Microenvironment-responsive self-releasing oxygen bio-3d printing ink, bio-3d printing scaffold and preparation method thereof

Abstract

The application discloses a microenvironment response self-releasing oxygen biological 3D printing ink, a biological 3D printing support and a preparation method. The preparation method of the biological 3D printing ink comprises the following steps: preparing polydopamine modified nanoparticles; mixing the polydopamine modified nanoparticles, tyramine modified natural polymer and mesenchymal stem cell suspension to obtain the biological 3D printing ink. The polydopamine modified nanoparticles are added to the biological ink containing the tyramine modified natural polymer and the mesenchymal stem cells, and the polydopamine has the microenvironment response self-releasing oxygen capability, so that the nanoparticles added to the biological ink containing the tyramine modified natural polymer and the mesenchymal stem cells can increase the self-releasing oxygen capability of the biological ink. The biological 3D printing ink is subjected to 3D printing to prepare the biological 3D printing support, and the support can protect the survival and proliferation of the mesenchymal stem cells under the oxidative stress condition.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a bio-3D printing ink, a bio-3D printing scaffold, and a preparation method thereof that respond to the microenvironment of bone defects and release oxygen spontaneously. Background Technology

[0002] Bone defects are often accompanied by local microvascular rupture, and regeneration and repair in the hypoxic microenvironment caused by blood supply interruption remains a challenge. On the one hand, hypoxia affects the proliferation and viability of endogenous stem cells; on the other hand, hypoxia induces reductive carboxylation in mitochondria, leading to the large-scale production of reactive oxygen species (ROS).

[0003] Bioprinting is a technology that uses a mixture of biological materials and cells as bio-ink to print cell-loaded scaffolds using 3D printing equipment. This technology provides a new strategy for tissue repair in tissue engineering. To ensure that the cells in the bio-ink can survive and proliferate normally after printing, bioprinting typically selects natural polymers as substrate materials, such as gelatin, alginate, collagen, and silk fibroin.

[0004] Currently, bio-3D printed scaffolds based on natural polymers have been extensively studied and widely applied in the field of biomedical materials. For example, patent application publication number CN117205364A discloses a 3D printed bio-ink, a functional scaffold, and its preparation method for bone defect repair, using gelatin and alginate as base materials; patent application publication number CN117159803A discloses a 3D printed bio-ink, its preparation method, and its application, using collagen as the base material. However, in these methods, natural polymer inks typically lack the ability to release oxygen in response to the microenvironment, resulting in poor cell survival in 3D printed cell-carrying scaffolds when facing the hypoxic microenvironment of the defect site.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a bio-3D printing ink, a bio-3D printing scaffold, and a preparation method that are responsive to the microenvironment of bone defects and release oxygen. This invention aims to solve the problem that existing natural polymer inks do not have the ability to release oxygen in response to the microenvironment, and that the cell survival of 3D printed cell-carrying scaffolds is poor when facing the hypoxic microenvironment of bone defect sites.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing a bio-3D printing ink that responds to the microenvironment of bone defects and releases oxygen spontaneously, comprising the steps of:

[0009] Preparation of polydopamine-modified nanoparticles;

[0010] The polydopamine-modified nanoparticles, tyramine-modified natural polymers, and mesenchymal stem cell suspensions are mixed to obtain a bio-3D printing ink.

[0011] Optionally, the nanoparticles are selected from one of hydrotalcite, manganese dioxide, cerium dioxide, and fullerene.

[0012] Optionally, the method for preparing polydopamine-modified nanoparticles includes the steps of: mixing nanoparticles and dopamine hydrochloride at a predetermined mass ratio in a Tris-HCl buffer solution or an alkaline solution (such as sodium hydroxide aqueous solution, ammonia water, etc.) with a pH of 7.5 to 9.0, and then stirring the mixture to obtain polydopamine-modified nanoparticles.

[0013] Optionally, the nanoparticles are mixed with dopamine hydrochloride at a mass ratio of 1:1 to 5:1, and the stirring reaction time is 2 to 12 hours.

[0014] Optionally, the step of mixing the polydopamine-modified nanoparticles, tyramine-modified natural polymers, and mesenchymal stem cell suspension to obtain bio-3D printing ink specifically includes: dissolving the tyramine-modified natural polymers in water under sterile conditions, then adding the polydopamine-modified nanoparticles, mixing evenly, and then adding the mesenchymal stem cell suspension to obtain bio-3D printing ink.

[0015] Optionally, the bio-3D printing ink contains 5% to 20% tyramine-modified natural polymer, 0.1% to 2% polydopamine-modified nanoparticles, and a concentration of 500,000 to 10,000,000 / mL of mesenchymal stem cells.

[0016] Optionally, the natural polymer is selected from one of gelatin, alginate, collagen, silk fibroin, and hyaluronic acid.

[0017] In a second aspect, the present invention provides a bio-3D printing ink that responds to the microenvironment of bone defects and releases oxygen spontaneously, wherein the ink is prepared by the method described in the present invention.

[0018] A third aspect of the present invention provides a method for preparing a bio-3D printed scaffold that responds to the microenvironment of bone defects and releases oxygen spontaneously, comprising the steps of: using a 3D printing device to print the bio-3D printing ink described in the present invention to obtain a bio-3D printed scaffold.

[0019] In a fourth aspect, the present invention provides a bio-3D printed scaffold that responds to the microenvironment of bone defects and releases oxygen spontaneously, wherein the scaffold is prepared by the method described in the present invention.

[0020] Beneficial Effects: This invention provides a microenvironment-responsive, self-releasing oxygen bio-3D printing ink for bone defects, a bio-3D printing scaffold, and their preparation method. Polydopamine-modified nanoparticles are added to a bio-ink containing tyramine-modified natural polymers and mesenchymal stem cells. Because polydopamine possesses microenvironment-responsive self-releasing oxygen capabilities, the addition of these nanoparticles to the tyramine-modified natural polymer bio-ink increases the self-releasing oxygen capacity of the bio-ink. The bio-3D printing ink, through 3D printing, can produce a bio-3D printing scaffold. This scaffold possesses microenvironment-responsive oxygen release capabilities and good biocompatibility, and can protect the survival and proliferation of mesenchymal stem cells within the bio-3D printing scaffold under oxidative stress conditions. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the biological 3D printed scaffold prepared in Example 1.

[0022] Figure 2 This is a schematic diagram showing the test results of the oxygen release capacity of the bio-3D printed scaffolds prepared in Example 1 and Comparative Example 1.

[0023] Figure 3 This is a schematic diagram showing the test results of the biocompatibility of the bio-3D printed scaffold prepared in Example 1.

[0024] Figure 4 This is a schematic diagram showing the test results of the cell protection ability of the bio-3D printed scaffolds prepared in Example 1 and Comparative Example 1 under oxidative stress. Detailed Implementation

[0025] This invention provides a bio-3D printing ink, a bio-3D printing scaffold, and a preparation method that respond to the microenvironment of bone defects and release oxygen spontaneously. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] The inventors discovered that existing technologies have the following drawbacks: a) Natural polymer bio-3D printing scaffolds do not have antioxidant capabilities; b) Natural polymer bio-3D printing scaffolds do not have the ability to release oxygen in response to the microenvironment; c) Natural polymer bio-inks cannot be used together with nanoparticles for cell-borne bio-3D printing; d) Natural polymer cell-borne bio-3D printing scaffolds cannot protect cell survival and proliferation under high oxidative stress.

[0027] This invention addresses the problems existing in the prior art by providing a bio-3D printing ink that can be used to address the microenvironmental response of bone defects. This bio-3D printing ink possesses a self-releasing oxygen capability in response to the microenvironment. Using this bio-3D printing ink to construct bio-3D printed scaffolds, the scaffolds can protect cells to survive and proliferate normally under oxidative stress through microenvironment-responsive self-releasing oxygen, laying the foundation for the widespread application of natural polymer bio-inks in various tissue engineering fields.

[0028] Natural polymers are organic substances that exist in nature and possess a high molecular weight structure. These polymers are typically composed of repeating monomer units and have a high molecular weight. Compared to synthetic polymers, natural polymers are more biocompatible and biodegradable. Natural polymers contain a large number of amino acid residues, allowing their properties to be improved through chemical modification. Tyramine is a polyphenol compound that can undergo a free radical-initiated oxidative polymerization reaction under the combined action of horseradish peroxidase and an oxidant (usually H₂O₂), catalyzing its oxidative polymerization.

[0029] Hydrotalcite is a type of compound with a special layered structure that can recombine divalent and trivalent metal ions. Under certain conditions, electron exchange can occur between these ions, and hydrotalcite can catalyze certain chemical reactions through this process. Dopamine (DA) is a type of catecholamine with the molecular formula C8H2O. 11 NO2, under alkaline conditions, deprotonates and oxidizes the dopamine monomer catechol to form dopaquinone. However, dopaquinone is structurally unstable and will undergo further oxidation, resulting in intramolecular rearrangement and cross-linking, forming dark brown polydopamine. Polydopamine can modify nanomaterials through the auto-oxidative polymerization of dopamine. Due to its adhesiveness, reducing properties, and good biocompatibility, polydopamine has been widely used in biomedical materials.

[0030] Mesenchymal stem cells (MSCs) are pluripotent stem cells that can be isolated from a variety of tissues, most commonly bone marrow and adipose tissue. These cells have the ability to self-renew and can differentiate into many different cell types, such as osteocytes, chondrocytes, and adipocytes.

[0031] Based on this, embodiments of the present invention modify nanoparticles (NPs) such as hydrotalcite, manganese dioxide, cerium dioxide, and fullerene with polydopamine (PDA). This modification allows the addition of these nanoparticles to bio-inks containing tyramine-modified natural polymers (including but not limited to gelatin, alginate, collagen, silk fibroin, hyaluronic acid, etc.) and mesenchymal stem cells, thereby endowing the bio-ink with a microenvironment-responsive self-releasing oxygen capability. Furthermore, by constructing a bio-3D printed scaffold using 3D printing equipment, the mesenchymal stem cells within the scaffold can be protected to migrate and proliferate normally under oxidative stress conditions such as inflammation, further expanding the application value of natural polymer materials in the field of tissue engineering.

[0032] Specifically, this invention provides a method for preparing a bio-3D printed scaffold that responds to the microenvironment of bone defects and releases oxygen spontaneously, comprising the following steps:

[0033] Preparation of polydopamine-modified nanoparticles;

[0034] The polydopamine-modified nanoparticles, tyramine-modified natural polymers, and mesenchymal stem cell suspensions are mixed to obtain a bio-3D printing ink.

[0035] In this invention, polydopamine-modified nanoparticles are added to a bio-ink containing tyramine-modified natural polymers and mesenchymal stem cells. Because polydopamine possesses a microenvironment-responsive self-oxygen release capability, this increases the self-oxygen release capacity of the bio-ink. The bio-3D printing ink is then used to prepare a bio-3D printed scaffold, which can protect the survival and proliferation of mesenchymal stem cells under oxidative stress conditions.

[0036] In one embodiment, the nanoparticles are selected from one of hydrotalcite, manganese dioxide, cerium dioxide, fullerene, etc.

[0037] Taking cobalt-based hydrotalcite (LDH) as an example, the preparation method of cobalt-based hydrotalcite includes the following steps: at room temperature, ZIF-67 is mixed with cobalt nitrate solution and reacted for 5 hours; the precipitate is collected by centrifugation; the collected precipitate is washed successively with deionized water and anhydrous methanol to obtain pure LDH.

[0038] In one embodiment, the method for preparing polydopamine-modified nanoparticles includes the steps of: mixing nanoparticles with dopamine hydrochloride in a Tris-HCl buffer solution or an alkaline solution (such as sodium hydroxide aqueous solution, ammonia water, etc.) with a pH of 7.5 to 9.0, and then stirring the mixture to obtain polydopamine-modified nanoparticles.

[0039] Preferably, the nanoparticles are mixed with dopamine hydrochloride at a mass ratio of 1:1 to 5:1 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1, etc.), and the stirring reaction time is 2 to 12 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.).

[0040] Taking polydopamine-modified LDH as an example, the preparation method of polydopamine-modified LDH includes the following steps: mixing LDH and dopamine hydrochloride (DA·HCl) in a certain mass ratio in Tris-HCl buffer solution with pH 8.5, reacting for a period of time, and then washing the product to obtain polydopamine-modified LDH (denoted as LDH@PDA nanomaterial).

[0041] In one embodiment, the step of mixing the polydopamine-modified nanoparticles, tyramine-modified natural polymers, and mesenchymal stem cell suspension to obtain bio-3D printing ink specifically includes: under sterile conditions, dissolving the tyramine-modified natural polymers in water, then adding the polydopamine-modified nanoparticles, mixing evenly (which can be achieved by stirring), and then adding the mesenchymal stem cell suspension to obtain bio-3D printing ink.

[0042] In one embodiment, the polydopamine-modified nanoparticles are added in the form of a polydopamine-modified nanoparticle suspension, which is prepared by dissolving the polydopamine-modified nanoparticles in deionized water, phosphate buffer, or Tris-HCl buffer to obtain the polydopamine-modified nanoparticle suspension.

[0043] In one embodiment, the mesenchymal stem cell suspension is obtained by the following method: mesenchymal stem cells are digested with trypsin for 1-5 minutes, then complete culture medium is added to stop the digestion, the mesenchymal stem cells are pipetted off and collected in a centrifuge tube, the complete culture medium is discarded after centrifugation of the centrifuge tube, and the cells are resuspended in complete culture medium to obtain the mesenchymal stem cell suspension.

[0044] In one embodiment, the bio-3D printing ink contains 5% to 20% by mass of tyramine-modified natural polymer (e.g., 5%, 8%, 10%, 15%, 20%), 0.1% to 2% by mass of polydopamine-modified nanoparticles (e.g., 0.1%, 0.5%, 1%, 1.5%, 2%), and 500,000 to 10,000,000 / mL by concentration of mesenchymal stem cells (e.g., 500,000 / mL, 1,000,000 / mL, 1,500,000 / mL, 2,000,000 / mL, 3,000,000 / mL, 4,500,000 / mL, 6,000,000 / mL, 7,000,000 / mL, 8,000,000 / mL, 9,000,000 / mL, 10,000,000 / mL).

[0045] In one embodiment, the natural polymer is selected from gelatin, alginate, collagen, silk fibroin, hyaluronic acid, etc.

[0046] This invention provides a bio-3D printing ink that responds to the microenvironment of bone defects and releases oxygen spontaneously, wherein the ink is prepared using the method described in this invention.

[0047] This invention provides a method for preparing a bio-3D printed scaffold that responds to the microenvironment of bone defects and releases oxygen spontaneously, comprising the steps of: using a 3D printing device to print the bio-3D printing ink described in this invention to obtain a bio-3D printed scaffold.

[0048] In one embodiment, the step of using a 3D printing device to print the bio-3D printing ink described in the embodiments of the present invention to obtain a bio-3D printed scaffold specifically includes:

[0049] Under aseptic conditions, bio-3D printing ink is loaded into a cartridge compatible with the 3D printing equipment. Printing parameters are set, and after adjustment, a plastic pad is placed on the printing platform, and the scaffold is printed according to the predetermined program. Once printing is complete, the scaffold is placed in an enzyme cross-linking agent for cross-linking, thus obtaining the bio-3D printed scaffold. The bio-3D printed scaffold is then transferred to a complete culture medium for cultivation.

[0050] This invention provides a bio-3D printed scaffold that responds to the microenvironment of bone defects and releases oxygen spontaneously, wherein the scaffold is prepared using the method described in this invention.

[0051] The present invention will be further described below through several embodiments.

[0052] Example 1

[0053] 1. Preparation of polydopamine-modified nanoparticles (the nanoparticles are hydrotalcite (LDH) nanoparticles), the specific preparation process is as follows:

[0054] (1) At room temperature, 10 mg ZIF-67 was mixed with 10 mL of 24 mM cobalt nitrate solution and reacted for 5 h. After the reaction was completed, the precipitate was collected by centrifugation and washed with deionized water and anhydrous methanol in sequence to obtain pure LDH nanoparticles.

[0055] (2) LDH nanoparticles and dopamine hydrochloride (DA·HCl) were mixed in a Tris-HCl buffer solution at a mass ratio of 1:2.5 at pH 8.5, and then the mixture was stirred for 3 h. The product was washed with deionized water and anhydrous methanol to obtain LDH@PDA nanomaterials. The LDH@PDA nanomaterials were dissolved in deionized water to obtain an LDH@PDA suspension with a concentration of 10 mg / mL.

[0056] 2. Preparation of bio-3D printing ink (using tyramine-modified gelatin as the base material) and enzyme cross-linking agent. The specific preparation process is as follows:

[0057] (1) Under sterile conditions, 0.5g of freeze-dried Gel-Tyr (referring to tyramine-modified gelatin) was placed in a 50mL centrifuge tube, 4.5mL of deionized water and 4.0mL of α-MEM culture medium were added to the centrifuge tube, and then the tube was placed in a water bath to dissolve. The water bath temperature was set to 40℃.

[0058] (2) After Gel-Tyr is completely dissolved, add 500 μL of LDH@PDA suspension and mix well to obtain cell-free Gel-Tyr-LDH@PDA bio-ink. Mesenchymal stem cells are digested with trypsin for 1 min, then digestion is stopped by adding complete culture medium. The cells are pipetted off and collected in centrifuge tubes. After centrifugation, the culture medium is discarded, and the cells are resuspended in complete culture medium to obtain a cell suspension. 500 μL of the cell suspension is added to the cooled cell-free Gel-Tyr-LDH@PDA bio-ink to obtain the bio-3D printing ink. The bio-3D printing ink contains 10% Gel-Tyr, 1% LDH@PDA, and a cell concentration of 1 million / mL.

[0059] (3) Preparation of enzyme cross-linking agent: Dissolve 400U horseradish peroxidase (HRP) in 20mL PBS (phosphate buffered saline solution), add 12μL 30% H2O2 solution, mix well and obtain enzyme cross-linking agent for later use.

[0060] 3. Fabrication of the biological 3D printing scaffold: The specific fabrication process is as follows:

[0061] (1) Load the prepared bio-3D printing ink into the cartridge that matches the printer, select a needle with a diameter of 400μm that matches the cartridge, and load it into the low-temperature nozzle.

[0062] (2) Set printing parameters: Set the bracket shape to a cuboid with a length of 13mm, a width of 13mm, a height of 0.25mm per layer, a total of 12 layers, and rotate the printing direction 90° after each layer is printed. Set the filament to emerge 300ms in advance.

[0063] (3) When printing, set the printing pressure to about 40 kPa and the barrel temperature to 23°C. Adjust the printing pressure and temperature according to the room temperature and the filament output during pre-extrusion until the filament output is smooth. Set the moving speed of the printing needle according to the filament output speed.

[0064] (4) After the parameters are adjusted, place the plastic pad on the printing platform and print the bracket according to the established procedure.

[0065] (5) After the scaffold is printed, first immerse the scaffold in the enzyme cross-linking agent on the pad for 5 minutes for pre-cross-linking.

[0066] (6) After pre-crosslinking is complete, gently separate the scaffold from the bottom plate with a spatula, place the scaffold in the enzyme crosslinking agent, and crosslink for 10 minutes to obtain the bio-3D printed scaffold. Figure 1 ).

[0067] Comparative Example 1

[0068] 1. Preparation of Gel-Tyr bio-3D printing ink: The specific preparation process is as follows:

[0069] (1) Under sterile conditions, place 0.5g of freeze-dried Gel-Tyr into a 50mL centrifuge tube, add 4.0mL of α-MEM culture medium to the centrifuge tube, dissolve it in a water bath, and set the water bath temperature to 40℃.

[0070] (2) After the Gel-Tyr is completely dissolved, add 500 μL of α-MEM medium and mix well to obtain cell-free Gel-Tyr bio-ink. Digest mesenchymal stem cells with trypsin for 1 min, then add complete medium to stop the digestion. Collect the cells by pipetting them into centrifuge tubes, centrifuge, discard the medium, and resuspend the cells in complete medium to obtain a cell suspension. Add 500 μL of the cell suspension to the cooled cell-free Gel-Tyr bio-ink to obtain the bio-3D printing ink. The bio-3D printing ink contains 10% Gel-Tyr by mass and a cell concentration of 1 million / mL.

[0071] 2. Fabrication of Gel-Tyr bio-3D printing scaffold: The specific fabrication process is as follows:

[0072] (1) Load the prepared bio-3D ink into the cartridge that matches the printer, select a needle with a diameter of 400μm that matches the cartridge, and load it into the low-temperature nozzle.

[0073] (2) Set printing parameters: Set the bracket shape to a cuboid with a length of 13mm, a width of 13mm, a height of 0.25mm per layer, a total of 12 layers, and rotate the printing direction 90° after each layer is printed. Set the filament to emerge 300ms in advance.

[0074] (3) When printing, set the printing pressure to about 40 kPa and the barrel temperature to 23°C. Adjust the printing pressure and temperature according to the room temperature and the filament output during pre-extrusion until the filament output is smooth. Set the moving speed of the printing needle according to the filament output speed.

[0075] (4) After the parameters are adjusted, place the plastic pad on the printing platform and print the bracket according to the established procedure.

[0076] (5) After the scaffold is printed, first immerse the scaffold in the enzyme cross-linking agent on the pad for 5 minutes for pre-cross-linking.

[0077] (6) After the pre-crosslinking is completed, gently separate the scaffold from the bottom plate with a medicine spoon, put the scaffold into the enzyme crosslinking agent, crosslink for 10 minutes, and you can obtain the Gel-Tyr bio-3D printed scaffold.

[0078] The oxygen release capacity of the bio-3D printed scaffolds prepared in Example 1 and Comparative Example 1 were tested respectively. The specific test steps are as follows: (1) After the bio-3D printed scaffolds were prepared, they were placed in a 100mM hydrogen peroxide solution to simulate an oxidative stress microenvironment. (2) The oxygen concentration in the solution was tested using a dissolved oxygen meter after 1 hour. (3) The oxygen concentration in the solution was tested using a dissolved oxygen meter after 24 hours. The results showed that the ability of the bio-3D printed scaffolds to release oxygen in response to the microenvironment was significantly improved (see Figure 2 (As shown in A and B).

[0079] The biocompatibility of the 3D-printed biological scaffold prepared in Example 1 was tested. The specific test steps are as follows: (1) After the 3D-printed biological scaffold was prepared, it was cultured in complete culture medium; (2) FDA / PI live / dead staining was performed on the cell-loaded 3D-printed biological scaffold at 1, 3, and 7 days; (3) The cell survival in the 3D-printed biological scaffold was observed using a confocal microscope. The results showed that the 3D-printed biological scaffold had good biocompatibility, and cells could survive and proliferate normally in it (see Figure 3 (As shown).

[0080] The ability of the bio-3D printed scaffolds prepared in Example 1 and Comparative Example 1 to protect cell survival and proliferation under oxidative stress was tested. The specific test steps are as follows: (1) After the bio-3D printed scaffolds were prepared, they were cultured in a complete culture medium containing 200 μmol H2O2; (2) FDA / PI live / dead staining was performed on the cell-loaded bio-3D printed scaffolds after 24 h of culture; (3) The cell survival in the bio-3D printed scaffolds was observed using a confocal microscope. The results showed that the bio-3D printed scaffolds had a strong ability to protect cell survival and proliferation under oxidative stress, and cells could survive and proliferate normally in them (see Figure 4 (As shown).

[0081] As shown above, the 3D printing ink with added polydopamine-modified nanoparticles significantly enhances the ability of the 3D printed scaffolds prepared with this ink to release oxygen under oxidative stress compared to the unmodified ink group. Furthermore, the 3D printed scaffolds prepared with this ink exhibit good biocompatibility. Additionally, the 3D printed scaffolds prepared with this ink demonstrate significantly improved ability to protect cell survival and proliferation under oxidative stress compared to the unmodified ink group.

[0082] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a bio-3D printing ink that self-releases oxygen in response to the microenvironment of bone defects, characterized in that, Including the following steps: Preparation of polydopamine-modified nanoparticles; The polydopamine-modified nanoparticles, tyramine-modified natural polymers, and mesenchymal stem cell suspensions are mixed to obtain a bio-3D printing ink. The method for preparing polydopamine-modified nanoparticles includes the following steps: mixing nanoparticles with dopamine hydrochloride in a Tris-HCl buffer solution or alkaline solution with a pH of 7.5 to 9.0, and then stirring the mixture to obtain polydopamine-modified nanoparticles. The nanoparticles are mixed with dopamine hydrochloride at a mass ratio of 1:1 to 5:1, and the stirring reaction time is 2 to 12 hours. The bio-3D printing ink contains 5%–20% tyramine-modified natural polymer, 0.1%–2% polydopamine-modified nanoparticles, and a concentration of 500,000–10,000,000 / mL of mesenchymal stem cells. The nanoparticles are hydrotalcite.

2. The method for preparing the self-releasing oxygen bio-3D printing ink responsive to the bone defect microenvironment according to claim 1, characterized in that, The steps of mixing the polydopamine-modified nanoparticles, tyramine-modified natural polymers, and mesenchymal stem cell suspension to obtain bio-3D printing ink specifically include: dissolving the tyramine-modified natural polymers in water under sterile conditions, then adding the polydopamine-modified nanoparticles, mixing evenly, and then adding the mesenchymal stem cell suspension to obtain the bio-3D printing ink.

3. The method for preparing the self-releasing oxygen bio-3D printing ink responsive to the bone defect microenvironment according to claim 1, characterized in that, The natural polymer is selected from one of gelatin, alginate, collagen, silk fibroin, and hyaluronic acid.

4. A bio-3D printing ink that self-releases oxygen in response to the microenvironment of bone defects, characterized in that, The bio-3D printing ink with self-releasing oxygen in response to the bone defect microenvironment, as described in any one of claims 1-3, was prepared.

5. A method for preparing a bio-3D printed scaffold that responds to the microenvironment of bone defects and releases oxygen spontaneously, characterized in that, The steps include: using a 3D printing device to print the bio-3D printing ink described in claim 4 to obtain a bio-3D printed scaffold.

6. A bio-3D printed scaffold that responds to the microenvironment of bone defects and releases oxygen spontaneously, characterized in that, The 3D-printed bio-scaffold with self-releasing oxygen in response to the bone defect microenvironment, as described in claim 5, was prepared.

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