CeO2 nano enzyme loaded SilMA hydrogel and application thereof in 3D biological printing tissue engineering trachea

Abstract

The invention discloses a CeO2 nano-enzyme loaded SilMA hydrogel and application thereof in a 3D biological printing tissue engineering trachea, and belongs to the technical field of biology.The preparation method of the CeO2 nano-enzyme loaded SilMA hydrogel comprises the steps that 1, CeO2 nano-enzyme is prepared, specifically, cerous nitrate hexahydrate, dextran and deionized water are stirred to be uniform, ammonia water is added, water bath heating is conducted, and the CeO2 nano-enzyme is prepared; carrying out aging, centrifugation, washing, vacuum drying and calcination on the obtained CeO2 nano-enzyme precipitation slurry to obtain CeO2 nano-enzyme powder; step 2, mixing CeO2 nano-enzyme into a 20 w / v% SilMA solution, so as to obtain a CeO2 nano-enzyme loaded SilMA hydrogel; step 3, taking 20% w / v of SilMA as a water phase and a paraffin oil solution containing 5% w / v of Span 80 as an oil phase, mixing, stirring under water bath heating to obtain SilMA hydrogel microspheres, and freeze-drying to obtain a porous structure; the preparation method comprises the following steps: adding CeO2 nano-enzyme into deionized water containing the SilMA hydrogel microspheres, so as to obtain a SilMA hydrogel microsphere solution loaded with the CeO2 nano-enzyme;

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically is a CeO2 nanoenzyme SilMA hydrogel loaded with CeO2 nanoenzyme and its application in 3D bioprinting tissue engineering trachea. Background Art

[0002] When the tracheal defect exceeds 1 / 2 of the total length of the trachea in adults and 1 / 3 of the total length of the trachea in children, the use of end-to-end tracheal anastomosis may cause serious postoperative complications such as tracheal rupture and anastomotic leakage due to excessive tension, and may even threaten the patient's life. At this time, the use of tracheal substitutes for tracheal reconstruction becomes an important treatment approach for long-segment tracheal injury, and tissue-engineered trachea is an ideal tracheal substitute for patients to achieve better long-term survival effects.

[0003] Whether the wound healing after tissue-engineered trachea transplantation is good or not plays a vital role in the survival rate of the body, and the local microenvironment is one of the key factors affecting wound healing. For example, the presence of oxidative stress (OS) damage in the local microenvironment will delay the progress of wound healing. The excessive local OS and inflammatory response caused by tissue-engineered trachea transplantation, the large amount of reactive oxygen species (ROS) and inflammatory factors produced, cause cell damage, and delay the process of vascularization and epithelialization.

[0004] The ideal preparation material for tissue engineering trachea needs to have good biocompatibility, be able to simulate a microenvironment similar to that of in vivo tissue, have good mechanical properties, suitable fluid dynamics characteristics, printability and high fidelity; be able to induce functionalized epithelial formation, and have a rich vascular network to meet the needs of clinical transplantation. Hydrogel is currently the most outstanding 3D printing bio-ink with a three-dimensional network structure formed by cross-linking of polymer chains. This structure gives it unique swelling properties and a three-dimensional skeleton. SilMA hydrogel has excellent biocompatibility and fluid dynamics characteristics, and has broad application prospects in the preparation of tissue engineering trachea. Nanozymes are nanomaterials with enzymatic activity that can catalyze related physiological reactions. They have certain similarities with natural enzymes in terms of total size, shape and surface charge. However, compared with natural enzymes, nanozymes also have the characteristics of high stability, low cost and simple large-scale preparation. Nanozymes can promote re-epithelialization, antibacterial, anti-inflammatory and antioxidant properties. In addition, when added to hydrogels, they can enhance the mechanical properties of hydrogels, thereby protecting surrounding tissues, and have great application potential in biomedical applications.

[0005] Cerium oxide (CeO2) nanozymes have broad application prospects in the biomedical field due to their excellent natural antioxidant enzyme-like catalytic activity and low cytotoxicity. 3+ and Ce 4+Coexistence, with dual catalytic effects of oxidation and reduction. When the body's reactive oxygen species (ROS) are overproduced, CeO2 nanozymes can protect tissues from OS damage, thereby regulating redox homeostasis. In addition, CeO2 nanozymes can produce oxygen while clearing excess ROS to relieve tissue hypoxia, reduce the release of proinflammatory factors to reduce inflammatory responses, and reduce the release of proinflammatory factors to reduce inflammatory responses and the occurrence of granulomas, thereby promoting wound healing, providing a good microenvironment for cell regeneration, and promoting tracheal tissue regeneration.

[0006] Hydrogel microspheres are spherical hydrogels with a diameter in the micrometer range. Traditional large-sized hydrogels (over 200 μm) will affect the diffusion efficiency of the loaded substances. However, hydrogel microspheres can improve the response speed of loaded bioactive substances and drugs due to their smaller size, and their porous structure can enhance the ductility and mechanical properties of the hydrogel itself. Currently, hydrogel microspheres have been used in biomedical applications such as preparing controlled-release carriers for therapeutic agents and constructing biomechanical structures. For example, hydrogel microspheres can encapsulate drugs or bioactive substances (cells, bioactive macromolecules, etc.), and can release therapeutic agents in a controlled manner locally to improve the microenvironment and relieve symptoms such as inflammation and OS. Summary of the invention

[0007] In order to reduce complications after tissue engineering trachea transplantation and promote wound healing, the present invention provides a CeO2 nanoenzyme SilMA hydrogel loaded with CeO2 nanoenzyme and its application in 3D bioprinting of tissue engineering trachea.

[0008] The present invention provides a CeO2 nanozyme SilMA hydrogel, and the preparation method thereof comprises the following steps:

[0009] Step 1: Preparation of CeO2 nanozyme:

[0010] Cerium nitrate hexahydrate and dextran were mixed in deionized water and stirred evenly, then ammonia water was added and heated in a water bath at 65° C. for 60 min, the obtained CeO2 nanozyme precipitate slurry was aged and centrifuged, washed with hot deionized water until the pH of the upper solution was 7, vacuum dried and calcined, and finally CeO2 nanozyme powder was obtained;

[0011] Step 2, preparing SilMA hydrogel loaded with CeO2 nanozyme;

[0012] The CeO2 nanozyme was mixed into a 20 w / v% SilMA solution to obtain a CeO2 nanozyme-loaded SilMA hydrogel, i.e., a CeO2 / SilMA hydrogel;

[0013] Step 3: Prepare the SilMA hydrogel microsphere solution loaded with CeO2 nanozyme, the steps are as follows:

[0014] Step 3.1, using 20% ​​w / v SilMA solution as the aqueous phase and 5% w / v Span 80 paraffin oil solution as the oil phase, mixing the aqueous phase and the oil phase solution, heating in a water bath at 70°C and continuously stirring for 30 min, washing repeatedly for multiple times to obtain SilMA hydrogel microspheres, and freeze-drying at -20°C for 48 hours to obtain a porous structure;

[0015] Step 3.2, add CeO2 nanozyme to deionized water containing SilMA hydrogel microspheres, stir magnetically and heat in a water bath at 60°C for 10 minutes, and after cooling, obtain a SilMA hydrogel microsphere solution loaded with CeO2 nanozyme.

[0016] Furthermore, in step 1, the mass ratio of the cerium nitrate hexahydrate, deionized water, and ammonia water is 0.1085:0.3255:2:0.4;

[0017] In step 2, the mass ratio of CeO2 nanozyme to SilMA is 1:10;

[0018] In the step 3.1, the volume ratio of the aqueous phase to the oil phase solution is 1:1; in the step 3.2, the mass ratio of CeO2 nanozyme, SilMA in SilMA hydrogel microspheres, and deionized water is 1:10:40.

[0019] Furthermore, the preparation method of the 20 w / v% SilMA solution is:

[0020] (1) Take 4 g of SilMA and put it into a centrifuge tube;

[0021] (2) Take 20 ml of the initiator standard solution and add it to the above centrifuge tube;

[0022] (3) Dissolve at room temperature for 0.5-1 h, stirring / shaking several times;

[0023] (4) Sterilize the obtained SilMA solution using a 0.22 μm sterile syringe filter;

[0024] The initiator standard solution is a 0.5% w / v initiator standard solution, and the preparation method is: take 20 ml of PBS, add it into a brown bottle containing 0.1 g of initiator LAP, heat and dissolve it in a 40-50° C. water bath for more than 15 minutes, and shake it several times during the process.

[0025] The application of the CeO2 nanozyme SilMA hydrogel in 3D bioprinting tissue engineering trachea is as follows:

[0026] Step 1: Cell culture and differentiation;

[0027] 1) Cultivate primary tracheal mucosal epithelial cells in vitro and perform subculture,

[0028] 2) Cultivate and differentiate bone marrow mesenchymal stem cells (BMSCs) in vitro to obtain chondrocytes and vascular endothelial cells;

[0029] Step 2: Preparation of bio-ink;

[0030] 2) Preparation of inner bio-ink: The obtained epithelial cells, BMSCs, and epidermal growth factor (EGF) were mixed into CeO2 / SilMA hydrogel as the inner bio-ink for constructing the epithelial cell layer;

[0031] 2) Preparation of outer bio-ink: Vascular endothelial cells, chondrocytes, BMSCs, growth factor TGF-β, and vascular endothelial growth factor VEGF were mixed into CeO2 / SilMA hydrogel as the outer bio-ink for constructing the chondrocyte layer;

[0032] Step 3: 3D printing polycaprolactone tracheal stent;

[0033] Based on the dynamic CT imaging data of the trachea, a tracheal stent was 3D printed using polycaprolactone as the material;

[0034] Step 4: constructing the tracheal structure through photocuring 3D bioprinting technology;

[0035] The inner layer of bio-ink is evenly 3D printed onto the inner wall of the tracheal stent obtained in step three, with a thickness of 0.5 mm; the outer layer of bio-ink is evenly 3D printed onto the outer wall of the tracheal stent obtained in step three, with a thickness of 0.5 mm.

[0036] Furthermore, the tracheal stent obtained in step three was immersed in the SilMA hydrogel microsphere solution loaded with CeO2 nanozyme at 37°C for 15 minutes, and fully cross-linked under blue light for 5 minutes, so that the SilMA hydrogel microsphere loaded with CeO2 nanozyme evenly wrapped the tracheal stent.

[0037] Beneficial effects: Under the premise of ensuring biocompatibility, the present invention can achieve long-term anti-inflammatory and antioxidant effects by mixing CeO2 nanozymes and SilMA hydrogels in a mass ratio of 1:10 in CeO2 / SilMA hydrogel, thereby promoting the re-epithelialization and vascularization of tissue-engineered trachea, and further improving the success rate of tissue-engineered tracheal transplantation and better long-term survival effects. The tracheal frame prepared by the hydrogel microspheres used in the present invention can improve the release and response speed of the loaded CeO2 nanozymes, and can improve the microenvironment in a locally controlled release of therapeutic agents, and relieve symptoms such as inflammation and OS. And its porous structure can enhance the ductility and mechanical properties of the hydrogel itself. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a picture of the CeO2 / SilMA hydrogel prepared in Example 1 after curing;

[0039] Figure 2 is a scanning electron micrograph of the CeO2 / SilMA hydrogel prepared in Example 1;

[0040] Figure 3 The 3D bioprinted tissue engineering trachea prepared in Example 2;

[0041] Figure 4 These are pictures of the New Zealand rabbit before 3D bioprinted tissue engineering tracheal transplantation and after long-segment tracheal resection in Example 2.

[0042] Figure 5 This is a picture of the New Zealand rabbit 3D bioprinted tissue engineered trachea transplantation in Example 2, showing that the 3D bioprinted tissue engineered trachea and the original trachea in the body are well anastomosed.

[0043] Figure 6 HE staining of New Zealand rabbits after long trachea transplantation in Example 2. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0045] Example 1: Preparation of CeO2 nanozyme SilMA hydrogel

[0046] Step 1: Preparation of cerium oxide (CeO2) nanozyme:

[0047] Cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and dextran were mixed in deionized water in a mass ratio of 1:3 and stirred evenly. Then, ammonia water (NH3·H2O) was added and heated in a water bath at 65°C for 60 min. The obtained CeO2 nanozyme precipitate slurry was aged and centrifuged for 5 min. After washing with hot deionized water until the pH of the upper solution was 7, it was vacuum dried and calcined to finally obtain CeO2 nanozyme powder.

[0048] The mass ratio of the cerium nitrate hexahydrate, deionized water and ammonia water is 0.1085:0.3255:2:0.4.

[0049] Step 2, preparing SilMA (methacrylylated silk fibroin) hydrogel loaded with CeO2 nanozyme;

[0050] CeO2 nanozyme was mixed into 20w / v% SilMA solution, and the mass ratio of CeO2 nanozyme to methacrylylated silk fibroin was 1:10, to obtain SilMA hydrogel loaded with CeO2 nanozyme, namely CeO2 / SilMA hydrogel.

[0051] The preparation method of 20w / v% SilMA solution is as follows:

[0052] (1) Take 4 g of SilMA and put it into a centrifuge tube;

[0053] (2) Take 20 ml of the initiator standard solution and add it to the above centrifuge tube; the initiator standard solution is a 0.5% (w / v) initiator standard solution, and the preparation method is: take 20 ml of PBS and add it to a brown bottle containing initiator LAP (containing 0.1 g LAP); heat and dissolve in a 40-50°C water bath for 15 minutes, shaking several times during the process.

[0054] (3) Dissolve at room temperature for 0.5-1 h, stirring / oscillating several times during the process (avoid severe ultrasound, high temperature and strong shear);

[0055] (4) Sterilize the SilMA solution using a 0.22 μm sterile syringe filter.

[0056] Step 3, preparing SilMA hydrogel microspheres loaded with nanozymes;

[0057] Step 3.1, 20% w / v SilMA was used as the aqueous phase, and a paraffin oil solution containing 5% w / v Span 80 was used as the oil phase. The aqueous phase and the oil phase solution were mixed and continuously stirred for 30 min in a 70°C water bath, and the mixture was centrifuged and washed three times with isopropanol and 75% ethanol to remove the surfactant Span 80 and residual paraffin oil, thereby obtaining SilMA hydrogel microspheres, and freeze-dried at -20°C for 48 hours to obtain a porous structure;

[0058] In step 3, the volume ratio of the aqueous phase to the oil phase solution is 1:1. In step 3, the preparation method of 20% (w / v) SilMA solution is as follows:

[0059] (1) Take 4 g of SilMA and put it into a centrifuge tube;

[0060] (2) Take 20 ml of the initiator standard solution and add it to the above centrifuge tube; the initiator standard solution is a 0.5% w / v initiator standard solution, and the preparation method is: take 20 ml of PBS and add it to a brown bottle containing the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite LAP (containing 0.1 g LAP); heat and dissolve in a 40-50°C water bath for 15 minutes, shaking several times during the process. The LAP standard solution can be stored at 4°C in the dark for 12 months.

[0061] (3) Dissolve at room temperature for 0.5-1 h, stirring / oscillating several times during the process (avoid severe ultrasound, high temperature and strong shear);

[0062] (4) Sterilize the SilMA solution using a 0.22 μm sterile syringe filter.

[0063] Step 3.2, add CeO2 nanozyme to deionized water containing SilMA hydrogel microspheres, stir magnetically at 300 rpm and heat in a water bath at 60°C for 10 minutes, so that the CeO2 nanozyme can be evenly dispersed inside or on the surface of the hydrogel microspheres. After cooling, a SilMA hydrogel microsphere solution loaded with CeO2 nanozyme is obtained.

[0064] The mass ratio of SilMA and deionized water in CeO2 nanozyme and SilMA hydrogel microspheres is 1:10:40.

[0065] After the CeO2 / SilMA hydrogel prepared in this example is cured, Figure 1 shown. Figure 2 is the scanning electron microscopy image of CeO2 / SilMA hydrogel. Figure 2 As shown, CeO2 nanozymes are evenly distributed in the microporous structure formed by SilMA hydrogel, which can increase the contact area between cells and nanozymes and exert anti-inflammatory and antioxidant effects.

[0066] Example 2: Preparation of 3D bioprinted tissue engineering trachea using CeO2 nanozyme SilMA hydrogel

[0067] In order to reduce complications after tissue-engineered trachea transplantation and promote wound healing, a method for preparing 3D bioprinted tissue-engineered trachea was provided, combined with the efficient and long-lasting antioxidant and anti-inflammatory functions of CeO2 nanozymes, to create a good microenvironment for epithelial cell growth and reduce the occurrence of granulomas and other postoperative complications.

[0068] Step 1: Cell culture and differentiation. The specific steps are as follows:

[0069] Step 1.1, tracheal epithelial cell culture:

[0070] The rabbit trachea was obtained using standard surgical methods, and the tracheal mucosal layer was torn off using forceps under a microscope. The primary tracheal mucosal epithelial cells were cultured in vitro using the tissue block method and subcultured.

[0071] Step 1.2: Cultivation and differentiation induction of bone marrow mesenchymal stem cells (BMSCs):

[0072] 1) Puncture the rabbit tibial plateau and extract about 2 ml of bone marrow. Then, whole bone marrow adherence screening and purification of BMSCs were carried out in an incubator containing 37°C and 5% CO. Routine culture was performed in L-DMEM medium containing 10% FBS (fetal bovine serum) and 1% AA (amino acid). The medium was replaced after 48 hours. When the cells grew to >80% confluence, they were digested with trypsin and passaged. The cells were used for subsequent induced differentiation at the second generation.

[0073] 2) Differentiation of BMSCs into chondrocytes: Second generation BMSCs were cultured at 1×10 4 The cells were seeded at a density of / ml in a culture dish and induced into chondrocytes using L-DMEM culture medium containing 10% FBS, 1% AA, IGF-150ng / ml, and TGF-β10ng / ml. The induction medium was replaced every 2-3 days. After 14 days of culture, chondrocytes were obtained.

[0074] 3) Differentiation of BMSCs into endothelial cells: Second generation BMSCs were cultured at 1×10 4 The cells were seeded at a density of / ml in a culture dish and induced into vascular endothelial cells using L-DMEM culture medium containing 10% FBS, 1% AA, IGF-120ng / ml, and EGF (epidermal growth factor) 30ng / ml. The induction medium was replaced every 2-3 days. After 14 days of culture, vascular endothelial cells were obtained.

[0075] Step 1.3, cell identification:

[0076] Epithelial cells: HE staining to observe morphology, CK-18 immunofluorescence staining, CK-18 immunocytochemical staining.

[0077] BMSCs: HE staining was used to observe the morphology, and flow cytometry was used to detect the surface markers of BMSCs (antibodies CD34, CD44, CD90, and CD105).

[0078] Chondrocytes: immunofluorescence staining of type II collagen, immunocytochemical staining of type II collagen, and PT-PCR detection.

[0079] Vascular endothelial cells: HE staining was used to observe the morphology, and flow cytometry was used to detect the expression of vascular endothelial cell markers (antibodies CD31, CD34, VEGF-R2, Tie-2 / Tek).

[0080] Step 2: Preparation of bio-ink. The specific steps are as follows:

[0081] Step 2.1, Preparation of inner layer bio-ink:

[0082] 0.5 ml of epithelial cells (1×10 5 / ml), 0.5 ml BMSCs (1×10 6 / ml), 0.5 ml of EGF (0.3 μg / ml) were mixed into 2 ml of CeO2 / SilMA hydrogel prepared in Example 1 as the inner layer bio-ink to construct the epithelial cell layer.

[0083] Step 2.2 Preparation of outer bio-ink:

[0084] 0.5 ml of endothelial cells (1×10 6 / ml), 0.5 ml chondrocytes (1×10 5 / ml), 0.5 ml BMSCs (1×10 6 / ml), 0.5 ml of growth factor TGF-β (0.1 μg / ml), and 0.5 ml of vascular endothelial growth factor VEGF (0.33 μg / ml) were mixed into 2 ml of CeO2 / SilMA hydrogel prepared in Example 1 as the outer layer of bio-ink to construct the chondrocyte layer.

[0085] Step 3: 3D Printing Polycaprolactone (PCL) Tracheal Stent

[0086] Based on the dynamic CT imaging data of the New Zealand rabbit trachea, a 3D printing STL file (CT scan data converted into a 3D model) was constructed. Polycaprolactone (PCL) was selected as the material, and a high-temperature printing nozzle was used to print on the rotating shaft. The printing temperature was set to 90°C: the rotating shaft rotated at a certain speed, and the printing nozzle moved back and forth along the rotating shaft. The printing speed was 5mm / s, and the bottom tracheal stent was first printed on the rotating shaft;

[0087] Then the rotating axis rotates in the opposite direction to that when printing the bottom layer, and prints the second layer of tracheal stent, which then forms a porous structure with the previous layer. The pore size of the tracheal stent can be adjusted by the rotation speed of the rotating axis and the travel speed of the print head, and the PCL tracheal stent is formed by bionic stacking in a layer-by-layer processing manner.

[0088] The PCL tracheal stent obtained in this embodiment has an outer diameter of 9-10 mm, an inner diameter of 7-8 mm, a wall thickness of 1-1.5 mm, a length of 30 mm, and a tube wall pore diameter of 200 μm.

[0089] Step 4: constructing a tracheal structure by using photocuring 3D bioprinting technology;

[0090] 1) The inner layer of bio-ink obtained in step 2 is evenly 3D printed onto the inner wall of the PCL tracheal stent obtained in step 3, with a coating thickness of about 0.5 mm.

[0091] 2) The outer layer of bio-ink obtained in step 2 is applied to the outer wall of the PCL tracheal stent obtained in step 3, and a 0.5 mm thick hydrogel coating is printed by 3D printing.

[0092] The final 3D bioprinted tissue engineered trachea is as follows Figure 3 As shown, the PCL tracheal stent is the middle layer and the CeO2 / SilMA hydrogel is the inner and outer layers.

[0093] Step 5: Perform tracheal replacement transplantation on New Zealand rabbits using the 3D bioprinted tissue-engineered trachea obtained in step 4.

[0094] Figure 4 These are pictures of New Zealand rabbits before 3D bioprinted tissue engineering trachea transplantation and after long-segment tracheal resection; Figure 5 This is a picture of the good anastomosis between the 3D bioprinted tissue-engineered trachea and the native trachea in the body after 3D bioprinted tissue-engineered trachea transplantation in New Zealand rabbits. Figure 6 HE staining of the tracheal epithelium after long-segment trachea transplantation in New Zealand rabbits. The staining results showed good re-epithelialization.

[0095] At present, after tissue-engineered trachea is transplanted into the body, inflammatory cells will be recruited at the wound to form a chronic inflammatory response, leading to oxidative stress, which is not conducive to the healing process of the wound. Under the premise of ensuring biocompatibility, CeO2 nanozymes in CeO2 / SilMA hydrogel and SilMA hydrogel in a mass ratio of 1:10 can achieve long-term anti-inflammatory and antioxidant effects, thereby promoting the re-epithelialization and vascularization of tissue-engineered trachea, which can further improve the success rate of tissue-engineered trachea transplantation and better long-term survival effects.

[0096] Example 3: Preparation of 3D bioprinted tissue engineering tracheal stent using CeO2 nanozyme SilMA hydrogel microspheres

[0097] By loading CeO2 nanozymes, the local microenvironment of the wound after tissue engineering trachea transplantation can be improved, OS and inflammatory response damage can be reduced, and good conditions can be provided for re-epithelialization and vascularization at the wound, thereby promoting wound healing.

[0098] The steps for preparing 3D bioprinted tissue engineering tracheal scaffolds using CeO2 nanozyme SilMA hydrogel microspheres are as follows:

[0099] 1. 3D printed polycaprolactone (PCL) tracheal stent;

[0100] Polycaprolactone (PCL) was selected as the material, and a high-temperature printing nozzle was used to print on a rotating shaft. The rotating shaft rotated at a speed of 5 mm / s, and the printing nozzle moved back and forth along the rotating shaft to first print the bottom layer of tracheal stent on the rotating shaft; then the rotating shaft rotated in the opposite direction to that when printing the bottom layer, and the second layer of tracheal stent was printed, which then formed a porous structure with the previous layer, thereby preparing a tracheal stent that matched the rabbit trachea structure.

[0101] 2. Preparation of composite scaffolds;

[0102] The obtained PCL scaffold print was immersed in the SilMA hydrogel microsphere solution loaded with CeO2 nanozyme at 37°C for 15 minutes, and fully cross-linked under blue light for 5 minutes, so that the SilMA hydrogel microsphere loaded with CeO2 nanozyme evenly wrapped the PCL scaffold to obtain the nanozyme-loaded light-cured hydrogel microsphere tissue engineering tracheal scaffold.

[0103] 3. Except for the different scaffold preparation method, other steps are the same as Example 2.

[0104] The tracheal stent prepared by the hydrogel microspheres used in Example 3 can improve the release and response speed of the loaded CeO2 nanozyme, and can release the therapeutic agent in a controlled manner to improve the microenvironment and relieve symptoms such as inflammation and OS. In addition, its porous structure can enhance the ductility and mechanical properties of the hydrogel itself. 3+ / Ce 4+ The redox pair balances the ROS homeostasis and reduces OS damage; and the natural antioxidant enzyme activity of CeO2 nanozymes can inhibit the release of pro-inflammatory factors, relieve inflammatory responses, and promote wound healing. Hydrogel microspheres are used as microcarrier platforms. By loading CeO2 nanozymes in hydrogel microspheres, the release efficiency of CeO2 nanozymes can be improved, cell proliferation and migration can be promoted, and rapid wound healing can be achieved. This nanozyme-loaded light-cured hydrogel microsphere tissue engineering tracheal stent shows the potential for anti-inflammatory and wound healing, and has broad application prospects in tracheal substitute transplantation.

[0105] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to the form and details without departing from the spirit and scope of the present invention.

Claims

1. A CeO2 nanozyme SilMA hydrogel, characterized in that: The preparation method thereof comprises the following steps: Step 1: Preparation of CeO2 nanozyme: The cerium nitrate hexahydrate and the dextran are mixed in deionized water and stirred evenly, and then ammonia water is added and heated in a water bath to obtain a CeO2 nanozyme precipitation slurry, and the precipitation slurry is aged and centrifuged, and then washed with hot deionized water, vacuum dried and calcined to obtain CeO2 nanozyme powder; Step 2, preparing SilMA hydrogel loaded with CeO2 nanozyme; The CeO2 nanozyme was mixed into a 20 w / v% SilMA solution to obtain a SilMA hydrogel loaded with CeO2 nanozyme, i.e., CeO2 / SilMA hydrogel; Step 3: Prepare the SilMA hydrogel microsphere solution loaded with CeO2 nanozyme, the steps are as follows: Step 3.1, using 20% ​​w / v SilMA solution as the aqueous phase and 5% w / v Span 80 paraffin oil solution as the oil phase, mixing the aqueous phase and the oil phase solution, heating in a water bath and continuously stirring for a period of time, washing repeatedly for multiple times, and obtaining SilMA hydrogel microspheres, which were freeze-dried to obtain a porous structure; Step 3.2, add CeO2 nanozyme into deionized water containing SilMA hydrogel microspheres, stir magnetically and heat in a water bath for a period of time, then cool to obtain a SilMA hydrogel microsphere solution loaded with CeO2 nanozyme.

2. The CeO2 nanozyme SilMA hydrogel loaded according to claim 1, characterized in that: In step 1, the mass ratio of cerium nitrate hexahydrate, deionized water, and ammonia water is 0.1085:0.3255:2:0.4, and the water bath heating temperature is 65° C. for 60 min; In step 2, the mass ratio of CeO2 nanozyme to SilMA is 1:10; In the step 3.1, the volume ratio of the aqueous phase to the oil phase solution is 1:

1. After the aqueous phase and the oil phase solution are mixed, they are continuously stirred for 30 minutes in a water bath heated at 70°C, and washed repeatedly to obtain SilMA hydrogel microspheres, which are then freeze-dried at -20°C for 48 hours to obtain a porous structure; In the step 3.2, the mass ratio of CeO2 nanozyme, SilMA in SilMA hydrogel microspheres, and deionized water is 1:10:40; the temperature of the water bath is 60°C, and the heating time is 10 minutes.

3. The CeO2 nanozyme SilMA hydrogel loaded according to claim 1, characterized in that The preparation method of the 20w / v% SilMA solution is as follows: (1) Take 4 g of SilMA and put it into a centrifuge tube; (2) Take 20 ml of the initiator standard solution and add it to the above centrifuge tube; (3) Dissolve at room temperature for 0.5-1 h, stirring and shaking several times; (4) sterilizing the obtained SilMA solution using a sterile syringe filter; The initiator standard solution is a 0.5% w / v initiator standard solution, and the preparation method is: take 20 ml of PBS, add it into a brown bottle containing 0.1 g of initiator LAP, and heat it in a 40-50° C. water bath to dissolve it.

4. Application of the CeO2 nanozyme SilMA hydrogel loaded according to claim 1 in 3D bioprinting tissue engineering trachea.

5. The use according to claim 4, characterized in that The steps include: Step 1: Cell culture and differentiation; 1) Cultivate primary tracheal mucosal epithelial cells in vitro and perform subculture, 2) Cultivate and differentiate bone marrow mesenchymal stem cells (BMSCs) in vitro to obtain chondrocytes and vascular endothelial cells; Step 2: Preparation of bio-ink; 1) Preparation of inner bio-ink: The obtained epithelial cells, BMSCs, and epidermal growth factor (EGF) were mixed into CeO2 / SilMA hydrogel as the inner bio-ink for constructing the epithelial cell layer; 2) Preparation of outer bio-ink: Vascular endothelial cells, chondrocytes, BMSCs, growth factor TGF-β, and vascular endothelial growth factor VEGF were mixed into CeO2 / SilMA hydrogel as the outer bio-ink for constructing the chondrocyte layer; Step 3: 3D printing polycaprolactone tracheal stent; Based on the dynamic CT imaging data of the trachea, a tracheal stent was 3D printed using polycaprolactone as the material; Step 4: constructing the tracheal structure through photocuring 3D bioprinting technology; The inner layer of bio-ink is evenly 3D printed onto the inner wall of the tracheal stent obtained in step three, with a thickness of 0.5 mm; the outer layer of bio-ink is evenly 3D printed onto the outer wall of the tracheal stent obtained in step three, with a thickness of 0.5 mm.

6. Use according to claim 5, characterized in that The tracheal stent obtained in step three was immersed in the SilMA hydrogel microsphere solution loaded with CeO2 nanozyme at 37°C for 15 minutes, and fully cross-linked under blue light for 5 minutes, so that the SilMA hydrogel microsphere loaded with CeO2 nanozyme evenly wrapped the tracheal stent.

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