Biological 3D printed tissue engineered trachea and construction method and application thereof

Abstract

The application relates to a biological 3D printed tissue engineering trachea and a construction method and application thereof, namely a cartilage / vascularized fiber tissue alternating tissue engineering trachea. The construction method comprises the following steps: firstly, cartilage specific biological ink is prepared by wrapping cartilage cells with cartilage tissue specific hydrogel; fiber specific biological ink is prepared by wrapping fibroblasts with fiber tissue specific hydrogel; then, through a double-needle extrusion type biological 3D printing technology, alternating printing is realized to form a ring-shaped tubular structure, and a cartilage / fiber tissue alternating tubular structure is constructed through multi-layer accumulation molding; further, vascularization of the fiber tissue is realized through a certain culture mode, and finally, a relatively mature cartilage / vascularized fiber tissue alternating tissue engineering trachea is obtained. The application also provides application of the cartilage / vascularized fiber tissue alternating tissue engineering trachea in tracheal defect repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical tissue engineering, and particularly relates to a biological 3D-printed tissue engineering trachea and a construction method and application thereof. BACKGROUND

[0002] Functional segmental tracheal reconstruction has been a great challenge in clinic, mainly because of the lack of ideal tracheal grafts. In recent years, the development of tissue engineering technology has provided a new treatment for tracheal defect reconstruction. Although the tracheal grafts constructed by using tissue engineering tubular cartilage have achieved certain repair effects in animal verification, the single and homogeneous tubular tracheal structure will inevitably lead to mechanical defects (such as lack of longitudinal stretching and transverse bending ability), and the lack of vascularized fibrous tissue between the cartilage rings provides necessary nutrients for cartilage regeneration, which is not conducive to the final clinical application. It is worth noting that the real tracheal structure is a heterogeneous structure with alternating cartilage rings and vascularized fibrous tissue rings, which is essential for the realization of the mechanical and physiological functions of the trachea.

[0003] Recently, a construction concept of assembling a tubular structure trachea based on cartilage rings has been reported in the literature, which is achieved by alternately stacking cartilage rings and high molecular rings (acellular) to form a tubular structure, which can simulate the physiological structure of the natural trachea to a certain extent (reference: Biomimetic Trachea Regeneration Using a Modular Ring Strategy Based on Poly(Sebacoyl Diglyceride) / Polycaprolactone for Segmental Trachea Defect Repair; Y. Xu, Y. Guo, Y. Li, Y. Huo, Y. She, H. Li, Z. Jia, G. Jiang, G. Zhou, Z. You, L. Duan, Adv. Funct. Mater. 2020, 30, 2004276.). However, the above-mentioned research fails to solve the following problems: 1) the synthetic material lacks ideal biocompatibility and tissue-specific microenvironment; 2) a long-term in vitro culture process is needed to achieve satisfactory cartilage formation for in vivo tracheal defect reconstruction; 3) the manually prepared tubular structure cannot realize the precise controllability of cell arrangement and alternating structure; 4) the vascularized fibrous tissue regeneration depends on random cell infiltration, which often leads to unstable tissue formation and poor vascularization; 5) the interface integration design of cartilage ring and vascularized fibrous ring is unreasonable, which is easy to fall off; 6) there is a lack of systematic functional evaluation of the regenerated trachea. Therefore, there is an urgent need to provide a new construction method of tissue engineering trachea. SUMMARY

[0004] In view of the performance defects of the tracheal grafts based on the existing technology of tissue engineering tubular cartilage construction in terms of mechanics, physiology and the like, the application provides a biological 3D printed tissue engineering trachea and a construction method and application thereof.

[0005] The object of the application can be achieved by the following technical solutions.

[0006] The application first provides a biological 3D printed tissue engineering trachea, which is an annular tubular structure formed by alternately stacking cartilage rings and vascularized fiber rings,

[0007] The cartilage ring is a cartilage-specific biological ink 3D printed by wrapping chondrocytes with a cartilage tissue-specific hydrogel,

[0008] The vascularized fiber ring is a fiber-specific biological ink 3D printed by wrapping fibroblasts with a fiber tissue-specific hydrogel,

[0009] The cartilage ring and the vascularized fiber ring have typical tissue characteristics, and the cartilage ring and the vascularized fiber ring are integrated structures.

[0010] In the application, the cartilage tissue-specific hydrogel refers to a hydrogel having chondrogenic directional induction function, and the cartilage-specific biological ink prepared by wrapping chondrocytes with the cartilage tissue-specific hydrogel refers to a biological ink having chondrogenic directional induction function.

[0011] In the application, the fiber-specific hydrogel refers to a hydrogel having fibroblast directional induction function, and the fiber-specific biological ink prepared by wrapping fibroblasts with the fiber tissue-specific hydrogel refers to a biological ink having fibroblast directional induction function.

[0012] In the application, the structure of the biological 3D printed tissue engineering trachea is characterized in that: 1) it has an annular tubular structure; 2) the cartilage ring and the vascularized fiber ring have an alternating structure; 3) the cartilage ring and the vascularized fiber ring have typical tissue characteristics; and 4) the cartilage ring and the vascularized fiber ring are integrated.

[0013] In an embodiment of the application, the annular tubular structure has an inner diameter of 0.1-40 mm, an outer diameter of 0.5-50 mm, and a height of 0.1-100 mm, preferably an inner diameter of 2-5 mm, an outer diameter of 4-10 mm, and a height of 5-20 mm.

[0014] In an embodiment of the application, the thickness ratio of the cartilage ring to the vascularized fiber ring is 10:1-1:10, preferably 5:1-1:1, and further preferably 2:1-1.5:1.

[0015] In one embodiment of the present application, the cartilage ring has the following histological characteristics: the regenerated cartilage exhibits typical cartilage lacuna structure, and has histological staining specific to cartilage, such as Safranin O and type II collagen.

[0016] In one embodiment of the present application, the vascularized fibrous ring has the following histological characteristics: the regenerated fibrous tissue exhibits clear blood vessel structure, and has specific staining of CD31 for angiogenesis and Masson and type I collagen for fibroblast.

[0017] In one embodiment of the present application, the interface bonding force of the cartilage ring and the vascularized fibrous ring is 1 kPa to 10 MPa, preferably 100 kPa to 1 MPa.

[0018] In one embodiment of the present application, the cartilage tissue-specific hydrogel is a cartilage tissue-specific hydrogel material prepared by dissolving a water-soluble polymer in a biocompatible medium and adding a cartilage tissue-specific component, and then cross-linking.

[0019] In one embodiment of the present application, the fibrous tissue-specific hydrogel is a fibrous tissue-specific hydrogel material prepared by dissolving a water-soluble polymer in a biocompatible medium and adding a fibrous tissue-specific component, and then cross-linking.

[0020] In one embodiment of the present application, the water-soluble polymer is selected from natural polymer materials or synthetic polymer materials.

[0021] In one embodiment of the present application, the natural polymer material includes natural polysaccharide substances and their modifications or degradation products, proteins and their modifications or degradation products.

[0022] In one embodiment of the present application, the natural polysaccharide substances include hyaluronic acid, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan, or chitosan quaternary ammonium salt.

[0023] In one embodiment of the present application, the proteins include various hydrophilic or water-soluble animal and plant proteins, collagen, serum proteins, silk fibroin, and elastin.

[0024] In one embodiment of the present application, the protein degradation products include gelatin or polypeptides.

[0025] In one embodiment of the present application, the synthetic polymer material includes two-arm or multi-arm polyethylene glycol, polyethylene imine, dendrimer, synthetic polypeptide, polylysine, polyglutamic acid, polyacrylic acid, polymethacrylic acid, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl alcohol, polyvinylpyrrolidone.

[0026] In one embodiment of the present application, the hydrogel material is preferably a natural polysaccharide or protein polymer, and more preferably hyaluronic acid or gelatin.

[0027] In one embodiment of the present application, the biocompatible medium is selected from distilled water, physiological saline, buffer solution or cell culture medium solution. Different biocompatible media can be selected according to different applications.

[0028] In one embodiment of the present application, after the water-soluble polymer is dissolved in the biocompatible medium, the concentration of the water-soluble polymer is 0.1% w / v to 60% w / v, and preferably 1% w / v to 20% w / v.

[0029] In one embodiment of the present application, the cartilage tissue-specific component includes cartilage acellular matrix, growth factors for promoting chondrogenesis, etc.

[0030] In one embodiment of the present application, the cartilage acellular matrix is derived from various cartilage-containing tissues in animals, and is preferably ear cartilage, articular cartilage, meniscus cartilage, costal cartilage, etc.

[0031] In one embodiment of the present application, the animals include pigs, cows, sheep, dogs, rabbits, mice, etc., and are preferably pigs, cows, sheep and dogs.

[0032] In one embodiment of the present application, the growth factors for promoting chondrogenesis include TGF-β, VEGF, FGF, etc.

[0033] In one embodiment of the present application, the fibrous tissue-specific component includes fibroblast acellular matrix, growth factors for promoting fibroblast differentiation, etc.

[0034] In one embodiment of the present application, the fibroblast acellular matrix is derived from various fibrous tissues in animals, and is preferably skin, dermis, muscle, etc.

[0035] In one embodiment of the present application, the animals include pigs, cows, sheep, dogs, rabbits, mice, etc., and are preferably pigs, cows, sheep and dogs.

[0036] In one embodiment of the present application, the growth factors for promoting fibroblast differentiation include TGF-β, VEGF, FGF, BMP-2-9, etc.

[0037] In one embodiment of the present application, when preparing the cartilage tissue-specific hydrogel or the fibrous tissue-specific hydrogel, the cross-linking method is selected from one or more of physical cross-linking, chemical cross-linking or photo-cross-linking, or a single cross-linking method is combined with multiple materials.

[0038] In one embodiment of the present application, the physical cross-linking is through entanglement or non-covalent bond cross-linking between polymer chains (reference document Xiangyu Liang, Pingguo Duan, Jingming Gao, et al. ACS Biomater. Sci. Eng. 2018, 4, 3506.). The physical cross-linking reaction includes thermal condensation (temperature-sensitive): polyisopropyl acrylamide (PNIPAAm), block copolymer (PEO-PPO-PEO, PLGA-PEG-PLGA, PEG-PLLA-PEG, PCL-PEG-PCL, etc.); self-assembly: hydrophilic-hydrophobic interaction, hydrogen bonding, host-guest interaction; ionic cross-linking: alginic acid and calcium ions; electrostatic interaction: chitosan and phosphoric substances.

[0039] In one embodiment of the present application, the chemical cross-linking is through covalent bond cross-linking between polymer chains (reference document Luping Cao, Bin Cao, Chengjiao Lu, et al. J. Mater. Chem. B 2015, 3, 1268.). The chemical cross-linking reaction includes thiol-Michael addition reaction, amide condensation reaction, Schiff base reaction, etc.

[0040] In one embodiment of the present application, the photo-cross-linking is through photochemical reaction to form covalent bond cross-linking (reference document Huitang Xia, Dandan Zhao, Hailin Zhu, et al. ACS Appl. Mater. Interfaces 2018, 10, 31704.).

[0041] In one embodiment of the present application, the photo-cross-linking constructed hydrogel material is prepared by photo-induced polymerization cross-linking reaction, that is, the free radicals generated by the photo-initiator under light source irradiation initiate the polymerization reaction of the double bond functional groups on the polymer derivatives containing methacrylate groups, thereby preparing the photo-cross-linking hydrogel.

[0042] In one embodiment of the present application, the photo-cross-linking hydrogel can be implemented as follows: the polymer derivatives containing methacrylate groups and the photo-initiator are dissolved in a biocompatible medium to prepare a hydrogel precursor solution with a certain concentration, and the photo-cross-linking hydrogel can be obtained under the irradiation of a light source with a wavelength of 254 nm-450 nm (preferably 365 nm or 405 nm).

[0043] In one embodiment of the present application, the methacrylate group-containing polymer derivative is selected from gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), chondroitin sulfate methacrylate (CSMA), elastin methacrylate (ElaMA), heparin methacrylate (HepMA), dextran methacrylate (DexMA), chitosan methacrylate (ChMA), cellulose methacrylate (CelMA), and the like.

[0044] In one embodiment of the present application, the photoinitiator can be selected from I 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) or LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate).

[0045] In the present application, the hydrogel material can be formed by one or more of physical crosslinking, chemical crosslinking or photo-crosslinking of water-soluble polymers, or by a combination of multiple materials crosslinked by a single crosslinking method.

[0046] In one embodiment of the present application, the hydrogel material can be a single-network hydrogel formed by a single crosslinking method, or a interpenetrating network hydrogel or double-network hydrogel formed by two or more crosslinking methods, or a composite crosslinking hydrogel formed by a combination of multiple materials crosslinked by the same crosslinking method.

[0047] In one embodiment of the present application, the chondrocyte cell density in the cartilage tissue-specific hydrogel is 10 4 ~ 10 10 / mL, preferably 10 7 ~ 10 8 / mL.

[0048] In one embodiment of the present application, the fibroblast cell density in the fibrous tissue-specific hydrogel is 10 4 ~ 10 10 / mL, preferably 10 7 ~ 10 8 / mL.

[0049] The present application also provides a preparation method of the biological 3D-printed tissue engineering trachea, which is a method for constructing a tissue engineering trachea based on biological 3D printing, comprising the following steps:

[0050] Preparation of cartilage-specific biological ink by wrapping chondrocytes with cartilage tissue-specific hydrogel;

[0051] Preparation of fibrous specific bio-ink by wrapping fibroblasts with fibrous tissue specific hydrogel;

[0052] Using cartilage specific bio-ink and fibrous specific bio-ink as materials, a cartilage ring and a vascularized fibrous ring are alternately printed by a double-needle extrusion type bio-3D printing technology, and the cartilage ring and the vascularized fibrous ring are alternately stacked to form a ring-shaped tubular structure.

[0053] The obtained ring-shaped tubular structure is cultured to realize vascularization of fibrous tissue, and a mature bio-3D printed tissue engineered trachea is obtained.

[0054] In an embodiment of the present application, the culture mode comprises in-vitro induced differentiation and culture, or in-vivo subcutaneous implantation culture, or in-vitro / in-vivo combined culture mode.

[0055] In an embodiment of the present application, the in-vitro induced differentiation and culture mode can be static culture or dynamic culture, wherein the static culture mode is to place the 3D printed tissue engineered trachea in a culture dish for culture, and the dynamic culture mode is to place the 3D printed tissue engineered trachea in a bioreactor for stirring or pressurized culture to promote the exchange of nutrients in the hydrogel.

[0056] In an embodiment of the present application, the in-vivo subcutaneous implantation culture is to implant the tissue engineered trachea without or with in-vitro culture into the subcutaneous tissue of an animal body. The animal body can be a nude mouse, an SD rat, a rabbit, a sheep, a dog or a pig, preferably a nude mouse or a sheep.

[0057] The histological characteristics of the bio-3D printed tissue engineered trachea provided by the present application are as follows: 1) the regenerated cartilage presents a typical cartilage lacuna structure, and has histological cartilage specific staining of safranin and type II collagen; and 2) the regenerated fibrous tissue presents clear blood vessel structure, and has hemangiogenesis specific staining of CD31 and fibroblast specific staining of Masson and type I collagen.

[0058] The structural characteristics of the bio-3D printed tissue engineered trachea provided by the present application are as follows: 1) the ring-shaped tubular structure; 2) the alternating structure of the cartilage ring and the vascularized fibrous ring; 3) the typical tissue characteristics of the cartilage ring and the vascularized fibrous ring; and 4) the integration of the cartilage ring and the vascularized fibrous ring.

[0059] The tissue engineered trachea constructed by the bio-3D printing of the present application realizes the recovery of mechanical and physiological functions: 1) the mechanical function reconstruction, such as longitudinal stretching and transverse bending ability; and 2) the physiological function reconstruction, wherein the vascularized fibrous tissue between the cartilage rings provides necessary nutrients for cartilage regeneration.

[0060] The present application also provides the application of the tissue engineered trachea constructed by the bio-3D printing.

[0061] The application provides application of a biologically 3D-printed tissue-engineered trachea in a tracheal defect repair material or a tissue-engineered tracheal graft.

[0062] The application scenarios of the biologically 3D-printed tissue-engineered trachea in the tracheal defect repair material include patch repair surgery and segmental tracheal reconstruction surgery.

[0063] In order to realize the construction of a bionic trachea, i.e., a tissue-engineered trachea with cartilage / vascularized fibrous tissue alternation, the application combines the advantages of biological 3D printing (personalization, precision, fast speed, etc.), and proposes a preparation method and application of a biologically 3D-printed tissue-engineered trachea.

[0064] Compared with the prior art, the application has the following advantages and beneficial effects:

[0065] (1) The natural hydrogel material has ideal biocompatibility and tissue-specific microenvironment, and can effectively promote specific tissue regeneration;

[0066] (2) The 3D-printed tissue-engineered trachea does not need to undergo a long-term in-vitro culture process, and can achieve satisfactory cartilage formation and in-vivo tracheal defect reconstruction;

[0067] (3) The annular tubular structure prepared by combining the biological 3D printing technology can realize accurate and controllable cell arrangement and alternating structure;

[0068] (4) The vascularized fibrous tissue regeneration depends on accurate three-dimensional cell arrangement (3D printing), which can promote tissue regeneration and vascularization formation;

[0069] (5) The interface integration of the cartilage ring and the vascularized fibrous ring can be realized by 3D printing, and is not easy to fall off;

[0070] (6) The regenerated tissue-engineered trachea realizes the recovery of mechanical and physiological functions. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 It is a schematic diagram of tissue-specific biological ink and a biologically 3D-printed tissue-engineered trachea.

[0072] Figure 2 It is an intuitive diagram of the effect of a biologically 3D-printed tissue-engineered trachea.

[0073] Figure 3 It is a general observation of the tissue-engineered trachea after being implanted subcutaneously in a nude mouse for 8 weeks.

[0074] Figure 4 It is a mechanical function evaluation of a tissue-engineered trachea.

[0075] Figure 5 Rabbit tracheal reconstruction surgery and repair of 8 weeks gross observation. DETAILED DESCRIPTION

[0076] The application provides a biological 3D printed tissue engineering trachea, which is an annular tubular structure formed by alternately stacking cartilage rings and vascularized fiber rings, the cartilage ring is prepared by 3D printing of cartilage-specific biological ink prepared by wrapping chondrocytes with cartilage tissue-specific hydrogel, the vascularized fiber ring is prepared by 3D printing of fiber-specific biological ink prepared by wrapping fibroblasts with fiber tissue-specific hydrogel, the cartilage ring and the vascularized fiber ring have typical tissue characteristics, and the cartilage ring and the vascularized fiber ring are integrated structures.

[0077] The structure of the biological 3D printed tissue engineering trachea provided by the application is characterized in that: 1) having an annular tubular structure; 2) the alternating structure of the cartilage ring and the vascularized fiber ring; 3) the cartilage ring and the vascularized fiber ring have typical tissue characteristics; 4) the cartilage ring and the vascularized fiber ring are integrated.

[0078] In some embodiments of the application, the annular tubular structure has an inner diameter of 0.1-40 mm, an outer diameter of 0.5-50 mm, and a height of 0.1-100 mm, preferably an inner diameter of 2-5 mm, an outer diameter of 4-10 mm, and a height of 5-20 mm. The thickness ratio of the cartilage ring to the vascularized fiber ring is 10:1-1:10, preferably 5:1-1:1, and further preferably 2:1-1.5:1.

[0079] In some embodiments of the application, the cartilage ring has the following tissue characteristics: the regenerated cartilage presents a typical cartilage lacuna structure, and has cartilage-specific staining of safranin and type II collagen in histology. The vascularized fiber ring has the following tissue characteristics: the regenerated fibrous tissue presents clear blood vessel structure, CD31 angiogenesis-specific staining, and Masson and type I collagen fibroblast-specific staining.

[0080] In some embodiments of the application, the interface bonding force of the cartilage ring and the vascularized fiber ring is 1 kPa-10 MPa, preferably 100 kPa-1 MPa.

[0081] In some embodiments of the application, the cartilage tissue-specific hydrogel is a cartilage tissue-specific hydrogel material prepared by dissolving a water-soluble polymer in a biocompatible medium and adding a cartilage tissue-specific component and cross-linking. The fiber tissue-specific hydrogel is a fiber tissue-specific hydrogel material prepared by dissolving a water-soluble polymer in a biocompatible medium and adding a fiber tissue-specific component and cross-linking.

[0082] In some embodiments of the present application, the water-soluble polymer is selected from natural polymer materials or synthetic polymer materials. The natural polymer materials include natural polysaccharide substances and their modifications or degradation products, proteins and their modifications or degradation products. The natural polysaccharide substances include hyaluronic acid, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan or chitosan quaternary ammonium salt. The proteins include various hydrophilic or water-soluble animal and plant proteins, collagen, serum proteins, silk fibroin, elastin. The protein degradation products include gelatin or polypeptides. The synthetic polymer materials include two-arm or multi-arm polyethylene glycol, polyethyleneimine, dendrimer, synthetic polypeptide, polylysine, polyglutamic acid, polyacrylic acid, polymethacrylic acid, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl alcohol, polyvinylpyrrolidone. The hydrogel material is preferably a natural polysaccharide or protein polymer, and further preferably hyaluronic acid or gelatin. The biocompatible medium is selected from distilled water, physiological saline, buffer or cell culture medium solution. Different biocompatible media can be selected according to different applications. After the water-soluble polymer is dissolved in the biocompatible medium, the concentration of the water-soluble polymer is 0.1% w / v to 60% w / v, and preferably 1% w / v to 20% w / v.

[0083] In some embodiments of the present application, the cartilage tissue-specific component includes cartilage acellular matrix, growth factors for promoting chondrogenic differentiation, etc. The cartilage acellular matrix is derived from various cartilage-containing tissues in animals, preferably ear cartilage, articular cartilage, meniscus cartilage, costal cartilage, etc. The animals include pigs, cows, sheep, dogs, rabbits, mice, etc., and preferably pigs, cows, sheep and dogs. In some embodiments of the present application, the growth factors for promoting chondrogenic differentiation include TGF-β, VEGF, FGF, etc.

[0084] In some embodiments of the present application, the fibrous tissue-specific component includes fibroblast acellular matrix, growth factors for promoting fibroblast differentiation, etc. The fibroblast acellular matrix is derived from various fibrous tissues in animals, preferably skin, dermis, muscle, etc. The animals include pigs, cows, sheep, dogs, rabbits, mice, etc., and preferably pigs, cows, sheep and dogs. The growth factors for promoting fibroblast differentiation include TGF-β, VEGF, FGF, BMP-2-9, etc.

[0085] In some embodiments of the present application, when preparing the cartilage tissue-specific hydrogel or the fibrous tissue-specific hydrogel, the cross-linking method is selected from one or more of physical cross-linking, chemical cross-linking or photo-cross-linking, or a single cross-linking method combined with multiple materials. The physical cross-linking is through the entanglement or non-covalent bond between polymer chains (reference Xiangyu Liang, Pingguo Duan, Jingming Gao, et al. ACS Biomater. Sci. Eng. 2018, 4, 3506.). The physical cross-linking reaction includes thermal condensation (temperature-sensitive): polyisopropyl acrylamide (PNIPAAm), block copolymer (PEO-PPO-PEO, PLGA-PEG-PLGA, PEG-PLLA-PEG, PCL-PEG-PCL, etc.); self-assembly: hydrophilic-hydrophobic interaction, hydrogen bonding, host-guest interaction; ionic cross-linking: alginic acid and calcium ions; electrostatic interaction: chitosan and phosphoric substances. The chemical cross-linking is through the covalent bond between polymer chains (reference Luping Cao, Bin Cao, Chengjiao Lu, et al. J. Mater. Chem. B 2015, 3, 1268.). The chemical cross-linking reaction includes thiol-Michael addition reaction, amide condensation reaction, Schiff base reaction, etc. The photo-cross-linking is through the photochemical reaction to form covalent bond cross-linking (reference Huitang Xia, Dandan Zhao, Hailin Zhu, et al. ACS Appl. Mater. Interfaces 2018, 10, 31704.). The photo-cross-linking constructed hydrogel material is prepared by photo-induced polymerization cross-linking reaction, that is, the free radicals generated by the photo-initiator under light source irradiation, which initiates the polymerization reaction of the double bond functional groups on the methacrylate group-containing polymer derivatives, thereby preparing the photo-cross-linked hydrogel. The photo-cross-linked hydrogel can be implemented in the following manner: dissolving the methacrylate group-containing polymer derivatives and the photo-initiator in a biocompatible medium to prepare a hydrogel precursor solution with a certain concentration, and then irradiating it under a light source with a wavelength of 254 nm-450 nm (preferably 365 nm or 405 nm), and the photo-cross-linked hydrogel can be obtained. The methacrylate group-containing polymer derivatives are selected from gelatin methacrylate (GelMA), hyaluronic acid methacrylate (HAMA), chondroitin sulfate methacrylate (CSMA), elastin methacrylate (ElaMA), heparin methacrylate (HepMA), dextran methacrylate (DexMA), chitosan methacrylate (ChMA), cellulose methacrylate (CelMA), etc.The photoinitiator can be I 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) or LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate).

[0086] In some embodiments of the present application, the hydrogel material can be formed by one or more of physical crosslinking or chemical crosslinking or photo-crosslinking of water-soluble polymers, or a combination of multiple materials crosslinked by a single crosslinking method. The hydrogel material can be a single-network hydrogel formed by a single crosslinking method, or an interpenetrating network hydrogel or a double-network hydrogel formed by two or more crosslinking methods, or a composite crosslinked hydrogel formed by a combination of multiple materials crosslinked by the same crosslinking method.

[0087] In some embodiments of the present application, the cell density of the chondrocytes in the cartilage tissue-specific hydrogel is 10 4 ~ 10 10 / mL, preferably 10 7 ~ 10 8 / mL. The cell density of the fibroblasts in the fibrous tissue-specific hydrogel is 10 4 ~ 10 10 / mL, preferably 10 7 ~ 10 8 / mL.

[0088] The present application also provides a method for preparing the biological 3D-printed tissue-engineered trachea, which is a method for constructing a tissue-engineered trachea based on biological 3D printing, comprising the following steps:

[0089] preparing cartilage-specific biological ink by wrapping chondrocytes in cartilage tissue-specific hydrogel;

[0090] preparing fibrous tissue-specific biological ink by wrapping fibroblasts in fibrous tissue-specific hydrogel;

[0091] using the cartilage-specific biological ink and the fibrous tissue-specific biological ink as materials, and forming cartilage rings and vascularized fibrous rings alternately by a double-needle extrusion type biological 3D printing technology, and stacking the cartilage rings and the vascularized fibrous rings alternately to form a ring-shaped tubular structure;

[0092] culturing the obtained ring-shaped tubular structure to achieve vascularization of the fibrous tissue, and obtaining a mature biological 3D-printed tissue-engineered trachea.

[0093] In some embodiments of the present application, the culture method comprises in vitro induced differentiation and culture, or subcutaneous implantation culture in vivo, or in vitro / in vivo combined culture method. The in vitro induced differentiation and culture method can be static culture or dynamic culture. Static culture means placing the 3D-printed tissue engineered trachea in a culture dish for culture, and dynamic culture means placing the 3D-printed tissue engineered trachea in a bioreactor for stirring or pressurized culture to promote the exchange of nutrients in the hydrogel. The subcutaneous implantation culture in vivo is to implant the tissue engineered trachea without or with in vitro culture into the subcutaneous tissue of an animal body. The animal body can be a nude mouse, an SD rat, a rabbit, a sheep, a dog, a pig, preferably a nude mouse or a sheep.

[0094] The histological features of the bio-3D-printed tissue engineered trachea provided by the present application are as follows: 1) the regenerated cartilage presents a typical cartilage lacuna structure, and has histological cartilage-specific staining of safranin and type II collagen; and 2) the regenerated fibrous tissue presents clear blood vessel structure, and has hemangiogenic-specific staining of CD31 and fibroblastic-specific staining of Masson and type I collagen.

[0095] The structural features of the bio-3D-printed tissue engineered trachea provided by the present application are as follows: 1) having a ring-shaped tubular structure; 2) an alternating structure of cartilage rings and vascularized fiber rings; 3) the cartilage rings and the vascularized fiber rings have typical tissue characteristics; and 4) the cartilage rings and the vascularized fiber rings are integrated.

[0096] The bio-3D-printed tissue engineered trachea constructed by the present application realizes the recovery of mechanical and physiological functions: 1) mechanical function reconstruction, such as longitudinal stretching, transverse bending ability, etc.; and 2) physiological function reconstruction, the vascularized fibrous tissue between the cartilage rings provides necessary nutrients for cartilage regeneration.

[0097] The present application also provides the use of the bio-3D-printed tissue engineered trachea.

[0098] The present application provides the use of the bio-3D-printed tissue engineered trachea as a tracheal defect repair material or tissue engineered trachea graft. The bio-3D-printed tissue engineered trachea as a tracheal defect repair material can be applied to scenarios such as patch repair surgery and segmental tracheal reconstruction surgery.

[0099] The present application will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0100] Embodiment one: preparation of cartilage tissue-specific hydrogel material

[0101] Preparation of the acellular cartilage matrix (ACM): Fresh cartilage tissue (e.g., ear cartilage, joint, meniscus, etc.) was obtained, and the surface excess tissue and fascia were removed. The cartilage was then cut into small pieces using scissors. The cleaned cartilage pieces were immersed in liquid nitrogen and ground into a powder using a cryogenic freezer mill for 5 min. The cartilage powder was then subjected to subsequent decellularization: 1) the cartilage powder was placed in a 0.5% w / v trypsin / phosphate buffer solution and shaken at 37°C for 24 h; 2) the supernatant was removed by centrifugation, and the cartilage was placed in a nuclease solution (containing 50 U / ml deoxyribonuclease and 1 U / ml ribonuclease A in 10 mM Tris-HCL, pH 7.5) and shaken at 37°C for 4 h; 3) the supernatant was removed by centrifugation, and the cartilage was placed in a 10 mM Tris-HCL solution containing 10 U / ml aprotinin and shaken at 37°C for 20 h; 4) the supernatant was removed by centrifugation, and the cartilage was placed in a 1% Triton X-100 / PBS (v / v) solution and shaken at 37°C for 24 h; and 5) the supernatant was removed by centrifugation, and the cartilage was washed multiple times in PBS. Finally, the cartilage was freeze-dried under vacuum to obtain the acellular cartilage matrix powder, which was stored in a desiccator.

[0102] Synthesis of methacrylated gelatin (GelMA): Gelatin (1 g) was dissolved in 10 mL PBS (pH 7.4) by heating to 50°C and stirring until completely dissolved. Then, 0.5 mL methacrylic anhydride was added, and the reaction was allowed to proceed for 2-3 h. After the reaction, the reaction solution was diluted with 40 mL PBS, and then transferred into a dialysis bag (MWCO 7000) for dialysis against deionized water for 2-3 d. The methacrylated gelatin (0.9 g) was obtained by freeze-drying. According to the hydrogen nuclear magnetic resonance spectrum, the content of double bonds was calculated to be about 75%.

[0103]

[0104] Synthesis of methacrylated chondroitin sulfate (CSMA): Chondroitin sulfate (1 g, 300 kDa) was dissolved in 100 mL deionized water, cooled to 0-4°C, and then 5 mL methacrylic anhydride was added. Then, 5 mL 5M NaOH was slowly added dropwise, and the reaction was allowed to proceed for 24 h. The reaction solution was then transferred into a dialysis bag (MWCO 7000) for dialysis against deionized water for 2-3 d. The methacrylated chondroitin sulfate (0.9 g) was obtained by freeze-drying. According to the hydrogen nuclear magnetic resonance spectrum, the content of double bonds was calculated to be about 60%.

[0105]

[0106] Preparation of the cartilage tissue-specific hydrogel material: 0.01 g of ACM, 0.05 g of GelMA, 0.02 g of CSMA and 2 mg of LAP were weighed and dissolved in 1 mL of PBS solution (pH = 7.4) to prepare a hydrogel precursor solution of ACM / GelMA / CSMA at 37°C. The solution was irradiated under a light source at a wavelength of 365 nm to achieve photo-crosslinking, and a cartilage tissue-specific hydrogel material of ACM / GelMA / CSMA was obtained.

[0107] Example II: Preparation of the fibrous tissue-specific hydrogel material

[0108] Preparation of the fibrous tissue decellularized matrix (DCM): Fresh dermis was taken and the surface excess tissue and fascia were removed, and then the dermis was cut into small pieces with scissors. The cleaned dermis tissue was immersed in liquid nitrogen and ground into a powder by a cryogenic freezer mill after sufficient pre-cooling for 5 min. Subsequent decellularization was performed in the following order: 1) The dermis powder was placed in a 0.5% w / v trypsin / phosphate buffer solution and shaken on a constant temperature shaker at 37°C for 24 h; 2) The supernatant was removed by centrifugation and placed in a nuclease solution (containing 50 U / ml deoxyribonuclease and 1 U / ml ribonuclease A dissolved in 10 mM Tris-HCL, pH = 7.5) and shaken at 37°C for 4 h; 3) The supernatant was removed by centrifugation and placed in a 10 mM Tris-HCL solution containing 10 U / ml aprotinin and shaken at 37°C for 20 h; 4) The supernatant was removed by centrifugation and placed in a 1% Triton X-100 / PBS (v / v) solution and shaken at 37°C for 24 h; 5) The supernatant was removed by centrifugation and washed repeatedly in PBS solution. Finally, the dermis decellularized matrix powder was prepared by vacuum freeze-drying and stored in a dry box for use.

[0109] Synthesis of methacrylated hyaluronic acid (HAMA): Hyaluronic acid (1 g, 340 kDa) was dissolved in 100 mL of deionized water, cooled to 0-4°C, and 5 mL of methacrylic anhydride was added, followed by slow dropwise addition of 5 mL of 5M NaOH. The reaction was allowed to proceed for 24 h, after which the reaction solution was poured into a dialysis bag (MWCO 7000) and dialyzed against deionized water for 2-3 d. The methacrylated hyaluronic acid (0.9 g) was obtained by freeze-drying. According to the hydrogen nuclear magnetic resonance spectrum, the content of double bonds was calculated to be about 60%.

[0110]

[0111] Preparation of fibrous tissue-specific hydrogel material: 0.01 g of DCM, 0.05 g of HAMA, 0.02 g of PEG-NHS (succinylated polyethylene glycol, 10 kDa) and 2 mg of LAP were weighed and dissolved in 1 mL of PBS solution (pH = 7.4) to prepare a hydrogel precursor solution of DCM / HAMA / PEG-NHS at 37°C. The light crosslinking was achieved under the irradiation of a light source at a wavelength of 365 nm, and the DCM / HAMA / PEG-NHS fibrous tissue-specific hydrogel material was obtained.

[0112] Example Three: In vitro induction evaluation of cartilage / fibrous tissue-specific hydrogel material

[0113] The experiment verified the rich cartilage-specific induction components in the hydrogel by co-culturing the cartilage tissue-specific hydrogel material prepared in Example One with mesenchymal stem cells (BMSCs). A certain concentration of ACM / GelMA / CSMA cartilage tissue-specific hydrogel solution (0.5% w / v) was co-cultured with BMSCs for 14 days, and the analysis of cartilage-specific expression was performed by alizarin red, type II collagen staining, and qPCR quantification. The experimental results showed that the cartilage tissue-specific hydrogel material had chondrogenic induction activity and could better promote cartilage regeneration.

[0114] The experiment verified the rich fibrous tissue-specific induction components in the hydrogel by co-culturing the fibrous tissue-specific hydrogel material prepared in Example Two with mesenchymal stem cells (BMSCs). A certain concentration of DCM / HAMA / PEG-NHS fibrous tissue-specific hydrogel solution (0.5% w / v) was co-cultured with BMSCs for 14 days, and the analysis of fibrous tissue-specific expression was performed by actin, type I collagen staining, CD31 immunohistochemical staining, and qPCR quantification. The experimental results showed that the fibrous tissue-specific hydrogel material had fibroblast induction activity and vascularization ability, and could better promote the regeneration of vascularized fibrous tissue.

[0115] Example Four: Preparation of cartilage / fibrous tissue-specific bio-ink

[0116] Preparation of cartilage-specific bio-ink: Cartilage cells were isolated from rabbit ears, routinely isolated, cultured and expanded, subcultured to the second or third generation, collected and adjusted to a final concentration of 10 x 10 6 / mL of cell suspension, and wrapped in the ACM / GelMA / CSMA hydrogel material prepared in Example One to obtain the cartilage-specific bio-ink.

[0117] Preparation of fibrous tissue-specific bio-ink: Fibroblasts were isolated from rabbit ears, routinely isolated, cultured and expanded, subcultured to the second or third generation, collected and adjusted to a final concentration of 5 x 106 / mL, encapsulated in DCM / HAMA / PEG-NHS hydrogel material prepared according to Example Two, to obtain the fibroblast-specific bio-ink.

[0118] Example Five: Preparation of Cartilage / Fibroblast-Specific Bio-Ink

[0119] Preparation of Cartilage-Specific Bio-Ink: Cartilage cells were isolated from rabbit ears, routinely isolated, cultured and expanded, subcultured to the second or third generation, and the cell suspension was collected and adjusted to a final concentration of 10 x 10 6 / mL, encapsulated in 10% w / v chitosan hydrogel precursor solution containing TGF-β, and chitosan hydrogel was prepared at 37°C to obtain the cartilage-specific bio-ink.

[0120] Preparation of Fibroblast-Specific Bio-Ink: Fibroblasts were isolated from rabbit ears, routinely isolated, cultured and expanded, subcultured to the second or third generation, and the cell suspension was collected and adjusted to a final concentration of 5 x 10 6 / mL, encapsulated in 10% w / v alginate hydrogel precursor solution containing VEGF, and 0.1% calcium chloride solution was added for crosslinking to prepare alginate hydrogel, to obtain the fibroblast-specific bio-ink.

[0121] Example Six: Preparation of Cartilage / Fibroblast-Specific Bio-Ink

[0122] Preparation of Cartilage-Specific Bio-Ink: Cartilage cells were isolated from rabbit ears, routinely isolated, cultured and expanded, subcultured to the second or third generation, and the cell suspension was collected and adjusted to a final concentration of 10 x 10 6 / mL, encapsulated in 10% w / v gelatin solution containing FGF, and 10% w / v oxidized hyaluronic acid solution was added, and gelatin / hyaluronic acid hydrogel was prepared by Schiff base crosslinking to obtain the cartilage-specific bio-ink.

[0123] Preparation of Fibroblast-Specific Bio-Ink: Fibroblasts were isolated from rabbit ears, routinely isolated, cultured and expanded, subcultured to the second or third generation, and the cell suspension was collected and adjusted to a final concentration of 5 x 10 6 / mL, encapsulated in 10% w / v borate-modified hyaluronic acid containing VEGF, and 10% w / v polyethylene glycol solution was added, and hyaluronic acid / polyethylene glycol hydrogel was prepared by borate- vicinal diol reaction to obtain the fibroblast-specific bio-ink.

[0124] Example Seven: Construction of 3D Printed Tissue-Engineered Trachea

[0125] The experiment first prepared cartilage and fibroblast tissue-specific bio-inks according to Example Four, and loaded them into the printing cartridge for use. As Figure 1As shown, by the double-needle extrusion type biological 3D printing technology, the alternating printing and the formation of the ring-shaped tubular structure are realized, and the cartilage / fibrous tissue alternating tubular structure (inner diameter 6 mm, outer diameter 8 mm, height 15 mm) is constructed by multi-layer accumulation molding. The printing parameters are line spacing: 500 μm; layer thickness: 160 μm; light crosslinking time: 30 seconds per layer; pneumatic pressure: 200-230 kPa; extrusion speed: 6 mm / s. After printing is completed, the cartilage / fibrous tissue alternating structure can be clearly observed, and has a certain mechanical strength to maintain its ring-shaped tubular structure. Figure 2

[0126] Example Eight: Construction of 3D Printed Tissue Engineering Trachea

[0127] The experiment first configures the cartilage and fibrous tissue specific biological ink according to Example Five, and fills it into the printing cylinder for use. By the double-needle extrusion type biological 3D printing technology, the alternating printing and the formation of the ring-shaped tubular structure are realized, and the cartilage / fibrous tissue alternating tubular structure (inner diameter 6 mm, outer diameter 8 mm, height 15 mm) is constructed by multi-layer accumulation molding. The printing parameters are line spacing: 500 μm; layer thickness: 160 μm; light crosslinking time: 30 seconds per layer; pneumatic pressure: 200-230 kPa; extrusion speed: 6 mm / s. After printing is completed, the cartilage / fibrous tissue alternating structure can be clearly observed, and has a certain mechanical strength to maintain its ring-shaped tubular structure.

[0128] Example Nine: Construction of 3D Printed Tissue Engineering Trachea

[0129] The experiment first configures the cartilage and fibrous tissue specific biological ink according to Example Six, and fills it into the printing cylinder for use. By the double-needle extrusion type biological 3D printing technology, the alternating printing and the formation of the ring-shaped tubular structure are realized, and the cartilage / fibrous tissue alternating tubular structure (inner diameter 6 mm, outer diameter 8 mm, height 15 mm) is constructed by multi-layer accumulation molding. The printing parameters are line spacing: 500 μm; layer thickness: 160 μm; light crosslinking time: 30 seconds per layer; pneumatic pressure: 200-230 kPa; extrusion speed: 6 mm / s. After printing is completed, the cartilage / fibrous tissue alternating structure can be clearly observed, and has a certain mechanical strength to maintain its ring-shaped tubular structure.

[0130] Example Ten: Evaluation of Nude Mouse Subcutaneous Regeneration Effect of 3D Printed Tissue Engineering Trachea

[0131] ​The experiment first constructs the 3D-printed tissue engineered trachea according to Example Seven, and then implants it subcutaneously in a nude mouse for in vivo culture. After 4 weeks and 8 weeks, the sample is taken out for detection and evaluation of various indicators related to cartilage and fibrous tissue regeneration in vivo. The experimental results show that the implanted 3D-printed trachea analog can effectively realize tracheal regeneration, and has an alternating structure of cartilage / vascularized fibrous tissue, which is basically similar to the normal tracheal structure Figure 3 ). At the same time, the regenerated trachea basically restores the mechanical function and can realize longitudinal stretching and transverse bending movement, which is of great significance for tracheal function reconstruction Figure 4 ).

[0132] Example Eleven: 3D-printed tissue engineered trachea is applied to rabbit tracheal reconstruction

[0133] New Zealand male rabbits are used to make a segmental tracheal defect model at the rabbit tracheal site to evaluate the in vivo reconstruction effect of the tissue engineered trachea. In the experiment, the tissue engineered trachea is constructed according to Example Seven, and is pre-embedded subcutaneously at the rabbit tracheal site for 8 weeks to obtain a relatively mature tissue engineered trachea. Then, a 15mm long segmental tracheal defect model is made at the rabbit tracheal site, and an end-to-end anastomosis surgery is performed. After 8 weeks of surgery, the rabbits in the experiment are killed by intravenous injection of air, and the repaired trachea is extracted to evaluate the experimental effect. The experimental results show that the 3D-printed tissue engineered trachea can effectively realize tracheal reconstruction, and basically restore the mechanical and physiological functions of the trachea Figure 5 ).

[0134] The above description of the embodiments is for the purpose of facilitating the understanding and use of the invention by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A bio 3D printed tissue engineered trachea, characterized in that, A ring-shaped tubular structure formed by alternately stacking cartilage rings and vascularized fiber rings, The cartilage ring is a cartilage-specific bio-ink 3D printed by wrapping chondrocytes with a cartilage tissue-specific hydrogel, The vascularized fiber ring is a fiber-specific bio-ink 3D printed by wrapping fibroblasts with a fibrous tissue-specific hydrogel, The cartilage ring and the vascularized fiber ring have typical tissue characteristics, and the cartilage ring and the vascularized fiber ring are integrated structures; The inner diameter of the ring-shaped tubular structure is 0.1-40 mm, the outer diameter is 0.5-50 mm, and the height is 0.1-100 mm; The cartilage tissue-specific hydrogel is a cartilage tissue-specific hydrogel material prepared by dissolving a water-soluble polymer in a biocompatible medium and adding a cartilage tissue-specific component and cross-linking; The fibrous tissue-specific hydrogel is a fibrous tissue-specific hydrogel material prepared by dissolving a water-soluble polymer in a biocompatible medium and adding a fibrous tissue-specific component and cross-linking; The water-soluble polymer is selected from natural polymer materials or synthetic polymer materials; The natural polymer material includes natural polysaccharide substances and their modified or degraded products, proteins and their modified or degraded products; The natural polysaccharide substances include hyaluronic acid, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan or chitosan quaternary ammonium salt; The proteins include various hydrophilic or water-soluble animal and plant proteins, collagen, serum proteins, silk fibroin, and elastin; the protein degradation products include gelatin or polypeptides; The synthetic polymer material includes two-arm or multi-arm polyethylene glycol, polyethyleneimine, dendrimer, synthetic polypeptide, polylysine, polyglutamic acid, polyacrylic acid, polymethacrylic acid, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone; The cartilage tissue-specific component includes a cartilage acellular matrix and a growth factor promoting chondrogenic differentiation; The cartilage acellular matrix is derived from cartilage-containing tissues in animal bodies; The growth factor promoting chondrogenic differentiation is selected from TGF-β, VEGF or FGF; The fibrous tissue-specific component is selected from a fibroblast acellular matrix or a growth factor promoting fibroblast differentiation; The fibroblast acellular matrix is derived from fibrous tissues in animal bodies; The growth factor promoting fibroblast differentiation includes TGF-β, VEGF, FGF, and BMP-2-9.

2. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The inner diameter of the ring-shaped tubular structure is 2-5 mm, the outer diameter is 4-10 mm, and the height is 5-20 mm.

3. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The thickness ratio of the cartilage ring to the vascularized fiber ring is 10:1-1:

10.

4. The bio-3D printed tissue engineered trachea according to claim 3, wherein, The thickness ratio of the cartilage ring to the vascularized fiber ring is 5:1-1:

1.

5. The bio-3D printed tissue engineered trachea according to claim 4, wherein, The thickness ratio of the cartilage ring to the vascularized fiber ring is 2:1-1.5:

1.

6. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The cartilage ring has the following tissue characteristics: the regenerated cartilage presents a typical cartilage lacuna structure, and has a cartilage-specific staining of safranin and type II collagen in histology; The vascularized fibrous ring has the following tissue characteristics: clear blood vessel structure in the regenerated fibrous tissue, hemangiogenic specific staining of CD31, and fibroblast specific staining of Masson and collagen type I.

7. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The interface bonding force of the cartilage ring and the vascularized fibrous ring is 1 kPa to 10 MPa.

8. The bio-3D printed tissue engineered trachea according to claim 7, wherein, The interface bonding force of the cartilage ring and the vascularized fibrous ring is 100 kPa to 1 MPa.

9. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The biocompatible medium is selected from distilled water, physiological saline, buffer or cell culture medium solution. The concentration of the water-soluble polymer in the biocompatible medium is 0.1% w / v to 60% w / v.

10. The bio-3D printed tissue engineered trachea according to claim 9, wherein, The concentration of the water-soluble polymer is 1% w / v to 20% w / v.

11. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The decellularized fibroblast matrix is derived from the skin, dermis and muscle in the animal body. The animal is selected from pigs, cows, sheep, dogs, rabbits and mice.

12. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The decellularized fibroblast matrix is derived from the skin, dermis and muscle in the animal body. The animal is selected from pigs, cows, sheep, dogs, rabbits and mice.

13. The bio-3D printed tissue engineered trachea according to claim 1, wherein, In the preparation of the cartilage tissue-specific hydrogel or the fibrous tissue-specific hydrogel, the cross-linking method is selected from one or more of physical cross-linking, chemical cross-linking or photo-cross-linking, or a combination of multiple materials in a single cross-linking method.

14. The bio-3D printed tissue engineered trachea according to claim 1, wherein, The chondrocyte has a cell density of 10 4 10 10 / mL in the cartilage tissue-specific hydrogel The fibroblasts have a cell density of 10 4 ~10 10 / mL in the fibrous tissue-specific hydrogel.

15. The bio-3D printed tissue engineered trachea according to claim 14, wherein, The chondrocyte has a cell density of 10 7 10 8 / mL in the cartilage tissue-specific hydrogel. The fibroblasts have a cell density of 10 7 10 8 / mL in the fibrous tissue-specific hydrogel.

16. The method of producing a bio 3D printed tissue engineered trachea according to any one of claims 1 to 15, wherein, The method comprises the following steps: The cartilage-specific bio-ink is prepared by wrapping the chondrocytes with the cartilage tissue-specific hydrogel. The fibrous tissue-specific bio-ink is prepared by wrapping the fibroblasts with the fibrous tissue-specific hydrogel. The cartilage ring and the vascularized fibrous ring are alternately printed by using the cartilage-specific bio-ink and the fibrous tissue-specific bio-ink as the material through the double-needle extrusion type bio-3D printing technology, and the cartilage ring and the vascularized fibrous ring are alternately stacked to form a ring-shaped tubular structure. The obtained ring-shaped tubular structure is cultured to realize the vascularization of the fibrous tissue, and a mature bio-3D printed tissue-engineered trachea is obtained.

17. A method for preparing a bio-3D printed tissue-engineered trachea according to claim 16, characterized in that, The culture method includes in vitro induced differentiation and culture, subcutaneous implantation culture in vivo, or in vitro / in vivo combined culture. The in vitro induced differentiation and culture method is selected from static culture and dynamic culture. The static culture method is to place the 3D printed tissue-engineered trachea in a culture dish for culture. The dynamic culture method is to place the 3D printed tissue-engineered trachea in a bioreactor for stirring or pressurized culture to promote the exchange of nutrients in the hydrogel. The subcutaneous implantation culture in vivo is to implant the tissue-engineered trachea without or with in vitro culture into the subcutaneous tissue of an animal.

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