A 3D printing scaffold with high bioactivity and high strength and a preparation method thereof

Abstract

This invention belongs to the field of bone tissue engineering, and particularly relates to a 3D-printed scaffold with improved bioactivity and high strength, and its preparation method. The method includes the following steps: 3D printing a bio-ink to form a scaffold; the bio-ink comprises an organic phase and an inorganic phase, the inorganic phase comprising osteoinductive calcium phosphate modified with at least one of tannic acid, gelatin, quaternized chitosan, and silk fibroin; the organic phase is selected from at least one of sodium alginate, polylactic acid, polyvinyl alcohol, polycaprolactone, and polyetheretherketone; the mass ratio of the inorganic phase to the organic phase is 1:(0.5-2). The scaffold prepared by this invention exhibits high strength and bioactivity.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering, and particularly relates to a highly bioactive and high-strength 3D-printed scaffold and its preparation method. Background Technology

[0002] The skeletal system, as one of the most important organs in the human body, is responsible for supporting daily movements, transporting nutrients, and delivering red blood cells. However, bone defects caused by tumors, arthritis, trauma, and osteoporosis seriously affect patients' health. While human bone tissue has a certain capacity for internal repair, once the defect exceeds a critical size, repair through methods such as bone grafting is necessary. Common bone grafts include allogeneic bone grafts and xenograft bone grafts, with xenograft bone grafts further divided into allogeneic and xenogeneic bone grafts.

[0003] Allogeneic bone grafting, using materials taken from the patient's own body, is the ideal method for bone repair due to its good biocompatibility. However, its availability is limited, and it can cause secondary damage to the harvesting site, thus restricting its application. Allogeneic bone grafting uses materials from donors, which is also limited in availability and carries the risk of triggering host immune responses and transmitting potential diseases. Xenogeneic bone grafting uses materials from different organisms, making it widely available, but it also faces serious host immune response issues. Therefore, synthetic bone repair materials have emerged. Tissue engineering, an emerging interdisciplinary field in recent years, refers to the technology of using the principles and methods of life sciences and engineering sciences to develop functional tissues / organs for in vivo transplantation to alleviate organ shortages or as in vitro models to study disease mechanisms and discover drugs.

[0004] The successful application of bone tissue engineering can overcome the limitations of current treatment options. As an important component of bone tissue engineering, scaffolds can mimic the properties of the extracellular matrix, providing templates for cell adhesion, growth, proliferation, and differentiation, and providing structural support for newly generated tissues. They also play an important role in bone tissue integration and angiogenesis. In addition, the interconnected porosity is very important for the continuous growth of bone tissue. The porous structure of bone tissue engineering scaffolds provides interconnected channels for cell migration, ion transport, cell-cell interactions, and the transport of oxygen, nutrients, and metabolites. Therefore, ideally, bone tissue engineering scaffolds should have the following characteristics: (1) an interconnected porous network; (2) biocompatibility and bioabsorbability with a controllable degradation and absorption rate to match the growth of cells / tissues in vitro and / or in vivo; (3) a surface suitable for cell attachment, proliferation, and differentiation; and (4) mechanical properties that match the mechanical properties of the implantation site tissue.

[0005] 3D printing technology, defined by the American Society for Testing and Materials (ASMT) as "a process of creating solid objects by connecting materials using three-dimensional model data, typically layer by layer, and is a process fundamentally different from subtractive manufacturing methods," offers advantages such as high printing precision, the ability to customize bio-devices for patient defects, and low cost. The greatest advantage of 3D bioprinting lies in its potential to spatially distribute cells within solid or semi-solid biomaterials, thereby optimizing tissue regeneration. By converting computed tomography (CT) or micro-CT data into printable images, implant models can be created for patient defects, reconstructing complex bone defects. Therefore, the development of 3D bioprinted bone tissue has significant implications for clinical practice.

[0006] Currently, 3D printing technology has been widely applied to tissue repair in various biological fields, including bone, cartilage, skin, vascular tissue, and the heart. Various biomaterials have been researched and developed for the repair of different tissues. In terms of composition, biomaterials mainly include metallic materials, ceramic materials, polymeric materials, and their composites. Metallic materials have outstanding mechanical strength and good wear resistance, but most metallic materials lack bioactivity and have issues such as short service life and the risk of requiring secondary surgery. Polymeric materials have good processing performance and bioinertness, but poor mechanical properties. Ceramic materials have good biocompatibility, high bioactivity, strong biodegradability, and certain osteoinductive and osteoconductive properties, but are brittle. Therefore, composite materials prepared by combining ceramic materials with other materials can integrate the advantages of various materials, opening up new avenues for the development of bone repair materials.

[0007] From a material composition perspective, natural bone tissue is a highly ordered tissue structure formed by the self-assembly of inorganic components (hydroxyapatite) and organic components (type I collagen). At the nanoscale, bone is composed of numerous structural proteins and polysaccharides, the main components of which are collagen fibrils with diameters between 35 and 60 nm and lengths up to 1 μm, exhibiting a periodicity of 67 nm and an interstitial gap of 40 nm. These fibrils are mineralized by anisotropic and extremely hard inorganic components—hydroxyapatite crystals located within the collagen interstitials. Therefore, bone tissue can be viewed as a composite of inorganic (hydroxyapatite) and organic phases (type I collagen). Organic / inorganic composite scaffolds can mimic the main components of bone tissue in terms of composition and often possess excellent biocompatibility and biodegradability. However, in addition to excellent biocompatibility and biodegradability, an ideal bone repair scaffold also requires mechanical properties that match those of the implantation site tissue. Currently, most organic / inorganic composite scaffolds are simply mixtures of organic and inorganic phases, often resulting in poor mechanical properties and limiting their application to repairing bone defects in non-load-bearing areas. This is because while current organic / inorganic composite scaffolds mimic bone tissue in terms of composition, they differ from the ordered arrangement and bonding of collagen and hydroxyapatite in natural bone tissue. The organic and inorganic components in current organic / inorganic composite scaffolds are often simply mixed without connecting bridges, which easily leads to separation of the inorganic and organic phases, reducing mechanical properties and thus limiting their applications. Developing a method to connect the inorganic and organic components is an ideal strategy to improve the mechanical properties of current organic / inorganic composite scaffolds.

[0008] In traditional surface modification strategies, most coupling agents are grafted onto powder surfaces via covalent bonds with surface hydroxyl groups. However, the number and reactivity of active hydroxyl groups on powder surfaces are limited, significantly hindering the application of these strategies. Mussels, marine mollusks, move along the seabed primarily by secreting large amounts of byssal proteins rich in L-3,4-dihydroxyphenylalanine and lysine, which adhere to rocks. These proteins connect rocks and mussels, aiding their movement in seawater. Inspired by the mussel adhesion mechanism, using a polymeric adhesive as a "bridge" to connect inorganic and organic phases has become a research hotspot. The main components of byssal proteins contain numerous catechol functional groups, which can modify non-specific surfaces such as rocks, a key aspect of the mussel adhesion mechanism. Tannic acid (TA), a plant polyphenol, contains numerous catechol groups in its structure and can strongly adhere to inorganic and metallic surfaces through surface bonding, hydrogen bonding, or the formation of catechol / gallic acid-metal complexes. Furthermore, due to its unique chemical structure and high functional group density, surface-fixed TA molecules can be further modified or bonded to polymers. This unique adhesion ability makes TA an ideal "bridging" molecule between inorganic and organic phases.

[0009] However, existing 3D-printed bone repair scaffolds still suffer from low mechanical strength and poor bioactivity. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a highly bioactive and high-strength 3D-printed bone repair scaffold and its preparation method, which can improve scaffold strength and enhance bioactivity by at least one of the following:

[0011] Specifically, the present invention provides the following technical solution:

[0012] Includes the following steps:

[0013] Specifically, the present invention provides the following technical solution: In a first aspect, the present invention provides a bio-ink, the bio-ink comprising an organic phase and an inorganic phase, the inorganic phase comprising bone-inducing calcium phosphate modified by at least one of tannic acid, gelatin, quaternized chitosan, and silk fibroin, the organic phase being selected from at least one of sodium alginate, polylactic acid, polyvinyl alcohol, polycaprolactone, and polyetheretherketone.

[0014] According to an embodiment of the present invention, the mass ratio of the inorganic phase to the organic phase is 1:(0.5-2), preferably 1:(1-1.5), for example 1:1.

[0015] According to an embodiment of the present invention, the ink further includes a first solvent, such as deionized water.

[0016] According to an embodiment of the present invention, the mass ratio of the inorganic phase, the organic phase and the first solvent is (4-6):(4-6):(150-300), and preferably the mass ratio of the inorganic phase, the organic phase and the first solvent is (4-6):(4-6):(180-250).

[0017] According to an embodiment of the present invention, the bone-inducing calcium phosphate is selected from one or more mixtures of tricalcium phosphate, hydroxyapatite, tetracalcium phosphate, calcium dihydrogen phosphate, and octacalcium phosphate, for example, octacalcium phosphate.

[0018] According to an embodiment of the present invention, the octacalcium phosphate is prepared using existing methods, such as co-precipitation.

[0019] As an example, the octacalcium phosphate is prepared by mixing ammonium dihydrogen phosphate, urea, calcium acetate and deionized water, and then obtaining octacalcium phosphate powder by co-precipitation.

[0020] According to an embodiment of the present invention, the molar ratio of ammonium dihydrogen phosphate, urea, calcium acetate and deionized water is 12:9:30:(15000-45000).

[0021] According to an embodiment of the present invention, the octacalcium phosphate is prepared by the following method: first, ammonium dihydrogen phosphate and urea are dissolved in deionized water, and then calcium acetate solution is added to carry out a co-precipitation reaction.

[0022] According to an embodiment of the present invention, the temperature of the coprecipitation reaction is 75-95°C, and the reaction time is 1-4 hours.

[0023] According to an embodiment of the present invention, the inorganic phase comprises tannic acid and bone-inducing calcium phosphate modified with quaternized chitosan.

[0024] According to an embodiment of the present invention, the organic phase is sodium alginate.

[0025] According to an embodiment of the present invention, the gelatin is selected from modified or unmodified gelatin, and the modified gelatin is preferably at least one of methacrylated gelatin and gelatin methacrylate.

[0026] Secondly, the present invention also provides a method for preparing the above-mentioned bio-ink, comprising the following steps: reacting at least one of tannic acid, gelatin, quaternized chitosan or silk fibroin with bone-inducing calcium phosphate to obtain an inorganic phase; mixing the inorganic phase with an organic phase and water and then freeze-drying to obtain a foamed solid; and mixing the foamed solid with a first solvent to obtain the bio-ink.

[0027] According to an embodiment of the present invention, the preparation method of the bio-ink includes the following steps: reacting tannic acid and octacalcium phosphate powder in a good solvent to obtain tannic acid-coated octacalcium phosphate powder; reacting quaternized chitosan with tannic acid-coated octacalcium phosphate powder to obtain quaternized chitosan and tannic acid-coated octacalcium phosphate powder; mixing with water and sodium alginate and then freeze-drying into a foamed solid; mixing the foamed solid with a first solvent to obtain the bio-ink.

[0028] As an example, reacting tannic acid with octacalcium phosphate powder in a second solvent to obtain tannic acid-coated octacalcium phosphate powder involves the following steps: dispersing octacalcium phosphate powder in a Tris-HCl buffer solution, dissolving tannic acid in the above solution, stirring and filtering to obtain tannic acid-coated octacalcium phosphate powder.

[0029] According to an embodiment of the present invention, the pH value of the tris-HCl buffer solution is 7.5-9.5.

[0030] According to an embodiment of the present invention, the mass ratio of the octacalcium phosphate powder, tannic acid, and Tris-HCl buffer solution is 1:(0.8-1.25):(50-100).

[0031] According to an embodiment of the present invention, the stirring temperature is room temperature and the stirring time is 12-48 hours.

[0032] As an example, reacting quaternized chitosan with tannic acid-coated octacalcium phosphate powder to obtain quaternized chitosan and tannic acid-coated octacalcium phosphate powder includes the following steps: dissolving quaternized chitosan in deionized water to prepare a quaternized chitosan solution; mixing the quaternized chitosan solution and tannic acid-coated octacalcium phosphate powder in deionized water, stirring and filtering to obtain quaternized chitosan and tannic acid-coated octacalcium phosphate powder.

[0033] According to an embodiment of the present invention, in the quaternized chitosan solution, the mass ratio of quaternized chitosan to deionized aqueous solution is 1:(500-2000).

[0034] According to an embodiment of the present invention, the mass ratio of the tannic acid-coated octacalcium phosphate powder, the quaternized chitosan solution, and the deionized water is 2:(50-200):(300-500).

[0035] According to an embodiment of the present invention, the stirring temperature is room temperature and the stirring time is 1-3 hours.

[0036] As an example, the process of obtaining bio-ink by adding water and sodium alginate to octacalcium phosphate powder coated with quaternized chitosan and tannic acid includes the following steps: mixing octacalcium phosphate powder coated with quaternized chitosan and tannic acid and sodium alginate powder evenly in deionized water, freeze-drying to obtain a foamed solid, and mixing the foamed solid with deionized water to obtain bio-ink.

[0037] According to an embodiment of the present invention, the mass ratio of the inorganic phase, the organic phase, and the deionized water is (4-6):(4-6):(150-300).

[0038] According to an embodiment of the present invention, the freeze-drying temperature is -(20-40)℃ and the time is 48-72h.

[0039] According to an embodiment of the present invention, the mass ratio of the foamed solid to deionized water is 1:(1-5).

[0040] According to an embodiment of the present invention, the filler density of the stent is 40%-60%.

[0041] Thirdly, the present invention provides a method for preparing a 3D printed scaffold, comprising the following steps:

[0042] The above-mentioned bio-ink was used to 3D print a scaffold.

[0043] According to an embodiment of the present invention, the preparation method includes the following steps: printing the above-mentioned bio-ink layer by layer using extrusion 3D printing technology, and placing the formed scaffold in a CaCl2 solution for cross-linking and curing.

[0044] According to an embodiment of the present invention, after the molded scaffold is cross-linked and cured in a CaCl2 solution, the following steps are further included: placing the cured scaffold in an oven to dry until completely dry, and then removing it to obtain a high-strength 3D-printed bone repair scaffold with an interconnected pore structure.

[0045] According to an embodiment of the present invention, the infill density in the printing is 40% to 60%, preferably 45% to 50%.

[0046] As an example, the printing method includes the following steps: using extrusion 3D printing technology to print ink layer by layer at a filling density, and placing the formed scaffold in a 5-20 wt% CaCl2 solution for cross-linking and curing for 3-12 hours, and drying at 20-80℃ for 6-48 hours to obtain a high-strength 3D printed bone repair scaffold.

[0047] Fourthly, the present invention also provides a stent prepared by the above method, wherein the compressive strength of the stent is ≥40MPa, preferably ≥42MPa.

[0048] According to an embodiment of the present invention, the support has a porous structure.

[0049] According to an embodiment of the present invention, after co-culturing the scaffold with the Calcein-AM / PI double staining kit for 3 days, the cell viability is ≥90%, preferably ≥95%, for example 98.7±9.6%.

[0050] Beneficial effects

[0051] 1) The method for preparing a highly bioactive and high-strength 3D printed scaffold in this invention involves modifying the osteoinductive calcium phosphate powder with tannic acid, quaternized chitosan, etc., resulting in the presence of catechol functional groups, hydroxyl groups, etc. on the surface of the osteoinductive calcium phosphate. The catechols can bind to biological tissues through hydrogen bonds, and the catechols can bind to each other and the hydroxyl groups, giving the scaffold good biocompatibility. At the same time, the prepared scaffold has a compressive strength ≥40MPa, exhibiting high strength. Attached Figure Description

[0052] Figure 1 The diagram shows the compressive strength test results of the stents prepared in Example 1, Comparative Example 1, and Comparative Example 2 in Test Example 1.

[0053] Figure 2SEM images of the stents prepared in Example 1, Comparative Example 1, and Comparative Example 2 in Test Example 1;

[0054] Figure 3 A is a fluorescent staining image of the scaffolds prepared in Example 2, Comparative Example 1, and Comparative Example 2 in Test Example 2; Figure 3 B is a cell activity graph of the scaffolds prepared in Example 2, Comparative Example 1, and Comparative Example 2 in Test Example 2. Detailed Implementation

[0055] The following will provide a more detailed description of the stent, its preparation method, and its application with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the invention and should not be construed as limiting the scope of protection of the invention. All technologies implemented based on the above description of the invention are covered within the scope of protection intended by the invention.

[0056] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0057] Example 1

[0058] (1) Mix 0.009 mol NH4H2PO4 and 0.03 mol CO(NH2)2 with 300 mL deionized water, and then stir magnetically at room temperature until completely dissolved to prepare solution A. Simultaneously, dissolve 0.0012 mol Ca(CH3COO)2·H2O in 300 mL deionized water to prepare solution B. Then, add solution B dropwise to solution A and stir for 5 min to form solution C. Then, heat solution C in a 90℃ water bath and stir vigorously for 2 h. After the reaction is complete, filter the obtained precipitate, wash it 3 times with deionized water and 2 times with anhydrous ethanol, and dry the filtered product at 60℃ for 24 h to obtain octacalcium phosphate powder.

[0059] (2) Prepare a 0.05M Tris-HCl buffer solution with pH=8.5. Take 8.88g of the prepared octacalcium phosphate powder and disperse it in 150mL of 0.05M Tris-HCl buffer solution with pH=8.5. Stir until completely dispersed to prepare solution D.

[0060] Dissolve 4g of tannic acid in 50mL of the above Tris-HCl buffer solution and stir until completely dissolved to form solution F.

[0061] Solution F was added dropwise to solution D while stirring to form solution G. Solution G was stirred at room temperature for 24 hours. After the reaction was completed, the resulting precipitate was filtered and repeatedly washed with deionized water until the filtrate was colorless. The filtered product was dried at 60°C for 24 hours to obtain tannin-modified octacalcium phosphate powder.

[0062] (3) Disperse 2g of the prepared tannic acid-modified octacalcium phosphate powder in 400mL of deionized water to obtain solution H. Dissolve 1g of quaternized chitosan in 1L of its ion-soluble aqueous solution to obtain a 1g / L quaternized chitosan solution, i.e., solution I. Add 100mL of solution I dropwise to 400mL of solution H, stir for 0.5h, filter and wash repeatedly with deionized water, take the precipitate, and obtain the quaternized chitosan and tannic acid-modified octacalcium phosphate powder, named quaternized chitosan and tannic acid-coated octacalcium phosphate powder.

[0063] (4) The "ink" required for printing was prepared using an in-situ composite method. Specifically, 6g of the prepared tannic acid and quaternized chitosan-modified octacalcium phosphate were dispersed in 100mL of deionized water and continuously stirred with a magnetic stirrer to ensure uniform dispersion. 4g of sodium alginate powder was added to the above solution, and stirring continued until the sodium alginate was completely dissolved, ensuring uniform mixing of sodium alginate with tannic acid and quaternized chitosan-modified octacalcium phosphate. The resulting solution was frozen at -20℃ for 12h, and then freeze-dried at -20℃ for 48h until completely freeze-dried. The resulting foamed solid was mixed with deionized water at a mass ratio of 1:3 and stirred until completely homogeneous to obtain the "ink" required for 3D printing.

[0064] (5) Cylindrical models with diameters of 15×5mm and 15×30mm were constructed using modeling software, and slicing files were exported using slicing software. The layer thickness was 0.6mm, and the infill density was 50%. Then, "ink" was printed layer by layer using an extrusion 3D printer under the control of gcode to obtain a porous scaffold. After printing, a quaternized chitosan and tannic acid modified octacalcium phosphate / sodium alginate scaffold was obtained. The quaternized chitosan and tannic acid modified octacalcium phosphate / sodium alginate scaffold was placed in a 10wt% CaCl2 solution for crosslinking for 6 hours until cured. The crosslinked scaffold was washed with deionized water and dried in an oven, named TOQ / SA.

[0065] Comparative Example 1

[0066] (1) Take 6g of the prepared octacalcium phosphate and disperse it in 100mL of deionized water. Use a magnetic stirrer to continuously stir it until it is evenly dispersed. Add 4g of sodium alginate powder to the above solution and continue stirring until the sodium alginate is completely dissolved, so that the sodium alginate and octacalcium phosphate are evenly mixed. Take the obtained solution and freeze it in a refrigerator at -20℃ for 12h. Then place it in a freeze dryer and freeze dry it at -20℃ for 48h until it is completely freeze-dried. Mix the foamed solid obtained after freeze drying with deionized water at a mass ratio of 1:3 and stir until it is completely uniform to obtain the "ink" required for 3D printing.

[0067] (2) Cylindrical models with dimensions of Φ15×5mm and Φ15×30mm were constructed using modeling software, and slicing files were exported using slicing software. The layer thickness was 0.6mm, and the infill density was 50%. Then, an extrusion 3D printer was used to print "ink" layer by layer under the control of gcode to obtain a porous scaffold. After printing, an octacalcium phosphate / sodium alginate scaffold was obtained. The octacalcium phosphate / sodium alginate scaffold was placed in a 10wt% CaCl2 solution for crosslinking for 6 hours until cured. The crosslinked scaffold was then washed with deionized water and dried in an oven, named OCP / SA.

[0068] Comparative Example 2

[0069] (1) Take 6g of the prepared tannic acid modified octacalcium phosphate and disperse it in 100mL of deionized water. Use a magnetic stirrer to continuously stir it until it is evenly dispersed. Add 4g of sodium alginate powder to the above solution and continue stirring until the sodium alginate is completely dissolved, so that the sodium alginate and tannic acid modified octacalcium phosphate are evenly mixed. Take the obtained solution and freeze it in a refrigerator at -20℃ for 12h. Then place it in a freeze dryer and freeze dry it at -20℃ for 48h until it is completely freeze-dried. Mix the foamed solid obtained after freeze drying with deionized water at a mass ratio of 1:3 and stir until it is completely uniform to obtain the "ink" required for 3D printing.

[0070] (2) Cylindrical models with diameters of 15×5mm and 15×30mm were constructed using modeling software, and slicing files were exported using slicing software. The layer thickness was 0.6mm, and the infill density was 50%. Then, an extrusion 3D printer was used to print "ink" layer by layer under the control of gcode to obtain a porous scaffold. After printing, a tannic acid-modified octacalcium phosphate / sodium alginate scaffold was obtained. The tannic acid-modified octacalcium phosphate / sodium alginate scaffold was placed in a 10wt% CaCl2 solution for crosslinking for 6 hours until cured. The crosslinked scaffold was washed with deionized water and dried in an oven, named TO / SA.

[0071] Experimental Example 1

[0072] The compressive strength of the stents prepared in Example 1, Comparative Example 1, and Comparative Example 2 was tested. The results were obtained using a universal testing machine with a diameter of 15×30mm, according to the Chinese National Standard GB / T8489-2006 (N=3, 3 samples per group). The morphology of these three groups of samples was also observed under a scanning electron microscope.

[0073] The experimental results show that the compressive strength of Example 1 reached 49.5±8.8MPa, the compressive strength of Comparative Example 1 reached 33.6±8.4MPa, and the compressive strength of Comparative Example 2 reached 38.2±5.7MPa, which shows that Example 1 has superior mechanical strength.

[0074] See Figure 1 As shown, compared with Comparative Examples 1 and 2, the organic / inorganic phases in the scaffold prepared in Example 1 exhibit better bonding.

[0075] Experiment Example 2

[0076] The cell viability of BMSCs cells cultured in the porous scaffold extract prepared in Example 2, Comparative Example 1, and Comparative Example 2 was determined by using a cell counting kit-8 (CCK-8) and a live / dead cell staining kit. The scaffold extract was prepared in accordance with ISO 10993-12:2017.

[0077] In the experiment, BMSCs were used at a rate of 1×10 5 cells / cm 2 The density of the scaffolds prepared in Example 1, Comparative Example 1, and Comparative Example 2 was co-cultured with the extracts in 96-well plates and placed in a cell culture incubator at 37°C and 5% CO2. After 24 hours of culture, 10 μL of CCK-8 solution was added to each well, and after incubation for 2 hours, the optical density (OD) of each well was measured at 450 nm using a microplate reader. Cell viability was calculated as follows:

[0078] Cell viability = (OD) 实验组 –OD 空白组 ) / (OD 对照组 -OD 空白组 )

[0079] When cell viability is above 70%, the scaffold is considered non-cytotoxic; when cell viability is below 70%, the scaffold is considered cytotoxic.

[0080] Live / Dead fluorescence staining was performed on BMSCs co-cultured for 3 days using the Calcein-AM / PI double staining kit. The relevant procedures were performed according to the instructions. After staining, the cells were incubated at 37°C in the dark for 30 minutes. Fluorescent staining images of BMSCs were obtained using an inverted fluorescence microscope.

[0081] See Figure 3 A live / dead cell fluorescence staining showed that, compared with Comparative Examples 1 and 2, BMSCs co-cultured for 3 days in Example 2 had greater cell viability.

[0082] See Figure 3 As shown in B, the cell viability of the porous scaffolds prepared in Example 2, Comparative Example 1, and Comparative Example 2 reached 98.7±9.6%, 89.0±8.4%, and 93.6±12.2%, respectively. This confirms that the scaffolds prepared using the ink of Example 2 are not biotoxic and have better biocompatibility than Comparative Example 2, showing potential for application as bone repair scaffolds.

[0083] The specific embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above exemplary embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a 3D printed scaffold, characterized in that, Includes the following steps: Bio-ink is used for 3D printing to form a scaffold; The bio-ink comprises an organic phase, an inorganic phase, and a first solvent, wherein the mass ratio of the inorganic phase, the organic phase, and the first solvent is (4-6):(4-6):(150-300), the inorganic phase comprises osteoinductive calcium phosphate modified with tannic acid and quaternized chitosan, the organic phase is sodium alginate, and the mass ratio of the inorganic phase to the organic phase is 1:(0.5~2). The bio-ink is prepared by the following method: tannic acid is reacted with octacalcium phosphate powder to obtain tannic acid-coated octacalcium phosphate powder; quaternized chitosan is reacted with tannic acid-coated octacalcium phosphate powder to obtain quaternized chitosan and tannic acid-coated octacalcium phosphate powder; water and sodium alginate are added and then freeze-dried to obtain a foamed solid; the foamed solid is mixed with a first solvent to obtain the bio-ink.

2. The method of claim 1, wherein the 3D-printed scaffold is prepared by, The bone-inducing calcium phosphate is selected from one or more of tricalcium phosphate, hydroxyapatite, tetracalcium phosphate, dihydrocalcium phosphate, and octacalcium phosphate.

3. The method for preparing a 3D printed scaffold according to claim 1, characterized in that, The process of obtaining bio-ink by adding water and sodium alginate to quaternized chitosan and tannic acid-coated octacalcium phosphate powder includes the following steps: mixing quaternized chitosan and tannic acid-coated octacalcium phosphate powder and sodium alginate powder evenly in deionized water, freeze-drying to obtain a foamed solid, and mixing the foamed solid with deionized water to obtain bio-ink.

4. A stent prepared by the method according to any one of claims 1-3, characterized in that, The support has a porous structure.

5. The bracket according to claim 4, characterized in that, The compressive strength of the support is ≥40 MPa.

6. The bracket according to claim 4, characterized in that, After co-culturing the scaffolds for 3 days using the Calcein-AM / PI double staining kit, the cell viability was ≥90%.

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