Tissue engineering integrated trachea regeneration construction method based on frame structure scaffold and application

Abstract

The invention discloses a tissue engineering integrated trachea regeneration construction method based on a frame structure scaffold and application. A multi-tissue-structure integrated gas pipe is constructed based on a 3D printing bioelastomer frame structure support, and the complex structure and physiological functions of a natural gas pipe are highly simulated. The preparation method comprises the following steps: firstly, by utilizing the characteristic that temperature-sensitive hydrogel Pluronic F-127 becomes semi-solid gel at 37 DEG C and is liquefied at a low temperature of 4 DEG C, and by adopting a strategy that the temperature-sensitive hydrogel is sacrificed after occupying, the patterned distribution of chondrocytes and fibroblasts on a two-dimensional plane is realized; secondly, simulating a three-dimensional tubular trachea structure through a 2D-3D (two-dimensional to three-dimensional) rolling assembly strategy, and realizing the ordered regeneration of C-shaped cartilage ring / O-shaped fiber ring alternate distribution and axial strip-shaped fiber bundle triple structures under the skin of the nude mouse. And finally, successfully extracting the airway mucosa epithelium, separating and amplifying enough airway basal cells in vitro, and loading the airway basal cells to the inner wall of the regenerated trachea by utilizing the photo-crosslinking hydrogel, thereby realizing the function simulation of the airway epithelium and enhancing the physiological functionality.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical regenerated trachea preparation, and relates to a tissue engineering integrated trachea regeneration construction method based on a frame structure scaffold and its application. Background Art

[0002] Clinically, segmental tracheal injuries have problems such as difficulty in direct anastomosis and easy scar formation after surgery. Therefore, a large number of artificial tracheal grafts are urgently needed to replace and anastomose the defective parts. Currently, most of the artificial tracheal grafts used in clinical practice are made of inert polymer materials such as polyester and polytetrafluoroethylene. However, this type of graft lacks biological activity and is implanted in the body for a long time as a substitute for foreign matter. It cannot interact well with cells or the biological environment and may even cause rejection reactions to form tissue scars and cause tracheal stenosis. Therefore, constructing an artificial trachea with biological activity and physiological functions is of great clinical significance for the repair of segmental tracheal defects.

[0003] With the development of biomaterials and tissue engineering, tissue-specific microenvironment bionic regulation, as well as composite tissue regeneration and functional reconstruction based on microenvironment bionic scaffolds, are expected to fundamentally solve the problem of repairing tracheal segmental defects. Tissue engineering regeneration is a new treatment method that uses modern biological techniques such as biomaterials, cell culture, and molecular biology, based on cells and materials, combined with the regeneration mechanism of organisms, to reconstruct or renew damaged diseased tissues. However, so far, only a small number of single types of tissue engineering skin and cartilage have been actually used clinically, and there has been no breakthrough progress in the trachea, a tissue with complex cellular components and structures.

[0004] In recent years, polymer materials have developed rapidly, and a series of biomaterials have been artificially synthesized, mainly polylactic acid (PLA), polyglycolic acid (PGA) and polycaprolactone (PCL) and their copolymers and derivatives. Although they are biocompatible and biodegradable, the scaffolds do not have elastic properties due to the nature of the materials themselves. However, most natural soft tissues have good mechanical elasticity and ductility, and are in a cyclic compression and recovery process. In a sense, the compatibility of biomaterials also includes the matching of mechanical properties with the host soft tissue, especially elastic dynamic tissues (such as elastic cartilage) and structural tissues (such as trachea). Therefore, for tracheal tissue engineering, the development of elastic biomaterials to simulate the complex elastic properties of tracheal tissue is a hot topic in the current field.

[0005] Poly(glycerol sebacate) (PGS) is a representative bioelastomer that has been widely used in the field of tissue regeneration. It was approved by the FDA for clinical implantation in 2021 and is also the bioelastomer material with the best clinical transformation prospects. PGS has good biocompatibility and degradability, and to a certain extent simulates the mechanical properties of many natural tissues. At the same time, it can adapt to the dynamic mechanical environment in the body. It is mainly concentrated in soft tissue engineering applications and has achieved remarkable results, including myocardial, vascular, cartilage, retinal tissue engineering, etc., and has been further expanded to applications such as drug sustained release and tissue adhesives. However, it is still in the early stages in the field of tracheal regeneration and is also an emerging research direction.

[0006] In recent years, 3D printing technology has been widely used in fields such as regenerative medicine due to its powerful capabilities in macro-morphological customization and precise control of microstructure, and has greatly promoted innovation and development in this field. However, PGS, as a thermosetting material, requires a long cross-linking process, which is difficult to match the continuous manufacturing method of 3D printing, greatly limiting the processing freedom and application direction. In the early stage, we developed thermosetting material 3D printing technology to solve the problem that PGS cannot be 3D printed, and in 2019 proposed a 4-axis 3D printed tubular scaffold based on PGS bioelastomer for tracheal cartilage regeneration research.

[0007] like Figure 1 As shown in the study, this study first proposed a new strategy for 4-axis 3D printing of tubular structures, which enabled the rapid construction of long-segment PGS tubular scaffolds with adjustable braided structures (Biomaterials 2020, 258, 120254), and used this technology to achieve tubular tracheal cartilage reconstruction (Science China Materials 2019, 62, 1910). This strategy is highly versatile and can be well applied to a very wide range of biomaterials, such as thermoplastics, thermosetting materials, and hydrogel materials. By adjusting the processing parameters, precise control of the macro and micro structures of the tubular scaffolds can be achieved.

[0008] This technology was combined with thermosetting PGS printing technology to achieve the preparation of PGS porous biological springs. The composite material obtained by mixing PGS prepolymer and salt particles in a ratio of 1:2 has good extrusion performance and can be continuously and evenly extruded into lines. The salt particles can be used as filling materials to maintain the stability of the material printing lines. The composite material lines can be attached to the surface of the metal rod. As the print head moves along the direction of the central axis of the metal rod and the metal rod rotates, the material lines present a spiral structure; after multiple reciprocating, the lines stack on each other to form an interwoven network structure. The overall shape of the PGS fiber interwoven structure does not change basically before and after curing. This is because the supporting effect of the salt particles ensures that the original printed macroscopic tubular structure and microscopic line structure can be maintained under high temperature conditions. After curing, chemical cross-linking occurs between the interwoven fibers, thereby forming a stable binding site and improving the overall structural stability of the scaffold. Due to the dual effects of PGS, a bioelastomer, and the corresponding tubular spring structure, the final prepared tubular scaffold has good elasticity and fatigue resistance, ensuring good compliance, matching and deformation recovery in the dynamic mechanical environment in the body.

[0009] The framework structure regenerative biological scaffold prepared by the above regenerative materials is used for regenerative tissue repair, especially for regenerating trachea, and there is still a lot of room for improvement. The problems faced mainly include the following aspects:

[0010] First, there are limitations on the currently available scaffold preparation processes and biomaterials. Currently, the most commonly used methods for preparing framework scaffolds include 3D printing, electrospinning, sol-gel method, etc. However, most of the materials that can be used in these preparation processes lack sufficient bioactivity and compatibility. The artificial synthetic biomaterials with biocompatibility and biodegradability, such as polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL) and their copolymers and derivatives, have the nature of the material itself, which determines that the scaffold does not have elastic properties and cannot match the mechanical properties of the host soft tissue, especially elastic dynamic tissues (such as elastic cartilage) and structural tissues (such as trachea). Although 3D printing technology has been widely used in fields such as regenerative medicine due to its powerful capabilities in macroscopic morphology customization and precise control of microstructure, and has greatly promoted innovation and development in this field, many bioelastic materials (such as PGS) require a long cross-linking process, which is difficult to match the continuous manufacturing method of 3D printing, greatly limiting the processing freedom and application direction of 3D printing.

[0011] Secondly, the current manufacturing precision of the frame structure bracket is not high. 3D printing is limited by the printer's electrical configuration, the printing speed is slow, and the conditions are relatively harsh. It cannot fully meet the needs of high-precision and high-resolution frame structures; electrospinning is limited by the nozzle design, the stability of the voltage electric field, and the properties of the spinning solution; the sol-gel method for preparing the frame structure bracket is limited by the mechanical strength and stability of the gel, and cannot form a stable, controllable, and fine frame structure. Therefore, it is difficult to construct a frame structure bracket that can simultaneously maintain its macroscopic structure and microscopic line structure requirements.

[0012] Finally, multi-tissue integrated regeneration is still a major difficulty in tissue engineering tracheal regeneration. Currently, only a small number of single types of tissue engineering skin and cartilage have been put into actual clinical application, and there has been no breakthrough progress for the trachea, a tissue with complex cellular components and structures. The natural trachea is a complex tissue with orderly multi-cell construction, including alternating structures of C-shaped cartilage rings and O-shaped vascularized fiber rings, dorsal strip fiber bundles, and the inner airway epithelium's quadruple tissue structure. Therefore, tissue engineering regeneration of the trachea requires multiple cell sources, and extraction, separation, and amplification are more difficult. The above-mentioned trachea constructed by 4-axis 3D printing tubular PGS scaffold is composed of a single cartilage, which inevitably causes the mechanical rigidity of the trachea, and it is difficult to achieve the precise construction of C-shaped cartilage and O-shaped fibers, and it is even more difficult to functionally integrate with airway epithelial cells.

[0013] Therefore, how to design and construct an integrated bionic gas pipe with physiological functions according to the multi-tissue structure of the gas pipe is a key scientific issue of this project and also a major challenge faced by this field. Summary of the invention

[0014] The present invention aims at the above problems, further studies the application of tubular PGS scaffolds in tracheal tissue engineering, and successfully prepares mature tissue engineering tubular cartilage by combining high-density chondrocytes and PGS / gelatin hybrid scaffolds in vitro. The tubular composite scaffold shows good biocompatibility with chondrocytes during in vitro culture, and mature tubular cartilage is prepared. After 8 weeks of in vitro culture, a clear tubular cartilage structure grows with the scaffold as a template, in which the yellow gelatinous cartilage tissue is coated on the outside of the white tubular scaffold to form a double-layer structure. This artificial tubular cartilage has good application prospects in tubular tracheal tissue regeneration.

[0015] The overall research purpose of this invention is to propose a universal method for tissue engineering tracheal regeneration and reconstruction based on a framework structure scaffold. On the framework structure scaffold printed by thermosetting PGS bioelastomer 3D, a strategy of thermosensitive hydrogel occupation and sacrifice is used to achieve the integrated bionic construction and regeneration of the trachea, a complex multi-tissue structure, and provide a new feasible solution and treatment method for clinical tracheal transplantation. The specific objectives mainly include the following three aspects:

[0016] 1. Realize the integrated construction and coordinated regeneration of multiple tracheal tissues. In the early stage, we used the tubular PGS material prepared by 4-axis 3D printing technology to achieve the regeneration of tissue-engineered tubular tracheal cartilage, but its disadvantage is that the tissue structure is too simple. The natural trachea is a complex tissue with orderly multi-cell structure, including C-shaped cartilage and vascularized fibrous connective tissue alternating structure, as well as the inner layer of airway epithelial tissue. We hope to achieve their integrated construction and synchronous regeneration for different types of tracheal tissues and their own special structures, so as to improve the overall effect of tracheal reconstruction.

[0017] 2. Based on the framework structure of the scaffold, establish a two-dimensional patterning technology to achieve precise inoculation of multiple cells on the scaffold and assembly from two-dimensional to three-dimensional. In view of the complex tissue structure of the natural airway (including alternating structures such as C-shaped cartilage rings and vascularized fibrous connective tissue, as well as the inner layer of airway epithelial tissue), we hope to establish a precise patterning construction technology to achieve controllable microstructure design and construction of the above-mentioned specific tissue structure in a two-dimensional plane, and then realize the integrated bionic construction of tracheal multi-tissue structure through assembly from two-dimensional to three-dimensional and in vivo molding and culture.

[0018] 3. Verify in animal models that the integrated tracheal tissue can maintain the multidimensional structure we designed and constructed in vitro. Achieve the regeneration of integrated tissue-engineered trachea in nude mice, overcome the current technical difficulties, and find corresponding solutions and strategies for truly effective tracheal vascularization, biological adaptability of polymer scaffolds, and graft rejection.

[0019] To achieve the above objectives, the present invention adopts the following technical solutions: Based on 3D printed PGS / PCL bioelastic framework structure scaffold, a multi-organizational integrated trachea (Integrated Trachea with Multi-Organizational Structure, IT-MOS) was successfully constructed, which highly simulated the complex structure and physiological function of the natural gas tube. First, by using the characteristics of thermosensitive hydrogel Pluronic F-127, which forms a semi-solid gel at 37°C and liquefies at a low temperature of 4°C, the strategy of thermosensitive hydrogel post-occupancy sacrifice (POS) was proposed for the first time, realizing the structure of patterned distribution of chondrocytes and fibroblasts on a two-dimensional plane. Secondly, through the 2D to 3D rolling assembly strategy, the three-dimensional tubular tracheal structure was simulated, and the orderly regeneration of the triple structure of alternating distribution of C-shaped cartilage rings / O-shaped fiber rings and axial strip fiber bundles was achieved under the skin of nude mice. Finally, we successfully extracted the airway mucosal epithelium and isolated and amplified a sufficient number of airway basal cells in vitro, and then used photo-crosslinked hydrogels to load them onto the inner wall of the regenerated trachea, achieving functional biomimetic of the airway epithelium and enhancing the physiological functionality of the tissue-engineered trachea ( Figure 2 A). In conclusion, the designed IT-MOS has elastic strength and quadruple histological structure similar to natural trachea, providing a new solution for the future clinical construction of functional tissue-engineered trachea.

[0020] The specific technical solution adopted by the present invention is as follows:

[0021] In a first aspect, the present invention provides a method for constructing integrated tracheal regeneration by tissue engineering based on a framework structure scaffold, comprising the following steps:

[0022] A. Preparation of bioelastic scaffolds

[0023] PGS or PGS with other biomaterials' prepolymers and inorganic salts are compounded into a multilayer structure through 3D printing, and then vacuum cured and cross-linked at high temperature to obtain a stable thermosetting PGS / PCL composite scaffold; the 3D printed scaffold is then immersed in a 1% gelatin solution and converted into a three-dimensional porous freeze-dried scaffold using a vacuum freeze dryer, so that a gelatin network with a smaller pore size is formed inside the PPG scaffold, and the PPG scaffold is cross-linked for 24 hours; the residual solution is completely removed in deionized water, and then a gelatin-modified PPG scaffold is obtained through vacuum freeze drying, which maintains the initial morphological characteristics of the PGS / PCL composite scaffold, and the gelatin is evenly filled into the interstitial area.

[0024] Preferably, the biomaterial is selected from one or more of PCL, PLGA, and PLA, and the mass ratio of PGS is 50-100%;

[0025] Preferably, the polymer material is further mixed with inorganic salt particles to form 3D printing ink, and 3D printing is performed to prepare the bioelastomer framework; wherein the mass ratio between the polymer material and the inorganic salt is 1:0.5 to 1:3, the salt particle size is 10 to 75 μm, and the inorganic salt is selected from any one or more of sodium chloride, potassium chloride, and sodium carbonate;

[0026] Preferably, the 3D printing process is as follows: select a print head with an inner diameter of about 100-1000 μm, a grid filling width of 1-2.5 mm, a grid interlacing angle of 90°, a layer height of 0.3-0.6 mm, a printing speed of 2-40 mm / s, and a printing temperature of 30-100°C; after 3D printing is completed, perform preliminary curing for 12 hours at 100°C and 0.5 bar vacuum conditions, then cross-link at 150°C and 1 bar vacuum for 24 hours to achieve complete curing, further immerse it in an ethanol-water mixed solution with an ethanol volume fraction of 10-75% to remove salt particles, and remove solvents including water and ethanol by air drying, heating, freeze drying, etc.

[0027] Preferably, after the 3D printed scaffold is immersed in a 0.5-3% gelatin solution, the vacuum freeze-drying conditions are as follows: -80°C, 5-50Pa, and converted into a three-dimensional porous freeze-dried scaffold; the three-dimensional porous freeze-dried scaffold is cross-linked with the PPG scaffold by the EDC / NHS method for 24 hours; after the scaffold is completely deionized to remove the residual solution, the vacuum freeze-drying conditions are as follows:

[0028] -80℃,5-50Pa.

[0029] B. Construction of multicellular two-dimensional patterning

[0030] At 37°C, the temperature-sensitive hydrogel Pluronic F-127 was coated on specific locations of the PPG scaffold, and then the chondrocyte suspension was seeded in the unoccupied areas to precisely distribute them in the strip-like structure. Subsequently, the temperature-sensitive hydrogel liquefied and disappeared at low temperatures, leaving behind the chondrocytes arranged in strips. Multiple cells were patterned and biomimetically constructed, laying the foundation for the subsequent construction of C-shaped cartilage rings in the 2D to 3D conversion.

[0031] Preferably, according to the three-dimensional structure of the natural gas pipe unfolded into a two-dimensional planar structure, the PPG framework structure scaffold is divided into areas corresponding to the distribution of cartilage tissue and fibroblast tissue, the thermosensitive hydrogel Pluronic F-127 is filled into the fibroblast tissue area to form a placeholder, and chondrocytes are seeded in the vacant area; after the cells adhere and grow, they are placed in a low-temperature environment of 4-10°C to remove the thermosensitive hydrogel, and the fibroblasts are seeded into the temporary placeholder area before the hydrogel and continue to be cultured.

[0032] Preferably, the seeding density of chondrocytes is 1-8*10 7 / ml, the seeding density of fibroblasts is 1-5*10 7 / ml.

[0033] C. Tissue engineering biomimetic trachea construction

[0034] The planar scaffold-cell complex constructed in vitro is rolled up and the two ends are anastomosed with absorbable sutures to construct a three-dimensional integrated bionic tissue-engineered trachea. After a period of in vivo culture in the subcutaneous or muscle flap, a regenerated tissue-engineered trachea is obtained. The airway mucosal epithelium is extracted and amplified in vitro to obtain a sufficient number of airway basal cells, which are loaded onto the inner wall of the regenerated trachea using photo-crosslinked hydrogels to achieve bionic airway epithelial function.

[0035] Preferably, a multi-cellular patterned scaffold / cartilage / fibroblast composite tissue is assembled and curled into a tubular structure, which is implanted in a subcutaneous or muscle flap for a period of time to form a vascularized regenerative tissue-engineered trachea; airway basal cells isolated, amplified, and differentiated and cultured in vitro are mixed into a photosensitive hydrogel, coated on the inner wall of the regenerative scaffold and cross-linked by ultraviolet light to form an epithelial layer; the photosensitive hydrogel is GelMA, and the concentration is 5-15%.

[0036] The method for isolating and expanding airway basal cells is as follows: the harvested mucosa is cut into 1 mm 2 The small pieces were washed thoroughly in PBS containing 1% antibiotics and digested with 0.15% collagenase NB4 at 37°C for 2 hours. Afterwards, the digestion solution was filtered through a filter and resuspended and centrifuged to obtain airway basal cells. The isolated airway basal cells were cultured in PneumaCult TM -Ex Plus medium, and when the cells reach 90% confluence, they are passaged and the second generation of airway basal cells are obtained.

[0037] In a second aspect, the present invention provides a tissue engineering integrated regenerated trachea, which is prepared by the method described above.

[0038] Two sections of the regenerated trachea were cut from the radial and axial directions. The radial sections corresponded to the C-shaped cartilage ring section (C ring) and the O-shaped fiber ring section (F ring) of the trachea, respectively, and the axial sections corresponded to the longitudinal cartilage fiber interlacing area (C band) and the full fiber tissue strip fiber bundle area (F bundle). Histological results showed that the radial C-shaped cartilage ring section showed a mature cartilage appearance, typical pit structure, and a large amount of specific ECM deposited inside the original scaffold compared to the fiber ring section. The fiber ring section was mainly covered by a large amount of fiber-like tissue; the axial cartilage fiber interlacing band still maintained the structure of alternating cartilage tissue and fibrous tissue, and the full fiber strip fiber band was basically covered by fiber-like tissue, which was consistent with the physiological structure of the natural trachea. This shows that our patterned integrated trachea can still maintain our original structural design in nude mice. So far, we have achieved multi-tissue integrated tracheal biomimetic at three levels: C-shaped cartilage ring, fiber ring, and axial strip fiber band.

[0039] Comparing the regenerated IT-MOS of the present invention with natural trachea, the high-density cell suspension inoculation makes the regenerated tracheal cartilage have a higher DNA content, and over time, the chondrocytes gradually secrete ECM, and the content of GAG and type II collagen in the regenerated trachea gradually increases and approaches the level of natural tracheal cartilage; the mechanical property test results show that the IT-MOS constructed by the present invention has stronger rigidity and elasticity than natural trachea, and can more effectively maintain mechanical compliance in the dynamic mechanical environment in vivo. In the comprehensive performance test, DNA, GAG, Col II, elastic modulus and elastic recovery rate were selected as 5 scoring criteria. The scoring results show that the various indicators of the regenerated trachea cultured in vivo for 8 weeks are comparable to those of the natural trachea; after 12 weeks of in vitro culture, the mechanical properties of the regenerated trachea are significantly better than those of the natural trachea, indicating that the tissue engineering regenerated trachea of ​​the present invention has similar comprehensive properties to the natural trachea.

[0040] Therefore, the third aspect of the present invention provides the use of the above-mentioned tissue engineering integrated tracheal regeneration in the treatment of tracheal segmental injuries.

[0041] A fourth aspect of the present invention provides a tracheal segmental injury regeneration and repair component, including the above-mentioned tissue engineering integrated tracheal regeneration.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The present invention establishes a technology for patterned seeding and distribution of multiple cells in vitro, and realizes the precise partitioned seeding of multiple cells on a 3D printed PGS two-dimensional planar scaffold, thereby realizing the planar alternating structure of C-shaped cartilage rings and O-shaped fibrous rings and the axial strip fiber bundle structure, that is, realizing the controllable microstructure design and construction of the above-mentioned specific tissue structure in a two-dimensional plane.

[0044] 2. Using a two-dimensional to three-dimensional assembly strategy, the planar patterned tissue-scaffold complex was constructed into a three-dimensional tubular multi-tissue integrated biomimetic trachea, which was then cultured subcutaneously in nude mice for 8 and 12 weeks to promote the maturation of tracheal cartilage and vascularization of fibrous connective tissue, thereby obtaining a tubular biomimetic trachea with alternating C-shaped cartilage rings and O-shaped vascularized fibrous rings.

[0045] 3. To verify the mechanical properties of IT-MOS, we compared the regenerated integrated trachea cultured in vivo for 8 and 12 weeks with natural trachea and found that the regenerated trachea had similar rigidity and elasticity to natural trachea.

[0046] 4. In order to verify the physiological structural characteristics of IT-MOS, two sections were cut from the radial and axial directions. The radial sections corresponded to the C-shaped cartilage ring section (C ring) and the O-shaped fiber ring section (F ring) of the trachea, and the axial sections corresponded to the longitudinal cartilage fiber interlacing area (C band) and the full fiber tissue strip fiber bundle area (F bundle). The histological results showed that the radial C-shaped cartilage ring section showed a mature cartilage appearance, a typical pit structure, and a large amount of specific ECM deposited inside the original scaffold, and the fiber ring section was mainly covered by a large amount of fibrous tissue; the axial cartilage fiber interlacing band still maintained the structure of alternating arrangement of cartilage tissue and fibrous tissue, and the full fiber strip fiber band was basically covered by fibrous tissue, which was consistent with the physiological structure of the natural gas tube. This shows that the integrated trachea of ​​the patterned design of the present invention can still maintain the original structural design in the nude mouse, so far realizing the multi-tissue integrated tracheal bionics at three levels of C-shaped cartilage ring, fiber ring, and axial strip fiber band.

[0047] 5. Finally, for the bionic construction of airway mucosal epithelium, primary airway basal cells were isolated and extracted from the rabbit tracheal mucosa and rapidly amplified in vitro. High concentrations of airway basal cells were loaded using GelMA photocrosslinked hydrogels and evenly inoculated onto the integrated tracheal inner wall constructed in the previous chapter, thereby preliminarily realizing the in vitro construction of airway epithelial structures. Epithelial tissue-specific staining can prove that there is an obvious ring structure loaded with airway epithelial cells on the inner side of the tube wall, proving that we have successfully constructed airway basal cells purified and cultured in vitro onto the inner wall of our tissue-engineered trachea, thus realizing the multi-tissue integrated tracheal bionics of the four-fold structure of C-shaped cartilage rings, O-shaped fiber rings, axial strip fiber bands, and tracheal inner wall mucosal epithelial layer. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The use of 4-axis 3D printing to construct a PGS tubular scaffold was shown, and this technology was used to achieve tubular tracheal cartilage reconstruction.

[0049] Figure 2The overall research design of the present invention and the related characterization of the scaffold material are shown: (A) Schematic diagram of the multi-tissue integrated trachea (IT-MOS) design. (B) Scaffold preparation flow chart: The 3D-printed PGS / PCL scaffold was modified with gelatin to prepare the PGS / PCL-Gelatin (PPG) scaffold. (C) Optical microscope image of the PPG scaffold: the green and red arrows represent the PGS / PCL framework and the gelatin fiber network, respectively. (D) Optical image of the 3D-printed PGS / PCL scaffold and the PPG scaffold. (E, F) Electron microscope structural images of the 3D-printed scaffold at different magnifications. (G) The macropore size of the PGS / PCL framework and the gelatin fiber network. (H) The micropore size inside the microwire. (I) Typical compressive stress-strain curve of the scaffold uniaxial compression test. (J) The scaffold was subjected to 10 cyclic compression tests with a maximum strain of 40%. (K) Comparison of compression modulus. (L) Water absorption of PGS / PCL and PPG scaffolds. (M) FTIR spectrum analysis of the scaffold. (N) Curve of dynamic water contact angle changing with time.

[0050] Figure 3 The process of constructing a two-dimensional regenerated trachea in vitro using patterned seeding technology is shown: (A) Schematic diagram of the precise distribution of multiple cell patterns using a thermosensitive hydrogel occupation and post-sacrifice strategy. (B) The state of thermosensitive hydrogel Pluronic F127 at 4°C and 37°C. (C) Precise pattern distribution of F127 on the scaffold. (D, E) The actual process and microscopic optical images of multi-cell patterned seeding. (F) Distribution map taken under a confocal microscope after live cell fluorescence staining. (G, H) Histological staining results of the cell-scaffold complex after 4 weeks of in vitro culture.

[0051] Figure 4 The following figure shows IT-MOS regenerated in nude mice for 8 and 12 weeks: (A) Schematic diagram of IT-MOS regeneration by 2D-3D assembly strategy. (B) Gross morphology of IT-MOS before and during subcutaneous culture in nude mice. (C) Gross morphology of IT-MOS after subcutaneous culture in nude mice. (DG) Histological staining results and vascularization indexes of IT-MOS in vivo at 8 / 12 weeks, four sections: radial cartilage ring (C ring) and fibrous ring (F ring); axial cartilage / fiber alternation zone (C / F band) and fiber band zone (F bundle). Ⅰ / Ⅱ are the results of safranin / fast green staining at 8 and 12 weeks. Ⅲ / Ⅳ are the vascularization indexes at 8 and 12 weeks. The red rectangle represents the cartilage area; the green rectangle represents the vascularized fibrous tissue area. (HJ) Statistical graphs of the average thickness, cartilage ratio, and vascularization ratio of the cartilage ring (C ring), fibrous ring (F ring), and axial fiber bundle (F bundle) at 8-12 weeks.

[0052] Figure 5Comparison of the composition and properties of IT-MOS and natural gas pipes: (AB) Side and top views of the regenerated air pipe under normal and compressed conditions. (CE) Quantitative analysis of the DNA content, GAG content, and COL II of the regenerated air pipe and natural gas pipe at 8 to 12 weeks. (F) Stress-strain curve of the uniaxial compression test (G) Stress-strain bar graph at compression strains of 20% and 40%. (H) Cyclic compression test with a maximum strain of 40%. (I) Curve of elastic recovery rate as a function of the number of compressions during the first 10 cycles of compression. The elastic rebound rate is the ratio of the rebound stress to the compressive stress in the same compression test. (J) Comprehensive evaluation diagram from five dimensions: DNA, GAG, ColII, elastic modulus, and elastic recovery rate.

[0053] Figure 6 The biomimetic model of the tracheal mucosal epithelium in IT-MOS is shown. (A) Schematic diagram of GelMA hydrogel loaded with airway basal cells simulating the tracheal epithelium. (B) Optical microscope image of airway basal cells cultured in vitro. (C) Immunofluorescence image of PCK and CK-5 specifically stained for airway epithelial cells. (D) Histological staining results of regenerated airway epithelium (orange arrow). (Red arrows are cartilage, green arrows are fibers, and black arrows are airway basal cells). (E) Immunofluorescence staining of PCK and CK-5 of regenerated airway epithelium (white arrows indicate airway basal cells). DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] 1. Preparation and characterization of PPG scaffolds

[0056] In previous studies, a salt composite printing strategy was first used to achieve 3D printing of bioelastomer PGS. Figure 2The PPG graded microporous scaffold shown in B. The scaffold is printed by mixing PGS and PCL to slow down the degradation rate of the PGS scaffold in the body to ensure compatibility with the cartilage regeneration rate. The PGS / PCL prepolymer and NaCl composite ink are 3D printed into a multilayer structure, and then vacuum cured and cross-linked at high temperature to obtain a stable thermosetting PGS / PCL composite scaffold. In order to improve the cell compatibility and cartilage growth activity of the PGS / PCL scaffold, the 3D printed scaffold is immersed in a 1% gelatin solution and converted into a three-dimensional porous freeze-dried scaffold using a vacuum freeze dryer. This process forms a gelatin network with a smaller pore size in the PPG scaffold ( Figure 2 C). In order to improve the stability and mechanical properties of the scaffold, the PPG scaffold was cross-linked by EDC / NHS method for 24 h. The residual solution was completely removed in deionized water, and the PPG scaffold was obtained by vacuum freeze drying. Finally, the PPG scaffold was gelatin-modified, still maintaining the initial morphological characteristics of the PGS / PCL scaffold, and the gelatin was evenly filled into the interstitial area ( Figure 2 D).

[0057] Scanning electron microscopy images showed that gelatin was present throughout the PGS framework and integrated in a well-organized hierarchical structure ( Figure 2 E and 2F). The PPG framework is composed of vertically stacked filaments with a diameter of approximately 700 μm.

[0058] (480.12±49.35μm), the pore size of the gelatin fiber network uniformly distributed between the PGS / PCL fibers was smaller (257.60±29.87μm) ( Figure 2 G). During cell inoculation and in vitro culture, cells were more likely to adhere and grow in the gelatin fiber network of the PPG scaffold. In addition, salt leaching resulted in a large number of cubic micropores on the surface and inside of the 3D printing filaments. The micropore sizes of the PGS / PCL and PPG scaffolds were (28.58±3.34)μm and (27.24±1.78)μm, respectively, which were determined by the size of the salt particles that could be adjusted by the grinding and sieving process during the preparation process ( Figure 2 H). These micropores provide channels for chondrocytes to migrate and grow in PGS / PCL microfilaments in vivo. In addition, this fibrous gelatin can modify the hydrophilicity of the PGS scaffold surface to mimic the natural ECM, which helps promote cell penetration, adhesion, proliferation and matrix secretion.

[0059] Due to the hydrogen bonding interaction between the amino and hydroxyl groups on the gelatin molecular chain, the addition of gelatin significantly improved the stiffness of the PGS scaffold. The elastic modulus of the PGS / PCL and PPG scaffolds measured by uniaxial compression tests were (138.25±21.97) kPa and (268.37±15.85) kPa, respectively. Figure 2 I and Figure 2 K). In addition, cyclic compression tests showed that PGS and PPG scaffolds have good elasticity and fatigue resistance under dynamic pressure ( Figure 2 J). The elasticity and fatigue durability of the scaffolds enable the regenerated cartilage tissue to withstand multiple dynamic deformations and maintain its original morphology after implantation.

[0060] In addition, the hydrophilicity of the PPG scaffold increased after gelatin modification. After water absorption, the weight gain percentage of the PPG scaffold (286.95±10.47%) was significantly higher than that of the PGS / PCL scaffold (56.95±4.98%) ( Figure 2 L). FTIR analysis further supports the enhanced hydrophilicity of PPG materials. -1 、1636cm -1 、1554cm -1 and 1238cm -1 The characteristic absorption peak at 1158 cm -1 and 1732cm -1 The characteristic absorption peak at represents the hydrophobic ester bond ( Figure 2 M). In addition, in the water contact angle experiment, the contact angle between the PPG scaffold and the water droplet reached 0 within 10 seconds (complete absorption), while the PGS / PCL scaffold group still maintained a contact angle of 61° after 5 minutes (incomplete absorption) ( Figure 2 N). These results indicate that gelatin-modified PPG scaffolds have better hydrophilicity and bioactivity and are more suitable for cell adhesion and proliferation.

[0061] 2. Constructing 2D IT-MOS in vitro

[0062] In order to solve the problem of alternating spatial structure between tracheal C-type cartilage rings and O-type fibers and axial strip fiber bundle structure, we first developed a post-occupancy sacrifice (POS) strategy of thermosensitive hydrogel to achieve multicellular patterned construction. Pluronic F-127 with a mass fraction of 30% was selected, which exhibits temperature-sensitive properties - liquid at 4°C and gel-like semi-solid at 37°C ( Figure 3 B). This gel-like semisolid can effectively hinder the distribution of cells on the PPG scaffold and dissipate into a liquid state at 4°C, thus playing a temporary blocking role during the cell seeding process. Through this POS strategy, the precise distribution of various complex patterns can be easily controlled ( Figure 3 C) and facilitates precise patterning on our PPG scaffolds ( Figure 3 D).

[0063] Figure 3 A shows the step-by-step process of multi-cell patterned distribution on PPG scaffolds. First, we used the temperature-sensitive hydrogel Pluronic F-127 to form a gel at high temperature (37°C) and occupy the predetermined space on the scaffold. At this stage, Pluronic F-127 is a semi-solid hydrogel that effectively occupies specific positions on the PPG scaffold. Subsequently, the chondrocyte suspension was seeded in the unoccupied area, so that it was precisely distributed in a strip-like structure. Subsequently, the thermosensitive hydrogel liquefied and eliminated at low temperature, leaving behind strip-like arranged chondrocytes. This arrangement laid the foundation for the subsequent construction of C-shaped cartilage rings in 2D to 3D conversion. After 3 weeks of in vitro culture of cartilage tissue, fibroblasts were introduced into the remaining positions (the original Pluronic F-127 positions), so that the C-shaped cartilage rings and O-shaped fiber rings were spatially cross-distributed.

[0064] To verify the accuracy of the method, we performed validation tests from three perspectives: optical microscopy, fluorescent cell labeling, and histological staining. During the cell seeding process, we captured microscopic images of each step using an optical microscope with an external light source ( Figure 3 E). These images clearly show distinct boundaries between the cell suspensions on gelatin, Pluronic F-127, and PPG scaffolds. This suggests that our POS strategy effectively achieves patterned distribution of cell suspensions. To further confirm the accuracy of pattern construction, we stained chondrocytes and fibroblasts with two different live cell fluorescent dyes (DiO (green) and Dil (red)) and then seeded them onto PPG scaffolds using the POS strategy as before ( Figure 3 F). The green area is DiO-labeled chondrocytes, the red area is Dil-labeled fibroblasts, and the black area is the scaffold. Under a fluorescence microscope, the two fluorescently labeled cells showed a clear pattern distribution, indicating that our POS strategy can achieve precise multicellular distribution on the PPG scaffold.

[0065] The next question to be addressed was whether our multicellular patterned constructs could maintain their morphology during in vitro culture. To investigate this, we cultured the scaffold-cell complexes in vitro for 4 weeks and then analyzed the morphology of the scaffolds by HE staining ( Figure 3 G) and SO / FG staining ( Figure 3H) The cross-section and longitudinal sections of the tissue were stained for verification. In the cross-section, the cartilage tissue in the area where chondrocytes were continuously seeded was mature and distributed continuously and evenly. The cartilage tissue also showed an initial regeneration state, characterized by the presence of typical pit structures unique to cartilage, accompanied by extracellular matrix deposition. In the longitudinal section, the areas where chondrocytes and fibroblasts were alternately seeded still maintained an alternating distribution structure. These findings indicate that our thermosensitive hydrogel POS strategy is effective for achieving multi-cellular patterned construction. In summary, these results indicate that our IT-MOS successfully achieved spatial structural bionics during the in vitro culture stage.

[0066] 3. 3D IT-MOS regenerates in nude mice for 8 and 12 weeks

[0067] After achieving the patterned structure on the two-dimensional plane, we further constructed a three-dimensional IT-MOS by rolling up the scaffold-cell complex constructed in vitro and anastomosing the two ends with absorbable sutures. It was then implanted under the skin of nude mice, with a silicone rod built in the middle to provide internal support to prevent the regenerated trachea from collapsing due to skin tension ( Figure 4 B) After 8 and 12 weeks of in vivo culture, regenerated IT-MOS were harvested ( Figure 4 A). The regenerated trachea maintains its original tubular structure, and the internal pores of the scaffold are gradually filled with new tissue. The tracheal structure is composed of porcelain-white C-shaped cartilage rings and O-shaped vascular fiber rings distributed alternately ( Figure 4 C) These findings suggest that we have successfully achieved the regeneration of IT-MOS by tissue engineering approach.

[0068] To evaluate the maturity of IT-MOS cartilage and determine whether the in vitro multi-tissue patterning structure can be maintained after in vivo culture, we performed histological staining on radial and axial sections of IT-MOS. The radial section corresponds to the C-shaped cartilage ring (C ring) and O-shaped fiber ring (F ring) of the trachea, and the axial section corresponds to the longitudinal cartilage / fiber interlaced area (C / F band) and strip fiber bundle area (F bundle). Figure 4 DG shows SO&FG staining (Ⅰ / Ⅱ) and vascular-specific immunofluorescence staining of vwf and α-sma (Ⅲ / Ⅳ) in 4 sections at 8 and 12 weeks. SO&FG staining showed that mature cartilage existed in the cartilage tissue area, with typical lacunar structure and a large amount of extracellular matrix (ECM) deposition, and the cartilage maturity at 12 weeks was significantly higher than that at 8 weeks. The cartilage matrix was basically distributed in a C-shape, which was consistent with the in vitro pattern structure ( Figure 4 D). The fibrous tissue area is mainly covered by fibrous tissue, with almost no cartilage tissue, especially in the F ring and F bundle sections ( Figure 4 E and 4G). The axial cross-section of the cartilage / fibrous region still maintains the alternating arrangement of cartilage and fibrous tissue ( Figure 4 F).

[0069] Angiogenesis is only seen in fibrous tissue areas, not cartilage areas. Therefore, we can also verify the distribution of cartilage and fibers by specific staining of vascularization indicators. Vascular-specific VWF and α-SMA immunofluorescence staining showed that the positive areas were mainly confined to fibrous tissue areas, namely, the F area in the C ring and C / F band, the F bundle ( Figure 4 DG-Ⅲ / Ⅳ).

[0070] The thickness, cartilage ratio, and vascularization ratio of the three parts were analyzed at 8 and 12 weeks. The trachea thickness in the cartilage area was significantly greater than that in the other two fibrous areas ( Figure 4 H). In addition, Figure 4 It can be seen that the proportion of cartilage in the cartilage ring area is much higher than that in the other two fiber areas, and it increases with the extension of in vivo culture time. This is consistent with the maturation of cartilage tissue and the deposition of extracellular matrix (ECM). Figure 4 J) It can be found that the degree of vascularization of the F ring and axial F bundle is significantly greater than that of the C ring. Therefore, it can be further proved that our IT-MOS successfully maintained the original in vitro structural design during the subcutaneous culture of nude mice.

[0071] 4. Comparison between regenerated IT-MOS and natural gas pipes

[0072] The regenerated IT-MOS had similar elasticity and rigidity to natural gas tubes, and the mechanical properties of the regenerated trachea at 12 weeks were significantly improved compared with those at 8 weeks ( Figure 5 A and 5B). We performed a comprehensive evaluation of the regenerated IT-MOs and compared them with natural tubes by quantitative analysis and elasticity testing. In the quantitative analysis, we compared the DNA content of IT-MOs cultured for 8 and 12 weeks with that of natural tubes ( Figure 5 C) GAG content ( Figure 5 D) and type II collagen content ( Figure 5 E). The results showed that high-density cell suspension inoculation resulted in a higher DNA content in the regenerated tracheal cartilage, and as time went on, chondrocytes gradually secreted ECM, and the content of GAG and type II collagen in the regenerated trachea gradually increased and approached the level of natural tracheal cartilage.

[0073] To investigate the mechanical properties of the regenerated trachea, uniaxial compression and cyclic compression tests were performed. Figure 5 F is the compressive stress-strain curve observed during a single compression process. It was observed that the simple PPG tubular stent did not have sufficient elasticity and mechanical structure. Therefore, we calculated and compared the elastic modulus of the other three tracheae under different strains and plotted the corresponding change curves ( Figure 5G). The results showed that the regenerated tubular bionic trachea had good rigidity and fatigue resistance: after 8 weeks of in vivo culture, the elastic modulus of the regenerated trachea was close to that of the natural trachea, while after 12 weeks, the elastic modulus of the regenerated trachea under larger strains even exceeded that of the natural trachea. Figure 5 H is the compressive stress-strain curve during the cyclic compression process. We analyzed the first 10 cycles and calculated the ratio of the rebound stress to the compressive stress when the compressive strain was 20% in each cycle, which is called the elastic recovery rate, which is used to measure the elasticity of the tissue and its ability to recover deformation ( Figure 5 I). Our experimental results show that the elastic recovery rate of the PPG scaffold with almost no elasticity after 8 weeks of regeneration is close to that of the natural gas tube, and the elastic recovery rate of IT-MOS after 12 weeks is higher than that of the natural gas tube, which indicates that our regenerated IT-MOS has better elastic properties than the natural gas tube under the same deformation conditions. Through uniaxial compression and cyclic compression tests, it can be proved that our IT-MOS has stronger rigidity and elasticity than the natural gas tube, and can more effectively maintain mechanical compliance in the dynamic mechanical environment in vivo.

[0074] In order to comprehensively evaluate the performance of our IT-MOS, we selected DNA, GAG, Col II, elastic modulus, and elastic recovery rate as five scoring criteria. In the quantitative evaluation, we gave the natural gas duct cartilage a full score of 100 and obtained the following Figure 5 The five-dimensional evaluation scale shown in J. The scoring results showed that the indicators of the regenerated trachea cultured in vivo for 8 weeks were comparable to those of the natural trachea; after 12 weeks of in vitro culture, the mechanical properties of the regenerated trachea were significantly better than those of the natural trachea. This indicates that our tissue-engineered regenerated trachea has similar comprehensive properties to those of the natural trachea.

[0075] 5. IT-MOS tracheal epithelial biomimetic construction

[0076] The biomimetic construction of the airway epithelium aims to replicate the natural structure and function of the respiratory tract. Airway basal cells are a type of multipotent stem cell located in the basal layer of the airway epithelium that has the ability to self-replicate and differentiate into different types of cells, including epithelial cells, mucus cells, and ciliated cells. These cells play a vital role in the growth and repair of the airway epithelium.

[0077] This study successfully isolated and extracted primary airway basal cells from rabbit tracheal mucosa and rapidly expanded them in vitro. We used photo-crosslinked GelMA hydrogel to load these airway basal cells onto the inner wall of our IT-MOS ( Figure 6 A), thus preliminarily realizing the bionic construction of tracheal epithelium. Figure 6B shows the morphological structure of airway basal cells isolated and amplified under different magnifications under an optical microscope. During in vitro culture, airway basal cells form a monolayer on the surface of the culture medium and continue to divide, proliferate and produce new cells. We can also see that they can secrete mucus to protect epithelial cells. In order to verify the reliability of the cell extraction and culture process, we performed immunofluorescence staining for total cytokeratin (PCK) and epithelial-specific cytokeratin 5 (CK-5). Figure 6 C. The staining results confirmed that the cultured airway basal cells had high purity and high cell activity.

[0078] To verify the effectiveness of airway epithelial construction, we performed histological staining (HE staining, SO&FG staining, Masson staining) of radial sections from IT-MOS ( Figure 6 D) and immunofluorescence staining (total keratin PCK and epithelial-specific keratin CK-5) ( Figure 6 E). The staining results showed that airway basal cells were observed to have successfully adhered to the inner wall of the IT-MOS tube and formed a ring-shaped structure similar to the airway mucosal epithelium. So far, we have achieved the integrated regeneration and reconstruction of four tissue structures in IT-MOS: C-shaped cartilage ring, O-shaped vascularized fibrous ring, axial strip fiber bundles and tracheal epithelial tissue.

[0079] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A tissue engineering integrated tracheal regeneration construction method based on a framework structure scaffold, characterized in that: The steps include: A. Preparation of bioelastic scaffolds PGS or PGS is compounded with prepolymers and inorganic salts of other biomaterials and then 3D printed into a multilayer structure, and then vacuum cured and cross-linked at high temperature to obtain a stable thermosetting PGS / PCL composite scaffold; the 3D printed scaffold is then immersed in a 1% gelatin solution and converted into a three-dimensional porous freeze-dried scaffold using a vacuum freeze dryer, so that a gelatin network with a smaller pore size is formed in the PPG scaffold, and the PPG scaffold is cross-linked for 24 hours; the residual solution is completely removed in deionized water, and then a gelatin-modified PPG scaffold is obtained by vacuum freeze drying, which maintains the initial morphological characteristics of the PGS / PCL composite scaffold, and the gelatin is evenly filled into the interstitial area; B. Construction of multicellular two-dimensional patterning At 37°C, the temperature-sensitive hydrogel Pluronic F-127 was coated on specific locations of the PPG scaffold, and then the chondrocyte suspension was seeded in the unoccupied areas so that they were precisely distributed in the strip-like structure; then, the temperature-sensitive hydrogel liquefied and disappeared at low temperatures, leaving behind the chondrocytes arranged in strips; Patterning a variety of cells into biomimetic structures lays the foundation for the subsequent construction of C-shaped cartilage rings in 2D to 3D transformation; C. Tissue engineering biomimetic trachea construction The planar scaffold-cell complex constructed in vitro is rolled up and the two ends are anastomosed with absorbable sutures to construct a three-dimensional integrated bionic tissue-engineered trachea. After being cultured in vivo in a subcutaneous or muscle flap for a period of time, a regenerated tissue-engineered trachea is obtained.

2. The tissue engineering integrated tracheal regeneration construction method based on the framework structure scaffold according to claim 1 is characterized by: in, In step A, the biomaterial is selected from one or more of PCL, PLGA, and PLA, and the mass ratio of PGS is 50-100%; The polymer material is further mixed with inorganic salt particles to form a 3D printing ink, and 3D printing is performed to prepare a bioelastomer framework; wherein the mass ratio between the polymer material and the inorganic salt is 1:0.5 to 1:3, the salt particle size is 10 to 75 μm, and the inorganic salt is selected from any one or more of sodium chloride, potassium chloride, and sodium carbonate; The 3D printing process is as follows: select a print head with an inner diameter of about 100-1000μm, a grid filling width of 1-2.5mmmm, a grid interlacing angle of 90°, a layer height of 0.3-0.6mm, a printing speed of 2-40mm / s, and a printing temperature of 30-100℃; After 3D printing, the film was initially cured at 100°C and 0.5 bar vacuum for 12 hours, and then cross-linked at 150°C and 1 bar vacuum for 24 hours to achieve complete curing. It was further immersed in an ethanol-water mixed solution with an ethanol volume fraction of 10-75% to remove salt particles, and the solvents including water and ethanol were removed by air drying, heating, freeze drying, etc.

3. The tissue engineering integrated tracheal regeneration construction method based on a framework structure scaffold according to claim 1, characterized in that: in, In step A, after the 3D printed scaffold is immersed in a 0.5-3% gelatin solution, the vacuum freeze-drying conditions used are as follows: -80°C, 5-50Pa, and converted into a three-dimensional porous freeze-dried scaffold; the three-dimensional porous freeze-dried scaffold is cross-linked with the PPG scaffold using the EDC / NHS method for 24 hours; after the scaffold is completely removed of the residual solution by deionized water, the vacuum freeze-drying conditions are as follows: -80°C, 5-50Pa.

4. The tissue engineering integrated tracheal regeneration construction method based on a framework structure scaffold according to claim 1, characterized in that: in, In step B, the three-dimensional structure of the natural gas pipe is unfolded into a two-dimensional planar structure, the PPG framework structure scaffold is divided into areas corresponding to the distribution of cartilage tissue and fibroblast tissue, the thermosensitive hydrogel Pluronic F-127 is filled into the fibroblast tissue area to form a placeholder, and chondrocytes are seeded in the vacant area; after the cells adhere and grow, they are placed in a low-temperature environment of 4-10°C to remove the thermosensitive hydrogel, and the fibroblasts are seeded into the temporary placeholder area before the hydrogel and continue to be cultured.

5. The tissue engineering integrated tracheal regeneration construction method based on a framework structure scaffold according to claim 1, characterized in that: in, In step B, the seeding density of chondrocytes is 1-8*10 7 / ml, the seeding density of fibroblasts is 1-5*10 7 / ml.

6. The tissue engineering integrated tracheal regeneration construction method based on the framework structure scaffold according to claim 1, Features: Among them, in step C, the multi-cellular patterned scaffold / cartilage / fibroblast composite tissue is assembled and curled into a tubular structure, and implanted into the subcutaneous or muscle flap for a period of time to form a vascularized regenerative tissue-engineered trachea; the airway basal cells isolated, amplified, differentiated and cultured in vitro are mixed into the photosensitive hydrogel, coated on the inner wall of the regenerative scaffold and cross-linked by ultraviolet light to form an epithelial layer; the photosensitive hydrogel is GelMA, and the concentration is 5-15%.

7. The tissue engineering integrated tracheal regeneration construction method based on a framework structure scaffold according to claim 1, characterized in that: in, The method for isolating and expanding airway basal cells is as follows: The harvested mucosa was minced into 1 mm 2 The small pieces were washed thoroughly in PBS containing 1% antibiotics and digested with 0.15% collagenase NB4 at 37°C for 2 hours. Afterwards, the digestion solution was filtered through a filter and resuspended and centrifuged to obtain airway basal cells. The isolated airway basal cells were cultured in PneumaCult TM -Ex Plus medium, and when the cells reach 90% confluence, they are passaged and the second generation of airway basal cells are obtained.

8. A tissue engineering integrated tracheal regeneration, characterized in that: The method is prepared by any one of claims 1 to 6.

9. Use of the tissue engineering integrated regenerated trachea according to claim 8 in the treatment of tracheal segmental injuries.

10. A tracheal segmental injury regeneration and repair component, characterized in that: Including the tissue engineering integrated regenerated trachea as described in claim 8.

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