Infectious bone defect bionic repair hydrogel scaffold and preparation method and application thereof

Abstract

The application discloses an infectious bone defect bionic repair hydrogel support and a preparation method and application thereof, and is characterized in that: the infectious bone defect bionic repair hydrogel support is composed of an upper antibacterial-vascularization layer simulating a bone membrane structure, a middle supporting layer simulating a bone cortex structure and a lower regeneration-regulation layer simulating a bone cancellous structure, the upper antibacterial-vascularization layer is made of a light response CuxO / SiC heterojunction powder, the middle supporting layer is made of silk fibroin, and the lower regeneration-regulation layer is made of HAP@Cur-CNDs-Pt NPs composite nanoparticles. Multi-dimensional structure and function integration realize the whole cycle regulation of 'antibacterial-barrier-regeneration', break through the limitation of traditional single function support, and provide an integrated solution for infectious bone defect repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical stent prosthesis engineering, in particular to an infectious bone defect biomimetic repair hydrogel stent and a preparation method and application thereof. BACKGROUND

[0002] Repair of infectious bone defects, such as large-area defects of oral and maxillofacial bone tissue caused by factors such as inflammation, trauma or surgery, is a major challenge in clinical practice. Such defects are often accompanied by bacterial infection, and the invasion of pathogenic bacteria not only inhibits the self-healing ability of bone, but also triggers excessive inflammatory response due to pathogen-associated molecular patterns (PAMPs) on the surface of the pathogen, which seriously hinders the regeneration process. The unique anatomical features of the maxillofacial bone, including porous structure, heterogeneous vascular distribution and mechanical load difference, shape a highly partitioned microenvironment, which precisely regulates the balance between bacterial colonization and immune response through the spatial distribution of oxygen gradient, mechanical stress and metabolic products. When the complex defect destroys the original three-dimensional structure, microorganisms are easy to break through the anatomical barrier to form abnormal colonization, leading to disordered distribution of immune cells, imbalance of pro-inflammatory / anti-inflammatory factors, and finally causing pathological bone resorption and fibrosis. Although the traditional bone graft combined with antibiotic regimen can partially solve the problem of anatomical reconstruction, it has inherent defects such as donor shortage, easy recurrence of infection and generation of drug-resistant bacteria. These clinical difficulties indicate that breaking through the existing treatment bottleneck requires seeking solutions from the dual dimensions of anatomical reconstruction and immune microenvironment regulation.

[0003] Bone tissue has a unique three-dimensional spatial hierarchical structure system, and its anatomical composition and functional positioning exhibit exquisite biomechanical adaptability. From the outside to the inside, it can be divided into three functional units: the outermost periosteum as a dynamic metabolic interface, rich in capillary network and nerve endings, not only undertakes more than 80% of the nutrient transport task of bone tissue, but also realizes the repair of bone damage through the proliferation and differentiation of inner layer osteogenic progenitor cells; the intermediate layer of bone cortex constitutes a mechanical barrier, which is composed of a regular arrangement of Haversian system. This layered structure enables the skeleton to effectively disperse impact energy, and the osteocyte lacuna-canalicular system constitutes a precise information transmission network; the core layer of cancellous bone presents a porous network structure, and the trabeculae are arranged in order along the stress line. This open structure not only reduces the weight of the bone, but also provides an ideal niche for mesenchymal stem cells. These stem cells have multilineage differentiation potential, regulate osteoclast activity by secreting factors, participate in immune surveillance, and form a unique bone immune microenvironment. The interaction network of this highly unified structure-function system realizes the integration of mechanical support, metabolic regulation and immune defense. Therefore, an ideal repair system needs to realize the organic unity of spatial reproduction of anatomical structure, dynamic transmission of mechanical signal and time sequence regulation of immune metabolism. SUMMARY

[0004] To solve the above technical problems, the first object of the present application is to provide a biomimetic repair hydrogel scaffold for infected bone defects, the second object is to provide a preparation method thereof, and the third object is to provide an application thereof. The multi-dimensional structure and function integration realizes the whole cycle regulation of "antibacterial-barrier-regeneration", breaks through the limitation of traditional single function scaffold, and provides an integrated solution for infected bone defect repair.

[0005] To achieve the above first object, the present application is implemented by the following technical scheme: a biomimetic repair hydrogel scaffold for infected bone defects, characterized in that: it is composed of an upper antibacterial-vascularization layer simulating the structure of periosteum, a middle support layer simulating the structure of bone cortex, and a lower regeneration-regulation layer simulating the structure of cancellous bone. The upper antibacterial-vascularization layer is made of light-responsive CuxO / SiC heterojunction powder, the middle support layer is made of silk fibroin, and the lower regeneration-regulation layer is made of HAP@Cur-CNDs-Pt NPs composite nanoparticles.

[0006] In the above scheme, the light-responsive CuxO / SiC heterojunction powder is prepared by the following method:

[0007] (1) Disperse 40-60 nm SiC nanoparticles in deionized water and ultrasonically treat to form a uniform suspension;

[0008] (2) Under stirring conditions, add a copper salt solution to the SiC suspension and continue stirring to allow copper ions to be fully adsorbed on the surface of SiC;

[0009] (3) Add a reducing agent solution drop by drop;

[0010] (4) Adjust the pH value of the mixed system to 8-9 with an alkaline solution;

[0011] (5) Heat the mixed system at 70-90°C for 6-12 hours;

[0012] (6) After the reaction is completed, collect the precipitate by centrifugation and repeatedly wash with deionized water, and finally freeze-dry to obtain CuxO / SiC heterojunction powder.

[0013] In the above scheme: the copper salt is one of copper chloride, copper sulfate and copper nitrate, and the mass ratio of the copper salt to SiC nanoparticles is 1:1; the reducing agent is L-ascorbic acid; the alkaline solution is sodium hydroxide solution. The amount of L-ascorbic acid added is 5-6.25 times the mass of SiC nanopowder and copper salt.

[0014] In the above scheme, the preparation method of the HAP@Cur-CNDs-Pt NPs composite nanoparticles is:

[0015] (1) Preparation of curcumin-derived carbon dots (Cur-CNDs): Curcumin was used as a precursor, and a high-temperature pyrolysis method was used for carbonization. Subsequently, the carbonized product was ground and dispersed in water, boiled, and then purified by centrifugation, filtration, and dialysis to obtain a Cur-CNDs aqueous solution with antioxidant activity.

[0016] (2) Preparation of CNDs-Pt: The Cur-CNDs solution was mixed with a chloroplatinic acid solution. Under alkaline conditions, the reducing property of Cur-CNDs was used as a carrier, and sodium borohydride was added to assist the in-situ reduction of Pt 4+ nanoparticles, which were loaded on the surface of Cur-CNDs to form a CNDs-Pt complex. The complex was purified by dialysis.

[0017] (3) HAP@Cur-CNDs-Pt NPs complexation: Nano-hydroxyapatite was dispersed in water, and the CNDs-Pt complex solution was added dropwise under stirring. Through electrostatic adsorption and coordination, CNDs-Pt was firmly loaded on the surface of HAP. After centrifugation and freeze-drying, the final composite nanoparticles were obtained.

[0018] In the above scheme, in step (1), curcumin was carbonized at 400-500°C. After filtration, the supernatant was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and the water was changed every 2 hours.

[0019] In the above scheme, step (2) is prepared as follows: Cur-CNDs solution is mixed with chloroplatinic acid solution, and the pH is adjusted to 9-11 with NaOH solution. Under stirring, sodium borohydride solution is slowly added, and the reaction is carried out at room temperature for 3-4 hours. After standing, the reaction is completed, and the solution is neutralized with hydrochloric acid and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain a CNDs-Pt solution.

[0020] The mass ratio of Cur-CNDs to chloroplatinic acid and sodium borohydride is 1:0.41-0.52:9-10.

[0021] In step (3), the mass ratio of CNDs to HAP is 1:5-1:1.

[0022] The second object of the present application is achieved as follows: The preparation method of the infectious bone defect biomimetic repair hydrogel scaffold is characterized by the following steps:

[0023] (1) Ink preparation: CuxO / SiC heterojunction powder, silk fibroin, and HAP@Cur-CNDs-Pt NPs composite nanoparticles are mixed with light crosslinking hydrogel prepolymer solution methylacrylated gelatin GelMA to prepare upper, middle, and lower biological inks.

[0024] (2) 3D printing and crosslinking: using a multi-nozzle extrusion type 3D printing system, according to the preset three-dimensional model and pore structure, the lower layer, the middle layer and the upper layer are printed in turn, and during and after printing, the light of a specific wavelength is irradiated to initiate the photoinitiator to make the GelMA and other prepolymers crosslink and solidify to form a stable three-dimensional hydrogel network, and a three-dimensional hydrogel scaffold with integrated structure and function is obtained.

[0025] In the above scheme: each layer is irradiated with 405 nm blue light for 30 seconds for preliminary crosslinking and solidification, and after printing is completed, the entire scaffold is irradiated under 405 nm blue light for 2 minutes to make it completely crosslink and solidify, and the final multilayer biomimetic repair hydrogel scaffold is obtained.

[0026] The application of a biomimetic repair hydrogel scaffold for infected bone defects in the preparation of medical devices or tissue engineering products for repairing conventional or infected bone defects.

[0027] In the above scheme: the infected bone defect is a maxillofacial bone defect or a skull defect caused by Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa infection.

[0028] The present application constructs a 3D structure scaffold from structural biomimicry to functional biomimicry to effectively cope with the various challenges of infected bone defects. In terms of structural biomimicry: the gradient pore structure is constructed by 3D printing to simulate the mechanical transmission characteristics and spatial framework of natural bone tissue to activate cell mechanical sensing signals; in terms of functional biomimicry, the layered 3D printed hydrogel scaffold can realize the integration of multiple functions: the upper layer simulates the periosteum as an antibacterial-vascularization layer, and loads a light-responsive CuxO / SiC heterojunction, which realizes controllable antibiosis through photocatalytic cascade reaction and photothermal effect under light; after stopping the light, the system utilizes the continuously released Cu 2+ and the trace amount of H2O2 generated by catalysis to activate key signaling pathways such as HIF-1α / VEGF, and to induce angiogenesis mildly and continuously; the middle layer simulates the high-strength cortical bone using silk fibroin; the lower layer simulates the spongiosa as a regeneration-regulation layer to play the role of osteogenesis and immune regulation, and nano-hydroxyapatite (HAP) loaded with antioxidant / immune regulation components (Cur-CNDs-Pt NPs) can remove residual ROS and promote osteogenesis-immune synergy; the layers of the hydrogel scaffold can realize dynamic synergy, and the ROS "on-demand activation" property of the light-responsive heterojunction (CuxO / SiC) and the continuous antioxidant function of HAP@CNDs-Pt NPs form a complement, with time-controllable property. This multi-dimensional integration of structure and function realizes the whole-cycle regulation of "antibiosis-barrier-regeneration", breaks through the limitations of traditional single-function scaffolds, and provides an integrated solution for infected bone defect repair.

[0029] This invention constructs a multi-layered 3D-printed hydrogel scaffold through two core strategies: "spatial partitioning" and "multi-enzyme cascade." Spatial partitioning refers to mimicking the structure of natural bone by positioning different functional materials at different levels of the scaffold; multi-enzyme cascade refers to utilizing the "light-switching" properties of the upper-layer photoresponsive material to perform antibacterial functions during the light-reflecting period and switch to a repair-promoting function during the non-light-reflecting period, thus complementing the continuous function of the lower layers.

[0030] Lower regeneration-regulation layer: mimicking cancellous bone, this layer serves as the primary interface for the scaffold to integrate with the host bone. It is designed as a three-dimensional interconnected porous structure with large pore sizes (e.g., 300-500 μm) to facilitate cell ingrowth, vascularization, and nutrient transport.

[0031] The mechanism of action of HAP@Cur-CNDs-Pt NPs composite nanoparticles is as follows:

[0032] ROS scavenging and antioxidation: Pt NPs have catalase-like and superoxide dismutase-like activities, and Cur-CNDs also have excellent antioxidant properties. The two work together to efficiently scavenge residual ROS that may have penetrated from the upper layer and excess ROS in the lesion site, alleviate oxidative stress, and protect mesenchymal stem cells (MSCs) and osteoblasts.

[0033] Immunomodulation: By reducing oxidative stress and the anti-inflammatory effects of curcumin derivatives, macrophages are induced to polarize from the pro-inflammatory M1 type to the anti-inflammatory M2 type, creating an immune microenvironment conducive to tissue regeneration.

[0034] Promoting bone differentiation: HAP itself provides a biomineralization template and osteogenic signal. At the same time, a healthy, low-ROS microenvironment can effectively promote osteogenic differentiation of MSCs, achieving high-quality bone regeneration.

[0035] Intermediate support layer: mimicking the bone cortex, this dense or minimally porous insulating layer provides primary mechanical support. A dense hydrogel network formed using silk fibroin provides a physical barrier.

[0036] Upper antibacterial-vascularized layer: mimicking the periosteum, this layer serves as the interface with soft tissue or the external environment. It also possesses a porous structure but is loaded with active ingredients that respond to external stimuli. The pore size is determined based on the characteristics of the soft tissue on the periosteum in different parts of the human body.

[0037] The mechanism of photoresponsive CuxO / SiC heterojunction is as follows:

[0038] Illumination Period (“On”): Under near-infrared light (808nm laser) irradiation, the CuxO / SiC heterojunction achieves efficient separation of photogenerated electron-hole pairs due to its built-in electric field. On one hand, these charge carriers react with H₂O / O₂ to generate reactive oxygen species (ROS), such as ·OH, through photodynamic therapy (PDT). 1 O2; on the other hand, heat is generated through the localized surface plasmon resonance (LSPR) effect to achieve photothermal therapy (PTT). The synergistic effect of PTT and PDT can rapidly and efficiently destroy bacterial biofilms and kill pathogens.

[0039] Non-illuminated period ("off"): After illumination is stopped, the heterojunction can continuously and slowly release low concentrations of Cu. 2+ And trace amounts of ROS. Low concentration of Cu 2+ It has been proven to be an effective pro-angiogenic factor, capable of upregulating the expression of genes such as HIF-1α and VEGF in vascular endothelial cells, thereby promoting angiogenesis. At this stage, its function shifts from "killing" to "repairing".

[0040] Compared with the prior art, the present invention has the following beneficial effects.

[0041] 1. Structural Biomimicry and Mechanical Adaptation: Hydrogel scaffolds that precisely simulate the spatial hierarchical structure and porosity characteristics of natural bone tissue (periosteum-cortical bone-cancellous bone) from macroscopic to microscopic levels, enabling them to achieve a good anatomical and biomechanical match with the host bone tissue. 2. Time-Controllable Synergistic Treatment: Achieving a time-sequential functional switch between "short-term highly effective antibacterial action" and "long-term orderly regeneration." Rapidly clearing biofilms and pathogens in the initial treatment phase, followed by precise regulation of the immune microenvironment, promoting angiogenesis and osteogenic differentiation during the repair phase, avoiding secondary damage to the regeneration process caused by traditional antibacterial strategies. 3. Multifunctional Integrated Design: Integrating seemingly contradictory functions into a single scaffold through spatial partitioning design, resolving the temporal and spatial contradictions of "antibacterial-anti-inflammatory-osteogenic" effects, and achieving synergistic effects. 4. Personalized Precision Repair: Utilizing 3D printing technology, the scaffold can be personalized according to the specific three-dimensional morphology of the patient's bone defect, achieving precise implantation. Attached Figure Description

[0042] Figure 1 A schematic diagram illustrating the overall construction concept and mechanism of action of the 3D-printed multifunctional hydrogel scaffold based on bone space structure biomimicry provided by the present invention.

[0043] Figure 2 The characterization results of the CuxO / SiC heterojunction are shown in the figures, including (A) high-resolution transmission electron microscopy (TEM) image, (B) scanning electron microscopy (SEM) image, (C) energy dispersive spectroscopy (EDS) elemental distribution map, (D) X-ray diffraction (XRD) pattern, and (EG) X-ray photoelectron spectroscopy (XPS).

[0044] Figure 3 The images show the multi-enzyme activity and photothermal properties of the CuxO / SiC heterojunction, including (A) peroxidase (POD) activity, (B) glutathione (GSH) oxidase activity, (C) singlet oxygen generation capacity, (D) UV-Vis absorption spectrum, (E1, E2) photothermal temperature rise diagrams and curves, and (F) photothermal stability.

[0045] Figure 4 Characterization images of HAP@Cur-CNDs-Pt NPs composite nanoparticles: (A) Morphology of nanoparticles observed by transmission electron microscopy; (B) Zeta potential; (C) SOD enzyme activity; (D) CAT enzyme activity.

[0046] Figure 5 The diagram shows the 3D printing process and physical property characterization of the multilayer hydrogel scaffold described in this invention, including (A) a schematic diagram of 3D printing, (B) a macroscopic photograph, and (C) a scanning electron microscope microstructure diagram.

[0047] Figure 6 The diagram and results of in vitro experiments to verify the functions of each layer of the scaffold include (A) the antibacterial effect of the upper CuxO / SiC heterostructure, (B) the angiogenesis-promoting ability, (C) the antioxidant effect of HAP@Cur-CNDs-Pt NPs, (D) the immunomodulatory effect, and (E) the osteogenic ability.

[0048] Figure 7 Micro-CT bone regeneration analysis results were used to verify the efficacy of the scaffold in treating infected bone defects in in vivo animal experiments. Detailed Implementation

[0049] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0050] Example 1:

[0051] Preparation and characterization of CuxO / SiC heterostructures

[0052] a. Prepare SiC nanoparticle (30-50nm) solution: Weigh 120mg of SiC nanoparticle powder, add it to 30mL of deionized water, and ultrasonically disperse for 30 minutes.

[0053] b. Prepare copper chloride solution: Add 120 mg of copper chloride dihydrate to 160 mL of deionized water.

[0054] c. Under stirring conditions, add copper salt solution to the SiC suspension and continue stirring for 30 minutes to allow copper ions to be fully adsorbed onto the SiC surface.

[0055] d. Dissolve 1500 mg of L-ascorbic acid in 30 mL of deionized water and slowly add it dropwise to the above mixture.

[0056] e. To prepare a 1M NaOH solution: Weigh 2g of NaOH and dissolve it in 50mL of deionized water.

[0057] f. Adjust the pH of the mixture to 8-9 with NaOH solution, then transfer it to a reaction vessel and react at 70-90℃ for 10 hours.

[0058] g. After the reaction is complete, the mixture is allowed to cool naturally to room temperature. The reaction suspension is then centrifuged, and the precipitate is washed three times with deionized water. After freeze-drying, CuxO / SiC heterojunction powder is obtained.

[0059] Copper salts can be selected from copper chloride, copper sulfate, and copper nitrate.

[0060] Characterization methods: Morphology and heterostructure were observed using high-resolution transmission electron microscopy and field emission scanning electron microscopy; phase composition and elemental chemical states were analyzed using X-ray diffraction patterns and X-ray photoelectron spectroscopy; light absorption performance was evaluated using ultraviolet-visible absorption spectroscopy; and the types and ability of reactive oxygen species generated under illumination were verified using specific fluorescent reagents. For example... Figure 2 As shown, high-resolution transmission electron microscopy (HR-TEM) and field emission scanning electron microscopy (FE-SEM) observations revealed that CuxO nanoparticles were successfully loaded onto the SiC surface, forming a clear heterogeneous interface. The lattice spacings of CuxO and SiC were measured to be 0.23 nm and 0.25 nm, respectively. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) results further confirmed the coexistence of Cu2O and CuO phases in the material. (See attached image) Figure 3 As shown, the UV-Vis absorption spectrum indicates that this heterojunction has a broad light absorption range (330-900 nm). Under irradiation with an 808 nm laser at 1 W / cm², the material exhibits a significant photothermal effect, rapidly heating to over 60°C within 5 minutes, demonstrating its excellent photothermal conversion capability. Performance tests show that the CuxO / SiC heterojunction possesses peroxidase-like (POD) activity, glutathione (GSH) oxidase activity, and singlet oxygen generation capability.

[0061] Preparation and characterization of HAP@Cur-CNDs-Pt NPs composite nanoparticles

[0062] (1) Preparation of curcumin-derived carbon dots (Cur-CNDs):

[0063] a. Place 200g of turmeric powder in a crucible, wrap it with tin foil, and place it in a muffle furnace. Heat at 400-500℃ for 2 hours.

[0064] b. After natural cooling, grind the obtained carbonized product, add it to deionized water and boil for 0.5-1.5 hours (repeat twice) to dissolve and break up large particles.

[0065] c. Cool the mixture, centrifuge, and collect the supernatant.

[0066] d. Dialyze the supernatant using a dialysis bag with a molecular weight cutoff of 3500 Da for 24 hours, changing the water every 2 hours, to finally obtain a purified Cur-CNDs aqueous solution.

[0067] (2) Preparation of CNDs-Pt:

[0068] A 600 μg / mL Cur-CNDs solution (80 mL) was mixed with a 10 mM chloroplatinic acid solution (6.15 mL), and the pH was adjusted to 9-11 with approximately 12.3 mL of 0.5 M NaOH solution. While stirring, a 10 mg / mL sodium borohydride solution (46 mL) was slowly added, and the mixture was allowed to react at room temperature for 3-4 hours, then allowed to stand overnight. After the reaction was complete, the solution was neutralized with hydrochloric acid and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain a CNDs-Pt solution.

[0069] (3) Recombination of HAP@Cur-CNDs-Pt NPs:

[0070] Weigh 60 mg of nano-hydroxyapatite (HAP) and disperse it in 20 mL of deionized water, then sonicate for 5-15 minutes. Add the prepared CNDs-Pt solution (CNDs to HAP mass ratio 1:5) dropwise to the HAP suspension and stir magnetically at room temperature for 12 hours. Finally, collect the precipitate by centrifugation and freeze-dry to obtain HAP@Cur-CNDs-Pt NPs composite nanoparticle powder.

[0071] Characterization methods: The morphology, size, and composite composition of the nanoparticles were observed using transmission electron microscopy; surface charge changes were analyzed using zeta potential; SOD enzyme activity was detected using a SOD enzyme kit, and catalase activity was detected using a dissolved oxygen analyzer. For example... Figure 4 The transmission electron microscopy (TEM) observations clearly show the morphology, size, and successful composite formation of the nanoparticles, revealing that Pt NPs and Cur-CNDs are uniformly loaded onto the HAP support surface. Zeta potential analysis shows that the surface potential of HAP@Cur-CNDs-Pt NPs is positive. This was confirmed by superoxide dismutase (SOD) assay. Figure 4 The composite nanoparticles exhibit significant SOD enzyme activity, effectively catalyzing the disproportionation reaction of superoxide anions. Simultaneously, their catalase-like activity was measured using a dissolved oxygen analyzer. Figure 4(D), proving that the material can efficiently catalyze the decomposition of hydrogen peroxide.

[0072] The above characterization results collectively demonstrate that the HAP@Cur-CNDs-Pt NPs composite nanoparticles were successfully constructed, possessing the activities of both SOD and CAT, two key antioxidant enzymes. This provides direct experimental evidence for their core function of continuously scavenging various reactive oxygen species (ROS) and alleviating oxidative stress in the lower layer, and serves as a material basis for constructing a stable microenvironment conducive to bone regeneration.

[0073] 3D printing:

[0074] (1) Ink preparation:

[0075] Lower layer ink: The prepared HAP@Cur-CNDs-Pt NPs powder was dispersed at a concentration of 1% (w / v) in a solution containing 5% (w / v) GelMA, and a photoinitiator (Irgacure 2959, concentration 0.5%) was added.

[0076] Intermediate layer ink: Mix silk fibroin (SF) solution with 5% (w / v) GelMA solution at a volume ratio of 1:1, and add photoinitiator (concentration of 0.5%).

[0077] Upper layer ink: The prepared CuxO / SiC heterojunction powder was dispersed at a concentration of 500 μg / mL in a 5% (w / v) GelMA solution, and a photoinitiator (concentration of 0.5%) was added. Upper layer bio-ink, middle layer bio-ink, and lower layer bio-ink were prepared.

[0078] (2) The scaffold was fabricated using a multi-nozzle extrusion 3D printing system. The pore structure of the scaffold was individually designed based on the anatomical characteristics of the bone tissue to be repaired.

[0079] First, using a printhead filled with lower-layer ink, a regeneration-regulation layer is printed according to a preset program. The pore size design of this layer mainly mimics the open network structure of cancellous bone to facilitate rapid cell ingrowth and vascularization. For rat cranial bone repair, this layer is printed as a cubic lattice structure with a side length of approximately 200-400 μm.

[0080] Subsequently, the printhead was replaced with one containing intermediate layer ink to print the barrier layer on top of the lower layer. This layer mimics the dense properties of bone cortex, achieving a dense structure with no or extremely low porosity by increasing the density of the printed paths, thus providing effective physical insulation.

[0081] Finally, the printhead is replaced with one loaded with the upper ink layer to print the antibacterial-vascularized layer. The pore size design of this layer is based on the biological characteristics of the periosteum and its surface soft tissue, ensuring both effective contact between the antibacterial components and the environment, and facilitating the adhesion of soft tissue cells. For example, in a rat model, this layer is printed as a small-pore cubic lattice structure with a side length of approximately 100-200 μm.

[0082] During the printing process, after each layer is printed, it is immediately irradiated with 405nm blue light for 20-30 seconds for preliminary cross-linking and curing to ensure the shape and adhesion between layers are maintained. After printing, the entire scaffold is then irradiated with 405nm blue light for 1-2 minutes to achieve complete cross-linking and curing, ultimately resulting in a structurally stable, firmly bonded multilayer biomimetic hydrogel scaffold (its macroscopic morphology and microstructure are shown in the attached figure). Figure 5 (As shown in A, B, and C of 5).

[0083] Example 2: In vivo animal experiments to verify the efficacy of the stent

[0084] (1) Establishment of an infectious bone defect model: Adult SD rats were selected and anesthetized to create a full-thickness bone defect with a diameter of 5 mm in the cranial region. A certain concentration of Staphylococcus aureus bacterial solution (e.g., 1×10^8 CFU / mL) was injected into the bone defect, and the wound was sutured to establish an infectious bone defect model.

[0085] (2) Grouping and treatment: The rats that successfully developed the model were randomly divided into three groups:

[0086] Experimental group: The multilayer biomimetic hydrogel scaffold prepared in this invention was implanted, and the implantation area was periodically irradiated with 808nm laser (1W / cm², 10 minutes) after the operation.

[0087] Control group: implanted with pure GelMA scaffolds without functional nanoparticles.

[0088] Blank group: No implantation treatment was performed.

[0089] (3) Results analysis: Figure 6 The diagram and results of in vitro experiments to verify the functions of each layer of the scaffold include (A) the antibacterial effect of the upper CuxO / SiC heterostructure, (B) the angiogenesis-promoting ability, (C) the antioxidant effect of HAP@Cur-CNDs-Pt NPs, (D) the immunomodulatory effect, and (E) the osteogenic ability.

[0090] from Figure 6 It can be seen that the upper CuxO / SiC heterostructure has good antibacterial and angiogenesis-promoting effects, while HAP@Cur-CNDs-Pt NPs have good antioxidant, mitochondrial function restoration, macrophage polarization towards repair-type M1 and improved results.

[0091] Eight weeks post-surgery, the animals were euthanized for analysis. (See attached image.) Figure 7 As shown, from left to right: blank group, control group, and experimental group. Micro-CT ( Figure 7 The results showed that the amount of new bone formation in the experimental group was significantly higher than that in the control group and the blank group.

[0092] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A biomimetic hydrogel scaffold for repairing infectious bone defects, characterized in that: It consists of an upper antibacterial-vascularization layer that simulates the periosteum structure, an intermediate support layer that simulates the cortical bone structure, and a lower regeneration-regulation layer that simulates the cancellous bone structure. The upper antibacterial-vascularization layer is made of photoresponsive CuxO / SiC heterojunction powder, the intermediate support layer is made of silk fibroin, and the lower regeneration-regulation layer is made of HAP@Cur-CNDs-Pt NPs composite nanoparticles. The biomimetic hydrogel scaffold for repairing infectious bone defects was prepared according to the following steps: (1) Ink preparation: CuxO / SiC heterojunction powder, silk fibroin, HAP@Cur-CNDs-Pt NPs composite nanoparticles were mixed with photocrosslinked hydrogel prepolymer solution methacryloyl gelatin (GelMA) to prepare upper layer bio-ink, middle layer bio-ink and lower layer bio-ink. (2) 3D printing and cross-linking: Using a multi-nozzle extrusion 3D printing system, the lower, middle and upper layers are printed sequentially according to the preset biomimetic three-dimensional model and pore structure. During and after printing, the GelMA prepolymer is cross-linked and cured by light of a specific wavelength to form a stable three-dimensional hydrogel network, thus obtaining a biomimetic scaffold with integrated structure and function.

2. The biomimetic repair hydrogel scaffold for infectious bone defects according to claim 1, characterized in that, The photoresponsive CuxO / SiC heterojunction powder is prepared according to the following method: (1) Disperse 40-60 nm SiC nanoparticles in deionized water and ultrasonically treat them to form a uniform suspension; (2) Under stirring conditions, add copper salt solution to SiC suspension and continue stirring to allow copper ions to be fully adsorbed on the SiC surface; (3) Add the reducing agent solution dropwise; (4) Adjust the pH of the mixture to 8-9 using an alkaline solution; (5) Heat the mixture at 70-90℃ for 6-12 hours; (6) After the reaction is complete, the precipitate is collected by centrifugation and washed repeatedly with deionized water. Finally, CuxO / SiC heterojunction powder is obtained by freeze drying.

3. The biomimetic repair hydrogel scaffold for infectious bone defects according to claim 2, characterized in that: The copper salt is one of copper chloride, copper sulfate, and copper nitrate, and the mass ratio of the copper salt to SiC nanoparticles is 1:1; the reducing agent is L-ascorbic acid; and the alkaline solution is sodium hydroxide solution.

4. The biomimetic repair hydrogel scaffold for infectious bone defects according to any one of claims 1-3, characterized in that, The preparation method of the HAP@Cur-CNDs-Pt NPs composite nanoparticles is as follows: (1) Preparation of curcumin-derived carbon dots (Cur-CNDs): using curcumin as a precursor, carbonization was carried out at 400-500℃ by high-temperature pyrolysis. The carbonized product was then ground and dispersed in water, boiled, and purified by centrifugation, filtration, and dialysis to obtain an aqueous solution of Cur-CNDs with antioxidant activity. (2) Preparation of CNDs-Pt: The above Cur-CNDs solution was mixed with chloroplatinic acid solution. Under alkaline conditions, using the reducing property of Cur-CNDs as a carrier, sodium borohydride was added to assist in the preparation of Pt. 4+ In situ reduction to platinum nanoparticles, which are then loaded onto the surface of Cur-CNDs to form a CNDs-Pt complex, which is then purified by dialysis. (3) Composite of HAP@Cur-CNDs-Pt NPs: Nano-hydroxyapatite was dispersed in water and sonicated. The CNDs-Pt composite solution was added dropwise under stirring. CNDs-Pt was firmly loaded onto the HAP surface by electrostatic adsorption and coordination. After centrifugation and freeze-drying, the final composite nanoparticles were obtained.

5. The biomimetic repair hydrogel scaffold for infectious bone defects according to claim 4, characterized in that: In step (1), curcumin is carbonized at 400-500℃; after filtration, the supernatant is dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and the water is changed every 2 hours.

6. The biomimetic repair hydrogel scaffold for infectious bone defects according to claim 5, characterized in that, Step (2) is prepared as follows: Cur-CNDs solution is mixed with chloroplatinic acid solution, and the pH is adjusted to 9-11 with NaOH solution; sodium borohydride solution is slowly added under stirring, and the reaction is carried out at room temperature for 3-4 hours. After standing, the reaction is completed, and the solution is neutralized to neutral with hydrochloric acid. The solution is then dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain CNDs-Pt solution.

7. The method for preparing the biomimetic repair hydrogel scaffold for infectious bone defects according to claim 6, characterized in that: Each printed layer is initially cross-linked and cured by irradiating with 405nm blue light for 30 seconds. After printing, the entire scaffold is then irradiated with 405nm blue light for 2 minutes to achieve complete cross-linking and curing, resulting in the final multilayer biomimetic repair hydrogel scaffold.

8. The use of the biomimetic repair hydrogel scaffold for infectious bone defects according to any one of claims 1-7 in the preparation of medical devices or tissue-engineered products for repairing conventional or infectious bone defects.

9. The application according to claim 8, characterized in that, The infectious bone defects are maxillofacial bone defects or skull defects caused by Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa infection.

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