all the alcohol. Place the bioreactor in a clean bench.

[0081] (4) Rinse the bioreactor and the double-layered support silicone tube assembly with sterile water, drain all excess water into a 1000ml beaker, and then place a sterile stirring rod into the bioreactor.

[0082] (5) Turn on the fan and ultraviolet light of the clean bench and let the bioreactor dry overnight.

[0083] (6) During the assembly process, a sterile culture dish is placed on the opening of the bioreactor to protect the internal double-layer scaffold silicone tube assembly from contamination.

[0084] (7) Connect the interfaces on both sides of the bioreactor with silicone tubes and insert both sides into sterile saline solution, then seal all connections.

[0085] Step three specifically involves:

[0086] like Figure 3 The image shows the actual cells cultured in the bioreactor. The detailed steps for cell inoculation and medium change are as follows:

[0087] (1) Inoculate porcine smooth muscle and chondrocytes (in a ratio of 7:3) onto the reactor and seal the reactor opening with a porous silica foam plug.

[0088] (2) Seal the holes on the silicone plug with a 0.22μm filter to ensure gas flow and provide a sterile environment, and seal the other parts with a sealing film.

[0089] (3) Connect the feeding port with a heparin cap, place the inoculated reactor into the incubator, and change its direction every 5 minutes.

[0090] (4) After 30 minutes, place the reactor on the clean bench, connect the 20ml syringe needle to the heparin cap in the opposite direction, put the back end of the needle into the culture medium, connect the silicone tube to the pressure pump, and let the culture medium flow into the reactor. After the culture medium submerges the support, stop the liquid inlet, seal the inlet of the bioreactor, and put it into the incubator.

[0091] (5) The medium should be changed every week thereafter. In short, connect the silicone tubing to the pressure pump to allow the culture medium to flow out, replace the heparin cap, and pump the fresh culture medium back into the bioreactor.

[0092] In this embodiment, the source of the inoculated smooth muscle cells can be replaced with other species that secrete abundant matrix (such as bovine aorta or human umbilical artery). The porcine smooth muscle cells used can be replaced with fibroblasts or other cell types that secrete the same matrix.

[0093] Step four is as follows:

[0094] The artificial blood vessels (cell matrix and its scaffold) in the bioreactor should be removed under aseptic conditions. Decellularization should also be performed under aseptic conditions, and the specific decellularization method is as follows:

[0095] The hollow tube of the artificial blood vessel was perfused with 1% SDS solution for 12 hours, then washed 3 times with PBS, 1% Triton X-100 solution was added for 2 hours, washed 3 times with PBS, and deoxyribonuclease was added to remove DNA while retaining the main matrix structure.

[0096] A cell-free tissue-engineered artificial blood vessel scaffold was obtained using the above method, with physical properties and collagen structure similar to human arteries. The decellularization process reduces the immune response while preserving the extracellular matrix and bioactive substances (such as extracellular vesicles). Figure 1 and Figure 4 As shown, the scaffold has a tubular structure, with an inner layer of micro / nanofiber network and an outer layer of tubular hollow mesh. The extracellular matrix cultured on it tightly integrates the inner and outer layers into a single unit.

[0097] The construction method of this invention utilizes in vitro culture, solving the problems of secondary trauma and long-term placement caused by in vivo embedding. It is simple and highly efficient. Furthermore, the artificial blood vessel provided by this invention has a short culture time and simple culture conditions. Compared to artificial blood vessels from Humacyte (USA), the cell source is readily available, and the culture time is shortened from 8 weeks to 4 weeks, improving production efficiency. In addition, this invention prepares decellularized extracellular matrix using decellularization technology. After engineered preparation, it can be immediately implanted. Based on pre-processing, it can be directly used for small-diameter vascular injuries in traumatic and emergency wounds, making it highly suitable for emergency treatment.

[0098] In the description of this invention, it should be understood that the cell source used is pig. Cells from other species (such as cattle, humans, etc.) should not be construed as a limitation on the cell source. Technical solutions obtained by changing the cell source or cell ratio when solving the same technical problem are also within the scope of protection of this patent.

[0099] In the description of this invention, it should be understood that porcine smooth muscle cells are inoculated, but they can be replaced with other cell types with vigorous matrix secretion, such as fibroblasts. Technical solutions obtained by changing the cell type or species source when solving the same technical problem are also within the scope of protection of this patent.

[0100] In the description of this patent, it should be understood that although small-diameter artificial blood vessels with diameters of 4 mm and 5 mm were cultured, decellularized matrix artificial blood vessels of different sizes can be cultured by changing the size of the reactor. Technical solutions obtained by changing the size of the reactor when solving the same technical problem are also within the scope of protection of this patent.

[0101] The following is an analysis of the relevant experimental results:

[0102] 1. For example Figure 4 As shown, the bioreactor cultured artificial blood vessels of two different diameters: 4 mm (Figures a and b) and 5 mm (Figures c and d). Gross images after culture show the generation of a large amount of extracellular matrix, which encapsulates the scaffold material and fuses it together. Figure 5 As shown in the figures, a and b are the scaffolds before decellularization, and c and d are the scaffolds after decellularization. Applying external force shows that the scaffolds have good mechanical strength before and after decellularization and will return to their original state during compression.

[0103] 2. For example Figure 6 and Figure 7 As shown, the scanning electron microscope images reveal collagen fibers and secreted extracellular vesicles, among other bioactive substances, before and after decellularization. Figure 6 Within it, it can be observed that the surface and cross-section are rich in extracellular matrix components. Figure 7 No cell structures were observed on the surface or in the cross-section, but collagen fibers and vesicle structures were observed, indicating that the decellularization effect was good and the extracellular matrix components were well preserved.

[0104] 3. For example Figure 8 As shown, HE and Safranin O staining revealed that the scaffold was uniformly enveloped by the extracellular matrix, with a large amount of matrix well anchored to the scaffold. Figure 9 As shown, Safranin O staining after decellularization revealed that cells were largely removed while the collagen matrix was well preserved.

[0105] 4. For example Figure 10 As shown, after the decellularized artificial blood vessel was implanted into the animal, the color Doppler ultrasound monitoring results showed that the blood flow in the transplanted body was smooth and no thrombus formation occurred.

[0106] The above examples illustrate the present invention only to aid in understanding it and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention.

Claims

1. A method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration, characterized in that: The method includes: A PCL near-field direct writing inner scaffold and a PGS / PCL hollow tube outer scaffold were fabricated and nested sequentially on a small-diameter silicone tube to obtain a double-layer scaffold-silicone tube assembly. The double-layer support silicone tube assembly was fixed inside the bioreactor. After soaking the bioreactor in an alcohol bath, the bioreactor was placed in a clean bench to dry. Porcine smooth muscle cells and porcine chondrocytes were inoculated onto a double-layered scaffold, and the culture medium was pumped into a bioreactor for culture. Under sterile conditions, the double-layer scaffold silicone tube assembly was removed from the bioreactor, the silicone tubes of the double-layer scaffold silicone tube assembly were removed, and decellularization was performed to retain the extracellular matrix and bioactive substances. A tissue-engineered artificial blood vessel for small-diameter arterial regeneration was obtained, whose PGS / PCL hollow conduit outer stent has a drug-loadable hollow perfusion conduit structure.

2. The method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration according to claim 1, characterized in that: PCL near-field direct writing inner scaffold is a micro / nano fiber network structure obtained by near-field direct writing technology using PCL.

3. The method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration according to claim 2, characterized in that: Fabrication of the outer support layer for PGS / PCL hollow pipes includes: Sucrose, dextran, fructose, glucose and ultrapure water were mixed and stirred until the color turned slightly yellow. After heating and drying, the mixture was 3D printed to obtain a caramel sacrificial template. PCL and PGS were added to a tetrahydrofuran solution to prepare a coating liquid, which was then coated onto the outside of a caramel sacrificial template to obtain a PGS / PCL caramel sacrificial template assembly. The PGS / PCL caramel sacrificial template assembly was placed in distilled water to dissolve the flexible sugar sacrificial carrier, thus obtaining the outer support of the PGS / PCL hollow pipe.

4. The method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration according to claim 3, characterized in that: The mixing ratio of sucrose, dextran, fructose, and glucose is 50:8:2:

25.

5. The method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration according to claim 4, characterized in that: The caramel sacrificial template is a grid-like circular tube structure. After being dissolved, the coating on its surface forms the outer support of the PGS / PCL hollow tube.

6. The method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration according to claim 5, characterized in that: The ratio of porcine smooth muscle cells and porcine chondrocytes seeded on the double-layer scaffold was 7:

3.

7. The method for constructing tissue-engineered artificial blood vessels for small-diameter arterial regeneration according to claim 6, characterized in that: The double-layer scaffold underwent decellularization, including: SDS solution is injected into the outer support of the PGS / PCL hollow pipe. Then wash with PBS and add Triton X-100 solution; Continue washing with PBS, add deoxyribonuclease to remove DNA while preserving the main matrix structure.

8. The tissue-engineered artificial blood vessel for small-diameter arterial regeneration obtained by the method as described in claim 7, characterized in that: The tissue-engineered artificial blood vessel includes a PCL near-field direct-write inner layer stent and a PGS / PCL hollow tube outer layer stent, and the whole is in the shape of a circular tube. The PCL near-field direct writing inner scaffold is a micro / nano fiber network structure; The outer support of the PGS / PCL hollow tube has circumferential and longitudinal channels that are interconnected, and the outer support of the PGS / PCL hollow tube is closely attached to the outer support of the PCL near-field direct writing inner support.

9. The tissue-engineered artificial blood vessel for small-diameter arterial regeneration obtained by the method according to claim 8, characterized in that: The tissue-engineered artificial blood vessel contains extracellular matrix and bioactive substances of porcine smooth muscle cells and porcine chondrocytes, tightly integrating the PCL near-field direct-write inner scaffold and the PGS / PCL hollow channel outer scaffold.

10. The tissue-engineered artificial blood vessel for small-diameter arterial regeneration obtained by the method according to claim 9, characterized in that: The hollow internal cavity of the outer support of the PGS / PCL hollow pipeline is a drug loading space.

Images