Multi-layer bionic trachea repair transplant and preparation method thereof

Abstract

The invention relates to a multi-layer bionic trachea repair graft and a preparation method thereof. The preparation method comprises the following steps: printing an electric writing net by using polycaprolactone particles; printing a planar caramel stent by using the moldable caramel raw material, and curling to form a C-shaped caramel stent; a C-type caramel stent is coated with polycaprolactone coating liquid, an electric writing net is attached to the inner side of the C-type caramel stent, the C-type caramel stent is dissolved by soaking the C-type caramel stent in distilled water, a C-type elastic hollow stent is formed, and therefore the nested multi-layer bionic tracheal stent is obtained. Mixing the primary cartilage cells and the human gingival mesenchymal stem cells, and inoculating the mixture to an electric writing net for culture; decellularizing the scaffold; and mixing the autologous cartilage particles with the allogenic adipose-derived mesenchymal stem cells, and injecting the mixture into a hollow pipeline of the C-shaped elastic hollow stent to obtain the multi-layer bionic trachea repair transplant. The multi-layer structure stent disclosed by the invention has biocompatibility and mechanical properties, promotes long-term survival and functional recovery of cells, improves vascularization and epithelialization effects, and has the potential of being converted into clinical application.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and particularly relates to a multi-layer biomimetic tracheal repair graft and a preparation method thereof. Background Art

[0002] Congenital tracheal stenosis, traffic accidents, industrial accidents, etc. may cause local or segmental defects of the trachea. As an important respiratory tract of the human body, the structural integrity and normal function of the trachea are crucial for maintaining life. Tracheal defects can lead to serious consequences such as dyspnea and infection, and even endanger life. Traditional tracheal repair methods mainly include autologous tracheal transplantation, allogeneic tracheal transplantation, and artificial tracheal implantation, etc., but these methods have many limitations, such as limited donor sources, large surgical trauma, many postoperative complications, and unsatisfactory repair effects, etc.

[0003] Driven by tissue engineering technology, various biomaterials, cells, and engineering technology means have been tried to construct tracheal substitutes to achieve the structural reconstruction and function restoration of the trachea. In terms of biomaterials, decellularized matrices, synthetic polymer materials, natural-derived materials, etc. have been widely studied in order to obtain good biocompatibility, mechanical properties, and degradation characteristics. In terms of cells, stem cells such as bone marrow mesenchymal stem cells and gingival mesenchymal stem cells have become the focus of tracheal repair research due to their strong proliferation and differentiation abilities. In terms of engineering technology, 3D printing technology, electrospinning technology, melt electrowriting technology, etc. provide new methods and ideas for the precise construction of tracheal scaffolds. However, despite some progress, the existing tracheal repair technologies still have many problems and challenges, such as it is difficult to balance the biocompatibility and mechanical properties of the scaffold materials, the long-term survival and function of cells are limited, the effects of vascularization and epithelialization are not good, the individual adaptability is poor, and the clinical transformation is difficult, etc.

[0004] Therefore, developing a new type, efficient, and safe tracheal repair technology has important clinical significance and broad application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide a multi-layer biomimetic tracheal repair graft and a preparation method thereof to solve the problems existing in the above-mentioned prior art.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A preparation method of a multi-layer biomimetic tracheal repair graft, the preparation method comprising:

[0008] Printing and electrowriting a network using polycaprolactone particles;

[0009] Printing a planar sugar scaffold using a plasticizable caramel raw material and curling it into a C-shaped caramel scaffold;

[0010] Prepare a polycaprolactone coating solution and coat it on the C-shaped caramel scaffold. Attach the electrospun mesh to the inner side of the C-shaped caramel scaffold, and immerse it in distilled water to dissolve the C-shaped caramel scaffold, forming a C-shaped elastic hollow scaffold, thereby obtaining a nested multi-layer bio-inspired tracheal scaffold;

[0011] Mix primary chondrocytes and human gingival mesenchymal stem cells, and inoculate them on the electrospun mesh of the nested multi-layer bio-inspired tracheal scaffold for culture;

[0012] Perform decellularization treatment on the nested multi-layer bio-inspired tracheal scaffold;

[0013] Mix autologous cartilage microparticles and allogeneic adipose mesenchymal stem cells, and inject them into the hollow tube of the C-shaped elastic hollow scaffold to obtain a multi-layer bio-inspired tracheal repair and transplantation body.

[0014] Furthermore, the electrospun mesh is printed by a MEW device, including a multi-layer interconnected grid structure.

[0015] Furthermore, the planar sugar scaffold is printed by a 3D printer, and the curled C-shaped caramel scaffold includes multiple C-shaped ring bodies.

[0016] Furthermore, the preparation process of the polycaprolactone coating solution is as follows:

[0017] Dissolve polycaprolactone particles in hexafluoroisopropanol, and add sodium chloride particles as a pore-forming agent to obtain a coating solution.

[0018] Furthermore, after attaching the electrospun mesh to the inner side of the C-shaped caramel scaffold, let it stand until the hexafluoroisopropanol volatilizes and dries, and then immerse the nested multi-layer bio-inspired tracheal scaffold in distilled water to dissolve the C-shaped caramel scaffold and sodium chloride particles.

[0019] Furthermore, before inoculating primary chondrocytes and human gingival mesenchymal stem cells on the electrospun mesh of the nested multi-layer bio-inspired tracheal scaffold, put the nested multi-layer bio-inspired tracheal scaffold outside a silicone tube, place it in alcohol, and irradiate it with ultraviolet light for disinfection.

[0020] Furthermore, after inoculating primary chondrocytes and human gingival mesenchymal stem cells on the electrospun mesh of the nested multi-layer bio-inspired tracheal scaffold, place the nested multi-layer bio-inspired tracheal scaffold in a high-glucose medium for culture;

[0021] The formula of the high-glucose medium is: 15% fetal bovine serum, 1% triple antibody, 1% glutamine, 1% Vc, and the balance is distilled water.

[0022] Furthermore, the decellularization treatment of the nested multi-layer bio-inspired tracheal scaffold includes:

[0023] Immerse the nested multi-layer bio-inspired tracheal scaffold in an SDS solution, and then place it in a Triton solution.

[0024] On the other hand, a multi-layer biomimetic tracheal repair and transplantation body obtained by the preparation method as described above is provided. The multi-layer biomimetic tracheal repair and transplantation body includes an electrospun mesh and a C-shaped elastic hollow scaffold, and the electrospun mesh is attached to the inner side of the C-shaped elastic hollow scaffold;

[0025] The electrospun mesh is inoculated with a composite cell composed of primary chondrocytes and human gingival mesenchymal stem cells;

[0026] Cartilage composite microparticles composed of autologous cartilage microparticles and allogeneic adipose mesenchymal stem cells are injected into the hollow pipeline of the C-shaped elastic hollow scaffold.

[0027] Furthermore, the C-shaped elastic hollow scaffold includes a plurality of C-shaped hollow ring bodies, which are arranged in parallel and have axially corresponding notches on one side. The circumferential ends of the plurality of C-shaped hollow ring bodies are connected and closed into one body through axially hollow line bodies. [[ID=--]]<000--

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The present invention provides a multi-layer biomimetic tracheal repair and transplantation body and a preparation method thereof. By combining 3D printing technology and melt electrospinning writing technology, a multi-layer structure scaffold with excellent mechanical properties and biocompatibility is prepared, improving the balance between biocompatibility and mechanical properties, promoting the long-term survival and functional recovery of the vascularized remodeling cartilage ring, improving the vascularization and epithelialization effects, enhancing the individual adaptability, and having the potential to be translated into clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0031] Figure 1 It is a schematic diagram of the MEW device used in the embodiment of the present invention.

[0032] Figure 2 It is a schematic diagram of the electrospun mesh printed and fabricated in the embodiment of the present invention.

[0033] Figure 3 It is a microstructural diagram of the electrospun mesh printed and fabricated in the embodiment of the present invention.

[0034] Figure 4 It is a microstructural diagram of the electrospun mesh printed and fabricated from another perspective in the embodiment of the present invention.

[0035] Figure 5Schematic diagram of a nested multi-layer bionic tracheal stent obtained by laminating a C-shaped caramel stent with an electrowriting mesh in an embodiment of the present invention.

[0036] Figure 6 Schematic diagram of cell inoculation in a nested multi-layer bionic tracheal stent according to an embodiment of the present invention.

[0037] Figure 7 Schematic diagram of the nested multi-layer bionic tracheal stent before and after decellularization in an embodiment of the present invention.

[0038] Figure 8 Schematic diagram of the injection of cartilage composite particles in an embodiment of the present invention.

[0039] Figure 9 This is a schematic diagram of the scaffold effect after injection of cartilage composite particles in an embodiment of the present invention.

[0040] Figure 10 Schematic diagram of a segmental tracheal defect graft repair according to an embodiment of the present invention. (a) shows a rabbit segmental airway defect and the implantation of a nested multilayer bionic tracheal stent during surgery, and (b) shows the "end-to-end" anastomosis of the nested multilayer bionic tracheal stent.

[0041] Figure 11 Schematic diagram of repairing a partial fenestration defect with a patch according to an embodiment of the present invention. In the figure, (a) is a schematic diagram of a fenestration defect in a rabbit's airway, and (b) is a schematic diagram of the effect of repairing the airway defect with a multi-layered patch.

[0042] Figure 12 The following are images of the repair results of a segmental tracheal defect at 6 weeks of surgery, according to an embodiment of the present invention. (a) is a fiberoptic bronchoscopic image of the segmental airway defect repair, (b) is a macroscopic image of the segmental airway defect repair (before sampling), (c) is a macroscopic image of the segmental airway defect repair (after sampling), and (d) is a quantitative comparison of the compressive strength of the nested tracheal graft and the native trachea before and after transplantation (0W / 6W).

[0043] Figure 13 The in vivo repair effect of the multilayer biomimetic tracheal repair grafts according to the present invention is shown in Figure 1. (a) shows a histological section of a defect repaired with a multilayer biomimetic tracheal repair stent; (b) shows a quantitative comparison of the number of capillaries per unit area in the nested tracheal graft (6W) and an autologous control; and (c) shows a quantitative comparison of the glycosaminoglycan content in the nested tracheal graft (6W) and an autologous control.

[0044] Figure 14 This is a demonstration of the in vivo epithelial reconstruction effect of a multi-layer biomimetic tracheal repair graft according to an embodiment of the present invention. In the figure, (a) is a DAPI fluorescence staining image (long axis section), and (b) is a keratin (CK) immunofluorescence staining image (long axis section). Detailed implementation mode

[0045] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0046] In the description of the present invention, it should be understood that all technologies and scientific terms used have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. When there is a contradiction, the definition in this specification shall prevail. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, the reagents used in the examples are commercially available products, and the devices used in the examples are existing devices. The limitation of the means, reagents or devices should not be construed as a limitation of the present invention. Means, reagents or devices of the same type that solve the same technical problems are within the protection scope of the present invention.

[0047] In the description of the present invention, it should be understood that when an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper limit preferred values and lower limit preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is separately disclosed. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0048] It should also be noted that although the order of steps is involved in the method description, in some cases, it can be executed in a different order from here and should not be construed as a limitation on the order of steps.

[0049] The present invention provides a preparation method of a multi-layer biomimetic tracheal repair graft, which is implemented by combining 3D printing technology and melt electrowriting technology. Polycaprolactone (PCL) is selected as the main construction material, which has excellent biocompatibility, memory, biodegradability, etc. The obtained multi-layer biomimetic tracheal repair graft can be applied to tracheal repair and reconstruction.

[0050] Specifically, the preparation method of the present invention includes the following steps:

[0051] S1: Printing and electrowriting a network using polycaprolactone particles.

[0052] Such as Figure 1, in this step, the electro-written network is printed by a MEW device (Engineering For Life Co., Ltd, EFL, MDW5800). First, PCL particles (Perstorp, CAPA 6800) are loaded into a material cylinder with a 35μm nozzle. The temperatures of the material cylinder and the nozzle are set to 80°C and 100°C respectively. The voltage between the nozzle and the electric field platform is 4.8 kV, the distance is 6 mm, and the printing speed is 1200 mm / min. The printing grid parameters are 3 cm × 1.6 cm, and the pore grid size is 250μm × 250μm. After heating and melting the PCL particles, printing begins. After printing, it is ready for use.

[0053] As Figures 2 - 4 , the printed electro-written network includes multiple interconnected grid structures and has abundant voids microscopically.

[0054] S2: Print a planar sugar scaffold using a plasticizable caramel raw material and curl it to form a C-shaped caramel scaffold.

[0055] In this step, the planar sugar scaffold is printed by a 3D printer. The plasticizable caramel raw material is loaded into a ParticleCloud 3D printer, and the parameters are adjusted as follows: the heating temperature of the material cylinder is 95°C, the temperature of the extrusion head is 100°C, the air pressure is 0.05 MPa, the printing rate is 5 mm / s, and the printing extrusion head is 0.4 mm. A planar sugar scaffold (2.5 cm × 1.2 cm, filling rate 12%) is printed according to a preset trajectory (generated from an STL file).

[0056] Then, the planar sugar scaffold is curled to obtain a C-shaped caramel scaffold. The C-shaped caramel scaffold includes multiple C-shaped ring bodies, which are arranged in parallel and have axially corresponding notches on one side. The circumferential ends of the multiple C-shaped ring bodies are connected and closed into one body through axial line bodies.

[0057] The plasticizable caramel raw material used in this step is an optimized composite, which is specifically obtained through the following process:

[0058] ① Mix sucrose, fructose, dextran, and glucose in proportion and dissolve them in distilled water. Continuously stir at a high temperature and boil to obtain a viscous solution.

[0059] Among them, the dextran specifically used is dextran 70 with a molecular weight of 70000, and the fructose used is D-fructose. The mass mixing ratio of sucrose, fructose, dextran, glucose, and distilled water is 10:1:1:2:10.

[0060] The temperature of continuous stirring at a high temperature is 300°C. Specifically, the mixture is continuously stirred with a glass rod under the condition of 300°C on an induction cooker until the solution becomes viscous and light yellow.

[0061] ② Place the viscous solution in a high-temperature environment for high-temperature cross-linking to obtain sugar printing material.

[0062] Specifically, the high-temperature cross-linking temperature is 100° C. and the time is 8 hours, which can be completed in an oven to finally obtain a melt-extruded printing material.

[0063] The above-mentioned special sugar mixing formula, as well as the special temperature range of boiling and cross-linking process, make the final flexible glycosyl retinoic acid flat substrate have good toughness and plasticity, soft and deformable, and can be cut into different required shapes at will. It can be manually squeezed, stretched, bent, kneaded, assembled, and bonded to obtain the required three-dimensional structure, and the required three-dimensional configuration can be maintained for a long time. If only sucrose is used to prepare the sugar printing material, a printing syrup can be formed under the action of high temperature, but the printing syrup will quickly cool down and crystallize, turning into a hard state and unable to deform. Therefore, when only sucrose is used to prepare the sugar printing material, it is necessary to directly print the three-dimensional solid structure. Such a printing mode inevitably requires 3D modeling and printing path planning. The present invention can quickly print the flexible glycosyl retinoic acid flat substrate as a two-dimensional planar structure, and then directly manually shape it to obtain the required three-dimensional structure. If necessary, some molds can also be used, which is more efficient and more accurate in shaping, and the soft three-dimensional structure can be fine-tuned at any time. After shaping the C-shaped caramel bracket, the C-shaped caramel bracket can be immersed in anhydrous ethanol to dehydrate and harden, and then dried. The C-shaped caramel bracket is no longer soft and has supporting strength.

[0064] S3: Prepare a polycaprolactone coating liquid and apply it to a C-shaped caramel bracket, attach the electrowriting mesh to the inner side of the C-shaped caramel bracket, immerse the C-shaped caramel bracket in distilled water to dissolve it, and form a C-shaped elastic hollow bracket, thereby obtaining a nested multi-layer bionic tracheal bracket.

[0065] In this step, the polycaprolactone coating liquid is also prepared from polycaprolactone. PCL particles (Perstorp, CAPA6800) are dissolved in hexafluoroisopropanol to prepare a 5% concentration of the coating liquid. Sodium chloride particles (sieved through a 500-mesh sieve) with a mass twice that of the PCL are added as a porogen. The mixture is thoroughly stirred and placed on ice for later use.

[0066] Then, the C-shaped caramel stent was clamped and coated with polycaprolactone coating liquid 4 times on it. After that, it was attached to the electrographing mesh. After standing for hexafluoroisopropanol to evaporate and dry completely, it was immersed in distilled water overnight. The C-shaped caramel stent was dissolved, leaving a C-shaped elastic hollow stent formed by the polycaprolactone coating. Together with the electrographing mesh, a nested multilayer bionic tracheal stent was obtained, such as Figure 5 In addition, sodium chloride particles were also dissolved by distilled water, forming abundant pores in the solid part of the C-shaped elastic hollow scaffold.

[0067] S4: Mix primary chondrocytes with human gingival mesenchymal stem cells, and inoculate them on the electrospun mesh of the nested multi-layer biomimetic tracheal scaffold for culture.

[0068] Before inoculating primary chondrocytes and human gingival mesenchymal stem cells on the electrospun mesh of the nested multi-layer biomimetic tracheal scaffold, place the nested multi-layer biomimetic tracheal scaffold in a custom-made glass reactor so that the scaffold can be suspended in the culture medium. At the same time, put the scaffold outside a silicone tube (inner diameter 6 mm, outer diameter 7 mm), place the glass reactor together in 75% alcohol, and irradiate with ultraviolet light for more than 5 hours for disinfection.

[0069] After that, mix primary chondrocytes (taken from the auricular cartilage of 4-week-old New Zealand rabbits and digested with type II collagenase overnight) with human gingival mesenchymal stem cells at a cell number ratio of 6:1, and inoculate them evenly on the surface of the electrospun mesh at a density of 1×10 7 / cm 2 . After standing for one hour until the cells adhere stably, add high-glucose medium (DMEM, 15% fetal bovine serum, 1% triple antibody, 1% glutamine, 1% Vc, and the balance is distilled water), and culture for two weeks, changing the medium once a week. After two weeks, take it out, wash it three times with PBS solution to form a biologically active tubular matrix layer for use.

[0070] S5: Perform decellularization treatment on the nested multi-layer biomimetic tracheal scaffold. Specifically, it includes:

[0071] Immerse the nested multi-layer biomimetic tracheal scaffold in 1% SDS solution, take it out after 10 h and then place it in 1% Triton solution for 1 h to complete the decellularization process, and wash it three times with PBS solution, as Figure 7 .

[0072] The decellularization process can prepare a decellularized matrix with high vesicle loading efficiency. This matrix retains the components and structure of the natural extracellular matrix, retains biologically active stem cell vesicles, and has good biocompatibility and biological activity.

[0073] S6: Mix autologous cartilage microparticles with allogeneic adipose mesenchymal stem cells and inject them into the C-shaped hollow pipeline of the nested multi-layer biomimetic tracheal scaffold to obtain a multi-layer biomimetic tracheal repair and transplantation body, as Figure 6 .

[0074] In this step, the preparation method of the injectable cartilage composite microparticles is as follows:

[0075] Take 1 cm×2 cm of auricular cartilage from the experimental animal (in this invention, 3 kg male New Zealand rabbits are used as an example of experimental animals), avoiding damaging the middle ear artery. Remove the skin, cut it into pieces after peeling off the connective tissue membrane, freeze it in liquid nitrogen for 10 min, and then grind it to 30 um in size with a ball mill. Mix it with allogeneic adipose mesenchymal stem cells at a ratio of 8:1 to form an injectable mixture in terms of properties, asFigure 8 , and then loaded into a syringe for later use.

[0076] In other embodiments, allogeneic adipose mesenchymal stem cells can be replaced with autologous nano-fat, which is easier to obtain and has a smaller immune rejection reaction.

[0077] After that, the injectable cartilage composite microparticles are injected into the C-shaped hollow pipe through a syringe to fill the C-shaped hollow pipe for later use, as Figure 9 .

[0078] This step involves a brand-new functional remodeling strategy. Through the "injectable cartilage microparticle technology" and the "rich vesicle matrix 3D construction" technology, injectable cartilage microparticles with high survival rate are prepared, avoiding the defect of long-term cell culture required by traditional tissue engineering technology for planting chondrocytes, enabling the intraoperative immediate construction and transplantation of C-shaped cartilage rings, and also promoting the vascularization and functional epithelial regeneration of the tracheal wall. Injecting and filling the cartilage composite microparticles into the hollow cavity of the nested implant can achieve the rapid filling of cells and adult tissue complexes in the scaffold, providing mechanical support and cell source for the structural reconstruction and functional recovery of the trachea.

[0079] The preparation method of the present invention innovatively combines 3D printing technology and melt electrospinning writing technology. 3D printing technology is used to construct the outer C-shaped hollow elastic scaffold, which can precisely control the shape and structure of the scaffold, achieve personalized customization to adapt to the anatomical structure differences of different patients. And the melt electrospinning writing technology is used to prepare the inner micro-nano structure electrospun mesh, which can precisely control the fiber diameter and pore structure, providing a good interface for cell adhesion and rich vesicle matrix growth.

[0080] Through the above preparation method, a multi-layer biomimetic tracheal repair implant is obtained. The implant includes an electrospun mesh and a C-shaped elastic hollow scaffold, and the electrospun mesh is attached to the inner side of the C-shaped elastic hollow scaffold. The electrospun mesh is inoculated with a composite cell composed of primary chondrocytes and human gingival mesenchymal stem cells, and the cartilage composite microparticles composed of autologous cartilage microparticles and allogeneic adipose mesenchymal stem cells are injected into the hollow pipe of the C-shaped elastic hollow scaffold. The C-shaped elastic hollow scaffold includes a plurality of C-shaped hollow ring bodies, which are arranged in parallel and have axially corresponding notches on one side. The circumferential ends of the plurality of C-shaped hollow ring bodies are connected and closed into one body through axially hollow line bodies.

[0081] The above structure is a biomimetic structure that mimics the natural trachea, which can better simulate the natural mechanical properties and physiological functions of the natural trachea, providing stable mechanical support for the structural reconstruction of the trachea. This design takes into account the multi-tissue structure of the trachea, including vascularized fascia, cartilage rings, and functional epithelium.

[0082] In addition, the above structure is prepared for use, and the cartilage composite microparticles for injection can be prepared immediately during the operation to achieve in-situ transplantation. This immediate construction method can handle the situation where acute tracheal injuries require transplantation and repair, and the digital printing technology ensures the precise matching of the transplanted body with the tracheal defect site of the patient.

[0083] The multi-layered biomimetic tracheal repair and transplantation body provided by the present invention can be used for tracheal repair and reconstruction, and a tracheal patch is prepared to repair local tracheal defects. Compared with the previous tracheal fistula defect patches mainly composed of membranes, the multi-layered structure of the present invention has better mechanical properties and can effectively avoid the problem of tracheal collapse.

[0084] Specifically:

[0085] (1) Dissect the trachea in the neck of New Zealand rabbits to expose it, and construct segmental (1.5 cm) or local fenestrated (7 mm × 1.0 cm) defects to verify the tracheal reconstruction effect of the multi-layered biomimetic tracheal repair and transplantation body.

[0086] (2) After hemostasis, perform "end-to-end" suture for segmental defects, and cover the fenestrated defect with a patch and suture it in place for local defects. Administer intramuscular injection of 100,000 units / day of penicillin for one week after the operation.

[0087] (3) Sacrifice by intravenous injection of air after 6W, take samples to observe histological results, and evaluate cartilage survival, vascularization level, and epithelial reconstruction effect.

[0088] Figure 10 (b) shows the "end-to-end" anastomosis effect of the transplanted body, indicating that the repaired tracheal structure is intact, without collapse or deformation, suggesting that the C-shaped elastic hollow scaffold (polycaprolactone material) can withstand physiological pressure after being wrapped by chondrocyte matrix, completing the decellularization process, and injecting cartilage microparticles, and its mechanical properties are close to those of the autologous trachea.

[0089] Figure 12 (a) The gross view of the fiber bronchoscope shows that the inner wall of the repaired airway is smooth, without granulation hyperplasia or scar contraction, indicating that epithelialization effectively avoids postoperative stenosis. Figure 12 (b) and (c) are gross views before and after sampling at 6 weeks after the operation, showing that the repaired site is tightly combined with the surrounding tissues, without mechanical fracture or mechanical failure caused by scaffold degradation. Figure 12 (d) Further quantitatively compare the compressive strength of the nested transplanted body and the autologous trachea to verify that its mechanical properties are superior to those of natural tissues after transplantation and remodeling, and can effectively resist neck movement and muscle compression of the surrounding tissues.

[0090] Figure 13 (a) (HE staining) shows that the cartilage microparticles in the pipeline are tightly integrated, and the lacuna structure is clear. There is no significant infiltration of inflammatory cells around the scaffold, and the interface between the newly formed tissue and the scaffold is well fused. At the same time, a large number of scattered new blood vessels can be seen inside and outside the hollow pipeline.Figure 13 (b) Further quantitatively compared the number of capillaries per unit area in the stratified structure graft (nested type) and autologous tracheal tissue. There was no statistical difference between the two, demonstrating excellent biocompatibility of the polycaprolactone material, which did not hinder transwall angiogenesis and could effectively nourish the cartilage microparticles in the conduit. Figure 13 (c) Quantitatively compared the glycosaminoglycan content in the stratified structure graft (nested type) and autologous trachea. The experimental group was much higher than the autologous control (p < 0.01), indicating the efficient integration and survival of the C-shaped rings of cartilage microparticles.

[0091] Figure 14 (a) DAPI fluorescence staining showed that the cell nuclei were evenly distributed and had a high density inside the graft, indicating that the vesicle-rich matrix wrapping the electrospun mesh was infiltrated by host cells and tissues during the remodeling process, forming vascularized fascia, which could effectively nourish the C-shaped rings of cartilage microparticles and resist airway contamination. Figure 14 (b) Keratin (CK) immunofluorescence staining showed that a continuous epithelial layer was formed in the repair area.

[0092] The present invention has the following technical advantages:

[0093] ① Improving the balance between biocompatibility and mechanical properties:

[0094] Existing tracheal repair materials are difficult to achieve an ideal balance between biocompatibility and mechanical properties. For example, some natural materials such as acellular matrix have good biocompatibility but poor mechanical properties and are difficult to withstand the normal physiological pressure of the trachea; while some synthetic materials such as polytetrafluoroethylene have good mechanical properties but poor biocompatibility and are prone to foreign body inflammatory reactions.

[0095] The present invention uses polycaprolactone material, combines 3D printing technology and melt electrospinning technology to prepare a stratified bio-inspired tracheal repair graft with excellent biocompatibility and mechanical properties. The outer C-shaped elastic hollow scaffold is made of polycaprolactone material, which has good mechanical properties and can withstand the normal physiological pressure of the trachea after filling with cartilage composite microparticles. The inner micro-nano structured electrospun mesh is also made of polycaprolactone material, which has good tissue permeability and biocompatibility, facilitating cell adhesion and the growth of extracellular matrix wrapping, thus achieving the balance between biocompatibility and mechanical properties, and no obvious material-induced inflammation occurs after transplantation.

[0096] ② Promoting the long-term survival and functional recovery of cells:

[0097] During tracheal repair, the long-term survival and function of cells are crucial for the restoration of tracheal function. Existing cell culture and transplantation techniques are difficult to ensure the long-term survival and function of cells in vivo. For example, chondrocytes may undergo dedifferentiation during in vitro culture, resulting in the loss of their function; the transplantation of epithelial cells is difficult to stably proliferate in a non-damaged airway environment. In short, due to reasons such as changes in the microenvironment and insufficient blood supply, the survival rate and function of cells are greatly affected.

[0098] Vascularization and epithelialization of the trachea are important links in achieving tracheal function reconstruction. However, existing technical means are not effective in promoting tracheal vascularization and epithelialization. For example, pre-vascularization techniques such as embedding tracheal scaffolds under the sternocleidomastoid muscle, greater omentum, or subcutaneous forearm to promote neovascularization have made some progress, but the surgical cycle is long and the long-term effects are not yet clear; in terms of epithelialization, although some studies have attempted to promote the epithelialization of tracheal substitutes by implanting airway basal cells, epithelial cells, or using cell membrane sheet technology, these methods have problems such as difficult material acquisition, difficult in vitro expansion, and easy senescence and apoptosis of transplanted cells.

[0099] The present invention uses an injectable cartilage microparticle technology to combine chondrocytes with a scaffold to achieve the intraoperative immediate construction and transplantation of C-shaped cartilage rings, providing mechanical support for tracheal transplants and avoiding the defect of traditional tissue engineering techniques that require long-term cell culture for chondrocyte implantation. At the same time, combined with a matrix-bound vesicle 3D culture technology, it loads gingival mesenchymal stem cell vesicles (GMSCs-EVs), and these vesicles have functions such as anti-inflammatory and promoting epithelial differentiation of cells, and can effectively combat inflammation, promote the differentiation of tracheal basal cells into ciliated columnar epithelium, and improve the cell survival environment. Experimental results show that this transplant can achieve the maturation of cartilage rings, rapid vascularization, fascia wrapping, and epithelial-like cell regeneration at the tracheal defect site. In addition, the construction of a rich vesicle matrix layer provides a stable interface environment for the functional regeneration of the airway mucosa, helps the regeneration of the airway ciliated epithelium, improves the effects of vascularization and epithelialization, thereby improving the level of tracheal function recovery, and enabling the repaired trachea to better restore its normal physiological functions such as breathing, secretion, and foreign body clearance.

[0100] ③Enhance individual adaptability:

[0101] Existing tracheal repair techniques have deficiencies in individual adaptability. Due to the great differences in the anatomical structures and tracheal defect conditions of patients, and the relatively fixed structures of existing tracheal repair materials and scaffolds, it is difficult to fully adapt to individual anatomical structure differences, resulting in poor repair effects. For example, some prefabricated tracheal scaffolds may not match the tracheal defect site of the patient after implantation, affecting the repair effect.

[0102] The present invention adopts digital printing technology, which can ensure the precise matching of the graft with the tracheal defect site of the patient. This method can better adapt to the individual anatomical structure differences, customize a tracheal repair graft that meets its specific needs, improve the success rate of the operation and the repair effect, and reduce the risk of postoperative complications.

[0103] ④ Promote clinical translation:

[0104] Although some progress has been made in laboratory research, the existing tracheal repair technologies face many difficulties in the process of clinical translation. For example, some technologies show good effects in animal experiments, but due to mechanical properties, their application in large animals still requires a large number of clinical trials to verify their safety and effectiveness; in addition, the preparation process of tracheal repair grafts is complex and the process of tissue engineering cell culture is long, which also limits their wide application in clinics.

[0105] Through the successful application verification in a variety of tracheal defect models, the research and development results of the present invention will provide a new and effective solution for clinical tracheal reconstruction, promote the development of tracheal repair technologies, emphasize the immediate intraoperative production of tracheal grafts and achieve in-situ transplantation, simplify the surgical process, reduce the operation time and complexity, quickly respond to clinical needs, and are especially suitable for the situation of acute tracheal injury that requires transplantation repair, having the potential to be translated into clinical applications.

[0106] The above uses specific examples to elaborate on the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the technical field to which the present invention pertains, based on the idea of the present invention, several simple deductions, deformations or substitutions can also be made.

Claims

1. Preparation method of a multi-layer biomimetic tracheal repair graft, characterized in that: The preparation method includes: Printing an electrospun mesh using polycaprolactone particles; Printing a planar sugar scaffold using a plasticizable caramel raw material and curling it to form a C-shaped caramel scaffold; Preparing a polycaprolactone coating solution and coating it on the C-shaped caramel scaffold, attaching the electrospun mesh to the inner side of the C-shaped caramel scaffold, and immersing it in distilled water to dissolve the C-shaped caramel scaffold, thereby forming a C-shaped elastic hollow scaffold, and obtaining a nested multi-layer biomimetic tracheal scaffold; Mixing primary chondrocytes and human gingival mesenchymal stem cells, and inoculating them on the electrospun mesh of the nested multi-layer biomimetic tracheal scaffold for culture; Performing decellularization treatment on the nested multi-layer biomimetic tracheal scaffold; Mixing autologous cartilage microparticles and allogeneic adipose mesenchymal stem cells, and injecting them into the hollow pipe of the C-shaped elastic hollow scaffold to obtain a multi-layer biomimetic tracheal repair graft.

2. The preparation method of the multi-layer biomimetic tracheal repair graft according to claim 1, characterized in that: The electrospun mesh is printed by a MEW device and includes a multi-layer interconnected grid structure.

3. The preparation method of the multi-layer biomimetic tracheal repair graft according to claim 2, characterized in that: The planar sugar scaffold is printed by a 3D printer, and the obtained C-shaped caramel scaffold after curling includes multiple C-shaped ring bodies.

4. The preparation method of the multi-layer biomimetic tracheal repair graft according to claim 3, characterized in that: The preparation process of the polycaprolactone coating solution is: Dissolving polycaprolactone particles in hexafluoroisopropanol and adding sodium chloride particles as a pore-forming agent to obtain a coating solution.

5. The preparation method of the multi-layer biomimetic tracheal repair graft according to claim 4, characterized in that: After attaching the electrospun mesh to the inner side of the C-shaped caramel scaffold, let it stand until the hexafluoroisopropanol volatilizes and dries, and then immerse the nested multi-layer biomimetic tracheal scaffold in distilled water to dissolve the C-shaped caramel scaffold and sodium chloride particles.

6. The preparation method of the multi-layer biomimetic tracheal repair graft according to claim 5, characterized in that: Before inoculating primary chondrocytes and human gingival mesenchymal stem cells on the electrospun mesh of the nested multi-layer biomimetic tracheal scaffold, put the nested multi-layer biomimetic tracheal scaffold outside a silicone tube, place it in alcohol, and irradiate it with ultraviolet light for disinfection.

7. The preparation method of the multi-layer biomimetic tracheal repair graft according to claim 6, characterized in that: After inoculating primary chondrocytes and human gingival mesenchymal stem cells on the electrospun mesh of the nested multi-layer biomimetic tracheal scaffold, place the nested multi-layer biomimetic tracheal scaffold in a high-glucose medium for culture; The formula of the high-glucose medium is: 15% fetal bovine serum, 1% triple antibody, 1% glutamine, 1% Vc, and the balance is distilled water.

8. The preparation method of the multi-layer biomimetic tracheal repair graft according to claim 7, characterized in that: The decellularization treatment of the nested multi-layer biomimetic tracheal scaffold includes: Immersing the nested multi-layer biomimetic tracheal scaffold in an SDS solution and then placing it in a Triton solution.

9. The multi-layer biomimetic tracheal repair graft obtained by the preparation method according to claim 1, characterized in that: The multi-layer biomimetic tracheal repair and transplantation body includes an electrospun mesh and a C-shaped elastic hollow stent, and the electrospun mesh is attached to the inner side of the C-shaped elastic hollow stent; The electrospun mesh is inoculated with composite cells composed of primary chondrocytes and human gingival mesenchymal stem cells; Cartilage composite microparticles composed of autologous cartilage microparticles and allogeneic adipose mesenchymal stem cells are injected into the hollow pipe of the C-shaped elastic hollow stent.

10. The multi-layer biomimetic tracheal repair and transplantation body according to claim 9, wherein: The C-shaped elastic hollow stent includes a plurality of C-shaped hollow ring bodies, which are arranged in parallel and have axially corresponding notches on one side. The circumferential ends of the plurality of C-shaped hollow ring bodies are connected and closed as a whole through axially hollow line bodies.

Images