3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold, and preparation method and application thereof

Abstract

The application discloses a 3D printing AgNPs / PLGA bacteriostatic bone tissue engineering scaffold and a preparation method and application thereof, and belongs to the technical field of bone tissue scaffolds. In the research, a direct ink writing (DIW) 3D printing technology is adopted, and a nano silver / poly lactic acid-co-glycolic acid (AgNPs / PLGA) material is used to prepare an antibacterial scaffold. Whether the scaffold is suitable for bone defect repair is verified by evaluating the micro morphology, mechanical properties and biological characteristics of the scaffold. The results show that HAP is deposited on the scaffold, and the mixing of the scaffold and the AgNPs is also verified. All the scaffolds with added silver nanoparticles have bacteriostatic activity. The cell toxicity experiment proves that the AgNPs / PLGA has good biocompatibility and can be used for the repair of bone tissue. The research shows that the AgNPs / PLGA scaffold has good mechanical properties and biocompatibility, and can effectively inhibit the growth of staphylococcus aureus and escherichia coli. The results show that the 3D printing AgNPs / PLGA scaffold has potential application in bone tissue engineering.

Description

Technical Field

[0001] This invention relates to the field of bone tissue scaffold technology, specifically to a 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold, its preparation method, and its application. Background Technology

[0002] In recent years, bone defect repair has become a major challenge and research hotspot in the global medical field. Every year, a large number of patients require bone defect repair due to congenital diseases, trauma, infection, and tumors. Large bone defects are often accompanied by a high risk of infection, and bacterial infection is one of the important factors leading to bone implantation failure in clinical practice. Pathogenic bacteria can significantly damage local tissues and bone regeneration capacity, causing local blood damage and osteonecrosis. To address this problem, the commonly used clinical treatment is the use of antibiotics. However, systemic antibiotics are difficult to achieve effective drug concentrations at the site of infection, which may lead to delayed bone healing, nonunion, or even amputation, and sometimes even death. To reduce the incidence of infection and ensure a good prognosis, the development of bifunctional biological bone scaffolds with antibacterial properties and bone defect reconstruction capabilities is urgently needed.

[0003] Charles Hull first reported 3D printing technology in 1986. With its development in biomedicine and tissue engineering, 3D printing has become one of the most promising methods for manufacturing bone scaffolds. 3D printing technology can utilize computer-aided design (CAD) models to produce scaffolds with specific geometries. Compared to traditional manufacturing techniques, 3D printing is increasingly widely used in tissue engineering and medical regeneration due to its rapid prototyping and personalized customization capabilities. It can precisely control the pore connectivity, porosity, and pore space distribution of the scaffold, which are crucial factors for bone tissue engineering scaffold applications, enabling cell permeability, fluid flow, and metabolic product removal. DIW (Digital Ink Wash) is the simplest and most cost-effective 3D printing technology, and is the most economical and widely used. A key characteristic of DIW is its ability to more freely customize inks, printing 3D structures with good precision at both the micro and mesoscopic scales. The rich potential of DIW technology has attracted extensive research. Many biomaterials can be printed using DIW, including polymers, ceramics, glass, cement, graphene, metals, and combinations thereof. For example, Guo et al. used the DIW (Distillation-Wash) fabrication method to create 3D geometries at room temperature by mixing soluble polymer polylactic acid with dichloromethane. Meanwhile, some researchers have successfully fabricated bone scaffolds using ceramic bioactive materials via DIW printing technology. For instance, Sun et al. successfully fabricated a bone scaffold for skull defect repair by mixing Wollastonite-magnesium powder with a polyvinyl alcohol solution using DIW technology. Furthermore, another important feature of DIW is its ability to uniformly mix materials; for example, carbon-based nanomaterials such as graphene can be uniformly added to polymer inks to print multifunctional 3D composite materials.

[0004] Polylactic-co-glycolic acid (PLGA) is a high-molecular-weight organic compound composed of lactic acid (LA) and glycolic acid (GA) monomers. It possesses good biocompatibility, degradability, and mechanical properties, and is non-toxic, making it widely used in bone tissue engineering. The main degradation pathways of PLGA are hydrolysis and autocatalytic degradation via ester bonds. In the human body, it is degraded into lactic acid (LA) and glycolic acid (GA), both of which are metabolic byproducts and can be naturally excreted. To meet the processing requirements of 3D printing, organic solvents such as acetone and 1,4-dioxane are generally used to dissolve PLGA in printing inks. Antibacterial properties are primarily imparted to scaffolds through antibiotic loading. For example, Visscher et al. developed a 3D-printed PCL scaffold containing macropores and micropores and coated with an antibiotic (cefazoline). This scaffold exhibited antibacterial activity against Staphylococcus aureus. Another group printed PCL / PLGA scaffolds loaded with tobramycin, which showed antibacterial activity against Staphylococcus aureus in vitro and successfully treated bone infections in a rat model. While this design holds great promise, the rapid development of antibiotic-resistant bacteria necessitates the development of alternatives to this approach.

[0005] Silver nanoparticles (AgNPs) have attracted considerable interest due to their low cytotoxicity, significant antibacterial properties, and relatively low drug resistance. They can be directly incorporated into inorganic materials or mixed with synthetic biopolymers to prepare 3D-printed antibacterial materials. Previous studies have suggested that AgNPs possess good antibacterial capabilities against bacteria, viruses, and other eukaryotic microorganisms. The antibacterial effects of AgNPs have been extensively studied, and their mechanisms are still being explored. Their antibacterial activity is primarily attributed to their oxidized form (Ag+), which can anchor and penetrate bacterial cell walls, leading to cell membrane disruption. AgNPs can also interact with the thiol groups of many important enzymes, inactivating them. Furthermore, silver ions act on the sulfur and phosphorus components of DNA, resulting in the inhibition of DNA replication. In addition, AgNPs cause the collapse of the cell membrane proton gradient and the disruption of many cellular metabolic mechanisms, leading to cell death. Due to their excellent antibacterial efficacy, they have become a treatment option for many diseases and are present in many commercial biomedical products, such as catheters and wound dressings.

[0006] Chinese patent application CN109938896A discloses a bone tissue engineering scaffold, comprising a hollow mesh-like scaffold and porous zinc-based metal spheres filled therein. The porous zinc-based metal spheres can be solid spheres with grooved surfaces or loose spheres made of rolled metal wires. Internal support columns can also be incorporated within the scaffold to provide enhanced mechanical properties. β-TCP particles can be filled to enrich bone marrow mesenchymal stem cells. This patent combines a mesh scaffold made of titanium or zinc-based metal with its internal support columns to create a bone tissue engineering scaffold with excellent mechanical properties. The porous zinc-based metal spheres promote osteogenic differentiation and inhibit osteoclast differentiation, providing support while promoting bone regeneration and ingrowth. The bone is then gradually replaced by bone tissue during degradation. The β-TCP particles enrich mesenchymal stem cells and promote bone repair. This invention can be applied to large-segment bone defects in load-bearing areas, promoting bone regeneration while providing mechanical support, thus achieving the therapeutic effect of repairing large-segment bone defects. However, the scaffold has poor antibacterial effects and poses a risk of infection, therefore further improvements are needed. Summary of the Invention

[0007] The technical problem to be solved by the present invention is how to provide a bone tissue engineering scaffold that simultaneously has antibacterial and bone repair-promoting properties.

[0008] The present invention solves the above-mentioned technical problems through the following technical means:

[0009] The first aspect of this invention provides a method for preparing a 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold, comprising the following steps:

[0010] (1) Preparation of AgNPs / PLGA composite material:

[0011] a. Place AgNPs (silver nanoparticles) powder and organic solvent in a centrifuge tube, and mix them evenly by ultrasonication to obtain an AgNPs solution;

[0012] b. Mix the AgNPs solution with PLGA, then add an organic solvent and stir until homogeneous to obtain an AgNPs / PLGA mixture;

[0013] (2) Fabrication of 3D printed AgNPs / PLGA scaffold: The AgNPs / PLGA mixture is printed using an FDM-based 3D printer. During the fabrication process, the AgNPs / PLGA mixture is extruded by air pressure and deposited onto the print bed through a nozzle to form filaments. The print head is moved along the X and Y axes and the print bed is lowered in the Z axis direction to perform layer-by-layer deposition to build a complete 3D structure.

[0014] Beneficial effects: This invention successfully printed an AgNPs / PLGA scaffold using DIW printing technology, uniformly mixing PLGA with nano-silver to achieve sustained release of nano-silver, thus realizing a long-term antibacterial effect. Furthermore, the scaffold exhibits good biocompatibility, no cytotoxicity, and can promote bone repair.

[0015] Preferably, the concentration of the AgNPs solution in step a is 0.1 mg / mL.

[0016] Preferably, the organic solvent in step (1) is chloroform.

[0017] Preferably, the PLGA in step b is model DG-85DLG300.

[0018] Preferably, in step b, the mass ratio of AgNPs solution to PLGA is 0.1-3:1.

[0019] Preferably, the 3D printer model in step (2) is Ultimaker 2+Extended.

[0020] Preferably, the printer nozzle model in step (2) is TPND-25G.

[0021] Preferably, the printer syringe model in step (2) is PSY-50E.

[0022] Preferably, the 3D printer parameters in step (2) are set as follows: printing speed: 35%, nozzle temperature: 3℃, platform temperature: 3℃, support layer height: 1mm, support width: 2cm, and support length: 2cm.

[0023] A second aspect of the present invention provides an AgNPs / PLGA antibacterial bone tissue engineering scaffold prepared using the above-described preparation method.

[0024] A third aspect of the present invention proposes the application of the AgNPs / PLGA antibacterial bone tissue engineering scaffold prepared by the above preparation method in bone defect repair.

[0025] The advantages of this invention are:

[0026] 1. This invention successfully printed an AgNPs / PLGA scaffold using DIW printing technology, uniformly mixing PLGA with nano-silver to achieve sustained release of the nano-silver, thus realizing a long-term antibacterial effect. Furthermore, this scaffold exhibits good biocompatibility, is non-cytotoxic, and can promote bone repair.

[0027] 2. SEM results showed that the scaffold had a uniform porous structure, and the pore size was conducive to the infiltration and adhesion of bone tissue cells. Furthermore, the mechanical properties of the scaffold were enhanced after the addition of silver nanoparticles to PLGA. XRD results showed the formation of HAP on the scaffold, indicating good direct biological activity and confirming the presence of silver nanoparticles on the scaffold. Attached Figure Description

[0028] Figure 1 This is a DLS diagram of the particle size of AgNPs powder used in this invention.

[0029] Figure 2 This is a SEM image of the particle size of AgNPs powder used in this invention.

[0030] Figure 3 The image shows a comparison of the distribution of AgNPs in the stent according to the present invention, where A is the SEM image of the stent and B is the EDS image of the stent.

[0031] Figure 4 Physical views and scanning electron microscope images of different supports of the present invention;

[0032] Figure 5 The diagram shows a comparison of the mechanical properties of different supports of the present invention, where A is the stress-strain curve of the support; B is the Young's modulus of the support (**, ## and && indicate p<0.01);

[0033] Figure 6 The images show actual specimens of the scaffolds with different AgNPs contents of the present invention after being immersed in SBF for 0, 1, 3, 7, and 15 days, respectively.

[0034] Figure 7 The in vitro silver strip release curves of the stents of the present invention with different AgNPs contents (0.1wt% (purple), 1wt% (green) and 3wt% (orange), respectively) on days 7, 14, 21 and 28.

[0035] Figure 8 The following are FTIR results for different supports and PLGA of this invention;

[0036] Figure 9 SEM micrographs (A) and XRD patterns (B) of HAP grown on different scaffold surfaces according to the present invention are shown, where (◆) represents HAP. Represents AgNPs;

[0037] Figure 10The diagram shows the qualitative analysis of the in vitro biological characteristics of different scaffolds of the present invention. A is a confocal laser image of MC3T3-E1 cells grown on the scaffold surface for 3 days after live (green) / dead (red) staining. B is a comparison of the absorbance values ​​of MC3T3-E1 cells adhering to different scaffolds after 1, 3, 5, and 7 days of culture, detected by the CCK-8 assay.

[0038] Figure 11 The diagrams show the antibacterial effects of different scaffolds according to the present invention. A represents the activity analysis of different AgNPs / PLGA scaffolds against *Escherichia coli*, B represents the activity analysis of different AgNPs / PLGA scaffolds against *Staphylococcus aureus*, C represents the antibacterial effect of different AgNPs / PLGA scaffolds demonstrated by plate coating, D represents the quantitative bacterial count by calculating the CFU of *E. coli* after scaffold treatment, and E represents the quantitative bacterial count by calculating the CFU of *Staphylococcus aureus* after scaffold treatment. (ns represents no statistical significance, * represents p < 0.05, **, ##and&& represent p < 0.01). Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1:

[0041] A method for preparing a 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold includes the following steps:

[0042] (1) Preparation of AgNPs / PLGA composite material:

[0043] a. Weigh 0.5g of AgNPs (silver nanoparticles) powder into a 10mL centrifuge tube, add 5mL of the organic solvent chloroform, and mix the two evenly by ultrasonication to obtain a 0.1mg / mL AgNPs solution;

[0044] b. Mix AgNPs solution with PLGA (DG-85DLG300) at a mass ratio of 1:1 (i.e., 1AgNPs / PLGA), then add chloroform and stir for 15 minutes with an ARV-310 stirrer (Thinky, Japan) to fully dissolve PLGA and obtain AgNPs / PLGA mixture.

[0045] (2) Fabrication of 3D-printed AgNPs / PLGA scaffolds:

[0046] The AgNPs / PLGA scaffold was fabricated using FDM technology. The scaffold model was created using Solidwork 2022 software and printed using an FDM-based 3D printer, Ultimaker 2+Extended (Ultimaker, Netherlands). The printing nozzle was a MUSASHI (TPND-25G), and the printing syringe was a MUSASHI (PSY-50E). During fabrication, the dissolved and uniformly mixed AgNPs / PLGA mixture was extruded using 250 kPa air pressure, forming filaments that were deposited onto the print bed through the nozzle. The print head moved along the X and Y axes, while the print bed was lowered along the Z axis to deposit layers sequentially to build the complete 3D structure. After each layer was deposited, the print bed was lowered, and the next layer was deposited on top of the pre-deposited layer. The 3D printing parameters were set as follows: print speed: 35%, nozzle temperature: 3℃, platform temperature: 3℃, scaffold layer height: 1 mm, scaffold width: 2 cm, scaffold length: 2 cm.

[0047] Example 2:

[0048] The difference between this embodiment and Embodiment 1 is that the phrase “AgNPs solution and PLGA (DG-85DLG300) at a mass ratio of 1:1 (i.e., 1AgNPs / PLGA)” is changed to “AgNPs solution and PLGA (DG-85DLG300) at a mass ratio of 3:1 (i.e., 3AgNPs / PLGA)”. The other steps are the same as in Embodiment 1.

[0049] Example 3:

[0050] The difference between this embodiment and Embodiment 1 is that "AgNPs solution and PLGA (DG-85DLG300) at a mass ratio of 1:1 (i.e., 1AgNPs / PLGA)" is changed to "AgNPs solution and PLGA (DG-85DLG300) at a mass ratio of 0.1:1 (i.e., 0.1AgNPs / PLGA)", while the other steps are the same as in Embodiment 1.

[0051] Characterization of AgNPs:

[0052] The particle size of the raw material AgNPs was measured using dynamic light scattering (DLS, Dynapro Titan TC). Figure 1 (As shown). AgNPs were uniformly coated on conductive adhesive, and the microstructure of the AgNPs was observed using a scanning electron microscope (SEM, FEI, Quanta FEG 250, United States). Figure 2 (As shown). From Figure 1, Figure 2 It can be seen that the particle size of nano-silver is around 100nm.

[0053] Characterization of AgNPs / PLGA scaffold physical specimens and morphology:

[0054] Bone scaffolds with different concentrations of AgNPs prepared in Examples 1-3 were prepared by mass percentage as 3AgNPs / PLGA, 1AgNPs / PLGA, and 0.1AgNPs / PLGA, respectively, and a control group of PLGA (0AgNPs / PLGA) was set up.

[0055] The microstructure and surface morphology of the bone scaffold samples prepared in Examples 1-3 and the control group PLGA samples were observed using scanning electron microscopy (SEM) at a scanning voltage of 5 kV. Before testing, the samples were sputter-coated with gold to increase their conductivity. Gold sputtering was applied to both the sides and cross-sections of the samples.

[0056] The results are as follows Figure 3 As shown, scanning electron microscopy (SEM) reveals that AgNPs are uniformly distributed within the AgNPs / PLGA scaffold (e.g., Figure 3 As shown in Figure A). Elemental analysis of the AgNPs / PLGA scaffold using EDS confirmed the presence of silver nanoparticles within the scaffold (e.g., ...). Figure 3 (as shown in B).

[0057] Our 3D model of the AgNPs / PLGA bracket, printed using DIW technology, exhibits a uniform porous structure with interconnected pores (e.g., ...). Figure 4 (As shown). The porous microstructure is an important feature of the bone scaffold because its large surface area to volume ratio promotes protein adsorption and provides anchoring sites for osteoblasts.

[0058] When the pore size of the scaffold is greater than 300 μm, it facilitates the infiltration, adhesion and migration of bone tissue cells, and also promotes blood vessel growth, thereby providing nutrients for bone tissue repair. The minimum acceptable size of the scaffold is about 100 μm

[38] , because pore sizes smaller than 100 μm may inhibit angiogenesis. Scaffolds with an average pore size of 550 μm are most conducive to bone tissue formation. Our scaffold has a pore size greater than 300 μm (e.g., Figure 4 (As shown in the front view).

[0059] Furthermore, the presence of gaps between each layer of the scaffold indicates that the scaffold is interconnected. In vivo and in vitro studies have shown that porosity has a positive impact on bone deposition rate and cell penetration depth

[40] . Microscopic images of the scaffold (e.g.) Figure 4The Micrograph shows that the fiber diameter is uniform and the internal composition of the fiber is compact, indicating that the 3D printed scaffold also has structural uniformity in terms of filament diameter.

[0060] Scanning electron microscope (e.g.) Figure 4 As shown in the figure, under the above printing conditions, the printed scaffold has high structural uniformity and controllable geometry, indicating that we can adjust the printing parameters according to the repair of bone tissue in different parts, thereby achieving the purpose of treatment.

[0061] Mechanical properties of the stent:

[0062] Scaffolds used in bone tissue engineering should possess mechanical properties suitable for the target tissue. Mechanical forces play a crucial regulatory role in bone remodeling and repair.

[0063] The tensile properties of the samples were measured using the AGS-X electromechanical test frame (SHIMADZU, Japan) according to the American Society for Testing and Materials (ASTM D638) standard. The Young's modulus and stress-strain curves of the AgNPs / PLGA stent were obtained through tensile testing to evaluate the mechanical properties of the stent.

[0064] Tensile testing, as a fundamental test for determining material strength, is widely accepted. The most commonly used test is the uniaxial tensile test, which involves uniformly stretching the specimen at a specified rate and recording the tensile force and elongation. Some material parameters, such as stress-strain curves and Young's modulus, can be derived. Therefore, we used tensile testing to characterize the mechanical properties of the support structure, with each sample repeated an average of three times.

[0065] like Figure 5 As shown in Figure A, the tensile strength of PLGA is 34.9 ± 0.23 MPa. Meanwhile, the addition of AgNPs significantly improves the tensile strength of the material. Specifically, the tensile strength of 0.1 AgNPs / PLGA is 45 ± 0.17 MPa, while the tensile strengths of 1 AgNPs / PLGA and 3 AgNPs / PLGA are 51.3 ± 0.67 MPa and 52.7 ± 0.51 MPa, respectively. Figure 5 As shown in Figure B, the Young's modulus of PLGA is 1.96±0.03 GPa. For the AgNPs / PLGA scaffold, the Young's modulus increases to 2.28±0.04 GPa, 2.44±0.08 GPa, and 2.46±0.08 GPa with increasing AgNPs concentration.

[0066] Typically, fillers restrict the flowability of polymer matrices and enhance scaffold rigidity. In this study, AgNPs were also used as fillers, improving scaffold rigidity because the addition of silver enhanced the Young's modulus of the AgNPs / PLGA scaffold, consistent with previous findings. However, the difference in Young's modulus between 1AgNPs / PLGA and 3AgNPs / PLGA was not statistically significant (2.44 vs. 2.46, p = 0.535), indicating that the Young's modulus of the scaffold may not increase indefinitely with increasing silver concentration. Here, the Young's modulus value of the scaffold falls between that of cancellous bone (0.02-0.5 GPa) and cortical bone (3-30 GPa), a result that supports the use of AgNPs / PLGA scaffolds in bone tissue engineering applications.

[0067] In vitro release of silver ions from the stent:

[0068] 0.1 g of AgNPs / PLGA scaffolds at different mass ratios were placed in 50 mL centrifuge tubes, and 20 mL of deionized water was added. The tubes were gently shaken until the scaffolds were completely submerged. The tubes were then incubated at 37°C. 3.5 mL deionized water samples were collected on days 7, 14, 21, and 28, and filtered through a 0.22 μm filter. The silver ion concentration in the solution was measured using inductively coupled plasma (ICP-7400, Thermo Fisher). Additionally, the scaffolds were soaked in deionized water, and color changes were observed to determine whether silver ions were being released from the scaffolds.

[0069] The release rate of silver ions from AgNPs / PLGA scaffold materials was measured to determine which silver ion concentration of AgNPs / PLGA scaffolds is more suitable for bone injury repair. Stable and sustained silver ion release is essential to maintain the long-term antibacterial effect of the scaffold. To provide effective antibacterial properties, it is necessary to maintain the release rate at a concentration level of at least 0.1 ppb.

[0070] like Figure 7 As shown, in the initial seventh day, the release rate of silver ions was rapid, with 0.23 ppm released by 3AgNPs / PLGA, and 0.035 ppm and 0.026 ppm by 1AgNPs / PLGA and 0.1AgNPs / PLGA, respectively. After seven days, the release rate of silver ions slowed down but still showed an increasing trend. On day 21, the release of silver ions reached a plateau. On day 28, silver ions continued to be released, indicating that silver ions were continuously released over time, which is essential for the long-term antibacterial effect of a bone tissue engineering repair scaffold. After soaking in deionized water, we observed that the color of the scaffold gradually lightened with prolonged soaking time (e.g., ...). Figure 6As shown in the image, this indicates that the scaffold releases silver ions. These results lay the foundation for our subsequent verification of the antibacterial effect.

[0071] Chemical structural characterization of the scaffold:

[0072] FTIR is used to detect the formation of chemical structures within the scaffold. As is well known, FTIR can detect chemical bond information. Figure 8 The FTIR spectra of PLGA, 0.1AgNPs / PLGA, 1AgNPs / PLGA, and 3AgNPs / PLGA scaffolds are shown. In the FTIR spectrum of the PLGA copolymer, we found a peak at 1753 cm⁻¹. -1 There is a strong peak at 1090 and 1182 cm⁻¹, which belongs to the tensile vibration of the C=O bond in the PLGA polyester structure. -1 The absorption peaks at 2885 and 2940 cm⁻¹ represent CO ether bonds. -1 The position represents an aliphatic CH2 group. All AgNPs / PLGA scaffolds, namely 0.1AgNPs / PLGA, 1AgNPs / PLGA, and 3AgNPs / PLGA, exhibited all the peaks corresponding to PLGA. No new peaks appeared in the AgNPs / PLGA scaffolds compared to PLGA. Therefore, no new chemical bonds were formed between AgNPs and PLGA.

[0073] Biological characterization of the scaffold:

[0074] The scaffold was immersed in SBF, and biological tests were performed on the samples, with three replicates for each sample. After 14 days, the scaffold was removed and dried overnight. The formation of HAP on the sample surface was observed under an electron microscope. The formation of HAP on the scaffold was further characterized by XRD to confirm the presence of HAP.

[0075] Biological properties are another important characteristic of bone implant materials. Microscopic images obtained by observing the surface under a scanning electron microscope after SBF immersion are shown below. Figure 9 As shown in Figure A, mineralized HAP growth was clearly visible on all scaffold surfaces. The HAP growth was further characterized by XRD, with results as shown in Figure A. Figure 9 As shown in Figure B, each scaffold exhibits a HAP characteristic peak at 31.89° in the XRD images. Furthermore, four peaks were observed at 38°, 44°, 65°, and 77° in the 1AgNPs / PLGA and 3AgNPs / PLGA scaffolds, representing Bragg reflections from the Ag(111), (200), (220), and (311) reflective planes, respectively. This also confirms the presence of AgNPs in the scaffolds. Due to the addition of a small amount of AgNPs in the 0.1AgNPs / PLGA scaffold, no characteristic peaks of AgNPs were observed.

[0076] Besides mechanical properties, biocompatibility is also a crucial performance characteristic of bone implant materials. This study evaluated the suitability of AgNPs / PLGA scaffolds for human bone tissue repair by verifying their cytotoxicity. MC3T3-E1 cells were used to assess the cell compatibility of the AgNPs / PLGA scaffold, and MC3T3-E1 is a good candidate for in vitro analysis of bone cells. Previous studies have shown that loading appropriate amounts of silver onto the material surface can exhibit good cell compatibility and proliferation rates. After culturing MC3T3-E1 cells for 3 days, cell viability was determined using a live / dead staining method. Live cells were stained with calcein-am (green), and dead cells were stained with propidium iodide (PI, red) under a fluorescence microscope. After 3 days of culture, we observed uniform cell attachment to the scaffold.

[0077] As the concentration of AgNPs increased, almost no dead cells were observed, indicating that at the highest AgNPs concentration in this experiment, there was no cytotoxicity, but it had a good antibacterial effect. Figure 10 A). The results of the CCK-8 experiment show that ( Figure 10 B) On days 1, 3, 5, and 7, cell viability was not significantly affected as the AgNP content increased. Neither the experimental nor the control group showed obvious cytotoxicity, which was consistent with the results of live and dead cell staining.

[0078] Studies have shown that excessive amounts of nanosilver can be toxic to the human body. In our study, the scaffold printed with the nanosilver content we incorporated was verified to be non-toxic through cell experiments. Figure 7 As can be seen, the silver release process of the scaffold is slow and continuous, thanks to the advantages of DIW printing technology, which avoids the cytotoxicity caused by sudden silver release. Therefore, the AgNPs-modified scaffold we printed has good biocompatibility and has the potential for application in bone tissue repair in the human body.

[0079] Cell proliferation:

[0080] 5mm diameter scaffolds were pre-soaked in alcohol for 2 hours, then washed three times with sterile PBS, and finally irradiated overnight with UV light. Cytotoxicity experiments were performed using mouse embryo osteoblast precursor cells (MC3T3-E1, Chinese Academy of Medical Sciences, China). The culture medium used was Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 5% penicillin / streptomycin (P / S, Biochrom AG), using T25 cell culture dishes. After cell resuscitation, the cells were incubated at 37°C in a 5% CO2 incubator. Once the cells had reached confluence, they were digested with trypsin (Gibco, United States), and the digested cells were counted. Cells were seeded in 96-well cell culture plates at a density of 1 × 10⁶ cells per well. 3 Cells. Five replicates were prepared for each scaffold group. The scaffolds were placed in 96-well plates before cell seeding. Cytotoxicity of the scaffolds was assessed using a CCK8 kit (BestBio, Shanghai, China) on days 1, 3, 5, and 7. Absorbance at 450 nm was measured using a microplate reader.

[0081] Fluorescent staining:

[0082] The scaffolds were cut to 1cm × 1.3cm size, pre-soaked in alcohol for 2 hours, washed three times with sterile PBS, and finally irradiated overnight with UV light. Cytotoxicity assays were performed using MC3T3-E1, similar to the CCK-8 assay. DMEM containing 10% FBS and 5% penicillin antibiotics was used as the culture medium. The cut scaffolds were placed in 24-well plates. Before adding the culture medium, a 6mm diameter iron ring was placed on top of the scaffold to ensure it sinks to the bottom. 1 × 10⁴ cells were seeded in each well, and 1 mL of DMEM was added. On day 3, Calcein AM·PI (BB-4126, Bestbio) was added for staining, and the cell status on the scaffolds was observed using a fluorescence confocal microscope.

[0083] Verification of antibacterial activity:

[0084] The antibacterial properties of the PLGA / AgNps scaffold were tested using *Staphylococcus aureus* (S. Aureus, ATCC 25923) and *Escherichia coli* (E. coli, ATCC 25922). Single colonies of each bacterium were picked and placed into 15 mL centrifuge tubes containing 5 mL of LB medium, and incubated overnight. The final bacterial concentration was diluted to 10⁵ CFU / mL. After scaffold printing, the scaffolds were dried in a 37°C oven. The scaffolds were then cut into 5 mm diameter discs. Before the antibacterial test, the scaffolds were soaked in 75% alcohol for 2 hours, followed by UV irradiation for 2 hours. The antibacterial test was performed using 96-well plates. First, sterile 5mm diameter scaffolds were placed in 96-well plates, with five replicates for each scaffold. Then, 100μL of bacterial solution was added to a final concentration, and the plates were incubated overnight (12 hours). The absorbance of the bacterial solution at 600nm was measured using a microplate reader (SPARK, Tecan) to estimate the bacterial growth status and thus evaluate the antibacterial effect of the scaffolds. Simultaneously, the bacterial solution in each group was diluted 105-fold, and colony counting was used to further verify the antibacterial effect of the scaffolds.

[0085] Ag concentrations within a certain range can kill bacteria without impairing mammalian cell function. AgNPs, with their broad antibacterial spectrum, are active against both Gram-positive and Gram-negative bacteria, and no bacterial resistance induced by AgNPs has been observed in clinically relevant pathogens. In our study, the decrease in OD600 absorbance indicates that the scaffold can inhibit the growth of *Escherichia coli* and *Staphylococcus aureus*.

[0086] As the concentration of AgNPs increased, its inhibitory effect on the growth of Escherichia coli became increasingly pronounced. Furthermore, the addition of AgNPs significantly inhibited the growth of Staphylococcus aureus. Figure 11 A&B). The antibacterial effects of 1 AgNPs / PLGA and 3 AgNPs / PLGA compared to PLGA were statistically significant (p < 0.001), while there was no statistically significant difference between 0.1 AgNPs / PLGA and PLGA (p = 0.107). When the concentration of AgNPs reached 3%, it could basically completely inhibit the growth of both Staphylococcus aureus and Escherichia coli, and there was no cytotoxicity at this concentration. Figure 10 ).

[0087] After incubating the diluted bacteria on MHA agar plates, we observed no sterile colonies growing on 3AgNPs / PLGA plates. Figure 11 C). The inhibitory effect increases with increasing AgNP concentration. Figure 11D and E, by CFU counting

[27] , the colonies of 0.1AgNPs / PLGA and 1AgNPs / PLGA scaffolds were less than those of PLGA and the control group, and the antibacterial ability of 3AgNPs / PLGA scaffold was significantly greater than that of the control group (p<0.01). There was no statistically significant difference in colony count between the PLGA group and the control group (p>0.05). This antibacterial effect is consistent with the antibacterial effect of OD600, further proving the antibacterial effect of AgNPs / PLGA scaffold. The antibacterial effect is achieved by releasing silver ions from the inside of the scaffold.

[0088] These results demonstrate the potential applications of the 3D-printed AgNPs / PLGA scaffolds of this invention in bone tissue engineering.

[0089] Statistical methods:

[0090] All data were analyzed using a two-tailed unpaired t-test. Furthermore, the differences between groups were as follows: ns p>0.05, * / # / &p<0.05,** / ## / &&p<0.01.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold, characterized in that, Includes the following steps: (1) Preparation of AgNPs / PLGA composite material: a. Place AgNPs powder and organic solvent in a centrifuge tube, and mix them evenly using ultrasound to obtain an AgNPs solution; the particle size of the AgNPs is 100 nm; the concentration of the AgNPs solution is 0.1 mg / mL; b. Mix the AgNPs solution with PLGA, then add an organic solvent and stir until homogeneous to obtain an AgNPs / PLGA mixture; the mass ratio of the AgNPs solution to PLGA is 0.1-3:1; (2) Preparation of AgNPs / PLGA scaffold for 3D printing: The AgNPs / PLGA mixture is printed using an FDM-based 3D printer. During the fabrication process, the AgNPs / PLGA mixture is extruded by air pressure and deposited onto the printing bed through a nozzle to form filaments. The print head is moved along the X and Y axes and the printing bed is lowered in the Z axis direction to perform layer-by-layer deposition to build a complete 3D structure.

2. The method for preparing the 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold according to claim 1, characterized in that, In step (1), the organic solvent is trichloromethane.

3. The method for preparing the 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold according to claim 1, characterized in that, The PLGA used in step b is model DG-85DLG300.

4. The method for preparing a 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold according to claim 1, characterized in that, In step b, the mass ratio of AgNPs solution to PLGA is 1:

1.

5. The method for preparing the 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold according to claim 1, characterized in that, In step b, the mass ratio of AgNPs solution to PLGA is 3:

1.

6. The method for preparing the 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold according to claim 1, characterized in that, In step b, the mass ratio of AgNPs solution to PLGA is 0.1:

1.

7. The method for preparing a 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold according to claim 1, characterized in that, The 3D printer used in step (2) is a Ultimaker 2+ Extended.

8. The method for preparing a 3D-printed AgNPs / PLGA antibacterial bone tissue engineering scaffold according to claim 1, characterized in that, In step (2), the printer nozzle model is TPND-25G and the printer syringe model is PSY-50E.

9. A bone tissue engineering scaffold prepared by the preparation method according to any one of claims 1-8.

10. The application of the bone tissue engineering scaffold prepared by the preparation method according to any one of claims 1-8 in the preparation of bone defect repair materials.

Images