Hydrogel bio-ink, preparation method thereof, 3D printing biodegradable airway stent prepared from hydrogel bio-ink and application of hydrogel bio-ink

Abstract

The invention discloses hydrogel bio-ink, a preparation method of the hydrogel bio-ink, a 3D printing biodegradable airway stent and application of the 3D printing biodegradable airway stent. The preparation method comprises the following steps that S1, lung tissue is subjected to freeze thawing, slicing, sequential decellularization of SDS and TritonX-100, air drying, smashing and cryopreservation, and a lung extracellular matrix is obtained; s2, performing sterilization and pepsin treatment on the lung extracellular matrix prepared in S1 to obtain a pretreated lung extracellular matrix; and S3, mixing methyl propionylated gelatin and an alginate solution, pouring the mixture into solid gel, gelatinizing the solid gel, and adding the pretreated lung extracellular matrix to prepare the hydrogel bio-ink. The prepared pig lung acellular extracellular matrix does not have an obvious nuclear structure, and DNA (Deoxyribonucleic Acid) determination shows that the concentration of an acellular group is obviously reduced (P is less than 0.01); along with the increase of the dECM concentration, the porosity is reduced, and the degradation time is prolonged; the airway stent is printed in a 3D biological printing mode, no obvious cytotoxicity (P is larger than 0.05) exists in a cytotoxicity experiment, and the mechanical property part of the airway stent is similar to that of a silicone stent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of hydrogel bio-ink, in particular to a hydrogel bio-ink, a preparation method thereof, and a 3D-printed biodegradable airway stent prepared therefrom and application thereof. BACKGROUND

[0002] Airway stenosis is caused by different causes, and the treatment methods mainly include respiratory intervention (such as stent placement) and surgery. In the past 20 years, with the continuous progress of materials science and the popularization of soft bronchoscopy in clinical practice, bronchoscopic stent placement as a minimally invasive treatment for airway stenosis has been widely used. In the long-term clinical application process, we found that most of the existing airway stents have certain problems, and even some patients have severe complications, which affect the prognosis and treatment effect. Therefore, a more ideal airway stent is needed, which can reduce the incidence of related complications as much as possible while maintaining the existing support.

[0003] Using 3D-printed customized patient-specific airway stents should significantly reduce stent migration, granulation tissue proliferation and other related problems. The introduction of 3D bioprinting technology is a major breakthrough in 3D printing in recent years, which uses biological ink as a bridge to use excellent biological materials as a good candidate for biological ink, and to realize the visualization of the stent by using 3D technology. Decellularized extracellular matrix is an important natural biological material at present, which has excellent biocompatibility. The decellularized extracellular matrix not only has high biocompatibility and low immunogenicity, but also has cell adhesion surface receptors.

[0004] A bone repair hydrogel stent and a preparation method thereof are disclosed in Chinese Patent No. CN117298337A. The bone repair hydrogel stent comprises the following components at the following concentrations: 10wt%-20wt% methacrylated gelatin, 10wt%-20wt% methacrylated sodium alginate, 100-200mM calcium-containing crosslinking agent, and 1-5wt% glutamine transaminase. In this scheme, the strength of the hydrogel is improved by triple crosslinking, and the crosslinking method is mild, effectively improving the strength of the hydrogel stent. SUMMARY

[0005] The present application aims to provide a hydrogel bio-ink, a preparation method thereof, a 3D-printed biodegradable airway stent prepared therefrom and application thereof. The 3D-printed biodegradable airway stent prepared from the hydrogel bio-ink has better anti-migration ability and biocompatibility.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] A preparation method of a hydrogel bio-ink, comprising the following steps:

[0008] S1, freeze-thaw, slice, SDS and Triton X-100 sequential cell removal, air-drying, crushing, and freezing of lung tissue to obtain lung extracellular matrix;

[0009] S2, sterilizing and pepsin treating the lung extracellular matrix prepared in S1 to obtain a lung extracellular matrix after pretreatment;

[0010] S3, mixing methyl methacrylate gelatin and alginate solution, perfusing into a solid gel, gelling, and adding the lung extracellular matrix after pretreatment to prepare a hydrogel bio-ink.

[0011] Airway stent implantation is one of the important interventional treatments for central airway stenosis of benign and malignant tumors. However, the current airway stent has limitations such as low biocompatibility, difficulty in removal, and long-term placement in the body leading to granulation tissue proliferation, so it is necessary to explore a new airway stent. The present application uses lung acellular extracellular matrix, methyl methacrylate gelatin (Gelatin Methacryloyl, GelMA) and sodium alginate (Alginate, ALG) to prepare a mixed bio-ink with high biocompatibility, and uses 3D bioprinting to prepare a new biodegradable airway stent, and studies the related properties of the stent.

[0012] According to the embodiments of the present application, the present application can be further optimized, and the following is the technical scheme formed after optimization:

[0013] In S3, the volume ratio of the mixture of methyl methacrylate gelatin and alginate solution is (1-2):(1-2).

[0014] In a preferred embodiment of the present application, in S3, the mass concentration of the methyl methacrylate gelatin is 8-12%, the mass concentration of the alginate solution is 1-3%, and the concentration of the lung extracellular matrix is 3-5%; preferably, the mass concentration of the methyl methacrylate gelatin is 8-10%, the mass concentration of the alginate solution is 2-3%, and the concentration of the lung extracellular matrix is 3-4%.

[0015] The stent prepared in this ratio range has better viscoelasticity, hardness and mechanical properties.

[0016] In a preferred embodiment of the present application, in S1, the sequential cell removal steps of SDS and Triton X-100 specifically include soaking in 1-3% SDS for 12-24 hours, washing with deionized water, soaking in 1-3% Triton X-100 for 12-24 hours, washing with deionized water and soaking for 12-24 hours.

[0017] In a preferred embodiment of the present application, in S2, the pepsin treatment comprises adding the lung extracellular matrix and pepsin in an acetic acid solution, and soaking, preferably, the concentration of the acetic acid solution is 0.2-1.5 mol / L, the volume ratio of the dECM and pepsin is (4-6):1, and the soaking time is 2-4 days.

[0018] In a preferred embodiment of the present application, in S2, the pretreatment in S2 further comprises neutralization, osmotic pressure adjustment, centrifugation, freeze-drying, and preservation, and the neutralization step is arranged after the pepsin treatment.

[0019] In a preferred embodiment of the present application, in S1, pressure is applied to the lung tissue during the sequential SDS and Triton X-100 cell removal steps, preferably, a pressure pot is used to apply pressure to the lung tissue.

[0020] In a preferred embodiment of the present application, in S3, the gelation is performed by irradiation with a 400-500 nm light source for 20-40 seconds.

[0021] The present application also discloses a hydrogel bio-ink prepared according to the preparation method.

[0022] The present application also discloses a 3D-printed biodegradable airway stent prepared using the hydrogel bio-ink, characterized in that the 3D-printed biodegradable airway stent has a hardness of 50-80 HA, a Young's modulus of 0.1-0.4 MPa, a modulus less than 1 MPa, and no cytotoxicity; preferably, the 3D-printed biodegradable airway stent has a hardness of 55-70 HA and a Young's modulus of 0.2-0.3 MPa.

[0023] The airway stent prepared by the present application has a hardness of 50-80 HA, while the hardness of a silicone airway stent material is 60-80 A, which is close to that of a silicone stent. The Young's modulus of the stent is 0.1-0.4 MPa, which is easy to deform, and is expected to be placed by a bronchoscope. The modulus of a human trachea reported in previous studies is in the range of 1-20 MPa, and the modulus in the present application is less than 1 MPa, so the stent does not exhibit mechanical properties incompatible with natural tissues. (The stent performance cannot be implanted in vivo, and some technical problems remain to be solved)

[0024] The present application also discloses a use of the hydrogel bio-ink in a 3D-printed biodegradable airway stent.

[0025] At present, dECM (decellularized Extracellular Matrix) has been studied as one of the components of bio-ink, but most of them are the construction of organoids, the establishment of in vitro disease models, etc. For airway diseases, most of them focus on the replacement of tissue-engineered trachea, and there is no related research in the field of airway stents.

[0026] Preferably, the sterilization step in S2 is to soak the dECM with hydrogen peroxide for 4-6 hours, and irradiate it with ultraviolet light for more than 6 hours.

[0027] Preferably, in S2, the neutralization step is to adjust the pH value of the dECM solution to between 7.3-7.4 with 1-3 mol / L alkali solution.

[0028] Preferably, in S2, the osmotic pressure adjustment step is to mix the dECM solution with 10X PBS at a volume ratio of 8-12:1 to balance the osmotic pressure.

[0029] Preferably, in S2, the centrifugation step filters the undigested part; the storage temperature is 2-6℃.

[0030] Preferably, in S1, the freezing and thawing temperature is -90 to -70℃, and the freezing and thawing time is 12-36 hours.

[0031] Preferably, in S1, the thickness of the lung tissue section is 1mm-2mm.

[0032] Preferably, in S1, the temperature of air drying is 30-40℃.

[0033] Preferably, in S1, the freezing temperature is -90 to -70℃.

[0034] Preferably, in S1, the lung tissue needs to remove the residual impurities and blood on the surface of the lung cells before freezing and thawing.

[0035] In the early stage of the application, perfusion combined with SDS (Sodium dodecyl sulfate) + TritonX-100 (polyethylene glycol octylphenyl ether) was used to successfully prepare lung decellularized extracellular matrix, which confirmed that it had little effect on the key proteins of lung extracellular matrix and the ultrastructure of the tissue.

[0036] The main role of airway stents is to restore and maintain airway patency. For patients with benign central airway stenosis, if the airway lumen is reduced by more than 50% due to any cause and related clinical symptoms occur, and other treatment methods are ineffective, airway stents should be considered. After stent placement, special attention should be paid to long-term complications (e.g., granulation tissue proliferation, microbial colonization, stent migration and rupture) and difficult removal. With the continuous development of bioengineering, biodegradable and personalized 3D printing may provide a new way to address the issues of re-extraction and stent migration. Emerging 3D bioprinting technology is a powerful tissue engineering technology with advantages such as controllable structure design and high utilization rate of biological materials. With the correct formulation and printing parameter settings, the key step of bio-ink can reproduce three-dimensional structures. In addition to meeting the basic biocompatibility characteristics, ideal bio-ink also needs to have printability, structural stability and other related characteristics to avoid structural collapse during printing. These requirements will strictly limit the selection of bio-ink materials, chemical reactions, and processing parameters. The initial inspiration for bio-ink design comes from ECM, and people hope to create favorable conditions for cell proliferation and growth by simulating the extracellular matrix of the target tissue. The main advantage of ECM is that it can maintain the biochemical and topological microenvironment of natural tissues and promote tissue remodeling and regulate cell function. Currently, it is difficult to be completely replaced, and most of the natural compounds or synthetic materials have limited interaction with cells, making it difficult for cells to adhere.

[0037] The dECM-based bio-ink printing method is a personalized treatment measure specific to tissues and organs. Therefore, despite the many limitations in the application process, 3D bioprinting based on dECM bio-ink is still a popular research topic, whether it is the innovation of bio-ink or the novel manufacturing method. For lung dECM, current research is mostly focused on the construction of disease models, organ chip research, cell culture, and organoid construction. In the field of airway stents, tissue engineering trachea is mainly constructed by using cartilage decellularized extracellular matrix, collagen and other materials, or the trachea is directly decellularized as a substitute for tissue engineering trachea. However, the lung extracellular matrix, which accounts for 50% of the non-alveolar components of the entire lung tissue, has no related research in airway stents. The pig lung in this invention is decellularized as a bio-ink material. The pig lung has a highly similar anatomical structure to humans and is widely available. Through 3D printing technology, a new type of airway stent with high biocompatibility and biodegradability is achieved.

[0038] The present application takes the freeze-thaw method as the first step of decellularization, but the effect is to be improved when used alone, therefore the present application takes the method of sequential freeze-thaw, SDS and TritonX-100 for decellularization, and the sequence of TritonX-100 at the end can help to remove the residual SDS in the tissue. At present, for large organs or organs with vascular system, perfusion method is mostly used for decellularization. The present application is also for lung, an organ with vascular system, but the ultimate goal is to make it one of the formulas of bio-ink, only the basic components of ECM need to be kept, not the advanced structure. The slice immersion method can make the tissue have a larger surface area, and may have a faster and higher yield decellularization process.

[0039] In addition, the lung tissue is different from other organs, as a gas-containing tissue, it often floats on the surface of the liquid. The present application creatively uses a pressure kettle to ensure that the lung tissue is always inside the liquid by applying a certain pressure, which is conducive to further improving the efficiency of decellularization.

[0040] In summary, the present application takes the slice immersion method combined with freeze-thaw and SDS+TritonX-100 for decellularization, from the gross appearance of the extracellular matrix, it can be clearly seen that after sequential immersion of reagents, the lung tissue changes from pale to translucent, and even the fine structure can be clearly seen. The present application proves by HE staining and DNA determination that most of the nucleated cells are successfully removed, effectively avoiding the related immune system response caused by the xenogeneic biological material, and providing conditions for the subsequent preparation of bio-ink material. In addition, the decellularized extracellular matrix produced by the present application has the advantages of high efficiency, convenience and yield, which is conducive to the realization of future mass production.

[0041] In the process of 3D bioprinting of bio-ink, the cross-linking between different polymers is also an important part. Cross-linking refers to the process of polymer changing from liquid to solid, and the density and mode of cross-linking determine the mechanical properties and physical properties of the final three-dimensional structure to some extent. The natural lung tissue-derived dECM cannot be directly used for bioprinting, and needs to be prepared into a pre-gel solution or powder for subsequent mixing with other materials after being digested by pepsin. Although the dECM powder prepared by pepsin pretreatment of human dECM is convenient for mixing, its mechanical properties will also be lost to some extent. And most studies show that the dECM gelation time is long and the cross-linking speed is slow. Therefore, it is difficult to construct specific shapes with single dECM in the printing process, and adopting multi-component bio-ink is a good strategy, which balances the advantages and disadvantages of different materials to configure a bio-ink that meets the requirements of printing and mechanics. At present, the materials commonly used for combination are mostly hydrogel materials, which mainly use physical or chemical methods for cross-linking to ensure their insolubility in water and provide a water-containing environment. According to the source, it can be divided into natural hydrogel and synthetic hydrogel. Collagen, gelatin and other natural substances have excellent biocompatibility and biodegradability, but most of them have poor mechanical properties, which limits their wide use. In contrast, synthetic hydrogels such as polyethylene glycol derivatives and polycaprolactone have high mechanical properties but poor biocompatibility, and often cause fibrous encapsulation after implantation. Natural hydrogels have been studied to improve or modify their shortcomings, for example: gelatin is modified by methacrylate to form GelMA to achieve light-induced covalent cross-linking. Recently, De Santis et al. found that the mixture of ECM solution and sodium alginate can accelerate cross-linking with the participation of calcium ions, and also realize the shear-thinning behavior, which is beneficial to the later extrusion 3D printing. Moreover, GelMA and sodium alginate are high-viscosity materials, and the mixed bio-ink can ensure stability and printability. At the same time, the covalent cross-linking of GelMA and the ionic cross-linking of sodium alginate can form an interpenetrating polymer network (IPN), which is beneficial to enhancing the mechanical properties of the gel. Therefore, considering the cross-linking speed, viscoelasticity and mechanical properties, the present invention selects these three materials as the components of the mixed bio-ink. In summary, the present invention converts the freeze-dried, crushed and pepsin-digested porcine lung extracellular matrix into a hydrogel precursor solution, and configures a GelMA / ALG / dECM bio-ink. Through mold pouring method and light curing combined with ionic cross-linking, the shaped hydrogel is successfully prepared. Through different concentration ratio experiments, the optimal ratio of GelMA and ALG is determined. It is found that the combination of 10% GelMA and 2% ALG has better viscoelasticity and hardness.The present application realizes the configuration of degradable bio-ink with excellent biocompatibility by providing receptors for cell adhesion through the addition of lung dECM. Both GelMA and ALG are porous materials. When we observed the microstructure of the mixed hydrogel, we found that the pore size decreased with the increase of dECM concentration, which is consistent with the findings of Agrawal P. This feature facilitates the determination of the dECM concentration later, enabling the scaffold to load cells and achieve its functionalization. In addition, the materials used to prepare the scaffold in the present application are biodegradable materials, and their degradation rate is generally related to the concentration of the bio-ink, the cross-linking mechanism, and the in vivo and in vitro environment. Through in vitro degradation experiments of mixed hydrogels with different proportions, we found that the mixed hydrogel prolongs the degradation time with the increase of dECM concentration. Its degradation products will eventually completely dissolve in complete medium, and the gel remains soft during degradation, which also indicates that the degradation products will not cause damage to the airway wall. As for the degradation time, the 3% dECM and 4% dECM groups all have a longer degradation time compared to the group without dECM. However, the uncontrollable degradation rate and the change in degradation mechanics are difficult problems that need to be solved in the future. The mixed bio-ink of the present application has high viscosity and shear thinning ability, and is especially suitable for extrusion type 3D printing, which is a simple and low-cost method. Through the pressure system, the bio-ink is extruded and deposited layer by layer to construct the structure we need, while being supplemented by light curing. Although its precision needs to be improved, it is sufficient for the preliminary design of the scaffold in the present application.

[0042] The appearance of the scaffold printed by the present application is similar to that of the silicone scaffold on the market. The evaluation of its biocompatibility has confirmed that the material leachate has no toxicity to cells. Combined with relevant mechanical experiments, we found that the new airway scaffold of 3% dECM / 10% GelMA / 2% ALG has a hardness of 56.5 HA, while the hardness of the silicone airway scaffold material is in the range of 60-80 A, which is close to that of the silicone scaffold. The Young's modulus of this scaffold is 0.298 MPa, which is easy to deform and can be placed by bronchoscopy. The modulus of human trachea reported in previous studies is in the range of 1-20 MPa, and the modulus in the present application is less than 1 MPa, so it does not exhibit mechanical properties that are not compatible with natural tissues. The dECM concentration is now about 3%-5% low concentration, because one of the main components of dECM, collagen, has a triple helix structure and intermolecular forces, which may change the cross-linking of the mixed hydrogel, so high dECM concentration will affect the mechanical properties of the mixed hydrogel. Poor mechanical properties are a common feature of scaffolds made of natural materials. In the future, we can consider further improving the material ratio or combination to further optimize it.

[0043] The application successfully prepares lung acellular extracellular matrix by using slice immersion method combined with physical and chemical methods, and innovatively uses lung acellular extracellular matrix, GelMA and alginate to prepare a mixed biological ink, and successfully prints a new type of biodegradable airway stent by using 3D biological printing technology. After biological and mechanical performance evaluation, the airway stent prepared by the application has good elasticity and toughness, and has no cytotoxicity and can be self-degraded within a certain period of time.

[0044] Conclusion:

[0045] 1. The application successfully prepares a new type of dECM / GelMA / ALG mixed hydrogel biological ink.

[0046] 2. The application uses the biological ink to successfully develop a new type of airway stent with high biocompatibility and good mechanical properties by using 3D biological printing technology.

[0047] Compared with the prior art, the application has the beneficial effects that:

[0048] The application successfully prepares a new type of dECM / GelMA / ALG mixed hydrogel biological ink, and uses the biological ink to successfully develop a new type of airway stent with high biocompatibility and good mechanical properties by using 3D biological printing technology. The stent is prepared by using mixed biological ink with excellent biocompatibility, lung acellular extracellular matrix, Gelatin Methacryloyl (GelMA) and sodium alginate (Alginate, ALG), and is formed by 3D biological printing technology.

[0049] Since the application uses lung acellular extracellular matrix-based mixed biological ink as a new type of biological ink, the 3D printed airway stent prepared by the application has better anti-migration ability and biocompatibility, and provides a new idea for the development of new airway stents. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is a schematic diagram of the change of the decellularized lung tissue in an embodiment of the application;

[0051] Figure 2 is a schematic diagram of the slice kettle immersion method in an embodiment of the application;

[0052] Figure 3 is a comparison diagram of HE staining and DNA content in the control group and the decellularized group in an embodiment of the application;

[0053] Figure 4 is a schematic diagram of the preparation of dECM freeze-dried powder in an embodiment of the application;

[0054] Figure 5is a schematic diagram of GelMA / ALG mixed bio-ink gel in an embodiment of the present application;

[0055] Figure 6 is the appearance and fluidity of GelMA / ALG / dECM hydrogel at room temperature at different concentrations;

[0056] Figure 7 is the scanning electron microscope and porosity of GelMA / ALG / dECM hydrogel at different concentrations;

[0057] Figure 8 is a schematic diagram of the in vitro swelling and degradation curves of each group of gels;

[0058] Figure 9 is the 3D printing process of dECM / GelMA / ALG hydrogel airway stent and the macrostructure of the stent;

[0059] Figure 10 is a schematic diagram of the extrusion experiment of dECM / GelMA / ALG hydrogel airway stent;

[0060] Figure 11 is the mechanical property detection of dECM / GelMA / ALG hydrogel airway stent;

[0061] Figure 12 is the cytotoxicity experiment of each group of stents.

[0062] Figure 13 is the general morphological structure of the stent prepared in the embodiment.

[0063] Figure 14 is the healing of the neck wound of the rabbit after the operation;

[0064] Figure 15 is the fistula condition of the rabbits in different groups;

[0065] Figure 16 is the general specimen of the trachea and the effect diagram of the stent filling the fistula after 2 weeks;

[0066] Figure 17 is the HE staining of the stent after 1 week of treatment. DETAILED DESCRIPTION

[0067] The present application will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0068] 1 Research materials

[0069] Pig lungs were purchased from the local market.

[0070] Human normal bronchial epithelial cells (Beas-2B) were preserved by the Central Laboratory of Xiangya Third Hospital, Central South University.

[0071] 2 Experimental reagents, instruments

[0072] The main experimental instruments in the experiment are shown in Table 2-1; the main reagents used in the experiment are shown in Table 2-2.

[0073] Table 2-1 Main materials and experimental instruments

[0074]

[0075]

[0076] Table 2-2 Main experimental reagents

[0077]

[0078]

[0079] Table 3 English abbreviation explanation

[0080]

[0081]

[0082] 3 Preparation and identification of lung acellular extracellular matrix

[0083] 3.1 Decellularization by soaking method

[0084] The present application uses freeze-thaw, SDS+TritonX-100 sequential soaking method to prepare pig lung acellular extracellular matrix. It includes the following steps:

[0085] Through the complete trachea of pig lung, tap water is perfused to remove surface impurities and try to flush residual blood. -80℃ for 24 hours. Remove the thicker tracheal part, and cut the remaining lung tissue part with a microtome, control the thickness to be 1mm-2mm. Sequential soaking at room temperature, the soaking order is: 1% SDS soaking for 12 hours - deionized water washing - 1% TritonX-100 soaking for 12 hours - deionized water washing and soaking for 12 hours. After constant temperature drying at 37 degrees Celsius, put it into a crusher for crushing. -80℃ freezing preservation.

[0086] 3.2 Evaluation of pig lung acellular extracellular matrix

[0087] Preparation of lung tissue paraffin section

[0088] Take the appropriate size of fresh lung tissue and acellular lung tissue, soak it in 4% paraformaldehyde, fixed for more than 1 day and perfect the label, then placed in dehydration box. The dehydration box is placed in the basket, then put into the dehydration machine for gradient concentration alcohol dehydration. After dehydration, immerse it in paraffin for the next procedure.

[0089] First, pour the melted paraffin into the embedding mold, then gently clamp the tissue block soaked in wax with a heated curved blunt forceps, mark the embedding surface in time, then bury it in the melted paraffin. Wait for a while, wait for the surface to solidify, then place it in a-20℃ accelerated solidification, after solidification, clamp and carefully remove the excess paraffin around the tissue block with a blade and try to trim it into a regular square. Place the trimmed wax block on the clamping table of the microtome, cut it into about 4μm thick slices, then store it at room temperature for later use. The slices are placed in a warm water bath at about 44℃, spread them on the liquid surface, gently clamp and attach them to the appropriate position on the glass slide, and bake the slices in a 60℃ oven for 2 hours.

[0090] Lung tissue section HE staining

[0091] According to the order, the control and experimental group slides are placed in xylene for 20 minutes, 3 times, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, distilled water for 5 minutes. Place the slides in hematoxylin reagent for about 5 minutes, rinse with tap water, then differentiate, quickly wash with water, and return to blue with PBS. Place the slides in eosin staining solution for 2 minutes. Place the slides in gradient alcohol and xylene again for dehydration, neutral resin mounting; observed under a microscope.

[0092] DNA content, DNA electrophoresis

[0093] According to the manufacturer's instructions, use the universal column type genomic DNA extraction kit to quantify the DNA content of the acellular tissue. DNA extraction includes the following steps:

[0094] Place 25 mg of control and experimental samples in a homogenizer, add 80 μΐ^of Buffer GTL, grind thoroughly and pour into a centrifuge tube, add 100 μΐ^of Buffer GTL. Add 20 μΐ^of Proteinase K. Mix the sample thoroughly using a vortex shaker. Place in a 56°C water bath until the tissue is completely lysed, every so often invert or shake the centrifuge tube to disperse the sample. Add 200 μΐ^of Buffer GL, mix the sample thoroughly using a vortex shaker, place in a 70°C water bath for 10 minutes, after a short centrifugation add 200 μΐ^of absolute ethanol to the centrifuge tube and mix thoroughly using a vortex shaker. Centrifuge briefly and pour the solution obtained into the Spin Columns DM that have been placed in a collection tube. Centrifuge at 12,000 rpm for 1 minute, after discarding the waste solution the Spin Column is reset. Add 500 μΐ^of Buffer GW1 to the Spin Column, centrifuge at 12,000 rpm for 1 minute, after discarding the waste solution the Spin Column is reset. Add 500 μΐ^of Buffer GW2 to the Spin Column, centrifuge at 12,000 rpm for 1 minute, after discarding the waste solution the Spin Column is reset. Then centrifuge at 12,000 rpm for 2 minutes, discard the waste solution. Dry the Spin Column. Place the Spin Column in a new centrifuge tube, add 50-200 μΐ^of sterile water to the middle part, leave at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, the DNA solution obtained is stored at -20°C.

[0095] Enzyme-free water is used to zero the micro-spectrophotometer, 2 μΐ^of the DNA solution is taken and dropped on the test platform, the absorbance at 260 nm and 280 nm is measured respectively, the absorbance in the range of 0.2-1.2 is ideal. The concentration is calculated.

[0096] 4 Preparation of bio-ink

[0097] 4.1 Pretreatment of decellularized extracellular matrix mainly includes:

[0098] The dECM is soaked in hydrogen peroxide for 4-6 hours and irradiated by ultraviolet light for more than 6 hours. Pepsin treatment: the dECM and pepsin are placed in a 0.5 mol / L acetic acid solution (the volume ratio of the two is 5:1), which is soaked at room temperature for 3 days until the dECM is basically dissolved. The pH value of the dECM solution is adjusted to 7.3-7.4 by using 1 mol / L NAOH solution. The dECM solution and 10X PBS are mixed at a volume ratio of 10:1 to balance the osmotic pressure. The undigested part is filtered. Freeze-drying treatment. Store at 4°C.

[0099] 4.2 Preparation of GelMA and photoinitiator

[0100] Preparation of 0.25% (w / v) initiator standard solution: Take 20 ml PBS and add it to a brown bottle containing initiator LAP (0.05 g LAP is contained in the bottle). Dissolve the solution in a 40-50 °C water bath for 15 minutes, with several shakes during the process.

[0101] Preparation of GelMA solution: Take the required amount of GelMA and place it in a centrifuge tube. Take the appropriate amount of initiator standard solution and add it to the centrifuge tube, shake it to ensure full saturation. Dissolve the solution in a 60-70 °C water bath for 20-30 minutes, with several shakes during the process. Keep it in the dark.

[0102] Preparation of alginate (ALG): Take the required amount of alginate and place it in a beaker. Add the alginate to the beaker in a certain proportion with deionized water, heat in a 70 °C constant temperature water bath, and stir while heating. Place it aside to defoam for later use.

[0103] 4.4 Preparation of bio-ink and evaluation of hydrogel properties

[0104] Mix 20% / 10% GelMA solution (w / v, g / ml) and 4% / 2% alginate solution (w / v, g / ml) in a 1:1 (v:v) ratio, pour it into a cylindrical solid gel, and irradiate it with a 405 nm light source for about 30 seconds to gel it. Then apply a 50 g weight for 30 seconds and observe whether it deforms or cracks after the pressure is removed to determine the optimal concentration ratio of GelMA to ALG. Then add 3%, 4%, and 5% dECM freeze-dried powder (w / v, g / ml) to prepare three different dECM / GelMA / ALG bio-ink hydrogels (A, B, and C) and test their properties.

[0105] Microstructure observation of the gel: Place the sample in 2.5% glutaraldehyde for 6-12 hours. Discard the fixing solution and rinse the sample with 0.1 mol / L pH 7.0 phosphate buffer for three times, each time for 15 minutes. Fix the sample with 1% osmium acid solution for 1-2 hours. Discard the previous solution and rinse the sample with 0.1 mol / L pH 7.0 phosphate buffer for three times, each time for 15 minutes. Perform gradient alcohol dehydration treatment: 30% ethanol solution for 15 minutes, 50% ethanol solution for 15 minutes, 70% ethanol solution for 15 minutes, 80% ethanol solution for 15 minutes, 90% ethanol solution for 15 minutes, 95% ethanol solution for 15 minutes, 100% ethanol solution for 20 minutes, and repeat twice. Treat the sample with a mixture of acetic acid and isoamyl acetate for 30 minutes. Discard the mixture from the previous step and treat the sample with pure isoamyl acetate for 1 hour, and perform carbon dioxide critical point drying treatment. Coat the film and observe it under a scanning electron microscope.

[0106] Gel water absorption rate and in vitro degradation rate: after the scaffold is solidified, the filter paper is wiped dry and weighed (W0), then immersed in complete medium, placed in a 37°C constant temperature water bath, and the gel is taken out at different times, the filter paper is wiped dry, weighed again (Wt), and the gel swelling rate and in vitro degradation rate are calculated.

[0107]

[0108] 5 3D printed airway scaffold

[0109] The biomaker system of Beijing Shangpu Biotechnology Co., Ltd. was used to print the scaffold. The preset printing parameters were: nozzle temperature: 21°C, platform temperature: room temperature, printing speed: 5mm 3 / s, starting layer speed: 5mm 3 / s, empty speed: 20mm 3 / s; extrusion speed: 1mm 3 / s; layer height: 0.2mm; blue light was continuously irradiated during printing, and the scaffold was immediately placed in a 2% calcium chloride solution for ion crosslinking after printing, and then washed with PBS for 3 times.

[0110] Scaffold mechanical property evaluation

[0111] Hardness test: the hardness of the material was tested using a pointer type Shore hardness tester. According to GB / T531.1-2008 method.

[0112] Tensile property test: standard mechanical sample was prepared, and universal material testing machine was used to perform GB / T13460-2008 method.

[0113] Scaffold biocompatibility evaluation

[0114] Cell culture: DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-amphotericin B was prepared, and Beas-2B cells were cultured at 37°C and 5% CO2. The culture medium was replaced every 2 days. When the cells covered 90% of the culture bottle, they were digested with 1ml 0.25% trypsin, and then cultured in 3 new culture bottles.

[0115] Cytotoxicity experiment: each scaffold was wiped dry and weighed, immersed in complete medium at a ratio of 0.2g / mL, and placed in a 37°C environment for 24 hours, then placed in a 4°C refrigerator for standby. Take Beas-2B cells, the cell density is 1X10 4 / mL, inoculated in 96-well plates, 100 μL of cell suspension per well, after incubation for 24 hours after adding complete medium to adhere, the medium was aspirated, and 100 μL of scaffold extract was added. CCK-8 detection was performed at the same time period after 24 hours and 48 hours; 10 μL of CCK-8 was added to each well at 10% (V / V), and the cells were further incubated in a 37°C cell incubator for 1 hour. The absorbance at 450 nm was measured by an enzyme-labeled instrument.

[0116]

[0117] 6Statistical methods: The measurement data were expressed as mean ± standard deviation (SD). Independent sample t-test was used to test the data between two groups. P<0.05 indicates that there is a significant difference between groups. All statistical analyses were performed using GraphPad Prism 9.4.1 software.

[0118] 7Results

[0119] Preparation and evaluation of porcine lung acellular extracellular matrix: The sliced porcine lung tissue was decellularized by the sequential soaking method of freeze-thawing + SDS + Triton X-100, and was laid flat on the surface of the reagent solution as much as possible. A certain pressure was applied to it by pressing the pot to make it fully contact with the solution, as shown in Figure 2 .

[0120] Observation of lung acellular tissue gross specimen: Morphological changes: Fresh porcine lung tissue structure is compact and elastic, most of which is suspended on the surface of the soaking solution. During the decellularization process, the tissue thickness becomes thinner than before, the volume becomes slightly larger than before, and part of the matrix can be seen in the trachea.

[0121] Color change: Figure 1 is a schematic diagram of the gross changes of the decellularized lung tissue in one embodiment of the present application, Figure 1 A is a lung without decellularization treatment; B is after 6 hours of SDS soaking; C is after 12 hours of SDS soaking; D is after 6 hours of Triton X-100 soaking; E is after all reagent soaking is completed. After the fresh porcine lung tissue is washed and stored at -80°C, the color becomes pale, the central part becomes milky white after 6 hours of SDS soaking, and the edge part or thinner part becomes translucent Figure 1 -B). The transparent part increased significantly after 12 hours Figure 1 -C), and small bubbles can be seen in the Triton X-100 soaking Figure 1 -D). The porcine lung acellular tissue is translucent Figure 1 -E) after soaking is completed.

[0122] Comparison of HE staining and DNA content of porcine lung acellular tissue and fresh lung tissue

[0123] Figure 3 Figure 1 is a comparison chart of HE staining and DNA content in the control group and the dECM group in one embodiment of the present application, Figure 3 Figure 1A is HE staining of the control group and the dECM group; Figure IB is a comparison of DNA content of the control group and the dECM group.

[0124] The control group: visible alveoli, airway and vascular structure, red-stained fibrous tissue, mixed with capillaries, no obvious red blood cell residues were found in the capillaries, and a large number of blue-stained cell nuclei were clearly visible. The dECM group: the structure is relatively loose, a large number of red-stained fibrous tissues can be seen, and blue-stained cell nuclei are not visible Figure 3 -A).

[0125] After decellularization by soaking method, the DNA concentration of untreated fresh lung tissue and dECM lung tissue measured by universal columnar genomic DNA extraction kit is as follows: untreated fresh lung tissue: 303.58±29.44; dECM tissue DNA concentration: 35.21±4.85 (unit: ng / mg dry weight tissue, n=3). Comparing the DNA content of the two groups, it was found that the DNA concentration of the decellularized group was significantly lower than that of the untreated group, with statistical difference (P<0.001) Figure 3 -B). The obtained dECM meets the standard: dsDNA <50 ng / mg (dry weight), and no obvious cell nucleus is found by HE staining.

[0126] 7.4 Preparation of dECM freeze-dried powder and determination of the optimal bio-ink ratio

[0127] Preparation of dECM freeze-dried powder: dECM after 72 hours of pepsin treatment is a mixed homogeneous white turbid liquid, in which flocculation can be occasionally seen. After freeze-drying treatment, it can be white powder, occasionally white flaky material, as shown in Figure 4 . Figure 4 Figure 1A is dECM without pepsin treatment; Figure IB is dECM after 72 hours of pepsin treatment; Figures 1C and 1D are freeze-dried powder prepared after freeze-drying treatment of the dECM solution in Figure IB.

[0128] Determination of the ratio of GelMA / ALG hydrogel bio-ink: Currently, most studies control the concentration of GelMA and ALG in the bio-ink combination to be between 5%-10% and 2%-4% respectively. Referring to the relevant literature, we configured various concentrations of GelMA / ALG bio-ink (as shown in Table 3-1), and compared the elastic properties of each group of hydrogels after cross-linking with a pressure of 50g, as shown in Figure 5 . Figure 5-1 20% GelMA+4% ALG hydrogel (group 2 in Table 3-1); Figure 5-220% GelMA + 2% ALG hydrogel (Group 3 of Table 3-1); Figure 5-3 20% GelMA hydrogel (Group 4 of Table 3-1); Figure 5-4 10% GelMA + 4% ALG hydrogel (Group 5 of Table 3-1).

[0129] Through the general evaluation of each group of gels, it was found that the appearance of Group 4, i.e. pure GelMA group, was transparent, the hardness and elasticity were better, but the touch was the driest. In the case of the same concentration of GelMA, as the ALG concentration increased, the elasticity and hardness of the gel were obviously improved. If the GelMA concentration is reduced, the hardness and elasticity will obviously deteriorate. Considering that the scaffold needs to have certain water absorption to keep it moist and good strength and elasticity, 20% GelMA and 4% ALG are selected, mixed according to the volume ratio of 1:1, i.e. the final concentration of 10% GelMA / 2% ALG as the final bio-ink ratio.

[0130] Table 3-1 GelMA / ALG different concentration ratio (the table is adjusted in order from low to high ALG concentration)

[0131]

[0132] 7.5 Mixed hydrogel evaluation

[0133] Macrostructure and microstructure: Referring to the experimental results described above, we finally determined and configured the bio-ink with a final concentration of 10% GelMA / 2% ALG after mixing, and added dECM freeze-dried powder at a ratio of 3%, 4%, and 5% (W / V, g / ml). After adding the freeze-dried powder, the liquid did not show obvious stratification. Three different concentrations of dECM / GelMA / ALG hydrogel bio-ink were prepared, and after 405 nm light irradiation, as shown in Figure 6 Figure 6 A: 10% G / 2% A hydrogel; Figure 6 B: 3% d / 10% G / 2% A hydrogel; Figure 6 C: 4% d / 10% G / 2% A hydrogel; Figure 6 D: 5% d / 10% G / 2% A hydrogel.

[0134] The control group has roughly the same color as the other groups, and the experimental group has a roughly uniform opaque gel-like appearance, with a small amount of flocculent material visible inside, no flow after light curing, and some elasticity.

[0135] As shown in Figure 7 Figure 7 ​​(Left): Fig. A is 10% G / 2% A hydrogel; Fig. B is 3% d / 10% G / 2% A hydrogel; Fig. C is 4% d / 10% G / 2% A hydrogel; Fig. D is 5% d / 10% G / 2% A hydrogel. Figure 7 (Right): Column chart of porosity of each group. Electron microscope (EM) images show that white fibrin monomers are attached to the three-dimensional network structure of the gel. The EM images are analyzed for porosity using imagej software. The porosity of 10% G / 2% A hydrogel is 21.64%, the porosity of 3% d / 10% G / 2% A hydrogel is 16.79%, the porosity of 4% d / 10% G / 2% A hydrogel is 9.85%, and the porosity of 5% d / 10% G / 2% A hydrogel is 8.85%, indicating that as the concentration of dECM increases, the porosity of the gel gradually decreases. Figure 7

[0136] Water absorption and in vitro degradation rate of mixed hydrogel: Considering that the mixed hydrogel needs to be placed in the body in the later stage, the materials themselves will interact in the body fluid environment in the body. In the ideal state, it can maintain its basic form for a certain period of time, and then it can degrade itself. Therefore, it is placed in complete medium with a temperature controlled at 37 degrees Celsius to simulate the in vivo environment and evaluate its in vitro degradation. As shown in Figure 8 Most of the high molecular hydrogel materials have swelling properties. Before 72 hours, as the concentration of dEMC increases, the swelling rate also gradually increases. After 72 hours, each group begins to gradually degrade. Taking 2 weeks as the observation period, it is found that the 4% dECM / 10% GelMA / 2% ALG group and the 3% dECM / 10% GelMA / 2% ALG group can slow down the degradation rate compared with the group without dECM. In addition, as the concentration of dECM increases, the degradation rate gradually slows down. However, considering that the higher the concentration of dECM, the higher the risk of pollution in the later stage, and the shorter the time for maintaining the mechanical properties of the scaffold, the final concentration of the scaffold bio-ink is determined to be 3% d / 10% G / 2% A.

[0137] 7.6 Preparation and mechanical evaluation of 3D printed biodegradable airway scaffold of dECM / GelMA / ALG

[0138] 3D printed airway scaffold: The mixed bio-ink is printed by extrusion molding. Figure 9 The 3D printing process of the mixed bio-ink and the macroscopic appearance of the airway scaffold are shown in Figure 9 Fig. A is a 3D Biomaker printer; Fig. B is a printing-related parameter setting; Fig. C is a schematic diagram of the printing process; Fig. D-F are, in turn, top, side, and oblique upper views of the mixed hydrogel (10% G / 2% A) airway scaffold; and Fig. G-I are, in turn, top, side, and oblique upper views of the mixed hydrogel (3% d / 10% G / 2% A) airway scaffold.

[0139] ​The stent is 14 mm in diameter, 1 mm in wall thickness, and 7 mm in height. The mixed hydrogel (10% G / 2% A) airway stent is white and translucent, and the hollow cylindrical shape is clear. The appearance of the stent with 3% is opaque and milky white. Both stents have smooth and continuous surfaces, and the thickness is roughly consistent.

[0140] Mechanical properties of 3D printed stents

[0141] Dynamic changes of the stent: The airway stent is placed in the airway with the help of a rigid bronchoscope or a soft bronchoscope. After radial pressure is applied to the 3D printed biodegradable airway stent, Figure 10 In the present application, Figures A-C are comparative diagrams of the mixed hydrogel (10% G / 2% A) airway stent before and after being squeezed by a straight blood vessel clamp; Figures D-F are comparative diagrams of the mixed hydrogel (3% d / 10% G / 2% A) airway stent before and after being squeezed by a straight blood vessel clamp. Figure 10 It is shown that after the pressure is removed, both groups can restore to the original state by themselves, and the elasticity is good. Therefore, it is possible to place it through a rigid bronchoscope.

[0142] Mechanical properties of the stent: For the two groups of samples, the elongation is measured by a universal material testing machine, and the stress-strain curve is drawn to evaluate the Young's modulus and the breaking elongation. The hardness of the 10% G / 2% A hydrogel airway stent is measured by a pointer-type Shore hardness tester, and the Shore hardness is 59 HA. Figure 11 In the present application, Figures A-C are comparative diagrams of the mixed hydrogel (10% G / 2% A) airway stent before and after being squeezed by a straight blood vessel clamp; Figures D-F are comparative diagrams of the mixed hydrogel (3% d / 10% G / 2% A) airway stent before and after being squeezed by a straight blood vessel clamp. Figure 11 The stress-strain curve of C shows the tensile properties: peak stress: 3.0 MPa; strain at break: 1069.238%; breaking elongation: 1064%. The calculated Young's modulus is 0.777 MPa. The Shore hardness of the 3% d / 10% G / 2% A hydrogel airway stent is 56.5 HA; the peak stress is 2.3 MPa; the strain at break is 1042.51%; the breaking elongation is 1068%; and the Young's modulus is 0.298 MPa. The hardness of the silicone airway stent material is 60-80 HA, and the elastic modulus is about 0.3 MPa. The hydrogel airway stent of the present application is similar to it, and in addition, its high breaking elongation proves that the stent is very tough.

[0143] Biocompatibility cell experiment of the stent: In order to determine the biocompatibility of the new airway stent, the stent is immersed in complete culture medium as the leaching liquid, and after leaching at 37°C for 24 hours, the material leaching liquid is co-cultured with Beas-2B cells for 24 hours and 48 hours. As shown in Figure 12We found that there was no significant difference in cell viability between the group without 3% dECM scaffold and the group with 3% dECM scaffold at the same time period (P>0.05).

[0144] Biocompatibility detection animal experiment

[0145] As shown in Figure 13 , the prepared scaffold containing 3% decellularized extracellular matrix (alginate: GelMA volume ratio 1:1) was generally milky white and columnar, and could be cut according to the size of the tracheal fistula of the rabbit.

[0146] Tracheal fistula and postoperative general condition of rabbits in different groups

[0147] As shown in Figure 14 , in which figure (a) 1 week after operation, the neck suture line was deformed, the wound was closed, and there was no redness; figure (b) 2 weeks after operation, the suture line was basically absorbed, and a small amount of scab was visible at the wound site. After tracheal fistula surgery, animals can breathe independently, eat and exercise normally, and defect group and gel composite group animals can survive to the specimen collection time. The postoperative weight of rabbits remained unchanged or decreased slightly on the original basis, which was considered to be caused by surgical consumption. The neck wound recovered well, and there was no sign of infection such as redness and pus Figure 14 -a, b).

[0148] Effect of new gel composite on tracheal fistula treatment

[0149] Anatomize the entire rabbit's anterior cervical skin to expose the trachea. Figure 15 Figure (a) A fistula with a length and width of about 3 mm (about 2 cartilage rings in size) was made under the rabbit's cartilage ring; figure (b) 1 week after operation, the healing of the fistula in the defect group; figure (c) 2 weeks after operation, the fistula in the defect group is completely closed, as shown in the yellow box; figure (d) 1 week after operation, the white covering of the gel composite group can be seen to block the fistula, as indicated by the blue arrow; figure (e) After removing the surface covering of the gel composite group, the fistula that is not completely closed can be seen (figure e is a picture without removing the gel), as indicated by the blue arrow; figure (f) 2 weeks after operation, the tracheal fistula in the gel composite group is completely closed, as shown in the yellow box. In the defect group, at 1 week ( Figure 15 -b) The tracheal fistula is clearly visible, but it is significantly smaller than before, and is covered with connective tissue, sealing the fistula; 2 weeks later ( Figure 15 -c), the fistula is completely closed and sealed by soft tissue, and the color is similar to that of normal tracheal soft tissue, most of which is granulation tissue, which is soft as a whole and has a clear boundary with the normal cartilage of the trachea.

[0150] And in the gel composite group, at 1 week ( Figure 15- d), the gel composite can be seen tissue ingrowth, completely cover the stoma, the overall white, similar to granulation tissue, generally no cartilage formation, after removing the surface tissue can be seen defect mouth Figure 15 - e), and a small amount of undegraded gel composite; 2 weeks ( Figure 15 - f), the stoma is completely closed, no residual gel composite, compared with the simple defect group, and the surrounding tracheal normal cartilage boundary gradually blurred, can be seen the formation of a similar surrounding normal cartilage ring structure, with ophthalmic forceps light touch, the quality is slightly hard.

[0151] Figure 16 In the figure (a) tracheal wall smooth, no granulation tissue ingrowth; figure (b) stent slightly larger than the stoma 2 weeks after the gel composite group repair site trachea gross specimen. Combined with the gel composite group stoma gross, it can be seen that the gel composite is not completely degraded at about 1 week, ingrowth stoma, can be a good closure of the defect; 2 weeks, incision neck skin, stent completely degraded, cut off the repair site trachea ( Figure 16 - a), the tracheal wall is smooth, no stenosis, no granulation tissue ingrowth to make the airway stenosis. 1 week, the stoma defect of the two groups were no significant difference in gross observation, were not completely healed, because each rabbit in the tracheal specimen when the neck stretch is inconsistent, by measuring the stoma to compare the defect size, there is error. 2 weeks, gel composite group repair area can be seen similar cartilage ring structure, stoma repair rate faster than the simple defect group, the gel composite to some extent accelerated the closure of the stoma, promote the formation of cartilage.

[0152] 7.6.4.2.2 Evaluation of the biocompatibility of residual gel composite

[0153] As Figure 17 shown, HE staining of the stent after 1 week of treatment, cells ingrowth stent, local visible small blood vessels, yellow arrow shown, magnification 100, undegraded gel composite group after 1 week of removal of HE staining ( Figure 17 ), can be seen remaining stent cells ingrowth, a small amount of capillary formation, stent internal staining is lighter than the periphery, the number of peripheral cells is more than the middle. The stent has a certain biocompatibility, the pore size of the stent is suitable for guiding seed cells distribution permeate into the stent.

Claims

1. A method for preparing a hydrogel bio-ink, characterized in that, Includes the following steps: S1. Lung tissue was frozen and thawed, sectioned, and sequentially decellularized with SDS and Triton X-100, then air-dried, pulverized, and cryopreserved to obtain lung extracellular matrix. S2. The lung extracellular matrix obtained in S1 is sterilized and treated with pepsin to obtain a pretreated lung extracellular matrix. S3. Mix methylpropionyl gelatin and alginate solution, pour into a solid gel, gelle it, and then add the pretreated lung cell extracellular matrix to obtain hydrogel bio-ink.

2. The method for preparing hydrogel bio-ink according to claim 1, characterized in that, In S3, the volume ratio of the methylpropionyl gelatin and the alginate solution is (1-2):(1-2).

3. The method for preparing hydrogel bio-ink according to claim 2, characterized in that, In S3, the mass concentration of the methylpropionyl gelatin is 8-12%, the mass concentration of the alginate solution is 1-3%, and the concentration of the lung extracellular matrix is ​​3-5%; preferably, the mass concentration of the methylpropionyl gelatin is 8-10%, the mass concentration of the alginate solution is 2-3%, and the concentration of the lung extracellular matrix is ​​3-4%.

4. The method for preparing hydrogel bio-ink according to claim 1, characterized in that, In S1, the sequential cell removal steps of SDS and Triton X-100 specifically include soaking in 1-3% SDS for 12-24 hours, rinsing with deionized water, soaking in 1-3% Triton X-100 for 12-24 hours, rinsing with deionized water and soaking for 12-24 hours.

5. The method for preparing hydrogel bio-ink according to claim 1, characterized in that, In S2, the pepsin treatment includes adding the lung extracellular matrix and pepsin to an acetic acid solution and soaking. Preferably, the concentration of the acetic acid solution is 0.2-1.5 mol / L, the volume ratio of the dECM to the pepsin is (4-6):1, and the soaking time is 2-4 days.

6. The method for preparing hydrogel bio-ink according to claim 1, characterized in that, In S2, the pretreatment also includes neutralization, osmotic pressure adjustment, centrifugation, freeze drying, and storage. The neutralization step is performed after the pepsin treatment.

7. The method for preparing hydrogel bio-ink according to claim 1, characterized in that, In S1, pressure is applied to the lung tissue during the sequential decellularization step using SDS and Triton X-100. Preferably, pressure is applied to the lung tissue using a pressure vessel.

8. A hydrogel bio-ink prepared by the preparation method according to any one of claims 1-7.

9. A 3D-printed biodegradable airway stent prepared using the hydrogel bio-ink of claim 8, characterized in that, The 3D-printed biodegradable airway scaffold has a hardness of 50-80 HA, a Young's modulus of 0.1-0.4 MPa, and a modulus of less than 1 MPa, and is non-toxic to cells; preferably, the 3D-printed biodegradable airway scaffold has a hardness of 55-70 HA and a Young's modulus of 0.2-0.3 MPa.

10. The application of the hydrogel bio-ink of claim 9 in a 3D-printed biodegradable airway stent.

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