A 3D printing bio-ink for bone defect repair, its preparation method and application

Abstract

This invention belongs to the field of bone tissue engineering, specifically relating to a 3D printing bio-ink for bone defect repair, its preparation method, and its application. The bio-ink comprises an organic phase and an inorganic phase. The inorganic phase includes osteoinducible calcium phosphate modified with at least one of tannic acid, gelatin, chitosan, and silk fibroin. The organic phase is selected from at least one of sodium alginate, polylactic acid, polyvinyl alcohol, polycaprolactone, and polyetheretherketone. The bio-ink of this invention can better promote bone repair by eliminating foreign body reactions.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering, specifically relating to a 3D printing bio-ink for bone defect repair, its preparation method and application, and particularly to a 3D printing bio-ink with anti-inflammatory and bone repair effects, its preparation method, and a method for self-assembling a 3D printing scaffold using the 3D printing bio-ink. Background Technology

[0002] The skeletal system, as one of the most important organs in the human body, is responsible for supporting daily movement, transporting nutrients, and delivering red blood cells. However, bone defects caused by tumors, arthritis, trauma, and osteoporosis severely impact patients' health. While human bone tissue has a certain internal repair capacity, once the defect exceeds a critical size, repair through bone grafting or other methods is necessary. Common bone grafts include autologous bone grafting and allogeneic bone grafting, with allogeneic grafting further divided into allogeneic and xenogeneic grafting. Autologous bone grafting has limited material sources and can cause secondary damage to the harvesting site, while allogeneic bone grafting has abundant material sources but faces the risk of severe host immune responses and the transmission of potential diseases. Therefore, synthetic bone repair materials have emerged. Tissue engineering, a rapidly developing interdisciplinary field, refers to the technology of using principles and methods from 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. 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.

[0003] The goal of tissue engineering is to combine scaffolds with living cells or bioactive molecules to promote tissue repair and regeneration. There are three main types of bioactive molecules involved in bone repair: 1. Growth factors that promote bone repair (e.g., bone morphogenetic protein BMP); 2. Antibacterial active molecules; 3. Active molecules with anti-inflammatory effects. The tissue microenvironment is mainly composed of growth factors, cells, and extracellular matrix (ECM) proteins. Signaling molecules are transmitted into the cell interior via growth factor receptors and integrase proteins on the cell surface, triggering intracellular signal transduction and ultimately promoting gene expression. Repairing infected bone defects is a major challenge in most bone grafts. Traditional repair methods involve systemic antibiotic injections after autologous or allogeneic bone grafting to control infection, but these subsequent treatments place a significant burden on patients. Therefore, it is essential to develop novel multifunctional materials with bone repair induction functions and in-situ antibacterial properties. Currently, most drugs used for repairing infected bone defects are antibiotics, and their potential systemic toxicity and poor penetration into necrotic tissue at the injury site cannot be ignored. 3D printing is a crucial molding technology in bone tissue engineering, offering advantages such as high printing precision, the ability to customize biodevices for specific patient defects, and low cost. The greatest advantage of 3D bioprinting lies in its potential for spatially distributing 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 tailored to patient defects can be created, reconstructing complex bone defects. Therefore, the development of 3D bioprinted bone tissue has significant and far-reaching implications for clinical practice. Octacalcium phosphate is a precursor to hydroxyapatite, an inorganic component of human bone tissue, possessing good biocompatibility and bioactivity, as well as a suitable degradation rate. However, the surface of octacalcium phosphate scaffolds lacks drug-responsive sites, preventing direct drug loading.

[0004] Puerarin is an isoflavone derivative extracted from the traditional Chinese medicine kudzu root. It can inhibit osteoclast differentiation, promote osteoblast proliferation, and promote macrophage polarization from the M1 phenotype to the M2 phenotype to promote bone defect repair. However, direct exposure of puerarin to body fluids can lead to drug inactivation, resulting in surgical failure.

[0005] Metal-organic frameworks (MOFs) possess well-defined crystal structures and flexible selectivity in binding to both organic and inorganic substances, offering numerous advantages over other drug carriers; among them, those composed of Zn... 2+ ZIF-8, composed of 2-methylimidazole, has a large specific surface area, excellent biocompatibility, and sensitivity to acidic environments, and therefore has been extensively studied in the field of drug delivery.

[0006] Nevertheless, there are still some key issues that prevent targeted drug delivery via ZIF-8 loading. Summary of the Invention

[0007] To address at least one of the problems of low mechanical strength and poor bioactivity in current 3D-printed bone repair scaffolds, this invention provides 3D-printed bio-inks for bone defect repair, their preparation methods, and applications, such as for the preparation of self-assembled 3D-printed scaffolds with anti-inflammatory and bone repair effects, which can better promote bone repair by eliminating foreign body reactions.

[0008] 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, chitosan, and silk fibroin, and the organic phase being selected from at least one of sodium alginate, polylactic acid, polyvinyl alcohol, polycaprolactone, and polyetheretherketone.

[0009] 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.

[0010] According to an embodiment of the present invention, the ink further includes a first solvent, such as deionized water.

[0011] 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).

[0012] 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.

[0013] According to an embodiment of the present invention, the octacalcium phosphate is prepared using existing methods, such as co-precipitation.

[0014] 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.

[0015] 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).

[0016] 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.

[0017] 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.

[0018] According to an embodiment of the present invention, the inorganic phase comprises tannic acid and chitosan-modified osteoinducible calcium phosphate.

[0019] According to an embodiment of the present invention, the organic phase is sodium alginate.

[0020] 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.

[0021] According to an embodiment of the present invention, the chitosan is selected from modified or unmodified chitosan, and the modified chitosan is preferably at least one of amino-modified chitosan, chitosan grafted with 2-methacrylate oxyethyl phosphate, chitosan grafted with sodium vinyl sulfonate, chitosan grafted with polyethylene glycol, and amidated chitosan, for example, quaternized chitosan.

[0022] 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, 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.

[0023] 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.

[0024] 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.

[0025] According to an embodiment of the present invention, the pH value of the tris-HCl buffer solution is 7.5-9.5.

[0026] 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).

[0027] According to an embodiment of the present invention, the stirring temperature is room temperature and the stirring time is 12-48 hours.

[0028] 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.

[0029] 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).

[0030] 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).

[0031] According to an embodiment of the present invention, the stirring temperature is room temperature and the stirring time is 1-3 hours.

[0032] 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.

[0033] 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).

[0034] According to an embodiment of the present invention, the freeze-drying temperature is -(20-40)℃ and the time is 48-72h.

[0035] According to an embodiment of the present invention, the mass ratio of the foamed solid to deionized water is 1:(1-5).

[0036] According to an embodiment of the present invention, the filler density of the stent is 40%-60%.

[0037] Thirdly, the present invention provides a method for preparing a 3D printed scaffold, comprising the following steps:

[0038] (1) The above-mentioned bio-ink was 3D printed to obtain a scaffold; bone repair drugs were encapsulated in the pores of MOF nanomaterials to obtain drug-containing MOF nanomaterials;

[0039] (2) Modify the surface of the scaffold with drug-containing MOF nanomaterials.

[0040] According to an embodiment of the present invention, the MOF nanomaterial is selected from zinc-based MOF nanomaterials, such as ZIF-8 nanoparticles.

[0041] According to an embodiment of the present invention, the bone repair drug is selected from at least one of quercetin, icariin, puerarin, etc., for example, puerarin.

[0042] According to an embodiment of the present invention, the encapsulation of bone repair drugs within the pores of MOF nanomaterials includes the following steps: dispersing MOF nanomaterials in a good solvent containing bone repair drugs and encapsulating agents to form a solution, condensing and refluxing to obtain drug-containing MOF nanomaterials.

[0043] According to an embodiment of the present invention, the good solvent is selected from at least one of methanol, ethanol, and deionized water, for example, methanol.

[0044] According to an embodiment of the present invention, the encapsulant is selected from materials that can encapsulate bone repair drugs within the pores of MOF nanomaterials, and that can be destroyed in the in vivo environment, allowing the bone repair drugs to leak out, and are non-toxic.

[0045] According to an embodiment of the present invention, the encapsulant is selected from at least one of dopamine, tannic acid, and Al2O3 ultrathin nanolayers, for example, dopamine.

[0046] As an example, ZIF-8 nanoparticles were dispersed in a methanol solution containing puerarin and dopamine hydrochloride, and then refluxed to obtain ZIF-8 nanoparticles with a dopamine coating encapsulated with puerarin.

[0047] According to an embodiment of the present invention, in the methanol solution, the concentration of the ZIF-8 nanoparticles is (0.1-0.5) mg / mL; the concentration of the puerarin is (10-100) μg / mL; and the concentration of the dopamine hydrochloride is (1-10) mmol / L.

[0048] According to an embodiment of the present invention, the condensation reflux time is 10-20 hours and the temperature is 30-90°C.

[0049] According to an embodiment of the present invention, in step (2), the concentration of the drug-containing MOF nanomaterial is (0.5-1.5) mg / mL, and the CaCl2 solution is (5-20) wt%.

[0050] According to an embodiment of the present invention, step (2) of modifying the surface of the scaffold with drug-containing MOF nanomaterials specifically includes the following steps: placing the scaffold in a CaCl2 solution containing drug-containing MOF nanomaterials for cross-linking and curing to obtain a self-assembled 3D printed scaffold.

[0051] According to an embodiment of the present invention, in step (2), the crosslinking curing is carried out under ultrasonic conditions, the ultrasonic time is 10-20 min, and the number of times is 10-30.

[0052] According to an embodiment of the present invention, after crosslinking and curing in step (2), the following step is further included: drying the scaffold.

[0053] According to an embodiment of the present invention, the drying time is 6-48 hours and the drying temperature is 20-60°C.

[0054] Fourthly, the present invention also provides a scaffold prepared by the method described above, wherein the surface of the scaffold has a layered structure formed by the self-assembly of drug-containing MOF nanomaterials.

[0055] According to an embodiment of the present invention, the thickness of the layered structure is 1 to 10 nm, for example, 3 nm, 5 nm, or 7 nm.

[0056] Beneficial effects

[0057] 1) The method for preparing a self-assembled 3D-printed scaffold with anti-inflammatory and bone repair effects in this invention involves loading anti-inflammatory drugs into the pores inside MOF nanomaterials, then encapsulating them with an encapsulating agent to prevent the anti-inflammatory drugs from leaking out in the natural environment and to maintain their medicinal properties. The MOF nanomaterials containing anti-inflammatory drugs are then self-organized on the surface of the scaffold to form a repair scaffold. After implantation, because the drugs loaded on the MOF nanomaterials are on the surface of the scaffold rather than inside it, they can directly contact body fluids. The encapsulating agent is destroyed upon contact with body fluids, allowing anti-inflammatory drugs such as puerarin to be released promptly, inhibiting inflammation and promoting bone repair. This method can better promote bone repair by eliminating foreign body reactions, providing a new approach for the development of tissue engineering and showing great application potential in the clinical treatment of bone defect repair.

[0058] 2) Encapsulating agents such as dopamine self-polymerize in MOF nanomaterials, not only encapsulating anti-inflammatory drugs, but also, due to the modification of osteoinductive calcium phosphate powder with tannic acid, quaternized chitosan, etc., the MOF nanomaterials and osteoinductive calcium phosphate surfaces have catechol functional groups, hydroxyl groups, etc. Catechols can bond with each other and with hydroxyl groups through hydrogen bonds, thereby connecting the scaffold and the drug-loaded MOF nanomaterials. At the same time, quaternized chitosan not only provides hydrogen bond reaction sites, but also makes the modified powder positively charged, which can electrostatically bond with the negatively charged nanoparticles, so that the drug-loaded MOF nanomaterials can self-assemble on the scaffold surface to form an outer layer structure with a large number of anti-inflammatory drugs. Attached Figure Description

[0059] Figure 1 This is a flowchart of the fabrication method of the 3D printed scaffold in Example 1;

[0060] Figure 2 Figure A shows the surface morphology of Comparative Example 1 and Comparative Example 2; Figure (B) shows the surface morphology of Experimental Example 1; Figure (C) shows the surface element distribution of Experimental Example 1.

[0061] Figure 3 A shows the fluorescence staining images of Example 2, Comparative Example 1, and Comparative Example 2; Figure 3 B shows the cell viability graphs for Example 2, Comparative Example 1, and Comparative Example 2.

[0062] Figure 4 A represents the plate count method used to test the antibacterial effects of Example 3, Comparative Example 1, and Comparative Example 2 against Staphylococcus aureus and Escherichia coli. Figure 4 B represents the inhibition zone method test of the antibacterial effects of Example 3, Comparative Example 1, and Comparative Example 2 on Staphylococcus aureus and Escherichia coli. Figure 4 C represents the inhibition rate of Staphylococcus aureus against Example 3, Comparative Example 1, and Comparative Example 2; Figure 4 D represents the inhibition rate of Escherichia coli against Example 3, Comparative Example 1, and Comparative Example 2;

[0063] Figure 5 A represents the expression levels of pro-inflammatory genes in Example 4, Comparative Example 1, and Comparative Example 2. Figure 5 B represents the expression levels of anti-inflammatory genes in Example 4, Comparative Example 1, and Comparative Example 2. Detailed Implementation

[0064] 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.

[0065] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0066] Example 1

[0067] A method for fabricating a 3D printed scaffold, see [link to documentation]. Figure 1 As shown, it includes the following steps:

[0068] (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.

[0069] (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.

[0070] Dissolve 4g of tannic acid in 50mL of the above Tris-HCl buffer solution and stir until completely dissolved to form solution F.

[0071] Solution F was added dropwise to solution D and stirred to form solution G. Solution G was stirred at room temperature for 24 hours. After the reaction was completed, the precipitate was filtered and washed repeatedly with deionized water until the filtrate was colorless. The filtered product was dried at 60°C for 24 hours to obtain tannic acid modified octacalcium phosphate powder.

[0072] (3) Take 2g of the prepared tannic acid-modified octacalcium phosphate powder and disperse it in 400mL of deionized water to prepare solution H. Take 1g of quaternized chitosan and dissolve it in 1L of its ionized water solution to prepare a 1g / L quaternized chitosan solution, i.e., solution I. Take 100mL of solution I and add it dropwise to 400mL of solution H, stir for 0.5h, filter and wash repeatedly with deionized water, take the precipitate, and you will get quaternized chitosan and tannic acid-modified octacalcium phosphate powder, named quaternized chitosan and tannic acid-coated octacalcium phosphate powder.

[0073] (4) Preparation of the "ink" required for printing using the 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, and stirring was continued until the sodium alginate was completely dissolved, so that the sodium alginate, tannic acid, and quaternized chitosan-modified octacalcium phosphate were mixed evenly. The resulting solution was frozen at -20℃ for 12h, and then placed in a freeze dryer at -20℃ for 48h until completely freeze-dried. The foamed solid obtained after freeze-drying was mixed with deionized water at a mass ratio of 1:3 and stirred until completely uniform to obtain the "ink" required for 3D printing.

[0074] (5) A cylindrical model with a diameter of 15×5mm was constructed using modeling software and a slice file was 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 code to obtain a porous scaffold. After printing, a quaternized chitosan and tannic acid modified octacalcium phosphate / sodium alginate scaffold was obtained and named TOQ / SA.

[0075] (6) ZIF-8 nanoparticles were prepared by room temperature method: Specifically, 0.7437 g of zinc nitrate was dissolved in 100 mL of methanol solution, and added to 100 mL of methanol solution containing 0.4106 g of 2-methylimidazole. After mixing evenly at room temperature, the mixture was allowed to stand for 6 h. After the reaction was completed, the mixture was centrifuged at 5000 rpm, washed 3 times with methanol solution, and dried at 60 °C to obtain ZIF-8 nanoparticles.

[0076] (7) Add 50 μg / mL puerarin to 100 mL of methanol solution containing 5 mmol dopamine hydrochloride, then add 0.3 g of ZIF-8 nanoparticles to the solution, and reflux at 60 °C for 18 h to obtain ZIF-8 nanoparticles loaded with puerarin and coated with dopamine (HPDA@Pur, abbreviated as HP).

[0077] (8) Disperse the 0.1g HP particles obtained in step (7) uniformly in a 10wt% CaCl2 solution, then immerse the TOQ / SA scaffold obtained in step (5) into the solution, sonicate for 15 min, and clean the surface of the TOQ / SA scaffold three times with 10wt% CaCl2. The entire process is recorded as one cycle. Repeat the process 15 times, and then dry at 60℃ for 24 h to obtain the TOQ / SA scaffold of HP nanoparticle self-assembly, denoted as TOQ@HP. 15 / SA.

[0078] See Figure 1 The diagram shows the fabrication process of a 3D-printed scaffold, in which the drug is encapsulated inside ZIF-8 and the outer layer is wrapped with PDA.

[0079] Comparative Example 1

[0080] This comparative example uses steps (1)-(5) in Example 1 to prepare a quaternized chitosan and tannic acid modified octacalcium phosphate / sodium alginate scaffold, denoted as TOQ / SA.

[0081] Comparative Example 2

[0082] (1) This step is the same as steps (1)-(6) in Example 1, and the printed quaternized chitosan and tannic acid modified octacalcium phosphate / sodium alginate scaffold (TOQ / SA) and nanoparticles ZIF-8 are obtained.

[0083] (2) 0.3 g of ZIF-8 nanoparticles were added to 100 mL of methanol solution containing 5 mmol of dopamine hydrochloride and refluxed at 60 °C for 18 h to obtain ZIF-8 nanoparticles (HPDA) with dopamine coating.

[0084] (3) The prepared 0.1g HPDA particles were uniformly dispersed in a 10wt% CaCl2 solution. The TOQ / SA scaffold from step (1) was then immersed in the solution and sonicated for 15 min. The scaffold surface was cleaned three times with 10wt% CaCl2. This process was recorded as one cycle. After repeating the process 15 times, the scaffold was dried at 60℃ for 24 h to obtain a TOQ / SA scaffold composed of HPDA nanoparticles layer by layer self-assembled, denoted as TOQ@H 15 / SA.

[0085] Experimental Example 1

[0086] The surface morphology and elemental distribution of the scaffolds prepared in Examples 1, 1, and 2 were obtained by measuring the Φ15×5mm scaffolds using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS).

[0087] Experimental results are as follows Figure 2 As shown, the scaffolds of Example 1 and Comparative Example 2 have a layer of nanoparticles attached to their surface with a thickness of approximately 3 μm.

[0088] Experiment Example 2

[0089] The stents (TOQ@HP) prepared in Example 1, Comparative Example 1, and Comparative Example 2 15 / SA, TOQ / SA, TOQ@H 15 Cell viability of BMSCs cultured in scaffold extract (SA) was determined using a cell counting kit-8 (CCK-8) and a live / dead cell staining kit; the scaffold extract was prepared according to ISO 10993-12 2017. In the experiments, BMSCs were cultured at a concentration of 1 × 10⁶ cells / year. 5 cells / cm 2The scaffolds prepared in Examples 1, 1, and 2 were co-cultured with their extracts in 96-well plates at 37°C and 5% CO2 for 24 hours. After incubation, 10 μL of CCK-8 solution was added to each well, and the plates were incubated for another 2 hours. The optical density (OD) of each well was then measured at 450 nm using a microplate reader. Cell viability was calculated as follows:

[0090] Cell vitality = (OD) 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )

[0091] When cell viability is above 70%, the current scaffold is considered non-cytotoxic; when cell viability is below 70%, the current scaffold is considered cytotoxic.

[0092] 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.

[0093] Experimental results are as follows Figure 3 As shown, Example 1, Comparative Example 1, and Comparative Example 2 are all non-cytotoxic, and the cell activity of Example 1 is better than that of Comparative Example 2. This confirms that Example 1 is non-cytotoxic and has the potential to be used as a bone repair scaffold in tissue engineering.

[0094] Experimental Example 3

[0095] The antibacterial properties of the scaffolds prepared in Example 1, Comparative Example 1, and Comparative Example 2 were determined by plate counting and inhibition zone methods. Staphylococcus aureus and Escherichia coli were inoculated onto nutrient agar plates and cultured at 37°C for 24 hours. A small amount of bacteria was dissolved in PBS solution and diluted to 1*10⁻⁶ using a colorimetric method. 8 cfu / mL, then diluted to 1*10 5 cfu / mL, for use in subsequent experiments.

[0096] Plate counting method: The scaffolds of Example 1, Comparative Example 1, and Comparative Example 2 were placed into the above bacterial solution at a ratio of 1g / 100mL, and after being incubated at 37℃ for 24h, they were taken out and plated for counting using the 10-fold dilution method. Each group was repeated 3 times, and the sterilization rate was calculated.

[0097] Sterilization rate = (average colony count in the blank group - average colony count in the experimental group) / average colony count in the blank group

[0098] The inhibition loop method: Use a pipette to transfer 50 μL of 1*10 5Spread the CFU / mL bacterial suspension evenly on an agar plate, place a porous support in the center of the agar plate, invert it in a 37°C oven and incubate for 24 hours, then remove it and compare the size of the inhibition zone.

[0099] Experimental results are as follows Figure 4 As shown, Example 1, Comparative Example 1, and Comparative Example 2 all have certain antibacterial effects against Staphylococcus aureus and Escherichia coli, with Example 1 showing the best antibacterial effect.

[0100] Example 4

[0101] The anti-inflammatory properties of the porous branches prepared in Example 1, Comparative Example 1, and Comparative Example 2 were determined by RT-qPCR. Macrophage Raw.264.7 cells were cultured in a medium and extract at a concentration of 1*10⁻⁶. 5 cells / cm 2 The cells were initially cultured at high density for 3 days. Total RNA was isolated and extracted using Trizol, and the RNA concentration was detected using an ultra-micro nucleic acid detector. After reverse transcription, cDNA was extracted and RT-qPCR was performed. The relevant operations were performed according to the standard protocol given in the instruction manual. The expression levels of pro-inflammatory genes (IL-6, CCL8) and anti-inflammatory genes (CD206, Arg1) were detected.

[0102] Experimental results are as follows Figure 5 As shown, compared with Comparative Example 1, Comparative Example 2 and Example 1 significantly downregulated the expression of pro-inflammatory genes (IL-6, CCL8) and significantly upregulated the expression of anti-inflammatory genes (CD206, Arg1), with Example 1 showing a significantly better effect than Comparative Example 2. This result indicates that the introduction of nanoparticles endows the scaffold with significant anti-inflammatory function, and the addition of puerarin further enhances the anti-inflammatory properties of the scaffold, confirming its potential application in early inflammatory responses.

[0103] 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, The method comprises the following steps:

1. 3D printing a bio-ink to obtain a scaffold; 2. dispersing ZIF-8 nanoparticles in a methanol solution containing puerarin and dopamine hydrochloride, condensing and refluxing to obtain dopamine-coated ZIF-8 nanoparticles encapsulating puerarin; 3. crosslinking and solidifying the scaffold in a CaCl2 solution of dopamine-coated ZIF-8 nanoparticles encapsulating puerarin to obtain a self-assembled 3D printed scaffold; In the methanol solution, the concentration of the ZIF-8 nanoparticles is 0.1-0.5 mg / mL; the concentration of the puerarin is 10-100 μg / mL; and the concentration of the dopamine hydrochloride is 1-10 mmol / L; The condensing and refluxing is performed for 10-20 h at a temperature of 30-90℃; The bio-ink comprises an organic phase and an inorganic phase, the inorganic phase comprises bone-inducing calcium phosphate modified by at least one of tannic acid, gelatin, chitosan and silk fibroin, and the organic phase is selected from at least one of sodium alginate, polylactic acid, polyvinyl alcohol, polycaprolactone and polyether ether ketone, and the mass ratio of the inorganic phase to the organic phase is 1:0.5-2.

2. The method of claim 1, wherein the 3D-printed scaffold is prepared by a method comprising: The bio-ink further comprises a first solvent, and the mass ratio of the inorganic phase, the organic phase and the first solvent is 4-6:4-6:150-300. The bone-inducing calcium phosphate is selected from one or more of tricalcium phosphate, hydroxyapatite, tetracalcium phosphate, calcium phosphate dihydrogen and octacalcium phosphate.

3. The method of claim 2, wherein the 3D-printed scaffold is prepared by, The inorganic phase comprises bone-inducing calcium phosphate modified by tannic acid and chitosan.

4. The method of claim 2, wherein the 3D-printed scaffold is prepared by, The preparation method of the bio-ink comprises the following steps: reacting at least one of tannic acid, gelatin, 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 freeze-drying to obtain a foamy solid, mixing the foamy solid with a first solvent to obtain the bio-ink.

5. The method of claim 4, wherein The freeze-drying is performed at a temperature of -40--20℃ for 48-72 h, and the mass ratio of the foamy solid to the first solvent is 1:1-5.

6. The method of claim 4, wherein the 3D-printed scaffold is prepared by, The preparation method of the bio-ink comprises the following steps: reacting tannic acid with octacalcium phosphate powder to obtain tannic acid-coated octacalcium phosphate powder, reacting quaternary ammonium chitosan with the tannic acid-coated octacalcium phosphate powder to obtain quaternary ammonium chitosan and tannic acid-coated octacalcium phosphate powder, mixing with water and sodium alginate, and freeze-drying to obtain a foamy solid, and mixing the foamy solid with a first solvent to obtain the bio-ink.

7. The preparation method according to claim 6, characterized in that, The preparation of the bio-ink from quaternary ammonium chitosan and tannic acid-coated octacalcium phosphate powder, water and sodium alginate comprises the following steps: uniformly mixing the quaternary ammonium chitosan and tannic acid-coated octacalcium phosphate powder and the sodium alginate powder in deionized water, freeze-drying to obtain a foamy solid, and mixing the foamy solid with deionized water to obtain the bio-ink.

8. A scaffold prepared by the method of any one of claims 1-7, wherein the surface of the scaffold has a layered structure formed by self-assembly of MOF nanomaterials containing drugs.

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